Site-directed Mutagenesis Of Sucrose Isomerase Pal? Derived From Klebsiella Sp. LX3 And Analysis Of Fermentational Products Of Pali Expressed By Bacillus Subtilis

Isomaltulose(?-D-glucosylpyranosyl-1,6-D-fructofranose)has excellent physiological functions,can be widely used as a sweetener,and has little in nature.Transformation of sucrose with sucrose isomerase(EC 5.4.99.11)to produce Isomaltulose is the main method of industrial production.Since sucrose isomerase catalyzes the sucrose conversion process,there is always a by-product monosaccharide,which is unfavorable to production.In this paper,the sucrose isomerase PalI derived from Klebsiella sp LX3 was used as the research object to explore the elimination of by-product monosaccharides in the product by site-directed mutagenesis and heterologous expression of Bacillus subtilis.The main research contents are as follows:(1)Since the main production of trehalulose-type sucrose isomerase is low in monosaccharides(glucose,fructose),we compared it with isomaltulose-type sucrose isomerase PalI and analyzed at secondary structure and three dimensionality.According to the multiple sequence alignment of the protein,the spatial difference of the active site and the surface hydrophobicity of the RDRDR loop region,six mutants of S459 A,R452A,S459A/R452 A,Lyo,del-D and Lyo/del-D were preliminarily designed.PalI was expressed by E.coli BL21(DE3)and purified by resin chromatographic column.The pure enzyme was reacted with the corresponding concentration of the sucrose substrate,and the product was analyzed by HPLC to obtain the corresponding product parameters and enzymatic parameters.The results showed that the ratios of isomaltulose to trehalulose peak area,trehalulose isomaltulose peak area to fructose glucose peak area of mutant S459 A,S459A/R452 A,and Lyo/del-D were significantly changed.The area ratio of isomaltulose to trehalulose peaks was 3.5,3.4,and 3.3,respectively,while the area ratio of trehalulose isomaltulose peaks to fructose glucose peaks were 40,49,23,respectively.Compared with the wild type enzyme,the isomaltulose to trehalulose peak area ratio was reduced by about 50 %,respectively,and compared with the trehalulose isomaltulose peak area to the fructose glucose peak area ratio increased by 18 %,48 % and reduced by 30 %.Kinetic analysis of three mutants,S459 A,S459A/R452 A and Lyo/del-D,and the Km value and Vmax were determined.Km was 171.6 ?mol/L,158.4 ?mol/L,160.4 ?mol/L,respectively,and Vmax was 21.3 ?mol/L/s,10.7 ?mol/L/s,4.6 ?mol/L/s,respectively.The conversion of each enzyme was calculated by reacting 4 % sucrose as substrate for 1 h.The conversion rates of S459 A,S459A/R452 A and Lyo/del-D were 71 %,37 % and 72 %,respectively.(2)Glucose and fructose,a by-product of sucrose isomerase,can be used as a rapid carbon source for microbial growth.The sucrose isomerase is constructed into pHT43 vector and introduced into the food grade expression vector Bacillus subtilis WB800 strain,and LSP(LB/Sucrose/Phosphate)medium was used to induce expression PalI at 30 °C.Analysis of various sugar components in the fermentation broth by HPLC revealed that the ratio of isomaltulose to trehalulose peak area,trehalulose isomaltulose peaks area to fructose glucose peaks area were mainly related to fermentation conditions.The concentration of sucrose and the fermentation time are crucial for composition of product.At 24 h of fermentation,the isomaltulose to trehalulose peak area ratio was less than 2,the trehalulose isomaltulose peak area to the fructose glucose peaks area ratio were 92:1.The maximum conversion effect of sucrose is that 20 % sucrose in the medium is completely converted in 24 h.