Rational Design To Improve The Catalytic Ability Of β-galactosidase And Its Application In Low-lactose Milk Preparation

Milk is a nutrient-rich beverage,while the principal carbohydrate,lactose,in milk could cause lactose intolerance.Nowadays,approximately 70%of the world population suffers from lactose intolerance.β-galactosidase is widely applied to produce the low-lactose product.β-galactosidase（LACA）derived from Aspergillus oryzae is regarded as safe for the food industry and shows outstanding advantages of high catalytic activity and strong extracellular secretion.However,the acidic pH optimum limits its catalytic efficiency in the production of low-lactose products with neutral pH such as milk.In the present study,β-galactosidase from A.oryzae in GH 35 family was heterologously expressed in P.pastoris X33 by genetic engineering.Mutants with higher pH optimum were obtained by protein engineering,and two mutants showed better hydrolytic ability in milk lactose hydrolysis.The reasons for the improvement of the mutants were analyzed.Finally,the heterologous expression level ofβ-galactosidase in P.pastoris was increased by co-expression with the antioxidant factor.The main results are as follows:（1）The gene ofβ-galactosidase from A.oryzae was optimized and cloned into the expression vector p PICZαA.The target gene was transferred into P.pastoris X33 by electroporation to get a recombinant strain that can expressβ-galactosidase efficiently.The amount of protein in the supernatant reached 1.1 mg·m L^-1,and theβ-galactosidase activity in the supernatant was 298 OHA·m L^-1 by shake-flask culture.（2）Surface charge engineering and catalytic domain engineering were applied to LACA to increase its pH optimum.Super Charge module in the Rosetta webserver was used to design10 mutation candidates which contributed to an increased overall number of positive charges on the surface.However,no beneficial mutants were obtained.The structure of LACA was aligned withβ-galactosidase in the GH 35 family with different pH optimums to select the mutation candidates that may influence the pH optimum of LACA.The Y138 and Y364 in the catalytic domain of LACA were chosen to be mutated.The Y138F,Y138V,Y138A,and Y138L mutants all increased the pH optimum from 4.5 to 5.5,and the Y364F mutant shifted the pH optimum to 6.0 by experimental verification.By p Ka prediction,the p Ka values of the acid/base residues,E200,in the Y138F and Y364F mutants increased from 6.1 to around 8.1 which may explain the reason for the higher pH optimums of these mutants.（3）The mutant enzymes were used to hydrolyze the lactose in milk.With the addition of the same activity units of the enzymes,the Y138F and Y364F hydrolyzed 60.3%and 82.5%,respectively,of the initial lactose in milk after 3 h of reaction which showed advantages over the wild-type（42.4%）and the commercial lactase（52.7%）.The lower end-product inhibition of the Y138F and Y364F mutants is another important reason for the high efficiency of lactose hydrolysis.We studied the galactose inhibition of theseβ-galactosidases.The results showed that the Y138F and Y364F mutants exhibited lower galactose inhibition than the wild-type enzyme.Molecular dynamics simulations were carried out to quantify the galactose-binding free energy of wild-typeβ-galactosidase,Y138F,and Y364F.The lower affinity for galactose of Y138F and Y364F may speed the galactose expulsion which leads to the lower galactose inhibition of Y138F and Y364F.（4）A total of 10 endogenous facilitators were co-expressed withβ-galactosidase in P.pastoris,including chaperones,antioxidant factors,and heat shock transcription factor HSF1.Among these facilitators,co-expressing the antioxidant gene,GLR1,coding for glutathione reductase withβ-galactosidase led to the expression ofβ-galactosidase increasing by 22.2%.The strain with the best expression ability termed X33-opt-GLR1-2 was fermented in 3 L bioreactor.The expression level ofβ-galactosidase in the 3 L bioreactor was 11.2-fold that in the shake flask.