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

被引:0
作者
Zhiwei Zhang
Fenghua Zhang
Liya Song
Ning Sun
Weishi Guan
Bo Liu
Jian Tian
Yuhong Zhang
Wei Zhang
机构
[1] Shanxi Agricultural University,College of Forestry
[2] Chinese Academy of Agricultural Sciences,Biotechnology Research Institute
[3] Beijing Technology and Business University,Beijing Key Lab of Plant Resource Research and Development
来源
3 Biotech | 2018年 / 8卷
关键词
β-Galactosidase; Galactose inhibition; Lactose hydrolysis; Molecular modification;
D O I
暂无
中图分类号
学科分类号
摘要
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.
引用
收藏
相关论文
共 77 条
[1]  
Dong YN(2013)Optimizing lactose hydrolysis by computer-guided modification of the catalytic site of a wild-type enzyme Mol Divers 17 371-382
[2]  
Wang L(2010)Recent advances refining galactooligosaccharide production from lactose Food Chem 121 307-318
[3]  
Gu Q(2008)Perspectives and applications of immobilised -galactosidase in food industry—a review Czech J Food Sci 26 1-14
[4]  
Chen H(1991)Applications of lactase in dairy foods and other foods containing lactose Food Technol 45 93-95
[5]  
Gosling A(2010)Engineering of a fungal beta-galactosidase to remove product inhibition by galactose Appl Microbiol Biotechnol 87 1773-1782
[6]  
Stevens GW(2011)Reduction of galactose inhibition via the mutation of beta-galactosidase from Biotechnol Lett 33 353-358
[7]  
Barber AR(2013) for lactose hydrolysis Int J Biol Macromol 60 109-115
[8]  
Grosová Z(2004)The crystal structure of acidic beta-galactosidase from Biotechnol Prog 20 1259-1262
[9]  
Rosenberg M(2006)Immobilization of lactase from J Agr Food Chem 54 4989-4998
[10]  
Rebroš M(2013) greatly reduces the inhibition promoted by glucose. Full hydrolysis of lactose in milk Protein Expr Purif 92 88-93