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Screening, Identification of Cellulose-Degrading Strains and the Construction of Genetic Engineering Lactococcus Lactis Which Produce Outer Endoglucanases

Author: LiFangZheng
Tutor: ZhaoHongKun
School: Shandong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Cellulose-degrading bacteria Appraisal Endo- cellulase Lactococcus lactis
CLC: S816.7
Type: Master's thesis
Year: 2011
Downloads: 292
Quote: 3
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Abstract


Cellulase as a feed additive to improve the nutritional value of feed utilization and roughage, reduce feed costs, effective in promote animal growth, reduce feces emissions, and improve the ecological environment and animal disease control, and cellulose-degrading bacteria fermentation straw, can be synthesized bacterial cells are rich in protein, these decomposition products and biomass can be used for single-cell protein feed. Lactic acid bacteria is a common bacteria within the intestinal tract of humans and animals, is recognized as the security level (Generally Recognized As Safe, GRAS) microorganisms. As a probiotic that can help maintain the ecological balance of intestinal flora micro, can activate the body's mucosal immune response, inhibiting the growth of pathogenic microorganisms in the intestinal tract, and enhance animal immunity and other biological functions. On the other hand, genetic engineering of lactic acid bacteria can set the prebiotic and the dual function of exogenous gene expression in one, in the field of functional foods, health care, labor oral vaccine has attractive prospect. This experiment from cows, goats, rumen contents and rotting forage by sodium carboxymethyl cellulose (CMC-Na) Tablet selection culture Congo red staining initial screening bacterial strains 18 strains able to produce clear transparent ring, the fungal strain 33 strains by measuring cellulase activity rescreening, isolated cellulose-decomposing fungi and bacteria each one as a follow-up experimental study by cloning fungal NL-3 of the 18S rDNA sequence and its homology to build Phylogenetic tree of related species. The results showed that the 18S rDNA sequences to have a total length of 504 bp, BLAST homology showed that the strain Penicillium; NL-3 strain on the evolutionary relationships and Penicillium (Penicillium) together into a family. Especially with the highest homology of Penicillium decumbens strain JU-A10, the sequence similarity of 99%. Based on strain morphology and 18S rDNA gene sequence analysis identified the isolates was identified as Penicillium genus Penicillium decumbens (Penicillium decumbens). Cloned bacterial 16S rDNA sequences of N9. Sequencing analysis showed that, 1514bp of its 16S rDAN and length of the sequence. BLAST homology of the strain Bacillus subtilis (Bacillus subtilis). Homology constructed a phylogenetic tree of related species, the Bacillus subtilis strain on the evolutionary relationships together into a family; combination of physiological and biochemical characteristics and 16S rDNA molecular identification, was identified as Bacillus subtilis. Published on GenBank Bacillus subtilis endo cellulase gene sequences (eg), primers were designed for of pMG36e plasmid cloning sites, subtilisin from Bacillus subtilis the N9 genomic DNA as a template, eg amplified gene sequences. The length of the sequence of the gene 1518bp sequence alignment analysis, eg with other sources reported endo-cellulase sequence similarity up to 92%. And subcloned into Lactococcus lactis expression plasmid pMG36e to construct the recombinant expression plasmid pMG36e-eg Lactococcus lactis. Electricity into the recombinant plasmid was transformed into Lactococcus lactis MG1614 competent cells successfully constructed cellobiohydrolase genetic engineering in the production of lactic acid bacteria. M17 tablet containing CMC can produce transparent circle this the recombinants point connected to the expression product is collected by SDS-PAGE and Western blot detected endo cellulase molecular weight of about 54kD, consistent with the expected results. Screened to multiple strains of cellulose-decomposing bacteria, and cloned endo cellulase genes (eg) build lactic acid bacteria expression vector pMG36e-eg, to build the recombinant endo cellulase gene engineering lactic acid bacteria. To the use of lactic acid bacteria expression system to obtain a high activity cellulase and development produced endo cellulase multifunction probiotics.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > General Animal Science > Feed > Feed additives
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