Molecular Modification And Characterization Of Thermostable β-galactosidase BgaB Mutants

β-Galactosidase is a member of glycosyl hydrolyse. The enzymeβ-galactosidase hydrolyses theβ-1, 4-D-galactosidic linkage of lactose, as well as those of related chromogens,o-nitrophenyl-β-D-galactopyranoside (oNP-Gal) and 5-bromo-4-chloro- 3-indolyl-β-d-galactoside (X-gal).β-Galactosidases have been used as tools in the dairy industry and could be useful in molecular biology. The application ofβ-galactosidases for the hydrolysis of lactose to its component monosaccharides, glucose and galactose, is one possible approach to overcome lactose intolerance.Thermostableβ-galactosidase BgaB from Geobacillus stearothermophilus is an ideal candidate for the hydrolysis of lactose in milk. Because BgaB has higher stability properties combined with neutral pH activity and favorable temperature activity optima. Relative to the hydrolysis reaction of the major commercial source ofβ-galactosidase, the use of thermostableβ-galactosidase would enable the hydrolysis process to take place at higher temperatures and offer certain important advantages, such as reducted risk of microbial growth, increased substrate solubility and reduced product inhibition.The hydrolysis activity of BgaB has been found lower than commercialβ-galactosidase in previous research. In the present study, the research purpose is to improvement the hydrolysis efficiency of the enzyme by function modified using modern molecular biotechnology. Meanwhile, since BgaB and mostβ-galactosidases in GH-42 are from the organisms which can survive in various extreme circumstances, the molecular modify study of BgaB will has reference meaning to other members of GH-42. The 3D structure of BgaB molecular has been simulated by homology modeling. According to the catalytic domain and non-catalytic domain, the hydrolysis efficiency of BgaB has been modified and improved from three different parts, including substrate inhibition, enzyme activity and thermostability. The mutant enzymes obtained at different aimino acid residues have been studied and the effect of residues mutants on the structure and function of enzyme has been investigated. In summary, the main results of this research including:(1) The study of the molecular structure of BgaB shows that this enzyme has a canonical TIM (Distored triosephosphate isomerase) barrel structure, which consists of an inner of eight parallelβ-strands surrounded by eightα-helices. Two putative catalytic residues Glu148 and Glu303 are located at the C-terminal ends ofβ-4 andβ-7 of the TIM barrel, respectively. The inhibitor binding sites have been predicted by molecular docking and molecular dynamics simulations. And the binding sites are Arg109, Phe341, Trp311, Asn147, Asn310, Try272 and His354. The role of these residues in inhibitor binding has been studied by molecular dynamics simulations and replacements with Ala in vitro. The results indicated that Phe341plays an important in the inhibiton of hydrolysis production galactosidase. The mutant F341T was obtained by site directed mutagenesis at residue Phe341, the Ki of F341T is 220 mM. This mutant has lower Ki than wild-type enzyme and other mutants. (2) We use semi-rational approach to engineer the hydrolysis activity of BgaB. Site-saturation mutagenesis libraries have been constructed and the mutants R109V, R109W, Y272S, Y272A, and E351R, which enzyme activity had increased, were obtained by screening. Mutant E351R had the highest enzyme activity, approximately 3.5-fold hgher than that of the wild-type enzyme. The result of screening of site-saturation library of Arg109 residue showed mutants at Arg 109 had low activity. Further study of site saturation mutant on Arg109 residue and the mutant enzymes properties shows that when Arg109 were replaced with non-polar amino acids Ala and Gly, BgaB enzymatic activity would complete loss. Our work shows that Arg109 plays a significant role in maintaining hydrolysis activity.(3) The catalytic function of BgaB is influenced when the enzyme was expressed in the prokaryotic host. In order to improve the thermostability of BgaB, some residues in the non-catalytic domain of BgaB have been modified by site-directed mutagenesis. These residues are the amino acids which conserved in thermostablyβ-galactosidase of GH-42, but mutational in BgaB sequence. Thermostility of BgaB has been improved when the residue Il42 wre replaced generating the mutant I42E. Compared with wild type BgaB, the half life of mutant I42E at 70oC increased from 10min to 40min. Analysis of molecular structure of BgaB show that Ile42 is located at the margin of the TIM barrel. The hydrogen can be generated between Glu42 and Ser39. Ile42 and Ser39 were replaced with Ala to study these tow residues’s founction. The study shows that the enzyme activity and thermotility of I42A/S39A are both decreased. Tm values of I42A, S39A, and I42A/S3A are lower than that of wild type enzyme. These results indicated that the hydrogen generating between Ile42 and Ser39 plays an important role on the thermostability. In this research, the results also show that a possible strategy of thermostable enzymes adopted may be improving the enzyme activity with loss of thermostility.(4)The accumulation mutations including substrate inhibition reduced mutants, hydrolysis activity and thermostability improved mutants were studied. The thermostability and substrate inhibition were simultaneous improved by accumulation mutations. However, the accumulation of the mutations within activity residues sites may cause negative affect on enzyme.