| Bacillus subtilis laccase has attracted more and more attention due to its wide range of substrates,good stability,non pollution and other natural advantages,and has been widely used in many industrial fields Our research group has previously extracted the wild-type laccase gene CAR2(GenBank accession number:MG571271)from Bacillus subtilis,and obtained its mutant CAH2 with high activity.By simulating the conformation of CAH2,we found that there is a large space of directed evolution in the area near the Cu I site(Cu2+I)and the substrate binding channel in the CAH2 molecule,and the direction of two kinds of CAH2 mutations was determined by further rational design.One is to mutate the proline sites of 212,217 and 222 on the substrate binding channel gate of laccase CAH2 to obtain mutant laccase A3(P212A,P217A,P222A)and G3(P212G,P217G,P222G);the other is to change the position of "hinge" on the substrate binding channel gate of laccase C AH2 to obtain mutant laccase SP(S360P).Primers were designed for different mutation sites of CAH2.Three mutant genes a3,g3 and sp were obtained by using the overlap extension PCR(SOE-PCR)method,and they were recombined with the expression vector pET-28a to obtain the corresponding recombinant expression vector.Finally,the corresponding mutant laccase A3,G3 and SP were obtained by induction expression and purification.Through the analysis of their enzyme activities,we found that the catalytic activities of the three mutants were significantly higher than that of the original laccase CAH2,especially laccase G3 and SP.Therefore,we further used G3 as a template and mutated S360P to obtain mutant laccase GSP.The results showed that the catalytic activity of laccase GSP was significantly increased.The basic properties and functions of original laccase CAH2 and its four mutants laccase A3,G3,SP and GSP were determined and compared.The results showed that:(1)the changes of amino acids in A3,G3,SP and GSP had no effect on the optimum temperature and pH value of laccase,but reduced the sensitivity of laccase to temperature or pH in a certain range.(2)The common metal ions Na+ and K+ in the sea and soil had little effect on laccase activity,while Mn2+and Mg2+had different degrees of activation or no effect on laccase activity,but Zn2+ and Ni2+ had obvious inhibition on laccase activity.(3)Compared with the original laccase CAH2,the kcat values of the four mutants were increased,which indicated that the reaction rates of these mutants were increased,and G3 was the most significant.The kinetic analysis showed that the mutation of Pro to Gly or ala(P212G/A,P217G/A,P222G/A)could reduce the steric hindrance of the substrate binding channel and facilitate the binding of the substrate to laccase,while the mutation of Pro toGly was more advantageous than the mutation of Pro to Ala;the mutation of Ser360Pro could enhance the conformational stability of the substrate binding channel and help the electron transfer from the substrate to T1.Therefore,the substrate binding efficiency and catalytic efficiency of mutant laccase G3,SP and GSP were improved,but SP showed higher substrate selectivity.Finally,the applicability of the five laccase was determined by dye decolorization and petroleum degradation.The results of decolorization of five dyes(indigo carmine,Congo red,crystal violet,methyl red and reactive brilliant blue)showed that the effects of five laccase on these dyes were similar,but the effects of different mutant laccase on different dyes were slightly improved.The decolorization of Congo red and indigo carmine dyes by the five laccase is ideal,and the decolorization rate can reach more than 80%without the mediation of 2,2 '-diazo bis(3-ethylbenzthiazoline-6-sulfonic acid,ABTS).In the presence of ABTS,the decolorization efficiency of crystal violet can be increased by more than 70%.Among them,mutant laccase A3(P212A,P217A,P222A)is beneficial to the decolorization of methyl red dyes,while mutant laccase SP(S360P)is beneficial to the decolorization of reactive brilliant blue dyes.The original laccase CAH2,mutant laccase G3 and GSP were selected to analyze their degradation of Petroleum(light oil 1 and light oil 2)and determine the corresponding degradation rate of petroleum.The results showed that:(1)when the ratio of oil volume fraction to enzyme activity unit was 1%:2U,10%:2U and 25%:0.5U respectively,the higher the ratio of oil volume fraction to enzyme activity unit,the higher the degradation rate of laccase to oil,indicating the effective degradation of laccase to oil;(2)the degradation rates of mutant laccase G3 and GSP were higher than those of original laccase 1 or 2 The degradation rate of enzyme CAH2 indicated that reducing the steric hindrance of substrate binding channel and enhancing the flexibility of gated peptide could improve the degradation of oil by laccase.(3)The degradation rates of light oil 1 and 2 by three laccase were different,because the oil was a mixture of multi-component hydrocarbons,and the components that could be degraded by laccase might be different.The higher the content of specific petroleum hydrocarbon components in petroleum degradation system,the higher the degradation rate.The results of this study suggest that 212,217,222 and 360 sites have different effects on the catalytic activity,stability and applicability of laccase,and their changes and rules have certain guiding significance for exploring the application potential of laccase and expanding the use value of laccase. |
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