Cloning And Expression Of Cellobiohydralase Gene Cbh? From Penicillium Oxalicum

Cellulose is very rich organic matter in nature, and its complete degradation process requires the participation of several enzymes. Cellulase with high catalytic efficiency and good security, is used widely in textile, washing and animal feed additives.The filter paper activity(FPA) of genetic stabable cellulase producing strain Penicillium oxalicum X10-279 was 16.7 U/m L, which was screened and bred by our labarotory. Cellulose was degraded to glucose under a variety of cellulase. CBH ? is the only one of the enzymes that can act on crystalline cellulose, which plays an important role in the degradation of cellulose. In this study, cloning and expression of the cellobiohydralase gene cbh? from Penicillium oxalicum X10-279 were carried out to obtain the active recombinant bacteria so as to improve the efficiency of cellulose degradation.According to the published sequences of cellulase genes in Gen Bank, forward primer with Eco R? cutting site of and reverse primer with Not? cutting site were devised. The cbh? gene was cloned by PCR amplification using the genomic DN A of Penicillium oxalicum X10-279 as template, ligating the purified PC R product with p MD18-T Vector, and transforming the recombinated plasmid into E.coli Top10 competent cells. Bioinformatics analysis showed that the cloned cbh? gene contains 1632 bp. The gene encoded a protein of 543 amino acids with an initiation codon ATG and a termination codon TAA. The sequence reported here was deposited in the Gen Bank database with nucleotide accession number KR269867 and protein accession number AKI32221. Homology analysis indicated that the anmino acids sequence of the cloned gene was near to Penicillium decumbens ACV95805 with the similarity of 98.70%. The predicted molecular weight o f the protein is 56.66 k Da, the theoretical isoelectric point of protein was 4.79, and the protein belongs to glycoside hydrolase fimily 7.The recombinant plasmid p MD18-T-cbh? and expression vector p ET-28a(+) were digested with Eco R? and Not?. The recovered cbh? gene and linearized plasmid vector p ET-28a(+)were ligated with T4 DN A ligase and transformed into E.coli BL21(DE3) competent cells. Recombinant bacteria of p ET-28a(+)-cbh?/BL21 was constructed successfully. Plasmids containing the foreign fragment were confirmed by gene sequencing. Objective protein was successfully expressed in the form of inclusion bodies in the periplasmic space of E. coli BL21(DE3). It is unlikely to produce soluble protein by changing the induction conditions. The optimal induction condition was determined as adding IPTG to a final concentration of 0.8 mmol/L when the bacterial density OD600 was 0.3~0.4, and then the recombinant bacteria was induced for 5 h at 37℃. The expression of the target protein reached 15.14 mg/L, which increased 50.49% than before.After purified the inclusion bodies by affinity chromatography with N i-colum, the expression yields of recombinant protein was 3.21 mg/L with the reco very of 21.2%. After renaturation, the p NPC activity reached 10.3 U/m L with a specific activity of 94.3 U/mg. The optimum p H and temperature of the recombinant enzyme were 5.0 and 55℃, respectively, and the protein showed good stability of temperature.