Screening And Identification Of Efficient Lignin-degrading Bacterial Strains And Study Of Laccase Properties

Lignin as the second polymer after cellulose in nature,is the basic structural unit of benzene propane through the C-C and C-O-C keys are connected into a complex,three-dimensional structure of amorphous,so it is difficult to be degraded.If microorganisms can be used,enzymes capable of producing a large amount of lignin to decompose and transform lignin can not only solve the problem of environmental pollution,but also can reasonably utilize natural lignin resources.Therefore,the use of reasonable methods to isolate and purify lignin-degrading bacteria,to explore enzyme-producing ability and fermentation conditions,and to construct highly efficient laccase gene-engineering bacteria and to apply them to production practice are particularly important,which will greatly speed up the pace of the lignin raw materials in the production of industry and agriculture,and it plays a key role in controlling environmental pollution.1.Screening and identification of laccase producing strains with high yieldIn order to obtain laccase-producing strains for the degradation of lignin,samples were collected from the places where rotten wood was deposited around Xinxiang,where straw was deposited,and paper mill wastewater was rich in lignin.This study using lignin as the sole carbon source,42 strains were screened out which has the ability to degrade lignin.The strains producing laccase were screened by guaiacol plate method to obtain 4 laccase-producing strains.The fungal strains were identified by physiological and biochemical analysis and 18 SrDNA sequence analysis.It was determined that strain No.2 was Myrothecium verrucaria,strain No.6 was strain Coriolopsis gallica,and strain No.8 was yellow.Phanerochaete chrysosporium,No.11 strain is White rot fungi.The laccase activity and lignin degradation ability were studied by liquid fermentation culture and ABTS method.2.Experiment two comparison of enzyme producing ability of target strains and exploration of their fermentation conditionsThe selected four fungal strains were picked in a liquid medium to prepare a crude enzyme solution,and the ABTS method was used to determine the enzyme activity in the supernatant every day.Experiments showed that after 6 days of culture,the laccase activity of No.8 strain reached the highest,which was 233.33 U/L.Next,the strain of Phanerochaete chrysosporium was selected as the object of study.The conditions of the culture medium and the conditions for enzyme production were optimized.The carbon source,nitrogen source,metal ion,pH,and temperature were used for the production of fermentation paint by liquid fermentation.The effect of enzyme on the laccase activity reached the highest level.The results showed that the optimal carbon source for this strain was lactose,the optimal nitrogen source was yeast powder,and the optimum copper ion concentration was 0.6 mmol/L.The temperature is 30 °C and the optimum pH is 4.Under this condition,the removal rate of lignin reached 84.7%,which has a good ability to degrade lignin,which lays the foundation for the further use of this strain for industrial production.3.Cloning and expression of three protein purification test of Phanerochaete Chrysosporium of the laccase geneIn order to obtain a high-yield laccase engineering strain,according to the known laccase gene sequence in Phanerochaete chrysosporium,specific primers were designed and PCR amplification was performed.The cds region was successfully obtained as a 1680 bp fragment,and the gene was found after sequencing.The sequence has a homology of 98% with the currently published laccase gene fragment,indicating that the gene has been successfully cloned.Nde I and Xho I were introduced into the laccase gene in two stages for digestion.The laccase gene was ligated into the pET-24 a vector and transformed into E.coli to obtain a genetically engineered bacterium.According to fungal laccase amino acid sequence of the amino acid sequence of the GeneBank log in: laccase has high homology with other fungi the laccase protein sequences of the amino acid sequence of SDS-PAGE;through whole cell lysates of recombinant expression pre,75 KDa band,After purification and recovery,the purity of laccase is above 98%;after successful pre-expression,the bacteria were collected and disrupted,and the effluent was loaded on the NI-NTA column for purification of the eluted mco1-c-His6.The protein was purified;in order to further verify that this band is the desired laccase band,we used the WesternBlot method to elute the 75 KDa target band in the purified and concentrated solution at the target site.The band is the induced laccase after purification.After purification and recovery,the purity of laccase is above 98%.By exploring the properties of the enzyme,comparison of gene engineering bacteria and Phanerochaete Chrysosporium cultured for different time enzyme activity,good dynamic construction of engineering bacteria has increased significantly than the original bacteria enzyme activity,increased by 39%,which lays a foundation for the further use of engineering strain for industrial production,but also pointed out the direction for future applications.