Potential application spectrum of microbial proteases for clean and green industrial production

Energy, Ecology and Environment, Sep 2017

Enzymes are the cornerstones of metabolism and constitute the fundamental basis for existence of life. However, recently enzymes are being implicated in diverse industrial processes because of their specific and fast action for efficient bioconversion of substrate to product, and their capability to save raw materials, energy and chemicals for various manufacturing processes. Enzymes are considered as environment-friendly (green) chemicals that may potentially help replacing completely or reducing the usage of hazardous chemicals for industrial processes, thus promising sustainable production and manufacturing. Among various industrial enzymes microbial proteases dominate the world enzyme market due to their multifaceted application potential in varied bioindustries like food, pharmaceutical, textile, photographic, leather and detergent. Promising applications of proteases in agricultural sector for instance may include biocontrol of pests, degumming of silk, selective delignification of hemp and wool processing. However, for successful industrial applications the proteases must be robust enough to suit the process conditions which are generally hostile. Proteases intended for industrial applications must have activity and stability over wide range of temperature and pH extremes for prolonged time periods and even in the presence of various potential enzyme inhibitors. Of various microbial proteases those from Bacillus spp. have got special significance because the latter are known for their ability to produce sturdy enzymes that might have suitability for industrial process conditions. The current article presents an interpretive summary of the recent developments on application potential of proteases for various industries.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://link.springer.com/content/pdf/10.1007%2Fs40974-017-0076-5.pdf

Potential application spectrum of microbial proteases for clean and green industrial production

Potential application spectrum of microbial proteases for clean and green industrial production Satbir Singh 1 2 Bijender Kumar Bajaj 0 1 2 0 Department of Biosciences, Durham University , South Road, Durham DH1 3LE , UK 1 & Bijender Kumar Bajaj 2 School of Biotechnology, University of Jammu , Jammu 180006 , India Enzymes are the cornerstones of metabolism and constitute the fundamental basis for existence of life. However, recently enzymes are being implicated in diverse industrial processes because of their specific and fast action for efficient bioconversion of substrate to product, and their capability to save raw materials, energy and chemicals for various manufacturing processes. Enzymes are considered as environment-friendly (green) chemicals that may potentially help replacing completely or reducing the usage of hazardous chemicals for industrial processes, thus promising sustainable production and manufacturing. Among various industrial enzymes microbial proteases dominate the world enzyme market due to their multifaceted application potential in varied bioindustries like food, pharmaceutical, textile, photographic, leather and detergent. Promising applications of proteases in agricultural sector for instance may include biocontrol of pests, degumming of silk, selective delignification of hemp and wool processing. However, for successful industrial applications the proteases must be robust enough to suit the process conditions which are generally hostile. Proteases intended for industrial applications must have activity and stability over wide range of temperature and pH extremes for prolonged time periods and even in the presence of various potential enzyme inhibitors. Of various microbial proteases those from Bacillus spp. have got special significance because the latter are known for their ability to produce sturdy enzymes that might have suitability for industrial process conditions. The current article presents an interpretive summary of the recent developments on application potential of proteases for various industries. Microbial enzymes; Protease; Bacillus; Applications; Eco-friendly; Pollution 1 Introduction Green chemistry also called sustainable chemistry aims at utilizing preferably renewable raw materials, avoiding toxic and hazardous chemicals, thereby producing minimum wastes during commercial production and manufacturing (Sheldon et al. 2007; Sharma and Bajaj 2017) . Furthermore, considering the ever-growing world energy demand, industrialization, rapidly depleting fossil-fuel reserves, environmental health issues, there is emphasis on development of sustainable industrial technologies that are environmentally safe and involve minimum emissions (Jegannathan and Nielsen 2013; Vaid and Bajaj 2017) . As a result there is a paradigm shift from traditional concepts of chemical-based production and manufacturing to eco-benign processes that are equally efficient and economic (Sarrouh et al. 2012; Nargotra et al. 2016) . Application of enzymes for industrial processes may contribute significantly towards development of environmentally benign processes (Bajaj and Jamwal 2013; Mhamdi et al. 2017) . Enzyme-based technologies promise efficient utilization of raw materials, generation of minimal or no wastes, and shun the usage of toxic chemicals (Singh and Bajaj 2016, 2017) . Enzymes are the fundamental molecules that govern various metabolic processes in living systems, and constitute the very foundation for the existence of life. However, recently, enzymes have become the focus of intense research by process engineers and production chemists mainly due to recognition of their application potential in various industries. Enzyme-based industrial processes are much more environmentally safer than the traditional chemical ones and involve less emissions (Sheldon et al. 2007) . But considering that enzymes are the biological molecules which are largely designed (by nature) to function under ambient conditions in the living systems, may not suit well for relatively hostile industrial process conditions (Bajaj et al. 2014) . Therefore, discovery of sturdy enzymes that are capable of functioning under industrial processes has gained considerable research impetus in recent years (Sharma and Bajaj 2017) . There is continuous and intense research focus on targeting processapt robust enzymes either by exploiting the enormous natural microbial diversity or by bioengineering (Singh and Bajaj 2015; Guleria et al. 2016) . Microbial enzymes may potentially be utilized for numerous industrial applications and may help replacing toxic and hazardous chemical catalysts (Nigam et al. 2012) . The continual exploration of application potential of enzymes has expanded the enzyme industrial market at annual growth rate of 7.6% (David et al. 2009) . Proteases are one of the most important groups of industrial enzymes that have been studied extensively during recent years (Sarrouh et al. 2012) . Proteases (EC 3.4) are a group enzymes which occur in all live forms, and execute a large variety of complex physiological and metabolic functions in living systems like cell division, signal transduction, digestion, blood pressure regulation, apoptosis and several others (Theron and Divol 2014). Proteases represent approximately 1–5% of the total gene content (Qazi et al. 2008) . Proteases constitute 60% of the global industrial market due to their huge application potential in diverse industries (Fig. 1). Proteases are used in pharmaceutical industry (Kumar et al. 2015) , food industry for peptide synthesis (Kumar and Bhalla 2005), leather industry for dehairing (Pillai et al. 2011; Singh and Bajaj 2017) , photographic industry for silver recovery (Joshi and Satyanarayana 2013) , detergent industry as an additive for detergent formulation (Giri et al. 2011) and in processing of keratin residues (Harde et al. 2011) . In addition, the proteases are also used for other applications such as contact lens cleaning (Pawar et al. 2009) , biofilm removal (Leslie 2011) , isolation of nucleic acid (Motyan et al. 2013) , pest control (Joshi and Satyanarayana 2013), degumming of silk (Mahmoodi et al. 2010) and selective delignification of hemp (Khan 2013) . The proteases intended for biotechnological processes must be robust enough and have the capability of kinetic and structural adaptations under extreme industrial microenvironments, e.g. extremes of temperatures, pH and presence of inhibitors (Singh et al. 2014) . Such proteases may be targeted from microbial sources, especially the extremophiles, i.e. microbes which are inhabitants of extreme ecological niches (Białkowska et al. 2016) . Microbial proteases account for approximately 40% of the total worldwide enzyme sales (Rani et al. 2012) . Microbes are the goldmines not only for proteases but also for other industrial products. Microorganisms represent the most preferred enzyme sources due to several unique advantages (Bajaj and Wani 2011; Sharma and Bajaj 2017) . Exploration of microbial diversity for targeting novel process-suitable proteases, has been an ongoing practice (Singh and Bajaj 2015, 2016) . Among various microbial groups, bacteria due to their enormous diversity and other special merits have become interesting source for industrial enzymes including proteases (Bajaj and Sharma 2011) . For industrial enzymes, strains of Bacillus spp. have gained much importance and account for 35% of total microbial enzyme sale (Jayakumar et al. 2012) . This is because of the ability of Bacillus spp. to produce enzymes that suit well the process conditions in industries (Priya et al. 2014; Rehman et al. 2017) . Enzymes from Bacillus spp. have poly-extremotolerance, i.e. capability of functioning under adverse process conditions like extreme of temperature, pH, presence of solvents, detergents and other potential enzyme inhibitors (Joshi and Satyanarayana 2013). Furthermore, Bacillus spp. may represent a model system for the heterologous expression of genes (Sadeghi et al. 2009) . For biotechnological processes involving protein production, several species of Bacillus, viz. B. subtilis, B. amyloliquefaciens and B. licheniformis and others, have become the most popular due to their excellent fermentation properties, high product yields and the complete lack of toxic by-products (Dijl and Hecker 2013; Singh and Bajaj 2016; Guleria et al. 2016) . The enzymes being marketed by Novo Industry, Denmark, are produced from Bacillus spp. (Georgieva et al. 2006; Jisha et al. 2013) . There are several reports of thermostable and wide range pH stable proteases from Bacillus spp. which have excellent compatibility for industrial processes (Baweja et al. 2016; Guleria et al. 2016) . The genus Bacillus is represented by Gram-positive, spore-forming, rod-shape bacteria that are obligate aerobes or facultative anaerobes (Dijl and Hecker 2013) . In view of the grave threat posed by the polluting chemical-based industries, development of sustainable and environmentally friendly technologies is being emphasized. Proteases are the lead enzymes from industrial application view point. The current article presents a recent research survey of robust proteases especially from Bacillus spp. that are apt for industrial process conditions. Fig. 1 Applications of proteases in different industrial sectors 2 Proteases in detergent industry Detergent enzymes are eco-friendly solution that is being used to improve the cleaning efficiency of conventional detergents (Kumar et al. 2008; Khajuria et al. 2015) . The industrial and economic importance of alkaline proteases, especially those from Bacillus spp. in the detergent industry, was realized during 1960s (Kazan et al. 2005). The use of enzymes in detergent formulations is now common in developed countries. Most of the currently marketed detergents contain enzymes (Kumar et al. 2008) . The use of proteases in the laundry detergents constitutes a big market share and accounts for approximately 25% of the total worldwide sale of enzymes (Tanksale et al. 2001) . Today, the enzymes are the staple ingredients of powder as well as liquid detergents, stain removers, laundry prespotters, automatic dishwashing detergents and other industrial and medical cleaning products. Detergent industry is one of the largest industries where enzymes usage is being practised. The enzymes enhance the efficacy of detergents for removing proteins from cloths soiled with sweat, grass, milk, egg, blood, etc. (Ida et al. 2016) . Proteases are the most extensively used enzymes in detergent formulations; however, amylases and lipases are also being added. Proteases work very efficiently as scissors to cut off the stains from cloths (Shankar and Laxman 2015) . Contrary to enzyme detergents, the non-enzymatic detergents are inefficient in removing proteins from cloths as organic stains adhere strongly to textile fibres, and become permanent due to oxidation and denaturation caused during bleaching and drying operations. Performance of protease in laundry detergents has been reported to be enhanced when applied in combination with lipase, amylase and cellulase (Khan 2013) . The ideal detergent protease should possess broad substrate specificity to facilitate removal of a variety of stains due to blood, food and other body secretions. Activity and stability at high pH and temperature (Giri et al. 2011; Padmapriya et al. 2012) and compatibility with other components of detergents like chelating and oxidizing agents (Nascimento and Martins 2006; Tekin et al. 2012) are among the major pre-requisites for the proteases intended for application in detergents (Giri et al. 2011) . Proteases from several Bacillus spp. are well characterized for usage as detergent additives, e.g. B. subtilis, B. cohnii, B. clausii, B. licheniformis and B. brevis (Guleria et al. 2016) . Alcalase was the first detergent protease developed by Novozyme during 1960s. Since then several other enzymes have been developed by Novozymes for removing protein stains, viz. Esperase, Savinase and Everlase. All of these are well suited to detergent formulations particularly at alkaline pH (Sumantha et al. 2006) . Protease from Bacillus pumilus MP 27 was considered as a good candidate for detergent industry due to its broad substrate range, activity over high and low temperatures and pH, and compatibility with surfactants and commercial detergents (Baweja et al. 2016) . Bacillus subtilis KT004404, an isolate from hydrothermal vents, produced a metalloprotease that has high resistance towards anionic surfactants, solvents and detergents. Additionally, the protease had good destaining properties which reflected its potential for application in detergent industries (Rehman et al. 2017) . Extremely pH stable and thermostable proteases have been reported from Micromonospora chaiyaphumensis S103 (Mhamdi et al. 2017) and from Aeromonas caviae (Datta et al. 2016) . Additionally, the proteases exhibited tolerance towards several organic solvents. Such proteases may have prospective for application as ideal additives for detergent formulations. Keratinolytic proteases (keratinases) have the ability to bind and hydrolyse solid substrates like hair (Singh et al. 2014) . Keratinolytic potential is an important property sought in detergent enzymes that may help removing protein substrates from solid surfaces. Keratinolytic proteases are attractive additives for detergents, especially for cleaning hard surfaces (Brandelli et al. 2010) . Furthermore, keratinases may also help in the removal of keratinous soils particularly from collars of shirts, on which most proteases fail to act (Gessesse et al. 2003) . Application of enzymes in detergents not only enhances cleansing efficacy, especially for rigid biomaterials, but also allows the washing to be accomplished at relatively low temperatures, thus saving energy and making the process economic (Rehman et al. 2017) . Supplementation of enzymes in detergents helps in reducing quantities of other hazardous chemical-based detergent components like soaps, oxidizing agents, chelating agents and surfactants, thus making detergents more eco-friendly. But there may be some limitations of enzymes usage in detergents, i.e. enzymes may be expensive to produce, may exhibit some allergic reactions, may get denatured at elevated temperatures and pH extremes and may digest some fabrics (wool). However, these limitations can be readily overcome by selecting the most apt detergent-compatible enzymes from the vast diversity of microbial proteases (Singh et al. 2014) . 3 Proteases in leather industry Vicious exploitation of natural resources, reckless usage of hazardous chemicals and consequential enormous environmental pollution have led to the concept of cleaner production. Tanneries represent one such industry that contributes heavily towards environmental degradation, and require serious attempts for mitigating its impact on the environment. Application of enzymes in leather processing may help developing eco-benign process that is less harmful to the environment (Pillai et al. 2011) . The studies on application of enzymes in leather processing and production were commenced in 1910. Since then a significant research has been undertaken, and currently several enzymes are being used at commercial level for leather processing. Leather processing involves several steps, viz. curing, soaking, liming, dehairing, bating, pickling, degreasing and tanning. The wastes or the effluents generated during these steps not only cause enormous environmental pollution and pose brutal threat to ecosystem, but may be severely health hazardous due to the presence of high concentrations of sulphide and chromium (Nilegaonkar et al. 2007) . In leather processing, the primary objective is to remove hair and open up the fibre structure suitably to get the desired properties in final finished leather. The conventional method of dehairing or depilation involves the usage of lime and sodium sulphide as it is more efficient and cheaper than other currently available technologies (Pillai et al. 2011) . Chromium salts are the most commonly used tanning agents. But this process is highly polluting and health hazardous due to the presence of residues of these chemicals in tannery waste. The process poses a serious health threat to tannery workers (Nilegaonkar et al. 2007) . The tannery effluent has high total dissolved solids (TDS) that contribute increased pollution load, i.e. BOD and COD in the wastewater. The use of enzymes for leather dehairing process involves proteolytic cleavage of cementing substances which holds the hair to the skin so that the hair can be removed without destruction. Enzymatic dehairing may involve application of several enzymes like proteases, amylases and lipases. Moreover, the application of enzymes for leather processing enhances the quality of leather and gives stronger and softer leather with less spots (Madhavi et al. 2011) . Enzymatic approach for leather processing is currently being explored as an eco-friendly option, especially for obviating or minimizing the conventional sodium sulphide-based processing (Sundararajan et al. 2011) . Different strategies have been used for the cost-effective production of dehairing proteases that may be used for developing greener and cleaner leather processing regime. Alkaline protease produced by B. subtilis strain VV under solid-state fermentation (SSF) on cow dung substrate, exhibited promising dehairing potential (Vijayaraghavan et al. 2012) . Production of alkaline protease that has potential for application in leather processing was increased by mutagenesis of B. licheniformis N-2 using UV irradiation and other chemical mutagens (Nadeem et al. 2010) . Leather industry waste was used as substrate (under SSF) for the production of alkaline protease from B. cereus 1173900 that has suitability for leather processing application (Ravindran et al. 2011) . Keratinolytic proteases with no collagenolytic activity and mild elastolytic activity are preferably used for dehairing process. Keratinase selectively breaks keratin tissue of the follicle, thereby pulling out intact hairs without affecting the tensile strength of leather (Macedo et al. 2008) . A metallokeratinase produced by Acinetobacter sp. R-1 had high keratinase activity and low collagenase activity. The keratinase showed reasonably good depilation on goat skin (Zhang et al. 2016b) . Additionally, the ability of keratinase to modify the wool surface showed its application potential in textile industry. Alkaline proteases from Bacillus spp. are the suitable candidates for efficient dehairing of hides in leather industry. There are several reports wherein dehairing potential of Bacillus proteases has been characterized, e.g. B. altitudinis GVC11 (Kumar et al. 2011) , B. cereus MCM B-326 (Nilegaonkar et al. 2007) , B. cereus VITSN04 (Sundararajan et al., 2011) , B. halodurans JB 99 (Shrinivas et al. 2012) , B. pumilus MCAS8 (Jayakumar et al. 2012) , B. subtilis (Sathiya 2013) and B. subtilis KT004404 (Rehman et al. 2017) . A thermostable alkaline protease from Bacillus amyloliquefaciens SP1 exhibited excellent dehairing (goat skin) potential (Guleria et al. 2016) . Considering the high risks associated with conventional leather processing, there is considerable emphasis on developing enzymatic approaches that are cleaner and safer. Such biobased processes would help reducing emissions. Thus, exploitation of enzymes for leather processing is beneficial in terms of improved process efficiency, highly specific enzyme-based catalysis, enhanced leather output and superior quality of leather. 4 Proteases for processing of keratin wastes Keratin-containing wastes are abundant in nature and have high protein content. However, accumulation of keratin wastes generally leads to environmental pollution as well as wastage of precious protein reserves (Mukherjee et al. 2011) . Keratin wastes could be transformed into protein hydrolysate that may have application as amino acid-rich animal feed supplement. Moreover, the protein hydrolysate can be used for the production of various essential amino acids such as lysine. Villa et al. (2013) reported the application potential of keratin hydrolysates produced from feathers for formulation of hair shampoos. Keratin in its native state structure is highly stable due to the presence of tightly packed helices and sheets with large number of disulphide bonds and is not easily degraded by common proteolytic enzymes like trypsin, papain and pepsin (Daroit et al. 2011) . Composition and molecular configuration of keratin, its constituent amino acids, disulphide bonds and cross-linkages are responsible for its hardness and insolubility (Parradoa et al. 2014) . Keratin occurs in nature mainly in the form of hair, horn, nails and cornified tissue (Gupta and Nayak 2015) . Feathers represent a rich protein source and contain about 90% of proteins and can be used for the production of protein-rich hydrolysate. Worldwide 24 billion chickens are killed annually, and around 8.5 billion tonnes of poultry feathers is produced. The poultry feathers are mostly discarded, and this not only adds to environmental pollution but also causes wastage of precious protein-rich reserve (Agrahari and Wadhwa 2010) . Furthermore, incineration of the huge amounts of keratin solid waste may have several ecological disadvantages (Deydier et al. 2005) . Conventional methods of feather processing are cost and energy intensive, involve harsh process conditions like high pressure and temperature and cause loss of nutritional value (Grazziotin et al. 2006) . Therefore, development of an eco-friendly enzyme-based technology for processing keratin waste is the need of the hour. Keratinases have been reported from several bacterial spp. (Singh et al. 2014) . Bacillus subtilis K-5 utilized a wide range of keratinous wastes, viz. diverse feather types, nails, hair and scales, for growth and keratinase production. Keratinase exhibited activity and stability over a broad pH (5–10) and temperature range (50–90 C) and showed multifarious application spectrum for blood stain removal from fabric, gelatin hydrolysis from waste X-ray films and dehairing of animal hide (Singh et al. 2014) . A thermostable and wide range pH stable protease from Micromonospora chaiyaphumensis S103 showed excellent potential for deproteinization of shrimp wastes for production of chitin (Mhamdi et al. 2017) . Bacterial keratinases, mostly from Bacillus spp., have been used to convert hard-to-degrade keratin into protein substitutes (Kainoor and Naik 2010; Harde et al. 2011) . Feather meal is nitrogen-rich, inexpensive and readily available source which may serve as potential substitute to guano (Brandelli et al. 2010) . The non-conventional sources like wastes from agriculture, poultry, meat and fish industry have been exploited to meet the demand of lowcost protein foods. Studies have shown that keratin hydrolysates may serve as good organic fertilizers (Gousterova et al. 2005) and as amino acid supplements for health foods and pet foods (Gupta and Ramnani 2006) . Two keratinases produced by B. licheniformis PWD-1, i.e. Versazyme and Cibenza DP100, have shown excellent results in broiler performance and growth of piglets (Odetallah et al. 2005; Wang et al. 2011) . Thus, the enzymatic hydrolysates produced by bioprocessing of keratin wastes may have potential for applications in food, feed and cosmetic industries. The process not only promises eco-friendly and sustainable approach for valorization of ‘wastes to wealth’, but opens new avenues for development of potentially novel biotechnological processes that are good for environmental health. 5 Fibrinolytic proteases as thrombolytic agents Fibrin is an insoluble protein derived from its soluble precursor, fibrinogen, which is involved in blood clotting. Fibrin plays a vital role in health and healing; however, formation of inappropriate clot especially under certain pathophysiological conditions in the body is a major risk factor for heart disorders (Bajaj et al. 2013, 2014) . Homoeostasis of formation and dissolution of fibrin maintain appropriate viscosity in the vascular system. A shift in balance towards fibrin overproduction leads to unwanted clotting resulting in cardiac complications like acute myocardial infarction, ischaemic heart diseases, valvular heart diseases, peripheral vascular diseases, arrhythmias, high blood pressure and stroke (Mine et al. 2005) . Various thrombolytic agents have been used for the removal of clots, such as plasminogen activators, like tissue-type plasminogen activator and urokinase, which trigger the conversion of plasminogen into active plasmin (Hwang et al. 2007) . Lumbrokinase from earthworm, fibrolase from snake venom and other plasmin-like proteins directly degrade fibrin, thereby dissolving thrombi rapidly and completely (Jayalakshmi et al. 2012) . Although plasminogen activators and urokinase are still widely used in thrombolytic therapy, their expensive prices and undesirable side effects have prompted researchers to target novel, cheaper and safer resources (Agrebi et al. 2010; Deepak et al. 2010) . Microbial fibrinolytic proteases have been reported from several microorganisms including Bacillus spp. (Kim et al. 2009; Jo et al. 2011a) . Production of fibrinolytic protease from a mutant strain of B. cereus GD55 was optimized using apple pomace as substrate (Raju and Goli 2014) . Bacillus spp. are well known for production of potent fibrin-degrading enzymes (Wang et al. 2009; Bajaj et al. 2013) as shown in Table 1. Bafibrinase, a non-toxic, nonhaemorrhagic fibrinolytic serine protease isolated from Bacillus sp., exhibited in vivo anticoagulant activity and thrombolytic potency (Mukherjee et al. 2012) . Process optimization techniques have been used for enhancing production of fibrinolytic proteases (Ashipala and He 2008; Agrebi et al. 2009; Mahajan et al. 2012) . Bacillus amyloliquefaciens UFPEDA 485 produced fibrinolytic protease that exhibited long-term stability, and activity at physiological conditions, and could be of therapeutic importance for human and veterinary applications (de Souza et al. 2016) . Streptomyces sp. CC5 produced a novel fibrinolytic protease that has high substrate specificity, strong thrombolytic activity, no toxicity and no prolonged bleeding time. Carrageenan-induced mouse tail (thrombosis model) was used for demonstrating excellent thrombolytic activity. Thus, the proteases may have potential for application in antithrombotic drug development (Sun et al. 2016) . A marine Streptomyces violaceus VITYGM produced an extracellular thrombolytic protease that had efficient blood clot lysis activity (Mohanasrinivasan et al. 2016) . This novel actinoprotease may have the potential for developing drug for the treatment of cardiovascular diseases. A fibrinolytic enzyme was purified (36-fold) from Cordyceps militaris, a medicinal mushroom. The enzyme was characterized for several properties and analysed for amino acid sequence. The fibrinolytic enzyme was capable of degrading a-, b- and c-chains of fibrinogen and activating plasminogen into plasmin. The enzyme can act as an anticoagulant and prevent clot formation by degrading fibrinogen. Thus, fibrin-degrading proteases may have great potential for prevention and treatment of thrombolytic diseases (Liu et al. 2017) . Keeping in view the fast emergence of cardiovascular problems including thrombosis, there is increased demand for chemotherapeutic thrombolytic agents. Most of the chemotherapeutics available are extremely expensive and have a range of side effects on the patients; therefore, there is a huge research impetus on investigating novel fibrinolytic agents that are safe, economic and effective for treatment. 6 Proteases for biofilm removal Biofilms also termed as ‘city of microbes’ are a structure formed by extracellular polymeric substances (EPS) and the bacteria enclosed within it (Leslie 2011) . The EPS consist mostly of polysaccharides, proteins and nucleic acids (Leroy et al. 2008) . These substances form structural matrix which help the bacteria to attach to the surface. EPS also facilitate survival in the adverse conditions and environments (Kostakioti et al. 2013) . Biofilm may be formed anywhere as long as the nutrients and a surface for adhesion are available. Biofilm is formed frequently in industrial systems associated with wastewater management, food processing, brewing, pulp and paper manufacturing and dairies. Harmful biofilms may cause grave economic losses due to reduced productivity, decreased product quality and loss of time and expenses for biofilm removal (Leslie 2011) . The durable microbial colonies in the biofilms impose an economic burden on companies as these may cause contamination by biofouling, i.e. the accumulation of microorganisms in aqueous environments. Bacterial adhesion is the initial step in colonization and is followed by increasing cell numbers and area of adherence, finally resulting in biofilm formation (Fig. 2). Biofilms are Douchi Traditional food in China 30 kDa, pH 8.0 and 50 C 30 kDa, pH 8.0 and 60 C Marine water Vector Control Research Centre, Puducherry Marine niches 28 kDa, pH 9.0 and 60 C 18.6 kDa 28 kDa, pH 9.0 and 50 C – 27 kDa Cheonggukjang Meju Wang et al. (2009) PMSF inhibited activity Wang et al. (2008) Soybean trypsin inhibitor inhibited Wang et al. (2006) activity Activity totally lost with PMSF Agrebi et al. (2009) Fibrinolytic activity was similar to that Balaraman and of streptokinase Prabakaran (2007) Activated by Ca2? and inhibited by Mahajan et al. Zn2?, Fe2?, Hg2? and PMSF (2012) Ashipala and He (2008) Lee et al. (2010) CCD used to optimize enzyme production Gene apr E86-1 was expressed in B. subtilis Jo et al. (2011b) Ghafoor and Hasnain (2010) Kim et al. (2009) Jayalakshmi et al. (2012) Hassanein et al. (2011) Yeo et al. (2011) Agrebi et al. (2010) Kim et al. (2006) Wang et al. (2011) Deepak et al. (2008) Bajaj et al. (2013) Ornamental plant nursery 27 kDa, pH 8.5 and 65 C Activity reduced by PMSF Cheonggukjang 27 kDa, pH 6.0 and 45 C Tryptic soy broth was best for enzyme production University of Madras, Chennai Soybean flour 24.6–33.0 kDa, pH 7.0–12 and 50 C 20.5 kDa, pH 9.4 and 40 C Inhibited by PMSF Organic solvent stable 27 kDa, pH 7.0 and 45 C Gene apr E5-41 was expressed in B. subtilis 37 kDa, pH 9.0 and 40 C Activity inhibited by PMSF Hwang et al. (2007) 28 kDa, pH 6.0–10 and 50 C 30 kDa, pH 9.0 and 60 C 29 kDa, pH 7.0 and 50 C 28–30 kDa, pH 8.0 and 40 C – pH 9.0 and 40 C PMSF, DIFP and TPCK reduced enzyme activity Mirabilis jalapa tuber powder used as complex media Enzyme was stable towards organic solvents Shrimp shell wastes used as sole C/N source CCRD was used to optimize medium Fe2? favours enzyme activity. Inhibited by Pb2? and Hg2? detrimental to both human life and industrial processes due to their association with infection, pathogen contamination, biofouling and slime formation. But sometimes biofilms are desirable and beneficial for example, in probiotics-gut adhesion (Gupta and Bajaj 2017) , environmental technologies and bioprocesses (Hori and Matsumoto 2010) . The most effective method for removing a biofilm is by the clean-in-place (CIP) method in combination with chemicals involving manual scrubbing of the affected area (Jessen and Lammert 2003) . But the method is impractical for larger structures where regions like joints, filters or gaskets are not easily accessible. Microbial enzymes may be safer and more efficient alternatives to traditional chemical means of biofilm removal. Proteases are the most commonly used biofilm removal agents like Savinase and Everlase; however, other hydrolases have also been exploited (Molobela et al. 2010) . Amylases, when used in combination with proteases, help to eliminate existing biofilms and prevent bacteria from adhering to surfaces (Deinhammer and Andersen 2011) . Considering the extreme conditions of industrial processes robust proteases that function optimally under such conditions are desired. Pernisine, a protease produced by Aeropyrum pernix, is optimally active at 90 C and could be used as potential biofilm-removing agent (Molobela et al. 2010) . Oulahal-Lagsir et al. (2003) examined the combined treatment which involved the application of ultrasound and enzyme preparations for removal of an E. coli model biofilm, made with milk on stainless steel sheets. Orgaz et al. (2006) demonstrated the use of enzymes from three different fungal sources, i.e. Aspergillus niger, Trichoderma viride and Penicillium spp., for their potential ability to remove Pseudomonas fluorescens biofilm. 7 Proteases for silk degumming Silk, also known as the ‘queen of fabrics’, is a composite material with two fibroin filaments surrounded by a cementing layer of sericin (Mahmoodi et al. 2010; More et al. 2013) . Silk processing from cocoons to the final finished clothing and articles involves several steps which include reeling, weaving, degumming, dyeing or printing and finishing (Zahn 1993) . Degumming is the process where sericin, i.e. the silk gum gluing the fibroin filaments, is totally removed in order to obtain silk with desirable properties (Fig. 3). Degumming of silk is traditionally carried out with soap or alkali. These methods face major limitations like degummed silk obtained is not of uniform quality, big loss of strength of silk, short shelf life of silk and highly hygroscopic nature of silk. Additionally, the usage of chemicals in the traditional methods causes environmental pollution and makes the process environmentally unsafe. Therefore, there is great impetus on developing enzyme-based silk-degumming process that is low energy demanding, uses least chemicals, maintains high strength of silk fibre and is environmentally healthy (Nakpathom et al. 2009) . Proteolytic enzymes, which can cleave the peptide bonds of sericin without destroying the fibroin, may have potential for application as degumming agents. Proteases are being projected as a replacement of the harsh and energy demanding chemicals for treatment process. Plant proteases like papain (Nakpathom et al. 2009) and bromelain (Devi 2012) are the effective cocoon cooking enzymes that are commonly used for the processing of cocoons. A bacterial enzyme Alkalase marketed by Novo has been found to be very effective in hydrolysing sericin. The combination of lipase and protease has been reported to be an effective approach for dewaxing and degumming. Furthermore, this enzyme cocktail provides positive effects on wettability of silk fibres (Freddi et al. 2003) . Thus, the enzyme-based technology could be used effectively for silk degumming in industries as an eco-friendly alternative. The production of silk-degumming protease from B. subtilis C4 was optimized to get an enhanced protease yield (Romsomsa et al. 2010) . Joshi and Satyanarayana (2013) reported a recombinant alkaline serine protease from a novel bacterium B. lehensis which enhanced the softness and shine of silk fibres. The protease could possibly be employed as an ecologically benign alternative to traditional harsh chemicals used in degumming processes. Furthermore, the nitrogen-rich discharges from enzymebased silk-degumming process may potentially be utilized as a nutrient substrate for microbial growth and protease Fig. 3 Structure of silk fibre and degumming of silk production. This degumming discharge has been used as substrate for protease production from B. licheniformis and Aspergillus flavus (Vaithanomsat et al. 2008) . Moreover, the degumming waste liquor that is rich in sericin content is being used as a raw material for the production of sericin powder. The sericin powder may have application in cosmetic industry as a moisturizer, for hair-care products and as a natural textile finish. Thus, enzyme-based silk degumming is an eco-friendly approach that may potentially help reducing usage of hazardous chemicals and undesired emissions due to usage of chemicals and fossil fuels. 8 Proteases in photographic industry The exposed X-ray films have approximately 5–15 g of silver per kg of film (Marinkovic et al. 2006) . Silver recovery from waste photofilms is a big business. Around 18–20% of the world’s silver needs are supplied by recycling photographic waste (Shankar et al. 2010) . Nearly 2.0 billion radiographs are taken each year, including chest X-rays, mammograms and CT scans (Cavello et al. 2013) . Since silver is linked to gelatin in the emulsion layer, it is possible to break the same and release the silver which could be used as source of secondary silver (Fig. 4). The conventional method for silver recovery involves burning the films directly. This method is the most primitive one and generates undesirable foul smell and causes enormous environmental pollution by producing undesired emissions. The chemical method for silver recovery from X-ray films involves usage of acid and alkali, and thus, it is quite harsh and environmentally hazardous (Marinkovic et al. 2006; Ekpunobi et al. 2013) . Furthermore, the polyester film on which emulsion of silver and gelatin is coated cannot be recovered by these methods. Enzyme-based method may be developed that may not only recover silver efficiently but might have minimal impact on the environment (Nakiboglu et al. 2003) . Proteases have been reported to possess excellent gelatinolytic activity for successful recovery of silver from X-ray films. Several Bacillus spp. proteases, viz. B. lehensis (Joshi and Satyanarayana 2013), B. subtilis (Nakiboglu et al. 2001; Kumaran et al. 2013) , B. cereus (Bajaj et al. 2013) and B. licheniformis (Pathak and Deshmukh 2012) , B. subtilis K-5 (Singh et al. 2014) and B. licheniformis K-3 (Singh and Bajaj 2017) , have been demonstrated to possess good gelatinolytic activity for efficient silver recovery from X-ray films. Proteases from other sources like Aspergillus versicolor (Choudhary 2013) and Purpureocillium lilacinum (Cavello et al. 2013) have also shown good gelatin hydrolysing ability. High temperature and slightly alkaline conditions favour stripping off of the gelatin layer. Thus, thermostable alkaline proteases from Bacillus spp. are well suited for the process (Nakiboglu et al. 2001). Nutritionally enriched medium was used for the production of proteolytic enzyme that was capable of efficiently hydrolysing X-ray-bound gelatin (Kumaran et al. 2013) . Considering the eco-unfriendly nature of chemicalbased methods of silver extraction from X-ray films, a greener approach that is based on application of enzymes is gaining attention. The enzyme-based silver extraction from X-ray films relies more on renewable energy resources than on fossil fuel and, thus, might offer an overall eco-safe process. Fig. 4 Gelatin hydrolysis for the release of silver from X-ray film 9 Proteases in food industry Proteases are used for a wide range of food processing applications, e.g. in dairy, bakery, fish and seafood processing, animal protein processing, meat tenderization, plant protein processing and generation of bioactive peptides. Major aim for application of enzymes in food processing is to enhance the nutritional and functional properties of foods such as improved digestibility, modifications of sensory quality, improvement of antioxidant capability and reduction of allergenic compounds (Tavano 2013) . However, the choice of enzyme and the desired degree of hydrolysis must be realized by taking into account the taste, solubility and specific application properties of the hydrolysate product. Microbial proteases have been exploited in the food industries in variety of ways. Primarily proteases have been used in the food industries for production of protein hydrolysates of high nutritional value. The protein hydrolysates can be used for blood pressure regulation, for infant food formulations, for specific therapeutic dietary products and for the fortification of fruit juices and soft drinks (Ray 2012) . However, the bitter taste of protein hydrolysates is a major barrier in their wide range utilization. Intensity of bitterness is proportional to the presence of hydrophobic amino acids in the hydrolysates (Sumantha et al. 2006) . Exopeptidases like leucine aminopeptidase and those that can cleave hydrophobic amino acids and proline are valuable in debittering protein hydrolysates (Sumantha et al. 2006) . Major application of proteases in dairy industry is the manufacture of cheese. The proteases produced by GRAS (generally recognized-as-safe) microbes help in hydrolysis of specific peptide bond to generate p-k-casein and macropeptides. Whey is an abundant liquid by-product of cheese-making process. Proteases convert whey into protein hydrolysate during whey bioconversion process. Proteases are used for chill-proofing of beer. The fresh beer produced may have haziness mainly due to the presence of complexes of proteins and tannins, which may be cleared by application of proteases. A metalloneutral protease from B. amyloliquefaciens SYB-001 had the ability to release more water-soluble proteins and may be suitable for brewing industry (Wang et al. 2013) . Application of exogenous proteases aids in the production of tender meat (Mageswari et al. 2017) . Tenderness is one of the most desirable characteristics of meat. Some of cuts from the big carcases may not be considered as prime quality meat cuts due to their toughness. However, the application of proteases may help transforming such meat pieces into quality cuts just like the prime ones by tenderization. The two most often used meat-tenderizing enzymes are papain and bromelain. To a lesser extent, ficin, derived from fig tree latex, is also used (Payne 2009). Microbial proteases (bacterial and fungal) have also been explored for meat tenderization application (Qihe et al. 2006; Ha et al. 2013) . Microbial proteases are also useful in baking process. Flour consists of gluten (glutenin and gliadin), starch, non-starch polysaccharides, lipids and trace amount of minerals. Protease-mediated weakening of gluten results in improved dough formation and enhanced dough rise mainly due to breakdown of complex network of glutenin and gliadin (Bajaj and Manhas 2012) . Application of proteases may help enhancing digestibility, solubility of proteins in foods and also upgrading the organoleptic properties of foods. Protease may be used for enzymatic synthesis of aspartame, a noncalorific artificial sweetener. The waste generated by food industry could be used as an inexpensive substitute for protease production by thermophilic strain B. caldolyticus DSM 405 (Jamrath et al. 2012) . The application of enzymes in various food processing units would not only substantially enhance the nutritional and functional attributes of foods but certainly help developing eco-friendly and sustainable bioprocesses that involve less usage of chemicals. 10 Proteases for prion degradation A prion in the scrapie form (PrPSc) is a misfolded infectious form of protein. They are causative agents of transmissible spongiform encephalopathies in a variety of mammals, including bovine spongiform encephalopathy in cattle (Prusiner 1998) . The name PrPSc is because of their discovery first in scrapie-affected sheep. PrPSc has high resistance to proteolytic digestion. Actually, the core of PrPSc, i.e. carboxy proximal, can withstand proteolysis even at very high level of proteinase-K. The presence of proteinase-K-resistant prion protein is considered as a definitive diagnostic test for prion diseases in humans and other species (Leske et al. 2017) . Recently, however, absence of protease resistance in PrPSc has been observed, and a mechanism for protease-sensitive prion infectivity has been proposed (Leske et al. 2017) . In humans, prions cause Creutzfeldt–Jakob disease, variant Creutzfeldt–Jakob disease, Gerstmann–Straussler– Scheinker syndrome, fatal familial insomnia and kuru. All known prion diseases affect the structure of the brain or other neural tissue, and all are currently untreatable and universally fatal (Prusiner, 1998) . Proteases may help degrading wrongly folded proteins. Yoshioka et al. (2007) identified a protease-producing Bacillus strain that was capable of degrading scrapie PrPSc. The protease (MSK 103) was also effective against dried PrPSc. Thermostable keratinase from B. pumilus KS12 has potential for degradation of Sup35NM (Rajput and Gupta 2013) . It may be envisaged that potentially novel proteases may be discovered that may help developing biobased therapeutics for prion diseases in humans and animals. 11 Proteases for biopolishing of wool Alkaline proteases are used for the manufacture of shrinkproof wool. Wool fibres are covered in overlapping scales pointing towards fibre tips, which could be hydrolysed by protease action. Wool is a special kind of keratin that has a high concentration of cysteine cross-links in the exocuticle of wool fibre. Proteases may have application potential for production of shrink-proof wool in an environmentally friendly process. However, protease treatment in general damages the wool by causing excessive loss of strength, and lowering the antifelting ability, which in turn may lead to additional damage to the fibre interior during wool processing (Wang et al. 2011) . Therefore, specific proteases whose action is limited to cuticle scale of wool fibre are desired (Shen et al. 2007) . Keratinase can be used in textile processing and may potentially replace the conventional physicochemical methods that are environmentally unhealthy. Thus, enzyme-based bioprocesses can be developed for efficient production of shrinkresistant fibre that have improved handling properties (Tahara et al. 2003) . A keratinase from Brevibacillus parabrevis CGMCC 10798 was purified and characterized for its excellent potential for wool processing (Zhang et al. 2016a) . A novel recombinant keratinase expressed in E. coli BL21 (DE3) exhibited high specificity towards some substrates including wool. The protease has the potential for application in wool processing (Su et al. 2017) . Several strains of bacteria like Bacillus, Exiguobacterium, Deinococcus and Micrococcus isolated from Patagonian Merino wool, were reported to produce wool-degrading enzymes. Bacillus sp. G51 exhibited the highest wool-keratinolytic activity. LCMS/MS analysis showed that two serine proteases of peptidase family S8 and a metalloprotease associated with Bacillolysin were responsible for hydrolysing keratin disulphide bonds. The enzyme substantially reduced the wool felting tendency without much weight loss. Thus, eco-friendly treatment approaches based on enzyme cocktail (with protease combination) may help designing the organic wool processing (Iglesias et al. 2017) . 12 Nematicidal activity of protease Nematoda is a diverse animal phylum inhabiting a broad range of environments. Depending on the species, a nematode may be beneficial or detrimental to plant health causing huge economic losses (Abad et al. 2008) . Strategies followed against pathogenic nematodes include use of chemical nematicides and different biocontrol agents like fungi and bacteria (Tian et al. 2007) . Traditional chemicalbased method not only causes a significant environmental pollution but also leads to the emergence of nematicide resistance (Yang et al. 2013) . The rhizobacteria have extensively been studied as biocontrol agents for the plantparasitic nematodes. Among these bacteria, numerous Bacillus spp. strains have been reported to express activities that suppress the pests and pathogens, including nematodes (Radnedge et al. 2003). There are several reports of proteases from non-Bacillus spp. being used as biocontrol agents against nematodes (Siddiqui et al. 2005; Ward et al. 2012) . Lian et al. (2007) demonstrated nematicidal activity of an extracellular cuticle-degrading protease Apr219 from Bacillus sp. strain RH219 isolated from rhizosphere. Alkaline protease from B. lehensis has been found to be useful as a biocontrol agent for plant pathogenic nematode Meloidogyne incognita (Joshi and Satyanarayana 2013) . Bacillus sp. B16 isolated from soil sample secreted extracellular cuticle-degrading protease that had remarkable nematotoxic activity against Panagrellus redivivus (Qiuhong et al. 2006) . A total of 120 bacterial strains of 30 species of the Bacillaceae and Paenibacillaceae were examined for nematicidal activities. Nine species, viz. Bacillus thuringiensis, B. cereus, B. subtilis, B. pumilus, B. firmus, B. toyonensis, Lysinibacillus sphaericus, Brevibacillus laterosporus and B. brevis, exhibited excellent nematicidal potential. Genome analysis was used for identification of potential virulence factors. One of the major mechanisms for nematicidal capacities was observed to be the ability of bacteria to produce proteases and/or chitinases (Zheng et al. 2016) . Thus, development of biobased strategies for controlling and managing the pests may contribute substantively towards environmental health as this would lead to reduced application of chemical-based pesticides. 13 Proteases for contact lens cleansing The tear film is a complex fluid composed mainly of water, lipids, proteins, sugars, mucin and carbohydrates. Films formed over the lenses provide a surface for the adhesion of opportunistic pathogens like Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis (Dutta et al. 2012) . Adhesion and colonization by microorganisms, particularly bacteria, on contact lenses have been implicated in several adverse events including microbial keratitis (Willcox and Holden 2001), contact lens-related acute red eye (Szczotka-Flynn et al. 2010) , contact lens peripheral ulcer (Wu et al. 2003) and infiltrative keratitis (Szczotka-Flynn et al. 2010) . Therefore, cleansing of contact lenses is utmost important. Proteases have been used for preparing contact lens cleaning solutions. Mainly plant and animal proteases have been used for preparing contact lens cleaning solutions. Recently, some microbial proteases have been shown to be promising agents for contact lens cleaning. Protease from Bacillus sp. 158 exhibited ability for cleaning of tear films and debris of contact lenses (Pawar et al. 2009) . The neutral protease isolated from Bacillus sp. 158 efficiently removed the protein deposits from contact lenses. The partially purified protease exhibited optimum pH and temperature for activity at pH 7.0 and 30 C, respectively. The enzyme could effectively be used to remove protein deposits from contact lenses and, thus, help increasing the transmittance of lenses (Pawar et al. 2009) . Clear-Lens Pro, currently used in contact lens cleaning formulations, marketed by Novozymes, Denmark, is of microbial origin. This preparation is used to remove protein-based deposits and protein films from contact lenses. The protease used in this preparation is from Bacillus sp. which hydrolyses the protein in the deposits and films (Sumantha et al. 2006) . Proteases from several Bacillus spp., viz. B. subtilis, B. licheniformis, B. thermophilus and B. cereus, exhibited excellent activity against artificial tear solution and, thus, could be of importance for contact lens cleansing. All the proteases had maximum activity at 40 C and pH 8 (Ismail et al. 2014) . Considering enormous campaign of bioeconomy, i.e. biobased products, processes and services, it is of course interesting to design and develop contact lens cleansing formulations using enzymes. 14 Future prospective of proteases Though proteases are extensively applied enzymes in several sectors of industrial biotechnology, further research is required for exploring the full application potential of proteases. Several processes like peptide synthesis and sequencing, digestion of unwanted proteins, cell culturing and tissue dissociation, preparation of recombinant antibody fragments, study of structure–function relationships, removal of affinity tags and proteolytic digestion of proteins, require immense research impetus. Moreover, with the application of recombinant DNA technology and protein engineering microbes can be manipulated to enhance the production of specific high priority industrial enzymes. Extremophilic organisms could be exploited for production of process-suitable novel enzymes. Furthermore, molecular intricacies of mechanisms involved for application of proteases in diverse processes need investigation. Environment assessment tools like life cycle assessment, carbon footprint, environmental impact assessment, global warming, acidification, eutrophication and photochemical ozone formation could be employed for determining the impact of cleaner enzymatic processes in place of conventional processes. 15 Conclusions Enzyme-driven industrial processes are the most appropriate alternatives to tedious, expensive and polluting traditional methods. Microbial proteases, especially from Bacillus spp., have enormously been exploited and constituted the backbone for several industries. The Bacillus spp. have the potential capability to produce industrially suitable enzymes which possess poly-extremotolerance, i.e. ability to withstand extremes of pH, temperatures, presence of organic solvents and a variety of other enzyme inhibitors. Thus, enzymes from Bacillus spp. meet the industrial process criteria. It is pertinent to refer Bacillus spp. as ‘microbial factories’ for industrial enzymes. Application of enzymes in detergents promises eco-friendly industrial processes which involve reduced usage of chemicals such as soaps, surfactants, bleach, oxidizing and chelating agents in the detergent formulation. Addition of enzymes in detergents augments their washing efficacy, especially for dirt/dust of biological origin. Furthermore, application of enzymes helps washing to be executed at lower temperatures, thus saving energy and environment. Application of enzymes in leather processing regime makes it more efficient, eco-benign and safer, i.e. free of sulphide and chromium usage. Enzymatic bioprocessing of keratin wastes envisages eco-friendly and sustainable valorization of wastes to wealth and offers huge potential for food, feed and cosmetic industries. Application of proteases in leather and textile industry not only improves the process economy but also improves product quality and makes the processes eco-benign and more efficient. Protease application in silk and wool industries promises high-quality silk/wool and a safer and eco-friendly process. Application of proteases helps developing green process for silver extraction from X-ray films, thus mitigating the enormous environmental pollution due to conventional process. Microbial proteases may potentially be developed as specific therapeutics for prion diseases in humans and animals. Microbial proteases are being investigated for development of potential thrombolytic agents considering the high cost and undesirable side effects of the available chemotherapeutics. Proteases may be developed as potential biocontrol agents that may help mitigating environmental pollution due to chemical-based pesticides. Thus, successful commercialization of proteases for several biotechnological processes in industries paves the way for development of clean, green and sustainable processes. Furthermore, recent advancements in the areas of molecular biology and protein engineering must be exploited to develop novel/tailor-made enzymes with greater efficacies under prevailing industrial process microenvironments. Acknowledgements Dr. Bijender Kumar (Bajaj) gratefully acknowledges the Institute of Advanced Study, Durham University, Durham, UK, for providing COFUND-International Senior Research Fellowship for ‘Research Stay’ at Department of Biosciences, Durham University, Durham, UK; Dr. Bijender Kumar (Bajaj) thanks the University Grants Commission, Government of India, for supporting Major Research Project (UGC MRP- Ref. F. No. 42-226/2013 (SR)), and Indian Council of Medical Research, Government of India, for Research Project (ICMR-Research Project Ref. No: 5/4/1/EXM/12NCD-II). Mr. Satbir Singh gratefully acknowledges the Council of Scientific and Industrial Research (CSIR) for Junior/Senior Research Fellowship. Authors thank the Director, School of Biotechnology, University of Jammu, Jammu, for laboratory facilities. Abad P , Gouzy J , Aury JM , Castagnone-Sereno P et al ( 2008 ) Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita . Nat Biotechnol 26 : 909 - 915 Agrahari S , Wadhwa N ( 2010 ) Degradation of chicken feather a poultry waste product by keratinolytic bacteria isolated from dumping site at Ghazipur poultry processing plant . Int J Poult Sci 9 : 482 - 489 Agrebi R , Haddar A , Hajji M et al ( 2009 ) Fibrinolytic enzymes from a newly isolated marine bacterium Bacillus subtilis A26: characterization and statistical media optimization . Can J Microbiol 55 : 1049 - 1061 Agrebi R , Hmider N , Hajji M et al ( 2010 ) Fibrinolytic serine protease isolated from Bacillus amyloliquefaciens An6 grown on Mirabilis jalapa tuber powders . Appl Biochem Biotechnol 162 : 75 - 88 Ashipala OK , He Q ( 2008 ) Optimization of fibrinolytic enzyme production by Bacillus subtilis DC-2 in aqueous two-phase system (poly-ethylene glycol 4000 and sodium sulfate) . Bioresour Technol 99 : 4112 - 4119 Bajaj BK , Jamwal G ( 2013 ) Thermostable alkaline protease production from Bacillus pumilus D-6 by using agro-residues as substrates . Adv Enzyme Res 1 : 30 - 36 Bajaj BK , Manhas K ( 2012 ) Production and characterization of xylanase from Bacillus licheniformis P11(C) with potential for fruit juice and bakery industry . Biocatal Agric Biotechnol 1 : 330 - 337 Bajaj BK , Sharma P ( 2011 ) An alkali-thermotolerant extracellular protease from a newly isolated Streptomyces sp . DP2. New Biotechnol 28 : 725 - 732 Bajaj BK , Wani MA ( 2011 ) Enhanced phytase production from Nocardia sp. MB 36 using agro-residues as substrates: potential application for animal feed production . Eng Life Sci 11 : 620 - 628 Bajaj BK , Sharma N , Singh S ( 2013 ) Enhanced production of fibrinolytic protease from Bacillus cereus NS-2 using cotton seed cake as nitrogen source . Biocatal Agric Biotechnol 2 : 204 - 209 Bajaj BK , Singh S , Khullar M , Singh K , Bhardwaj S ( 2014 ) Optimization of fibrinolytic protease production from Bacillus subtilis I-2 using agro-residues . Braz Arch Biol Technol 57 : 653 - 662 Balaraman K , Prabakaran G ( 2007 ) Production and purification of a fibrinolytic enzyme (thrombinase) from Bacillus sphaericus . Indian J Med Res 126 : 459 - 464 Baweja M , Tiwari R , Singh PK , Nain L , Shukla P ( 2016 ) An alkaline protease from Bacillus pumilus MP 27: functional analysis of its binding model toward its applications as detergent additive . Front Microbiol 7 : 1195 Białkowska A , Gromek E , Florczak T , Krysiak J , Szulczewska K , Turkiewicz M ( 2016 ) Extremophilic proteases: developments of their special functions, potential resources and biotechnological applications . In: Rampelotto P (ed) Biotechnology of extremophiles. Springer, Switzerland, pp 399 - 444 Brandelli A , Daroit DJ , Riffel A ( 2010 ) Biochemical features of microbial keratinases and their production and applications . Appl Microbiol Biotechnol 85 : 1735 - 1750 Cavello IA , Hours RA , Cavalitto SF ( 2013 ) Enzymatic hydrolysis of gelatin layers of X-ray films and release of silver particles using keratinolytic serine proteases from Purpureocillium lilacinum LPS # 876 . J Microbiol Biotechnol 23 : 1133 - 1139 Choudhary V ( 2013 ) Recovery of silver from used X-ray films by Aspergillus versicolor protease . J Acad Ind Res 2 : 39 - 41 Daroit DJ , Anna VS , Brandelli A ( 2011 ) Kinetic stability modelling of keratinolytic protease P45: influence of temperature and metal ions . Appl Biochem Biotechnol 165 : 1740 - 1753 Datta S , Menon G , Varughese B ( 2016 ) Production, characterization and immobilization of partially purified surfactant-detergent and alkali-thermo stable protease from newly isolated Aeromonas Caviae . Prep Biochem Biotechnol. doi:10.1080/10826068 . 2016 . 1244688 David L , Vierros M , Hamon G , Arico S , Monagie C ( 2009 ) Marine genetic resources: a review of scientific and commercial interest . Mar Policy 33 : 183 - 194 de Souza FA , Sales AE , Silva PE , Bezerra RP et al ( 2016 ) Optimization of production, biochemical characterization and in vitro evaluation of the therapeutic potential of fibrinolytic enzymes from a new Bacillus amyloliquefaciens . Macromol Res 24 : 587 - 595 Deepak V , Kalishwaralal K , Ramkumarpandian S , Babu SV et al ( 2008 ) Optimization of media composition for nattokinase production by Bacillus subtilis using response surface methodology . Bioresour Technol 99 : 8170 - 8174 Deepak V , Ilangovan S , Sampathkumar MV , Victoria MJ et al ( 2010 ) Medium optimization and immobilization of purified fibrinolytic URAK from Bacillus cereus NK1 on PHB nanoparticles . Enzyme Microb Technol 47 : 297 - 304 Deinhammer R , Andersen C ( 2011 ) Methods for preventing, removing, reducing, or disrupting biofilm . US Patent , 20110104141 Devi RY ( 2012 ) Biotechnological application of proteolytic enzymes in post cocoon technology . Int J Sci Nat 3 : 237 - 240 Deydier E , Guilet R , Sarda S , Sharrock P ( 2005 ) Physical and chemical characteristics of crude meat and bone meal combustion residue: waste or raw material . J Hazard Mater B 121 : 141 - 148 Dijl JM , Hecker M ( 2013 ) Bacillus subtilis: from soil bacterium to super-secreting cell factory . Microb Cell Fact 12 : 1 - 6 Dutta D , Cole N , Willcox M ( 2012 ) Factors influencing bacterial adhesion to contact lenses . Mol Vis 18 : 14 - 21 Ekpunobi UE , Okwukogu OK , Anozie AI et al ( 2013 ) Deposition and characterization of silver oxide from silver solution recovered from industrial wastes . Am Chem Sci J 3 : 307 - 313 Freddi G , Mossotti R , Innocenti R ( 2003 ) Degumming of silk fabric with several proteases . J Biotechnol 106 : 101 - 112 Georgieva D , Genov N , Voelter W , Betzel C ( 2006 ) Catalytic efficiencies of alkaline proteinases from microorganisms . Zeitschrift fu¨r Naturforschung C 61 : 445 - 452 Gessesse A , Rajni HK , Gashe BA ( 2003 ) Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather . Enzyme Microb Technol 32 : 519 - 524 Ghafoor A , Hasnain S ( 2010 ) Purification and characterization of an extracellular protease from Bacillus subtilis EAG-2 strain isolated from ornamental plant nursery . Pol J Microbiol 59 : 107 - 112 Giri SS , Sukumaran S , Sen SS , Olive M , Banu N , Jena PK ( 2011 ) Oxidizing agent stable alkaline protease from a newly isolated Bacillus subtiis VSG-4 of tropical soil . J Microbiol 49 : 455 - 461 Gousterova A , Braikova D , Goshev I , Christov P et al ( 2005 ) Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis . Lett Appl Microbiol 40 : 335 - 340 Grazziotin A , Pimentel FA , de Jong EV , Brandelli A ( 2006 ) Nutritional improvement of feather protein by treatment with microbial keratinase . Anim Feed Sci Technol 126 : 135 - 144 Guleria S , Walia A , Chauhan A , Shirkot CK ( 2016 ) Purification and characterization of detergent stable alkaline protease from Bacillus amyloliquefaciens SP1 isolated from apple rhizosphere . J Basic Microbiol 56 : 138 - 152 Gupta M , Bajaj BK ( 2017 ) Functional characterization of probiotic lactic acid bacteria isolated from kalarei and development of probiotic-fermented oat flour . Probiotics Antimicrob Proteins. doi:10.1007/s12602-017-9306-6 Gupta P , Nayak KK ( 2015 ) Characteristics of protein-based biopolymer and its application . Polym Eng Sci 55 : 485 - 498 Gupta R , Ramnani P ( 2006 ) Microbial keratinases and their prospective applications: an overview . Appl Microbiol Biotechnol 70 : 21 - 33 Ha M , Bekhit AE , Carne A , Hopkins DL ( 2013 ) Comparison of the proteolytic activities of new commercially available bacterial and fungal proteases toward meat proteins . J Food Sci 78 : 170 - 177 Harde SM , Bajaj IB , Singhal RS ( 2011 ) Optimization of fermentative production of keratinase from Bacillus subtilis NCIM 2724 . Agric Food Anal Bacteriol 1 : 54 - 65 Hassanein WA , Kotb E , Awny NM , El-Zawahry YA ( 2011 ) Fibrinolysis and anticoagulant potential of a metallo protease produced by Bacillus subtilis K42 . J Biosci 36 : 773 - 779 Hori K , Matsumoto S ( 2010 ) Bacterial adhesion: from mechanism to control . Biochem Eng J 48 : 424 - 434 Hwang Kyung-Ju , Choi KH , Kim MJ et al ( 2007 ) Purification and characterization of a new fibrinolytic enzyme of Bacillus licheniformis KJ-31, isolated from Korean traditional Jeot-gal . J Microbiol Biotechnol 17 : 1469 - 1476 Ida E ´ L, da Silva RR , de Oliveira TB et al ( 2016 ) Biochemical properties and evaluation of washing performance in commercial detergent compatibility of two collagenolytic serine peptidases secreted by Aspergillus fischeri and Penicillium citrinum . Prep Biochem Biotechnol. doi:10.1080/10826068 . 2016 .1224247 Iglesias MS , Sequeiros C , Garc´ıa S , Olivera NL ( 2017 ) Newly isolated Bacillus sp. G51 from Patagonian wool produces an enzyme combination suitable for felt-resist treatments of organic wool . Bioprocess Biosyst Eng. doi:10.1007/s00449-017-1748-4 Ismail KS , Jadhav AA , Harale MA et al ( 2014 ) Study of protease enzyme from Bacillus species and its application as a contact lens cleanser . Br Biomed Bull 2 : 293 - 302 Jamrath T , Lindner C , Popovic MK , Bajpai R ( 2012 ) Production of amylases and proteases by Bacillus caldolyticus from food industry wastes . Food Technol Biotechnol 50 : 355 - 361 Jayakumar R , Jayashree S , Annapurna B , Seshadri S ( 2012 ) Characterization of thermostable serine alkaline protease from an alkaliphilic strain Bacillus pumilus MCAS8 and its applications . Appl Biochem Biotechnol 168 : 1849 - 1866 Jayalakshmi T , Krishnamoorthy P , Ramesh Babu PB , Vidhya B ( 2012 ) Production, purification and biochemical characterization of alkaline fibrinolytic enzyme from Bacillus subtilis strainGBRC1 . J Chem Pharm Res 4 : 5027 - 5031 Jegannathan KR , Nielsen PH ( 2013 ) Environmental assessment of enzyme use in industrial production-a literature review . J Clean Prod 42 : 228 - 240 Jessen B , Lammert L ( 2003 ) Biofilm and disinfection in meat processing plants . Int Biodeterior Biodegrad 51 : 265 - 269 Jisha VN , Smitha RB , Pradeep S et al ( 2013 ) Versatility of microbial proteases . Adv Enzyme Res 1 : 39 - 51 Jo HD , Kwon GH , Park JY et al (2011a) Cloning and overexpression of aprE3-17 encoding the major fibrinolytic protease of Bacillus licheniformis CH 3-17 . Biotechnol Bioprocess Eng 16 : 352 - 359 Jo HD , Lee HA , Jeong SJ , Kim JH ( 2011b) Purification and characterization of a major fibrinolytic enzyme from Bacillus amyloliquefaciens MJ5-41 isolated from Meju . J Microbiol Biotechnol 21 : 1166 - 1173 Joshi S , Satyanarayana T ( 2013 ) Characteristics and applications of a recombinant alkaline serine protease from a novel bacterium Bacillus lehensis . Bioresour Technol 131 : 76 - 85 Kainoor PS , Naik GR ( 2010 ) Production and characterization of feather degrading keratinase from Bacillus sp . JB 99. Indian J Biotechnol 9 : 384 - 390 Kazan D , Denizei AA , Kerimakonu MN , Erarslan A ( 2005 ) Purification and characterization of a serine alkaline protease from Bacillus clausis GMBAE 42 . J Ind Microbiol Biotechnol 32 : 335 - 344 Khajuria V , Sharma K , Slathia P et al ( 2015 ) Production of a detergent-compatible alkaline protease from Bacillus cereus K-3 . J Mater Environ Sci 6 : 2089 - 2096 Khan F ( 2013 ) New microbial proteases in leather and detergent industries . Innov Res Chem 1 : 1 - 6 Kim SB , Lee DW , Cheigh CI et al ( 2006 ) Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean , Tempeh. J Ind Microbiol Biotechnol 33 : 436 - 444 Kim GM , Lee AR , Lee KW et al ( 2009 ) Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from Cheonggukjang . J Microbiol Biotechnol 19 : 997 - 1004 Kostakioti M , Hadjifrangiskou M , Hultgren SJ ( 2013 ) Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era . Cold Spring Harb Perspect Med 3 : 010306 Kumar D , Bhalla TC ( 2005 ) Microbial proteases in peptide synthesis: approaches and applications . Appl Microbiol Biotechnol 68 : 726 - 736 Kumar D , Savitri Thakur N , Verma R , Bhalla TC ( 2008 ) Microbial proteases and application as laundry detergent additive . Res J Microbiol 3 : 661 - 672 Kumar EV , Srijana M , Kumar KK , Harikrishna N , Reddy G ( 2011 ) A novel serine alkaline protease from Bacillus altitudinis GVC11 and its application as a dehairing agent . Bioprocess Biosyst Eng 34 : 403 - 409 Kumar RS , Rajesh R , Gokulakrishnan S , Subramanian J ( 2015 ) Screening and characterization of fibrinolytic protease producing Bacillus circulans from mangrove sediments Pitchavaram, South East Coast of India . Int Lett Nat Sci 1 : 10 - 16 Kumaran E , Mahalakshmipriya A , Rajan S ( 2013 ) Effect of fish waste based Bacillus protease in silver recovery from waste X-ray films . Int J Curr Microbiol Appl Sci 2 : 49 - 56 Lee Ran A , Kim GM et al ( 2010 ) Cloning of aprE86-1 gene encoding a 27-kDa mature fibrinolytic enzyme from Bacillus amyloliquefaciens CH86-1 . J Microbiol Biotechnol 20 : 370 - 374 Leroy C , Delbarre C , Ghillebaert F et al (2008 ) Effects of commercial enzymes on the adhesion of a marine biofilm-forming bacterium . Biofouling 24 : 11 - 22 Leske H , Hornemann S , Herrmann US et al ( 2017 ) Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein . PLoS ONE 12:0170503 Leslie A ( 2011 ) Preventing biofilm formation using microbes and their enzymes . Basic Biotechnol 7 : 6 - 11 Lian LH , Tian BY , Xiong R et al ( 2007 ) Proteases from Bacillus: a new insight into the mechanism of action for rhizobacterial suppression of nematode populations . Lett Appl Microbiol 45 : 262 - 269 Liu X , Kopparapu NK , Li Y et al ( 2017 ) Biochemical characterization of a novel fibrinolytic enzyme from Cordyceps militaris . Int J Biol Macromol 94 : 793 - 801 Macedo AJ , Silva WOB , Termignoni C ( 2008 ) Properties of a non collagen-degrading Bacillus subtilis keratinase . Can J Microbiol 54 : 180 - 188 Madhavi J , Srilakshmi J , Raghavendra Rao MV , Sambasiva Rao KRS ( 2011 ) Efficient leather dehairing by bacterial thermostable protease . Int J Bio Sci Bio Technol 3 : 11 - 26 Mageswari A , Subramanian P , Chandrasekaran S et al ( 2017 ) Systematic functional analysis and application of a cold-active serine protease from a novel Chryseobacterium sp . Food Chem 217 : 18 - 27 Mahajan PM , Nayak S , Lele SS ( 2012 ) Fibrinolytic enzyme from newly isolated marine Bacillus subtilis ICTF-1: media optimization, purification and characterization . J Biosci Bioeng 113 : 307 - 314 Mahmoodi NM , Moghimi F , Arami M , Mazaheri F ( 2010 ) Silk degumming using microwave irradiation as an environmentally friendly surface modification method . Fiber Polym 11 : 234 - 240 Marinkovic J , Korac M , Kamberovic Z , Matic I ( 2006 ) Recycling of silver from exposed X-ray films . Acta Metall Slovaca 12 : 262 - 268 Mhamdi S , Ktari N , Hajji S et al ( 2017 ) Alkaline proteases from a newly isolated Micromonospora chaiyaphumensis S103: characterization and application as a detergent additive and for chitin extraction from shrimp shell waste . Int J Biol Macromol 94 : 415 - 422 Mine Y , Wong AHK , Jinag B ( 2005 ) Fibrinolytic enzymes in Asian traditional fermented foods . Food Res Int 38 : 243 - 250 Mohanasrinivasan V , Subathra CD , Yogesh S et al ( 2016 ) In vitro thrombolytic potential of actinoprotease from marine Streptomyces violaceus VITYGM . Cardiovasc Hematol Agents Med Chem. doi:10.2174/1871525715666161104112553 Molobela IP , Cloete TE , Beukes M ( 2010 ) Protease and amylase enzymes for biofilm removal and degradation of extracellular polymeric substances (EPS) produced by Pseudomonas fluorescens bacteria . Afr J Microbiol Res 4 : 1515 - 1524 More SV , Khandelwal HB , Joseph MA , Laxman RS ( 2013 ) Enzymatic degumming of silk with microbial proteases . J Nat Fiber 10 : 98 - 111 Motyan JA , Toth F , Tozser J ( 2013 ) Research applications of proteolytic enzymes in molecular biology . Biomolecules 3 : 923 - 942 Mukherjee AK , Rai SK , Bordoloi NK ( 2011 ) Biodegradation of waste chicken-feathers by an alkaline b-keratinase (Mukartinase) purified from a mutant Brevibacillus sp . strain AS-S10-II. Int Biodeterior Biodegrad 65 : 1229 - 1237 Mukherjee AK , Rai SK , Thakur R et al ( 2012 ) Bafibrinase: a nontoxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency . Biochimie 94 : 1300 - 1308 Nadeem M , Qazi JI , Baig S ( 2010 ) Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing . Braz Arch Biol Technol 53 : 1015 - 1025 Nakiboglu N , Toscali D , Yasa I ( 2001 ) Silver recovery from waste photographic films by an enzymatic method . Turk J Chem 25 : 349 - 353 Nakiboglu N , Toscali D , Nisli G ( 2003 ) A novel silver recovery method from waste photographic films with NaOH stripping . Turk J Chem 27 : 127 - 133 Nakpathom M , Somboon B , Narumol N ( 2009 ) Papain enzymatic degumming of Thai Bombyx mori silk fibers . J Microsc Soc Thail 23 : 142 - 146 Nargotra P , Vaid S , Bajaj BK ( 2016 ) Cellulase production from Bacillus subtilis SV1 and its application potential for saccharification of ionic liquid pretreated pine needle biomass under one pot consolidated bioprocess . Fermentation 2 : 19 Nascimento WCA , Martins MLL ( 2006 ) Studies on the stability of protease from Bacillus sp . and its compatibility with commercial detergent . Braz J Microbiol 37 : 307 - 311 Nigam VK , Singhal P , Vidyarthi AS ( 2012 ) Studies on production, characterization and applications of microbial alkaline proteases . Int J Adv Biotechnol Res 3 : 653 - 669 Nilegaonkar SS , Zambare VP , Kanekar PP et al ( 2007 ) Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326 . Bioresour Technol 98 : 1238 - 1245 Odetallah NH , Wang JJ , Garlich JD , Shih JCH ( 2005 ) Versazyme supplementation of broiler diets improves market growth performance . Poult Sci 84 : 858 - 864 Orgaz B , Kives J , Pedregosa AM et al ( 2006 ) Bacterial biofilm removal using fungal enzymes . Enzyme Microb Technol 40 : 51 - 56 Oulahal-Lagsir N , Martial-Gros A , Bonneau M , Blum LJ ( 2003 ) Escherichia coli-milk biofilm removal from stainless steel surfaces: synergism between ultrasonic waves and enzymes . Biofouling 19 : 159 - 168 Padmapriya B , Rajeswari T , Nandita R , Raj F ( 2012 ) Production and purification of alkaline serine protease from marine Bacillus species and its application in detergent industry . Eur J Appl Sci 4 : 21 - 26 Parradoa J , Rodriguez-Morgado B , Tejada M et al ( 2014 ) Proteomic analysis of enzyme production by Bacillus licheniformis using different feather wastes as the sole fermentation media . Enzyme Microb Technol 57 : 1 - 7 Pathak AP , Deshmukh KB ( 2012 ) Alkaline protease production, extraction and characterization from alkaliphilic Bacillus licheniformis KBDL4: a lonar soda lake isolate . Indian J Exp Biol 50 : 569 - 576 Pawar R , Zambare V , Barve S , Paratkar G ( 2009 ) Application of protease isolated from Bacillus sp. 158 in enzymatic cleansing of contact lenses . Biotechnology 8 : 276 - 280 Payne CT ( 2009 ) Enzymes . In: Tarte R (ed) Ingredients in meat products: properties, functionality and applications . Springer, New York, pp 173 - 198 Pillai P , Mandge S , Archana G ( 2011 ) Statistical optimization of production and tannery applications of a keratinolytic serine protease from Bacillus subtilis P13 . Process Biochem 46 : 1110 - 1117 Priya JDA , Divakar K , Prabha MS et al ( 2014 ) Isolation, purification and characterisation of an organic solvent-tolerant Ca2?-dependent protease from Bacillus megaterium AU02 . Appl Biochem Biotechnol 172 : 910 - 932 Prusiner SB ( 1998 ) Prions . Proc Natl Acad Sci 95 : 13363 - 13383 Qazi JI , Jamshaid H , Nadeem M , Ali SS ( 2008 ) Production of proteases by Aspergillus niger, through solid state fermentation . Punjab Univ J Zool 23 : 037 - 046 Qihe C , Guoqing H , Yingchun J , Hui N ( 2006 ) Effects of elastase from a Bacillus strain on the tenderization of beef meat . Food Chem 98 : 624 - 629 Qiuhong N , Xiaowei H , Baoyu T et al ( 2006 ) Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor . Appl Microbiol Biotechnol 69 : 722 - 730 Radnedge L , Agron PG , Hill KK et al ( 2003 ) Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis . Appl Environ Microbiol 69 : 2755 - 2764 Rajput R , Gupta R ( 2013 ) Thermostable keratinase from Bacillus pumilus KS12: production, chitin crosslinking and degradation of Sup35NM aggregates . Bioresour Technol 133 : 118 - 126 Raju EVN , Goli D ( 2014 ) Effect of physiochemical parameters on fibrinolytic protease production by solid state fermentation . World J Pharm Pharm Sci 3 : 1937 - 1954 Rani K , Rana R , Datt S ( 2012 ) Review on latest overview of proteases . Int J Curr Life Sci 2 : 12 - 18 Ravindran B , Kumar AG , Aruna Bhavani PS , Ganesan Sekaran G ( 2011 ) Solid-state fermentation for the production of alkaline protease by Bacillus cereus 1173900 using proteinaceous tannery solid waste . Curr Sci 100 : 726 - 730 Ray A ( 2012 ) Protease enzyme-potential industrial scope . Int J Technol 2 : 01 - 04 Rehman R , Ahmed M , Siddique A et al ( 2017 ) Catalytic role of thermostable metalloproteases from Bacillus subtilis KT004404 as dehairing and destaining agent . Appl Biochem Biotechnol 181 : 434 - 450 Romsomsa N , Chim-anagae P , Jangchud A ( 2010 ) Optimization of silk degumming protease production from Bacillus subtilis C4 using Plackett-Burman design and response surface methodology . Sci Asia 36 : 118 - 124 Sadeghi HMM , Rabbani M , Naghitorabi M ( 2009 ) Cloning of alkaline protease gene from Bacillus subtilis 168 . Res Pharm Sci 4 : 43 - 46 Sarrouh B , Santos TM , Miyoshi A et al ( 2012 ) Up-to-date insight on industrial enzymes applications and global market . J Bioprocess Biotech S 4 :002. doi: 10 .4172/ 2155 - 9821 . S4 - 002 Sathiya G ( 2013 ) Production of protease from Bacillus subtilis and its application in leather making process . Int J Res Biotechnol Biochem 3 : 7 - 10 Shankar S , Laxman RS ( 2015 ) Biophysicochemical characterization of an alkaline protease from Beauveria sp. MTCC 5184 with multiple applications . Appl Biochem Biotechnol 175 : 589 - 602 Shankar S , More SV , Laxman RS ( 2010 ) Recovery of silver from waste X-ray film by alkaline protease from Conidiobolus coronatus . J Sci Eng Technol 6 : 60 - 69 Sharma M , Bajaj BK ( 2017 ) Optimization of bioprocess variables for production of a thermostable and wide range pH stable carboxymethyl cellulase from Bacillus subtilis MS 54 under solid state fermentation . Environ Prog Sustain Energy. doi:10 .1002/ ep.12557 Sheldon RA , Arends I , Hanefeld U ( 2007 ) Green chemistry and catalysis . Wiley, Weinheim Shen J , Rushforth M , Cavaco-Paulo A , Guebitz G , Lenting H ( 2007 ) Development and industrialization of enzymatic shrink-resist process based on modified proteases for wool machine washability . Enzyme Microbial Technol 40 : 1656 - 1661 Shrinivas D , Kumar R , Naik GR ( 2012 ) Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity . J Ind Microbiol Biotechnol 39 : 93 - 98 Siddiqui IA , Haas D , Heeb S ( 2005 ) Extracellular protease of Pseudomonas fluorescens CHA0, a bio-control factor with activity against the root-knot nematode Meloidogyne incognita . Appl Environ Microbiol 171 : 5646 - 5649 Singh S , Bajaj BK ( 2015 ) Medium optimization for enhanced production of protease with industrially desirable attributes from Bacillus subtilis K-1 . Chem Eng Commun 202 : 1051 - 1060 Singh S , Bajaj BK ( 2016 ) Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid state fermentation . Prep Biochem Biotechnol 46 : 717 - 724 Singh S , Bajaj BK ( 2017 ) Agroindustrial/forestry residues as substrates for production of thermoactive alkaline protease from Bacillus licheniformis K-3 having multifaceted hydrolytic potential . Waste Biomass Valoriz 8 : 453 - 462 Singh S , Gupta P , Sharma V et al ( 2014 ) Multifarious potential applications of keratinase of Bacillus subtilis K-5 . Biocatal Biotransform 32 : 333 - 342 Su C , Gong JS , Zhang RX et al ( 2017 ) A novel alkaline surfactantstable keratinase with superior feather-degrading potential based on library screening strategy . Int J Biol Macromol 95 : 404 - 411 Sumantha A , Larroche C , Pandey A ( 2006 ) Microbiology and industrial biotechnology of food-grade proteases: a perspective . Food Technol Biotechnol 44 : 211 - 220 Sun Z , Liu P , Cheng G et al ( 2016 ) A fibrinolytic protease AfeE from Streptomyces sp. CC5, with potent thrombolytic activity in a mouse model . Int J Biol Macromol 85 : 346 - 354 Sundararajan S , Kannan CN , Chittibabu S ( 2011 ) Alkaline protease from Bacillus cereus VITSN04: potential application as a dehairing agent . J Biosci Bioeng 111 : 128 - 133 Szczotka-Flynn LB , Pearlman E , Ghannoum M ( 2010 ) Microbial contamination of contact lenses, lens care solutions, and their accessories: a literature review . Eye Contact Lens 36 : 116 - 129 Tahara M , Mabuchi N , Takagishi T ( 2003 ) Shrink proofing of wool fabrics by pulse corona discharge and enzymes . Sen-I Gakkaishi 59 : 153 - 157 Tanksale A , Chandra PM , Rao M , Deshpande V ( 2001 ) Immobilization of alkaline protease from Conidiobolus macrosporus for reuse and improved thermal stability . Biotechnol Lett 23 : 51 - 54 Tavano OL ( 2013 ) Protein hydrolysis using proteases: an important tool for food biotechnology . J Mol Catal B Enzym 90 : 1 - 11 Tekin N , Cihan AC , Takac ZS et al ( 2012 ) Alkaline protease production of Bacillus cohnii APT5 . Turk J Biol 36 : 430 - 440 Theron LW , Divol B ( 2014 ) Microbial aspartic proteases: current and potential applications in industry . Appl Microbiol Biotechnol 98 : 8853 - 8868 Tian B , Yang J , Lian L et al ( 2007 ) Role of an extracellular neutral protease in infection against nematodes by Brevibacillus laterosporus strain G4 . Appl Microbiol Biotechnol 74 : 372 - 380 Vaid S , Bajaj BK ( 2017 ) Production of ionic liquid tolerant cellulase from Bacillus subtilis G2 using agroindustrial residues with application potential for saccharification of biomass under one pot consolidated bioprocess . Waste Biomass Valor 8 : 949 - 964 Vaithanomsat P , Malapant T , Apiwattanapiwat W ( 2008 ) Silk degumming solution as substrate for microbial protease production . Kasetsart J (Nat Sci) 42 : 543 - 551 Vijayaraghavan P , Vijayan A , Arun A et al ( 2012 ) Cow dung: a potential biomass substrate for the production of detergentstable dehairing protease by alkaliphilic Bacillus subtilis strain VV . SpringerPlus 1 : 1 - 9 Villa ALV , Aragao MRS , dos Santos EP et al ( 2013 ) Feather keratin hydrolysates obtained from microbial keratinases: effect on hair fiber . BMC Biotechnol 13 : 15 Wang CT , Ji BP , Nout BLR et al ( 2006 ) Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional douchi . J Ind Microbiol Biotechnol 33 : 750 - 758 Wang SH , Zhang C , Yang YL et al ( 2008 ) Screening of a high fibrinolytic enzyme producing strain and characterization of the fibrinolytic enzyme produced from Bacillus subtilis LD-8547 . World J Microbiol Biotechnol 24 : 475 - 482 Wang SL , Chao CH , Liang TW , Chen CC ( 2009 ) Purification and characterization of protease and chitinase from Bacillus cereus TKU006 and conversion of marine wastes by these enzymes . Mar Biotechnol 11 : 334 - 344 Wang SL , Wu YY , Liang TW ( 2011 ) Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007 . New Biotechnol 28 : 196 - 202 Wang J , Xu A , Wan Y , Li Q ( 2013 ) Purification and characterization of a new metallo-neutral protease for beer brewing from Bacillus amyloliquefaciens SYB-001 . Appl Biochem Biotechnol 170 : 2021 - 2033 Ward E , Kerry BR , Manzanilla-Lopez RH et al ( 2012 ) The Pochonia chlamydosporia serine protease gene vcp1 is subject to regulation by carbon, nitrogen and pH: implications for nematode biocontrol . PLoS ONE 7 : 1 - 12 Willcox MD , Holden BA ( 2001 ) Contact lens related corneal infections . Biosci Rep 21 : 445 - 461 Wu P , Stapleton F , Willcox MD ( 2003 ) The causes of and cures for contact lens-induced peripheral ulcer . Eye Contact Lens 29 : 63 - 66 Yang J , Liang L , Li J , Zhang KQ ( 2013 ) Nematicidal enzymes from microorganisms and their applications . Appl Microbiol Biotechnol 97 : 7081 - 7095 Yeo WS , Seo MJ , Kim MJ et al ( 2011 ) Biochemical analysis of a fibrinolytic enzyme purified from Bacillus subtilis strain A1 . J Microbiol 49 : 376 - 380 Yoshioka M , Miwa T , Horii H et al ( 2007 ) Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein . J Appl Microbiol 102 : 509 - 515 Zahn H ( 1993 ) Silk . In: Willey VCH (ed) Ullmann's encyclopedia of industrial chemistry, vol A24 . VCH Verlagsgescllschaft , Weinheim, pp 95 - 106 Zhang RX , Gong JS , Su C et al ( 2016a ) Biochemical characterization of a novel surfactant-stable serine keratinase with no collagenase activity from Brevibacillus parabrevis CGMCC 10798 . Int J Biol Macromol 93 : 843 - 851 Zhang RX , Gong JS , Zhang DD et al (2016b) A metallo-keratinase from a newly isolated Acinetobacter sp . R -1 with low collagenase activity and its biotechnological application potential in leather industry . Bioprocess Biosyst Eng 39 : 193 - 204 Zheng Z , Zheng J , Zhang Z , Peng D , Sun M ( 2016 ) Nematicidal spore-forming Bacilli share similar virulence factors and mechanisms . Sci Rep. doi:10.1038/srep31341


This is a preview of a remote PDF: https://link.springer.com/content/pdf/10.1007%2Fs40974-017-0076-5.pdf

Satbir Singh, Bijender Kumar Bajaj. Potential application spectrum of microbial proteases for clean and green industrial production, Energy, Ecology and Environment, 2017, 1-17, DOI: 10.1007/s40974-017-0076-5