A Thermostable Glucoamylase from Bispora sp. MEY-1 with Stability over a Broad pH Range and Significant Starch Hydrolysis Capacity

November A Thermostable Glucoamylase from Bispora sp. MEY-1 with Stability over a Broad pH Range and Significant Starch Hydrolysis Capacity Huifang Hua 0 2 Huiying Luo 0 2 Yingguo Bai 0 2 Kun Wang 0 2 Canfang Niu 0 2 Huoqing Huang 0 2 Pengjun Shi 0 2 Caihong Wang 0 2 Peilong Yang 0 1 2 Bin Yao * 0 2 0 Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences , Beijing, 100081 , P. R. China, 1 CAAS-ICRAF Joint Laboratory on Agroforestry and Sustainable Animal Husbandry , Beijing, 100193 , P. R. China 2 Editor: Y-H Percival Zhang , Virginia Tech , United States of America Background: Glucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme) producer, Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation. Methodology/Principal Findings: A glucoamylase gene (gla15) was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia) emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70C. The enzyme was stable over a broad pH range (2.2-11.0) and at high temperature up to 70C. It hydrolyzed the breakages of both a-1,4- and a-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H1-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of a-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger. - Conclusion/Significance: GLA15 has a broad pH stability range, hightemperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups. Starch is a polysaccharide carbohydrate of plant cells, and is composed solely of aglucose units that are linked by a-1,4- or a-1,6-glycosidic bonds. It has two homopolysaccharide types: amylopectin and amylose. Amylopectin is an a-1,4linked D-glucose polymer with approximately 5% of a-1,6-linked branches, whereas amylose is a linear polymer essentially consisting of a-1,4-linked glucopyranose residues [1]. Starch is one of the most important renewable natural resources and is used in many industries including textile, laundry, pharmaceutics, paper making and food manufacture. In food industry, starch is mainly used to produce glucose, which can be further utilized to produce high-fructose syrups, bioethanol, organic acids and amino acids [2, 3]. Mass production of glucose from starch is a two-stage process, involving aamylase and glucoamylase. In the first step, starch slurry is gelatinized, followed by liquefaction rapidly by a thermostable a-amylase at 95105 C and pH 6.06.5 [4]. In the following saccharification step, the obtained dextrin is further hydrolyzed to b-D-glucose by glucoamylase. Currently, Aspergillus niger glucoamylase is widely used in industry. It has the temperature and pH optima at 6065C and 4.04.5, respectively, and retains only 10% of initial activity after incubation at 70C for 30 min [5]. Thus adjustment of temperature and pH from liquefaction to saccharification process is necessary, which not only requires additional equipments but also causes higher production cost. Great efforts have been attempted to improve the performance of glucoamylase, including screening of glucoamylase-producing thermotolerant and thermophilic organisms [5], site-directed mutagenesis [6], and directed evolution [7, 8]. Unfortunately, these works only made limited contribution to the improvement of economical industrial glucoamylases. Exploration of novel glucoamylases is another strategy. Glucoamylases from thermoacidophilic and thermophilic bacteria have been reported, including Clostridium thermohydrosulfuricum [9], Clostridium thermosaccharolyticum [10], and Thermoanaerobacter tengcongensis MB4 [11]. In comparison with fungal glucoamylases, these enzymes are active and stable at high temperatures (6070C), but have low production yields. Archaea growing at temperatures higher than 60C is another promising microbial source of biocatalysts for industrial starch processing. Some glucoamylases from hyperthermophilic Sulf (...truncated)