| China is a big country in sugarcane bagasse cultivation,and there is a large amount of sugarcane bagasse as a by-product in sugarcane processing,but no effective treatment or reuse approach has been formed.Taking bagasse as raw material and releasing monosaccharide through enzymatic hydrolysis as raw material for carbon supply in fermentation industry is one of the important application directions of bagasse resource reuse,which is helpful to improve the economic benefit and society benefit of sugarcane comprehensive processing industry.Breeding new strains with direct hydrolysis and utilization of bagasse to produce high value added chemicals or materials monomers will contribute to the large-scale application of bagasse enzymatic interpretation of monosaccharide with low cost and high efficiency.Therefore,this study based on the results of previous research in a laboratory,used the hydrolases exo glucanase(Cbh C),endo glucanase(Egl A)and β-glucosidase(Bg A),which are effective in assisting the hydrolysis of bagasse,as target enzyme molecule.Taking the L-lactic acid producing strain as the starting strain,recombinant E.coli with the ability to secrete and express these enzymes was constructed by genetic engineering,a new system of mixed fermentation and a new technology of consolidated bioprocess to produce Llactic acid were established.The main research contents of this paper are as follows:(1)Recombinant E.coli producing exo-glucanase(Cbh C),endo-glucanase(Egl A)and β-glucosidase(Bg A)was successfully constructed.Using the recombinant plasmids p PIC9K-cbhc,p PIC9K-egla and p PIC9K-bga obtained in the early stage of the research group and carrying exogenous genes derived from Aspergillus niger as templates,the complete sequence was cloned by PCR amplification The recombinant plasmids p ND-cbhc,p ND-egla and p ND-bga were obtained by cloning the target genes into downstream of the promoter Pamy L and signal peptide SPamy of expressing the plasmid p ND-113.The above recombinant was transformed into E.coli B0013-090 B,a strain with high production of L-lactic acid monomer,and constructed the recombinant E.coli 090B-cbhc,090B-egla and 090B-bga,which secrete and express exo-glucanase,endo-glucanase and β-glucoside successfully.(2)The recombinant E.coli 090B-cbhc,090B-egla and 090B-bga expressed and secreted exo-glucanase,endo-glucanase and β-glucosidase successfully.The results showed that there are obvious transparent circles around the spotting holes of the fermentation broth of the three recombinant bacteria on the Congo Red plate.Using CMC-Na as substrate,the enzyme activity of 090B-cbhc and 090B-egla were 0.220U/m L and 0.530 U/m L.The enzyme activity of the recombinant strain 090B-bga was determined to be 0.070 U/m L by using cellobiose as substrate.(3)A new mixed-bacteria fermentation process using bagasse as raw material was established,and realize the direct fermentation to synthesize L-lactic acid using bagasse as raw material.Recombinant bacteria are aerobic cultured to produce enzymes.The fermentation broth of 090B-cbhc,090B-bga and 090B-egla is based on the amount of bacteria 1:2:1,it is connected to the bagasse medium to enter the anaerobic fermentation.At the end of fermentation,the output of lactic acid is 15.0 g/L,the conversion rate of bagasse to lactic acid is 43.5%,and the conversion rate of lactic acid is 0.417 g/(L·h),compared with the 090 B,the lactic acid content increased by30.43%.A new process for synthesis of L-lactic acid from bagasse by consolidated bioprocessing with recombinant Escherichia coli mixed system was established. |
