Site-directed mutation of β-galactosidase from Aspergillus candidus to reduce galactose inhibition in lactose hydrolysis

3 Biotech (2018) 8:452 https://doi.org/10.1007/s13205-018-1418-5 ORIGINAL ARTICLE Site-directed mutation of β-galactosidase from Aspergillus candidus to reduce galactose inhibition in lactose hydrolysis Zhiwei Zhang1 · Fenghua Zhang2 · Liya Song3 · Ning Sun2 · Weishi Guan2 · Bo Liu2 · Jian Tian2 · Yuhong Zhang2 · Wei Zhang2 Received: 31 May 2018 / Accepted: 1 September 2018 © The Author(s) 2018 Abstract β-Galactosidase is widely used for hydrolysis of whey lactose. However, galactose inhibition has acted as a major constraint on the catalytic process. Thus, it is sensible to improve upon this defect in β-galactosidase through protein modification. To reduce the galactose inhibition of Aspergillus candidus β-galactosidase (LACB), four amino acid positions were selected for mutation based on their molecular bindings with galactose. Four mutant libraries (Tyr96, Asn140, Glu142, and Tyr364) of the LACB were constructed using site-directed mutagenesis. Among all of the mutants, Y364F was superior to the wild-type enzyme. The Y364F mutant has a galactose inhibition constant (Ki) of 282 mM, 15.7-fold greater than that of the wild-type enzyme (Ki = 18 mM). When 18 mg/ml galactose was added, the activity of the wild-type enzyme fell to 57% of its initial activity, whereas Y364F activity was maintained at over 90% of its initial activity. The wild-type enzyme hydrolyzed 78% of the initial lactose (240 mg/ml) after 48 h, while the Y364F mutant had a hydrolysis rate greater than 90%. The β-galactosidase Y364F mutant with reduced galactose inhibition may have greater potential applications in whey treatment compared to wild-type LACB. Keywords Aspergillus candidus · β-Galactosidase · Galactose inhibition · Lactose hydrolysis · Molecular modification Introduction Zhiwei Zhang and Fenghua Zhang contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13205-018-1418-5) contains supplementary material, which is available to authorized users. Approximately, 85% of the milk used for manufacturing cheese is discarded as whey (Panesar and Kennedy 2012). Recovery of whole whey solids as ingredients for human or animal food has been a common approach adopted by large industrial processors. Since the main component (70–72%) Jian Tian * Yuhong Zhang Wei Zhang Zhiwei Zhang Fenghua Zhang 1 Liya Song College of Forestry, Shanxi Agricultural University, Taigu, Shanxi 030801, People’s Republic of China 2 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People’s Republic of China 3 Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University, Beijing 100048, People’s Republic of China Ning Sun Weishi Guan Bo Liu 13 Vol.:(0123456789) 452 Page 2 of 7 of whey powder is lactose, direct utilization of whey is impeded by its poor sweetening power, low solubility, and lactose intolerance. Hydrolysis of lactose to monosaccharides, however, significantly increases the options to producing various by-products from whey. For example, hydrolyzed lactose has greater sweetening power and capability to replace saccharose or starch syrup in confectionery and ice-cream industries (Panesar et al. 2006). Hydrolyzed lactose can act as a substrate to produce d-tagatose, an important hexoketose monosaccharide sweetener with health-care functions (Oh 2007), which can greatly increase the additional output of whey in dairy processes. β-Galactosidase (E.C. 3.2.1.23), also known as lactase, has been suggested for hydrolyzed-lactose milk production and whey hydrolysis (Panesar et al. 2006). However, complete hydrolysis at high lactose concentrations is difficult due to inhibition by galactose and glucose, which can slow the hydrolysis process or even stop the reaction (Park et al. 2010b). Galactose acts as a competitive inhibitor of microbial β-galactosidases by forming galactosyl–enzyme intermediates with β-galactosidase, preventing lactose from entering the active site (Gosling et al. 2010). The mutation of β-galactosidase from Caldicellulosiruptor saccharolyticus can clearly reduce galactose inhibition in lactose hydrolysis (Kim et al. 2011). However, β-galactosidase derived from Caldicellulosiruptor saccharolyticus must pass a series of assessment before it can be applied to industrial food practices. The β-galactosidases of commercial interest are isolated mainly from Kluyveromyces spp., Candida kefyr yeast and the Aspergillus spp. fungi (Holsinger and Kligerman 1991; Grosová et al. 2008). Aspergillus candidus β-galactosidase (LACB, Uniprot entry: Q8TFE6) has excellent enzymatic properties, including high thermostability, high specific activity, and a wide pH range for enzymatic reactions compared to the commercial enzyme from Aspergillus oryzae ATCC 20,423 (Zhang et al. 2002). However, LACB is also seriously inhibited by galactose during whey lactose hydrolysis, and a large amount of galactose is produced during the process. For this reason, it is worth attempting to modify LACB for applications in the whey industry. The crystal structure of Aspergillus oryzae β-galactosidase (LACA, Uniprot entry: Q2UCU3) has been determined at a 2.60 Å resolution, and four galactose-binding sites were suggested to exist on the enzyme (Maksimainen et al. 2013). As LACB and LACA share high sequence similarity and both belong to the glycoside hydrolase 35 (GH-35) family, the latter was explored as a template to determine the galactose-binding residues in LACB. In this study, the predicted residues were engineered to reduce galactose inhibition. 13 3 Biotech (2018) 8:452 Materials and methods Strains, plasmids and media Escherichia coli Trans1-T1 cells (TransGen Biotech, Beijing, China) and Pichia pastoris GS115 (Invitrogen, CA, U.S.A) were used as gene cloning and expression hosts, respectively. The P. pastoris–E. coli shuttle expression vector pPIC9-lacb was previously constructed in our laboratory (Zhang et al. 2002). Media, including minimal dextrose (MD) medium, minimal methanol (MM) medium, and yeast peptone dextrose (YPD) medium, were prepared according to instructions in the Pichia expression kit (Invitrogen). Fermentation basal salts (FBS) medium and Pichia trace metal (PTM) complied with Pichia fermentation guidelines (Invitrogen). Construction of the mutation library The to-be-mutated sites in Aspergillus candidus β-galactosidase (LACB) were determined by sequence alignment between LACB and Aspergillus oryzae β-galactosidase (LACA). These two enzymes are both in the CAZy (http:// www.cazy.org/) GH-35 family with high sequence similarity (99.3%) and a close evolutionary relationship. The galactose-binding sites in the LACA have been previously reported (Maksimainen et al. 2013). Residues Tyr96 (Y96), Asn140 (N140), Glu142 (E142) and Tyr364 (Y364) in LACB that were similar to galactose-binding sites in LACA (...truncated)