Genome Mining,Heterologous Expression And Semi-rational Engineering Of Endo-inulinase

With the development of people's health awareness,fructooligosaccharides?FOS?have attracted wide attention.FOS is a kind of prebiotics which can stimulate the growth of probiotics in gut,improve overall health of man and reduce the risk of diseases.Thus,FOS has high values in food industry.Endo-inulinase can act on the?-2,1 glycosidic bonds of inulin to produce FOS.Compared with traditional method using fructosyl transferase,the application of inulinase in FOS production has several advantages,such as low by-product proportion and high production efficiency.However,the sources of endo-inulinases are very limited,mainly from molds and bacteria.Meanwhile,the low yield and poor enzymatic properties of existing endo-inulinases also unfavored the application of this enzyme in industries.Therefore,it is important to find novel endo-inulinases from different microbial sources with good properties.In this study,the first yeast-derived endo-inulinase gene was discovered through rational gene mining and then was solubly expressed in Escherichia coli.The enzymatic properties and the effect of this enzyme on FOS production was tested in lab.Furthermore,the yield of this novel endo-inulinase in E.coli was improved through fermentation optimization.Finally,its catalytic activities were improved through molecular docking and site-directed mutation.The main results of the paper are as follows:1.Genome mining and eterologous expression of endo-inulinase.Three putative endo-inulinase genes from different microbial sources were found through rational genome mining.Lipomyces starkeyi NRRL Y-11557 was the only one likely to produce endo-inulinase after enzymatic activity verification,but the yield is rarely low.Through gene sequence alignment,transcription analysis,enzymatic activity and SDS-PAGE analysis,inu3B was confirmed as the key functional gene,and its soluble expression was achieved with pET-22b?+?in E.coli BL21?DE3?.2.Biochemical properties of INU3B.The specific activity of INU3B against inulin was2262.8±82.3 U·mg^-1,which was the highest reported to date.The optimal temperature and pH of INU3B were 70°C and pH 5.0-6.0,it also show good stability against high temperature and broad pH.The K_m and V_max values were 10.6 mg·m L^-1 and 2564.1?mol·mg^-1·min^-1,respectively.The main products were DP3,DP4 and DP5 with no by-products?glucose and fructose?when INU3B was applied in FOS production in lab.3.Fermentation optimization was conducted in flask scale.The optimized conditions were as follows:recombinant E.coli harboring inu3B gene was cultured in glucose 8 g·L^-1,yeast extract 24 g·L^-1,peptone 12 g·L^-1 and phosphate 200 mM at 37°C,180 r·min^-1 for 2 h,and then induced by 0.1 mM IPTG at 20°C for 36 h.The maximal enzymatic activity reached 1316.3U·m L^-1,which was 2.13 times as high as that not optimized.4.Semi-rational engineering to improve catalytic activity.The three-dimensional protein structure of INU3B was constructed by homology modeling and molecular docking was conducted for INU3B and GF4.Based on the results of molecular docking,alanine screening was done targeting the 7 key amino acids(M⁴⁶,F¹⁰⁴,T¹⁰⁵,V²³⁹,T²⁶⁰,S³²⁶,Y³²⁷)existing in substrate binding pocket of INU3B.The specific enzymatic activity of M46A mutant was 1.26times higher than that of wild-type while the activities of other six single mutants decreased.This indicated the important roles of those amino acids in endo-inulinase catalysis.