| Bean dregs, soybean by-products, are excellent plant protein carbohydrate material, and cellulose content of which is up to about 52 %, and contain a variety of nutrients. The okara hydrolysis will increase its efficiency, the present study constructed by okara engineered bacteria capable of hydrolyzing biotechnological, which lays foundation for the improved yield of soluble dietary fiber and oligosaccharide in the okara as well as soybean isoflavones, providing a new way for enhancing the added value of bean dregs.The key to improving the content of soluble substances is to improve the utilization of its cellulose, but cellulase enzyme cellobiohydrolase crucial in cellulase system, which is the only component of the enzyme hydrolysis of cellulose crystalline regions. The key to improving the content of soluble substances is to improve the utilization of its cellulose, but cellulase enzyme cellobiohydrolase crucial in cellulase system, which is the only component of the enzyme hydrolysis of cellulose crystalline regions.The primer was designed according to T.reesei CBH II gene(GenBank accession number: M55080) and the gene cDNA was obtained by PCR method with the sequence length of approximately 1440 bp. Then the whole CBHⅡ gene was synthesized after the codon of were optimized according to lactic acid bacteria preference; Prokaryotic expression vector pMG36e-CBH II was constructed through connection between target gene and shuttle vector pMG36 e. First, the CBH II(cellobiohydrolase) gene from Trichoderma viride expression was study by E. coli expression system, The expression vector pMG36e- CBH II into E. coli BL21 with heat shock method, construct recombinant bacteria cells and cultivation of recombinant bacteria to extract proteins, The recombinant proteins was determinated by SDS-PAGE electrophoresis and displayed a molecular weight of the protein is approximately 53 kD. The enzyme activity of cellulose is 16.4 U/ mL in cell supernatant by DNS assay and supernatant when the culture to 16 h, there is almost no activity in cell lysate. The results showed that cellobiohydrolase gene in E. coli expression was successfully.The cellobiohydrolase gene from Trichoderma viride expression was studyed by Lactobacillus expression system. A 81 bp signal peptide sequence was added in the upstream gene in order to secretory expression of the target protein. Then the whole CBH II gene was synthesized after the codon of were optimized according to lactic acid bacteria preference; Prokaryotic expression vector pMG36e-S-CBH II was constructed through connection between target gene and shuttle vector pMG36 e. The method of electric transformation was used to get recombinant lactic acid bacteria on lactic acid bacteria competent cells. The recombinant proteins was determinated by SDS-PAGE electrophoresis and displayed a molecular weight of the protein is approximately 53 kD which consistent with the expected size. Enzyme activity of Lactococcus lactis engineering bacteria in different temperature, different pH, different culture time were studied. The enzyme activity of cellulose in the appropriate temperature and pH were 50 ℃ and 5 respectively,Which is 16.7 U/ mL in culture supernatants by DNS assay when the culture to 16 h. There is almost no activity in cell lysate and supernatant. The results showed that the signal peptide sequence was correctly identified by lactic acid bacteria and the extracellular expression was realized successfully, which laid the foundation for further experimental research.In this study, lactic acid bacteria fermented bean dregs project at 30 ℃, pH natural conditions,we measured the water-soluble substance fermented of bean dregs. To analyze data Used SPSS 21.0 statistical analysis software, we used independent sample t-test to recombinant bacteria fermentation bean dregs and without restructuring bacteria fermented bean curd quality difference analysis of water-soluble substances, results show that the P < 0.05, the difference significant. The essential nutrients determination of bean dregs after fermentation, results show that the total fiber increased by 3.89 %, crude fat increased by 17.43 %, the total amino acid increased by 1225 %, 63.9 % increase of total sugar, soluble dietary fiber increases by 138.05 % :The test results show that the construction of engineering strain of Lactococcus lactis can reach the effect of fermented soybean dregs and improve the content of soluble substances purpose... |
PDF Full Text Request