Expression,Characterization And Thermostability Modification Of Endo-Polygalacturonase From Penicillium Sp.

Endo-polygalacturonase is one of the most widely studied pectinases belonging to the glycoside hydrolase 28 family,which can specifically hydrolyze polygalacturonic acid chain by acting uponα-1,4-glycosidic bonds to generate oligomeric galacturonic acid with different degrees of polymerization.Endo-polygalacturonase is suitable for the preparation of pectin oligosaccharides and is of great significance to improve the added value of pectin.In this study,two endo-polygalacturonase genes from P.rolfsii BM6 and P.arizonense were cloned and heterologous expressed in K.phaffii;the recombinant enzymes were purified and characterized,and composition and biological activities of the enzymatic products were determined;finally,the thermostability of one of the enzymes was modified by rational design.The main contents of this study were as follows:（1）Gene mining,heterologous expression and enzymatic characterization of the endo-polygalacturonases from Penicillium sp..The genes pe PGA and pe PGB from P.rolfsii BM6 and P.arizonense were heterologously expressed in K.phaffii,respectively.The recombinant enzymes were purified and characterized;the enzyme pe PGA showed the maximum activity at60°C in p H 6.0 and pe PGB had the maximum activity at 65°C in p H 5.0;the recombinant enzymes had good stability in the p H range of 3.5-8.0;The recombinant enzyme pe PGA remained 75.8%residual activity after 1 h incubation at 45°C,and pe PGB remained stable after1 h treatment at 50°C.The enzyme activities of pe PGA and pe PGB in the supernatant were 1572U·m L^-1 and 1662 U·m L^-1 by shake-flask cultivation,respectively.The recombinant enzyme pe PGB was expressed by high-density fermentation in a 50 L bioreactor.After 108 h induction by methanol,the protein concentration and enzyme activity in the fermentation supernatant were 2.33 mg·m L^-1 and 12144 U·m L^-1,respectively,which were 6.3 times and 7.3 times higher than those by shake flask.（2）Composition analysis and biological activities assay of the products hydrolyzed by the recombinant endo-polygalacturonases.The hydrolysis products of the recombinant enzymes were analyzed by high-performance anion-exchange chromatography.The results demonstrated that both enzymatic products contained more oligomeric galacturonic acid,but contained a small amount of mono-galacturonic acid.The pectin oligosaccharides pe PGA-POS and pe PGB-POS were prepared by using recombinant enzymes,and the biological activities of the two kinds of oligosaccharides were determined.Oligosaccharides pe PGA-POS and pe PGB-POS showed obvious antibacterial activity against the gram-negative bacteria（E.coli JM 109）and the gram-positive bacteria（B.Subtilis 168 and S.aureus）,however,pe PGA-POS and pe PGB-POS had almost no inhibition against fungi C.albicans.The oligosaccharides pe PGA-POS and pe PGB-POS had good antioxidant activities,and the DPPH free radical scavenging rate was74.0%and 71.6%,respectively.In addition,the growth of probiotics P.acidilactici,L.plantarum and L.paracasei were significantly promoted by oligosaccharides pe PGA-POS and pe PGB-POS.What is more,pe PGA-POS and pe PGB-POS were more conducive to the growth of P.acidilactici than fructooligosaccharides.（3）The website Disulfide by Design 2.0 was used to predict potential disulfide bond mutation sites,in which the thermostability of the mutant E257C/N286C was improved,and the residual enzyme activity was 1.5 times higher than that of the wild-type after 20 min of treatment at 55°C.The online prediction tools ABACUS and Fire Prot were used to predict the thermostability sites of pe PGB,in which the thermostability of the mutant D207N was improved,and the residual enzyme activity was 2.3 times higher than that of the wild-type after20 min of treatment at 55°C.The sites were combined to obtain the three-point mutant E257C/N286C/D207N,the residual enzyme activity was 2.9 and 11.6 times higher than that of the wild-type after 20 min and 60 min of treatment at 55°C.Through molecular dynamics simulation,it was shown that the thermostability of the mutant E257C/N286C/D207N was improved by the combined effects of disulfide bond and electrostatic interaction.