Optimization Of Cellulase Gene And Its Expression In Lactobacillus Plantarum

Cellulose is the largest recycled resource in nature,and it has a long history used by people.If these resources can be used effectively,it will have practical significance for humankind to solve energy crisis,food shortages and environmental pollution.At present,using microorganisms and cellulase to break down cellulose is an efficient and environmentally friendly method.Cellulase as a feed additive can significantly increase feed digestibility and availability,improve feed quality and animal production performance,which has a broad application prospect in animal husbandry,but its application has been restricted by low enzyme activity and high cost.More attention has been aroused in modifying cellulase gene by directed evolution technology in vitro and constructing genetic-engineering microbes for high-activity cellulase production.In this study,exogluconase CBH I gene was amplified from DNA library of Trichoderma koningii and Aspergillus niger.DNA shuffling was used to reform cellulase gene,which was connected with plasmid p MG36 e to construct recombinant vector of p MG36e-CBH I,and then transferred into Lactobscillus plantarum for expression.The main contents were as follows.(1)The CBH I gene from Trichoderma koningii and Aspergillus niger was successfully selected by RT-PCR,and the homology between the two genes and cellulase gene published in NCBI was greater than 99%.(2)Using DNA shuffling to reform cellulase genes,and then the mutant genes were cloned in p MD19-T vector and transformed into DH5α.We got g1 and g2.In g1,the bases were changed from G to A at site 172,from A to G at site 636,and the amino acids were changed from Arg to Try,Pro to Ser;in g2,the bases were changed from G to A at site 172,from G to A at site 554,from T to C at site 1114,and the amino acids were changed from Arg to Try,Pro to Leu,but the amino acid wasn’t changed at site 1114,it was Glu.The mutant cellulase genes were cloned in p MG36 e vector for further gene transformation.(3)The vectors were transformed into Lactobscillus by electroporation,and selected by erythromycin resistance screening.The result indicated that the cellulase activity of g1 was 4.83 and 4.19 U/L in MRS medium with low glucose.The optimal temperature was 50 ℃ for both mutant cellulases,and the optimal p H value was 5.0 for g1 and 5.5 for g2.