| As an agricultural country in our country the production of straw is very high every year, but only a few of them are used in industrial production, most of them were wasted by directly geting into the environment, thus formed the very big pollution and wasting.The straw is used to make silage is one of the effective ways to make the straw into treasure,whether the straw can be effectively used or not depend on whether the lignin can be degradation.Lignin as silage additives it can greatly improve its utilization,but due to its low production cannot meet the demand of industry and agriculture.According to reports that laccase has been expression in a variety of host cells,in order to increase its production.The lactic acid bacteria is a kind of food grade security microbial, if it can be used as the host cell heterologous expression of laccase,building the engineering lactic acid bacteria as a new microbial agents added in silage,it Will play the double function of lactic acid bacteria with laccase,the laccase is the most enzyme to degradation lignin, if the restructuring lactic acid bacteria which have the ability to secrete laccase is used for silage fermentation,it can greatly improve the utilization rate of feed and feed quality,so it has a broad application prospect.This study by lactobacillus apricot bao mushroom laccase secretion expression, to study the nature and function of the laccase enzymology,which to add it in silage to provide the theory of basic basis.This lab has made apricot bao mushroom laccase genes into food-grade lactic acid bacteria as host cells, with build restructuring project lactic acid bacteria successful.This study will be to expand training engineering strains of lactic acid bacteria, obtain recombinant bacteria secrete laccase, studied the enzymatic properties of laccase. We carried out the following experiment:1. In order to select a single transformant, re-engineering lactic acid bacteria liquid coating to the solid MRS culture medium containing erythromycin then pick, white colony directly for PCR, screening of positive restructuring.2. And extract the filter to the positive recombinant plasmid to further verified the PCR3. let the verified exists laccase gene that positive recombinant child coating to contain 0.04% guaiacol MRS in solid culture medium for culture, and express product testing experiment with flat color method, Restructuring specific chromogenic reaction, if lactic acid bacteria colony color into red brown suggests that have the ability to secrete laccase then pick color changes deeper colony, measure its enzyme activity.4. Compare the ability of production acid between lactic acid bacteria restructuring project and lactic acid bacteria.Successful screening to lactic acid bacteria in the restructuring of the capacity of producing laccase engineering, studied the optimum temperature and ph.If lactic acid bacteriawith has the ability to secrete can be used as silage additives, will greatly improve the efficiency and the quality of silage, this experiment provides the fine bivalent strains for our country’s animal husbandry development lay the foundation. |
