Cloning,Expression And Application Of A Highly Active Trehalase

Trehalase hydrolyze trehalose into glucose and is widely used in industrial fermentation,food production and biomedicine.In the field of industrial fermentation,the addition of trehalase improves the utilization rate of carbohydrate raw materials and the yield of target fermentation products.However,the research on trehalase in China is relatively backward,with few reports on trehalase resources and low overall enzyme activity expression level.It is urgent to explore new trehalase strains and coding genes,construction of high expression system and enzyme application research.In this paper,therefore,we focus on the selection and identification of trehalase-producing strains in soil,the cloning and recombinant expression of trehalase gene and the study of its enzymatic properties,protein engineering modification of trehalase and its fermentation,and application characteristics of the enzyme.The main research contents are as follows:(1)Breeding of trehalase-producing bacteria and cloning and expression of coding genes.A strain of P.cypripedii was screened from nature,and its trehalase coding sequence gene PCTre was cloned and expressed in Escherichia coli and Bacillus subtilis systems respectively.The recombinant E.coli BL21(DE3)/p ET-3b(+)-PCTre had high enzyme production capacity,and the maximum fermentation enzyme activity was 289 U/m L.The recombinant strains were isolated and purified from the fermentation broth.SDS-PAGE analysis showed that there was a single band at about 63 k Da,consistent with the theoretical molecular weight,and the specific enzyme activity was 943 U/mg.The enzymatic properties study showed that PCTre hydrolyze trehalose specifically.Its optimum p H and temperature are 5.5 and 35°C respectively and PCTre shows good stability in the range of 25-55°C and p H 5-8.5.PCTre has good tolerance to organic solvents.After 24 h treatment in 15% ethanol environment,there was still 60% of residual activity.It is speculated a good potential in ethanol fermentation production.(2)Protein engineering modification and on-tank fermentation of trehalase.In order to enhance the application potential of the recombinant trehalase PCTre,a site-directed mutagenesis strategy was used to modify the molecule of the enzyme.Based on the threedimensional model and bioinformatics analysis of PCTre,43 mutants with potential impact on enzyme activity or stability were screened and designed.The mutant strains were constructed and trehalase activity and enzymological properties were determined by fermentation.The experimental results showed that the enzyme activity of the mutant strain A308 E increased by46% to 422 U/m L and specific activity was 1251 U/mg.Its stability was improved at p H 5.5compared with the wild type.The stability of mutant strains T477 A,S521T,P196 C and Q255 K was also improved within 25-35°C.The mutant strain was fermented in a 5 L fermenter and the highest enzyme activity reached 2150 U/m L at 28 h by fed-batch fermentation method,which was 5.58 times higher than that of the shaker bottle;The highest enzyme activity of 4130 U/m L was obtained at 28 h by constant rate feeding fermentation strategy,which was 1.92 times that of batch fermentation.(3)Application of trehalase.In order to explore the application value of trehalase,the hydrolysis of trehalose in yeast extracts by trehalase was investigated.The results showed that trehalose in the yeast extract could be completely hydrolyzed within 20 min,and the glucose content of the samples increased after hydrolysis.Among them,the glucose content of Procelys Yeast Extract 7 increased from 1.45% to 10.31%,which the application value of trehalase in enhancing yeast extracts.In the simulated ultra-high gravity ethanol fermentation system,the hydrolysis of trehalase on trehalose was investigated.Addition of 50 U trehalase could completely hydrolyze trehalose within 12 h in a 100 m L reaction system containing 5%trehalose and 15% ethanol.The good hydrolysis effect of trehalase in simulating ethanol fermentation system laid the foundation for its further application in industrial ethanol production.