Screening, Identification Of Cellulose-Degrading Strains And The Construction Of Genetic Engineering Lactococcus Lactis Which Produce Outer Endoglucanases

Cellulose as a feed additive can improve feed utilization rate and nutritional value of fodder and reduce feed costs. It is obvious in the promotion of animal growth, reduction of dejecta, the ecological environment improve and animal disease control. Meanwhile, through the fermentation of cellulose-degrading strains bacterial cells rich in protein are synthesized which can be used for single cell protein feed.Lactococcus lactis is the beneficial bacteria in annimal and human being's gut, it is generally acknowledged as a security microorganism, on the one hand, it could maintain the ecological balance in the gut, stimulate the mucous membrane immune response of the organism, on the other hand, the recombinant gene Lactobacillus could be useful an resource for the development of functional foods, medical care and oral vaccine.Congo-red staining method was used to isolated 33 fugues and 18 bacteria strains of cellulose-decomposing from the rumen of the cows and sheep and rotten fodder. According to the detection of cellulase activity,one fungi and one bacteria strain with high efficient cellulose decomposing were isolated. The results showed that the complete 18S rDNA gene sequence was 504 bp. A phylogenetic tree was constructed by comparing the 18S rDNA sequence with other relative fungus species in the GenBank database. In the phylogenetic tree the strain NL-3, Penicillium decumbens strain JU-A10 constitute a branch with the similarity value 99%. According to morphologica characteristics and phylogenetic analysis, the strain NL-3 was identified as Penicillium decumbens. A phylogenetic tree was constructed by comparing the 16S rDAN sequence of the strain with other relative bacteria species in the GenBank database. According to Physiological and biochemical characteristics and phylogenetic analysis, the strain N9 was identified as Bacillus subtilis.According to the sequence of eg published on GenBank, eg gene was PCR amplified from N9 genome DNA by primer designed for Multiple cloning site of pMG36e. The complete gene sequence was 1518bp. By sequence analysis, the similarity value betwwen eg and other endo-cellulase was 92%. Recombinant Lactococcus lactis plasmid vectors pMG36e was constructed by subcloning eg gene. Recombinant genetic engineering Lactococcus lactis producing endoglucanases was constructed by transforming recombinant plasmid into competent cell of Lactococcus lactis MG1614. The recombinant transformants vaccinated to the M17 plates containing CMC could produce degradation halo. The sizes of expressed recombinant proteins was 54 KD by SD-PAGE analysis and Western blot analysis.Screening of high effective cellulose degradation strains, cloning of eg gene, constructing of Lactobacillus expression vector pMG36e-eg and recombinant genetic engineering Lactococcus lactis producing endoglucanases were completed in the study. It is expected that highly active cellulase are produced to use Lactobacillus expression system , and multi-functional microecological products producing endo cellulose are developed.