Research On The Enzymatic Characteristics And Dye Decolorization Of Bacillus Sphaericus Laccase

Laccase（EC 1.10.3.2）is a kind of environment-friendly polyphenol copper oxidase,which can catalyze the oxidation of the substrate by O₂ and generate water at the same time.It has the characteristics of high catalytic oxidation activity and wide range of substrates,especially it can catalyze the degradation of aromatic dyes in textile printing and dyeing wastewater and organic chlorides in paper bleaching wastewater.It has attracted much attention in the field of green environmental protection.Compared with fungal laccase,bacterial laccase,especially Bacillus laccase,has attracted more concern in recent years due to its high temperature resistance,alkali resistance,abundant resources and easy expression in industrial host organisms.In the early stage of this study,it was found that Bacillus sphaericus C3-41 had the ability to decolorize dyes of broad-spectrum.The strain was identified to produce three kinds of proteins with laccase activity,of which were named Cot A,Cue O and Mcue O respectively.In this paper,the gene was heterologously expressed in E.coli BL21,and the enzymatic properties and dye decolorization of the expression products were studied.Three kinds of laccases with high specific enzyme activity can be obtained by salting out,histag protein affinity chromatography and dialysis concentration of the cell broken solution of heterologous expression strains.The activity of purified Mcue O protein was 198.81U/mg,that of purified Cue O protein was 260.97U/mg,and that of purified Cot A was 329.72U/mg.SDS-PAGE and Western Blot analysis showed that the molecular weight of three laccase was about32KDa for Mcue O,65KDa for Cue O and 100KDa for Cot A.The influence factors of enzyme activity were studied.The results showed that the concentration of metal ions in the additive at 0.2m M,Cu²⁺and Zn²⁺promoted the activity of three laccases,Ba²⁺had slight effect on the activity of three laccases,Fe³⁺inhibited the activity of three laccases.The laccases of Bacillus sphaericus have good thermal stability.After 30minutes of water bath treatment at 80℃,the activity of laccases can maintain more than 50%of the initial activity.At the same time,they possess excellent alkaline stability.After 5 hours of treatment in p H10,the activity of the three laccases is greatly improved.The dye decolorization properties of the three kinds of purified laccase were researched.The results indicated that the single laccase had a certain decolorization effect on triphenylmethane dyes and anthraquinone dyes,and the decolorization ratio of laccase Cot A and Cue O on Malachite Green was over 95%at p H10.The addition of 0.2m M Cu²⁺promoted the decolorization on most of the dyes by Mcue O,Cue O and Cot A.The mediators that promoted laccase dye decolorization were preliminarily screened.With the addition of 40μM mediator,AS（Acetosyringone）promoted the decolorization of Crystal Violet by laccase Cot A,Cue O and Mcue O,and the decolorization rate of single laccase increased from 30.4%,38.0%and 26.0%to 57.3%,60.8%and 47.0%respectively.ABTS[2,2-diazo-bis（3-ethylbenzothiazole-6-sulfonic acid）]promoted the decolorization of Methylene Blue by three laccases,and the decolorization rate increased from from 10.0%,2.0%and 12.0%of laccase alone to 86.5%,85.0%and 65.0%.The three laccases performed different decolorization characteristics on different dyes,indicating that Bacillus sphaericus laccases have a good potential and application prospect in decolorization of printing and dyeing wastewater.