Analysis Of Functions And Properties Of Laccase From Bacillus Pumilus And Study On Its Application

Laccase（E.C.1.10.3.2）is a copper-containing polyphenol oxidase.Due to its broad substrate specificity and the production of only water as the product,laccases have been used in many biotechnological applications,including production of laccase-based biosensors and organic materials,transformation of various lignin-related compounds,multiple Degradation of phenolic pollutants,pulp bleaching,beverage processing,dye decolorization and textile wastewater,etc.Currently commercial laccases are all derived from fungi,but fungal laccases are unstable under some conditions,such as high temperature and alkaline conditions,thus limiting their application.In contrast,most bacterial laccases,which can function at higher temperatures and a wider p H range,are rapidly growing in industrial applications.Therefore,the discovery and research of bacterial laccase is of great significance.The main contents and results of this study are as follows:（1）A novel laccase gene lac was obtained from Bacillus pumilus TCCC 11568,with a fragment length of 1530 bp,encoding 509 amino acids.The 3D structure of LAC was obtained by homology modeling,and its active centers were T1Cu（His419,Cys492,His497 and Met502）,T2Cu（His103 and His422）and T3Cu（His105,His151,His493and His153,His424 and His491）.（2）Recombinant laccase（rLAC）was prepared by Escherichia coli,and its enzymatic properties were analyzed.The optimum temperature of rLAC is 80°C,and the optimum p H value is 6.0;rLAC has good stability in the range of 0-70°C and p H3.0-10.0;the activity of rLAC is hardly affected by the metal ions K⁺,Ca²⁺,Cu²⁺,Mg²⁺,Zn²⁺,Ba2+,Ni²⁺,Fe²⁺and the effect of inhibitor SDS.（3）The thermostable mechanism of rLAC was investigated by molecular dynamics simulations.The results showed that compared with the tertiary structure at 50°C,rLAC maintained its structural integrity at 80°C,while the overall conformation formed a more open channel,which facilitates the binding of the substrate to the active site,resulting in elevated binding energy.（4）Degradation of bisphenol A（BPA）by rLAC,identification of five degradation intermediates:benzoic acid,hydroquinone,p-hydroxyphenylacetic acid,p-hydroxypropiophenone and 3,4-dimethylbenzoic acid.（5）Using Pichia pastoris to display LAC,prepare LAC whole-cell catalyst（d LAC）,and use d LAC to degrade BPA,which still has a relative degradation rate of60%compared with the initial BPA degradation rate after repeated use for 4 times,so the d LAC whole-cell catalyst can be reused at least 4 time.