Engineering Of Transglycosylation Products Specificity And Fermentation Optimization Of β-galactosidase From Bacillus Circulans ATCC 31382

Galacto-oligosaccharide（GOS）,as a prebiotic widely used in the food,health products and pharmaceutical industries,has the functions of stimulating intestinal peristalsis,improving immunity,promoting the value of probiotics and preventing osteoporosis.The market demand on GOS increased continuously in recent years.At present,the industrial production of GOS mainly usesβ-galactosidase for enzymatic preparation.Therefore,the performance ofβ-galactosidase is the key to affect the production efficiency of GOS.In this study,β-galactosidaseβ-Gal-Ⅱderived from Bacillus circulans ATCC 31382 was heterologously expressed in Escherichia coli to study its enzymatic properties and performance to prepare GOS.Besides,five dominant variant s were obtained through molecular modification.Then the wild-type and dominant variants were heterologously expressed in Bacillus subtilis.The fermentation conditions were optimized,and high-density fermentation was carried out in a 3-L bioreactor.The main findings of this paper are as follows:（1）Heterologous expression,enzymatic properties and performance in GOS preparation ofβ-Gal-Ⅱin E.coli.Theβ-galactosidase gene（β-Gal-Ⅱ）derived from B.circulans ATCC31382 was chemically synthesized,and transformed into E.coli BL21（DE3）-Δlac Z expression strain.The enzyme activity of the broken wall supernatant was 6.57 U·m L^-1,and the specific activity was 15.59 U·mg^-1.Its optimum temperature and p H were 60℃and 5.0.It had a half-life of about 3 h at 50℃,but was almost completely inactivated when incubated at 60℃for 30min.And it maintained more than 95%of its activity when incubated at 40℃for 24 h.It also exhibited excellent stability in the p H range of 5.0-7.0,maintaining 99%activity after 24 h.The reaction were optimized for the preparation of GOS using lactose as the substrate,and the maximum conversion of 61.59%was finally obtained under the optimal reaction conditions(50℃,enzyme addition of 2.5 U·m L^-1,40%substrate concentration,p H 5.0)for about 24 h.Among all transglycosylation products,the yield of allolactose and long-chain GOS（DP≥3）reached 23.33%and 31.93%,accounting for 37.88%of the total GOS,respectively.（2）Construction of screening system,screening of variants and analysis of enzymatic conversion products.By constructing a screening system with allolactose as the effector and the fluorescence intensity of e GFP as the detection signal,the mutant library ofβ-Gal-Ⅱwas constructed and screened.Two variants Y406F and Y563F were finally obtained.Comparing the composition of the enzymatic conversion products of these two mutants and the wild type,the allolactose conversion rates of Y406F and Y563F increased from 23.33%of the wild type to 29.12%and 30.09%.（3）Rational design ofβ-Gal-Ⅱto improve long-chain GOS conversion.By analyzing the structure of the active center ofβ-Gal-II,five residues were selected for mutation and sixteen variants were constructed.Finally,R333M,N467S and R441K were identified to be the dominant variants.The enzyme conversion products of these three mutants were analyzed,and the results showed that the conversion rate of long-chain GOS of R333M,N467S and R441K was significantly improved compared with the wild type,from 31.93%to 41.36%,43.96%and38.55%.（4）Heterologous expression ofβ-Gal-II in B.subtilis,optimization of fermentation conditions,and high-density fermentation in 3-L bioreactor.The coding gene ofβ-Gal-Ⅱwas transferred into B.subtilis WS9 strain to obtain recombinant bacterium WS9/β-Gal-II.After 24h of shake flask fermentation,the enzyme activity was 3.63 U·m L^-1.The fermentation nitrogen source was optimized at the shake flask level in terms of type,concentration,and concentration and ratio of the combined nitrogen source.The results showed that when 7.5 g·L^-1 of yeast extract fermentation（YEF,Angel）and 7.5 g·L^-1 corn steep powder（Angel）were used as the compound nitrogen source,the total enzyme activity reached the highest value of 6.82 U·m L^-1,which was 1.5 times of TB fermentation and 2-2.5 times of single nitrogen source fermentation.After that,the fermentation temperature was optimized in the 3-L bioreactor,the results showed that 37℃was better than 33℃,and the total enzyme activity was up to 138.29 U·m L^-1,which was 20.3 times of the shake flask fermentation.The WS9/N46S and WS9/Y563F was fermented in 3-L bioreactor and the highest total enzyme activity was 19.3 times and 24.1 times of the shake flask fermentation,respectively.