A Preliminary Study Of Cloning And Characteristics Of New Laccase Gene Of Fosmid Library From Bovine Rumen Microbial Domesticated By Miscanthus

The rapid development of economy resulted in the overexploitation and over-use of fossil fuels, including oil and natural gas, it would caused energy shortage problem and serious pollution about environment. Owing to dwindling of fossil fuels,more and more people pay attention to the renewable biomass energy.Nowadays, Cellulose becomes the mainly raw material of biomass energy, it could be degraded to biological fuel ethanol by fermentation. However, we discovered that the existence of lignin seriously limits the hydrolysis action of cellulose molecules in the cellulose degradation process. For the sake of increasing the efficient utilization of cellulose, we have to firstly solve the degradation of lignin. Enzymatic degradation is one of the best way to realize the resource utilization and environmental protection in all the method of degradation of lignin. It is the most outstanding study that the exploration of laccase among enzyme which the lignin is degradated.This study obtained16bacterial strains with high laccase activities from20160clones of Fosmid metagenomic library which achieved from Miscanthus domesticated bovine rumen microbial via functional screening.lt is successfully to constructe a subclone library containing234clones.4high activity trains was screened from the subclone by functional screening method, the maximum activity strain134B2was selected and sent to BGI for sequencing.The sequencing result suggested that the length of clone was2864bp. we obtained a1521bp ORF by ORF Finder software,then,it was named unlac134B2which encode506amino acids by means of the online software prediction, then, we know the molecular weight of unlac134B2protein is54037.2Da, pI3.78, unstable coefficient23.12. It is suggested that the protein is very stable. The Blast comparisons in NCBI database indicate that it’s very low about the similarities between unlac134B2and reported laccase gene,which indicated that the gene is a novel gene.We constructed a prokaryotic expression vector that the unlac134B2gene insected in pET30a(+) vector. Transferring the expression vector into the competent cells BL21in order to getting the conditions of induced expression. The optimum inducing condition is that:IPTG concentration is0.5mmol/L, induction temperature is30℃, induction time is10h. The resraech of enzymatic protein of unlac134B2indicated that the optimal reaction temperature is45℃; the optimum pH value is4.5; it is stable when the temperature is between30℃-40℃, pH value is between4.5-6, the activity is still maintained at more than80%; the impact of Cu2+on unlac134B2’s enzyme activity is obviously positive, it’s the most significant about the inhibition of Fe3+. but K+, Na+have little influence about its enzyme activity, whatmore,it’s also have some negative influence by Mg2+、Ca2+、Zn2+、Mn2+.In short, the results suggested that it was prosperous to screen a laccase gene named unlac134B2by the metagenome library from bovine rumen microbial. According to the unlac134B2gene,it encoded protein which has effective laccase activity. It provided a favorable conditions to exhaustive study the degradation of lignin and the diversification of laccase properties.