Expression And Application Of Key Enzyme Genes Involved In Pulp Bleaching From Saccharomonospora Viridis

Saccharomonospora viridis is a thermophilic actinomycete belonging to Saccharomonosporagenus，Pseudonocardiaceae family, which has good heat and alkali resistance and can degrade naturalwood cellulose and hemicellulose. Therefore, the strain S. viridis and its lignocellulolytic enzymes haveimportant research signiifcance in the fields of resoucres and environment and also have potentialapplication values in the fldds ofbiomass energy and pulp bleaching.This study was carried out around the core of key enzymes involved in bio-bleaching pulp. Thegenes of dye-decoloirzing peroxidase and xylanase rfom S. viridis were cloned. The recombinantenzymes were expressed, purified and characteirzed. Besides, the effects of different recombinantenzymes in pulp bleaching were investigated.Bioinformatics’ analysis revealed that a DyP-type peroxidase gene existed in the genome of S.viridis DSM43017. This gene，svidyp (the registration number on GenBank database was KF444221)，consists of1,215bp encoding a polypeptide of404amino acids. The gene encoding SviDyP was clonedby the double temperature PCR method. The recombinant enzyme SviDyP was expressed inEscherichia coli and purified. The optimal reaction temperature and pH of SviDyP were70°C and7.0，respectively. Its optimal substrate was bright green. The recombinant protein could eiffcientlydecolorize several tirarylmethane dyes, anthraquinonic and azo dyes under neutral conditions. It washighly thermostable (>60%activity atfer incubaiton at70。C，pH7.0for2h) and alkali resistant (>80%activity atfer incubation at pH5—10for1h at37°C).The svidyp gene was cloned by polymerase chain reaction method, and then transformed into Pichia pastor is. The expression vector p PI CZaA vidyp was constructed. Using electrotransformationmethod, the recombinant plasmid p PI CZaA-s vidyp was transformed into R past oris GS115. Then thestrain P. pastoris i pPICZaA-^v/c/y/? was obtained. Under the induction of0.5percent of methanol,SviDyP was secreted up to the maximum xylanase activity of24.6U/mg. The purity of recombinantenzyme reached gel electrophoresis level only through membrane bag puriifcation。The xylanase gene from S. viridis was cloned by polymerase chain reaction method, and thentransformed to P. pastoris. The recombinant plasmid pP\C9-svixynlOA was constructed, and thentransformed into P. pastoris GS115by electrotransformation method. Atfer that the strain P. pastoris ipPICZaA-^v/fryp was obtained. Atfer induced for48h by0.5percent of methanol, the xylanase activityof the recombinant enzyme SviXyNlOA reached to7.53U/mL. The optimal reaction temperature andpH of SviXyNlOA were at70。C，pH7.0. Its optimal substrate is oat spelt xylan, showing substantialactivity at wide range of pH (pH5.0-9.0). Furthermore, the xylanase showed excellentthermo-alkali-stability (>90%activity at60°C,3h;>80%activity at pH4.0-10.0for1h) and almost nocellulase activity.The bleaching effects of eucalyptus kraft pulp treated by different recombinant enzymepreparations were different. The enzymes SviDyP expressed by E. coli and P. pastoris had acceleratedactions during the pre-bleaching of eucalyptus kraft pulp, resulting in an increase of1.5%,2.7%,6.6%and4%in brightness, respectively. Atfer the subsequent DED bleaching, the birghtness of the pulppaper pre-bleached by the compound enzyme (Xylanse and SviDyP expressed by P. pastoris) wasincreased vastly up to89.67%ISO.