Study On Directed Evolution Of Bacterial Laccases

Laccase,with low storage requirements and a wide range of catalytic substrates,is an environmentally friendly enzyme in nature,whose only by-product after reaction is water.As people’s awareness of environmental protection gradually increased,the research on laccase was more and more extensive in recent years.With spatial conformational analysis of the substrate binding channel site of the primary laccase CAH2（Car2,Gen Bank number AYD68845.1）,this paper found that the region rich in Pro residues（P212,P215,P217,P220,P222,P226,P384）made the conformation of the site too rigid,which means that the ring-like side chain structures relatively firmly hold the orientation of the peptide chain at this site.However,our primary evolved laccases A3（P212A,P217A,P222A）and G3（P212G,P217G,P222G）we designed earlier had increased the flexibility of the laccase substrate binding channel to a limited extent while the primary evolved laccase GSP（S360P）moleculehad a more favorable arrangement of the two related structural domains for substrate binding than CAH2,but still limited due to the rigid structure of the local peptide chain caused by the cyclic side chain structure of residue P384.Thus,there is still much evolution room in these laccase molecules between nearby areas close to the type I copper(T1 Cu²⁺)site and the substrate binding channel site.To further reveal the influence of the conformation of the above mentioned sites on the catalytic activity and application properties of laccase,further evolutionary design of laccases A3,G3 and GSP were carried out in this paper respectively.It includes obtaining mutant laccases A6（P215A P220A P226A）,G6（P215G P220G P226G）by mutating proline at sites 215,220 and 226 near the substrate binding channel of A3 and G3 to alanine and glycine respectively and obtaining mutant laccase GSL（P384L）by mutating proline at site 384 in the substrate binding channel near type I copper(T1 Cu²⁺)of GSP to leucine.The following main results were obtained through the research of this paper:1.Corresponding primers were designed for the different mutational directions of laccases A6,G6 and GSL,then,the corresponding mutant genes a6,g6 and gsl were obtained by overlapping extension PCR（SOE-PCR）,and the recombinant expression vectors of laccase were further constructed.The mutant laccases A6,G6,and GSL were obtained with induced expression and purification of the target protein.Compared with the primary laccases A3,G3 and GSP,the specific activity of laccases A6 and GSL was increased,in which the latter’s specific activity was increased significantly.2.The enzymatic properties of laccases A6,G6 and GSL were determined.Its results showed that:（1）The amino acid changes of the related sites in above three laccases had little effect on the optimal temperature and p H,but changed the sensitivity of the laccases to environmental p H and temperature to a certain extent.（2）The metal ions Na⁺and K⁺had no effect on the laccase activity and Mn²⁺and Mg²⁺activated the three laccase activities,while Zn²⁺and Ni²⁺inhibited it.（3）Different concentrations of organic solvents also affected the activity of the three laccases,among which 10%dimethyl sulfoxide and 1%and 10%ethanol inhibited the catalytic activity of laccase A6.（4）kinetic parameters of laccases A6,G6 and GSL were determined and analyzed.The results showed that:A6,G6 and GSL were similar to A3,G3 and GSP respectively in K_mand k_cat value.Among them,the K_m value of GSL was the smallest and the k_catvalue of G6 is the largest,the k_cat/K_m value of laccase GSL was the largest,indicating that P384L mutation had a significant effect on improving the catalytic efficiency of laccase.3.In this paper,the degradation effect of the optimized laccases on the five common dyes in the textile industry were studied.Its results showed that（1）With or without ABTS mediator in the catalytic system,the decolorization rates of indigo carmine,erythrosine,and congo red by the three laccases can all reach over 80%,indicating that these two dyes are more suitable substrates for laccase.（2）The degradation of crystal violet by three laccases was significantly dependent on the presence of ABTS mediator.（3）The degradation effect of the three laccases on methyl red and reactive brilliant blue is poor with or without ABTS mediator in the system.All the three laccases can degrade petroleum（light oil No.1 and light oil No.2）in a time-dependent manner.Among them,the degradation effects of laccases G6 and GSL on petroleum were in order of GSL>G3>G6（with G3 as the control）,and the highest degradation rate of G6 and GSL is about 56%when the enzyme activity unit to petroleum volume fraction was 0.5U:25%（1:50）.In this paper,the enzymatic properties and functions of three laccases A6,G6 and GSL were determined and analyzed.Its results showed that the functions of laccases were closely related to their structural diversity,and sites 215,220,226 and 384 of laccases had a certain degree of effect on the enzymatic properties and functions of laccases.In this paper,the enzymatic properties and functions of three laccases A6,G6 and GSL were determined and analyzed.The results showed that the functions of laccases were closely related to their structural diversity,and sites 215,220,226 and 384 of laccases had certain effects on the enzymatic properties and functions of laccases.