Isolation And Gene Cloning Of Cellobiohydrolase Produced By Penicillium Purpurogenum HBZ003 From Mangrove

Cellobiohydrolase family (CBHs) is the key enzyme of cellulase system. It played an important role in cellulose degradation through disrupting the crystalline region of cellulose. A high-yield cellulase strains Penicillium purpurogenum HBZ003 which isolated from Mangrove soil was used to study its cellobiohydrolase (CBH) activity. The optimum enzyme production medium and fermentation conditions of cellobiohydrolase (CBH) activity was determined. And then, the purification, enzymatic properties and gene cloning of cellobiohydrolase were researched in order to reveal its relation with ocean Penicillium and other mold at gene level.Results of the optimum culture medium and the improvement optimum fermentation conditions to CBH from Penicillium purpurogenum HBZ003:1.6% bran,0.4% CMC,0.5% (NH4)2SO4,0.3% KNO3,0.6% NaCl and 0.03% CaCl2, 0.05%Tween 60, initial pH 4.0, with 80 mL/250 mL liquid medium volume and 10% inoculum size, under 30℃, at 160 r/min speed table for 5d, At last have the CBH enzyme activity up to 5.54 U.To minimize impurity proteins, we ultimately selected range of 20-45% ammonium sulfate saturation so that circumscribed cellulose enzyme can get sufficient preliminary purification; Then go on the dialysis and Sephadex G-100 column chromatography purification steps, eventually producing department and purified Penicillium purpurogenum HBZ003 cellulase components with CBH activity, the purified CBH activity is increased to 2058 u/mg than vitality, and the recovery rate is 3.85%;Have the three tube of enzyme liquid that one with the highest CBH enzyme activity,one with the highest protein content and one between them get SDS-PAGE gel electrophoresis, then CBH enzyme in the tube with highest CBH enzyme activity is shown as a single stripe, it illustrate that the molecular weight of CBH enzyme is 75-80 kD; In enzymology properties study can found that if put CBH enzyme in 30℃ to 55℃ temperature insulation for 0.5 h,it still can maintain 90% enzyme activity, it is important to note that after 0.5 h under 100℃ heat preservation, it is still have about 20% remaining energy; In addition, the suitable temperature range is 35 "C to 55℃; The suitable pH range is pH4.0-6.0;During common metal ions in the lab, Fe2+ and Mn2+ have a strong activation on CBH enzyme, Ca2+, Co2+, Cu2+ have a weak effectt, while Mg 2+, Zn2+ and Al3+ showed inhibitory effect on CBH enzyme.Designing primers for CBH I, it has made headway through preliminary studying and obtained an specificity stripe with PCR, the related results shown in the article after sequencing for it.According to the function of catalytic reaction sites, CBH was divided into CBHⅠ and CBH Ⅱ, the former role in the reducing end while the latter in nonreducing end. Currently reports to penicillium CBHⅠ gene sequence is given priority to, CBH Ⅱ gene sequences of short duration is not reported. According to the known sequence, primers was designed for HBZ003 CBH genes cloning, has got 2 pieces, the sequencing results are 646 bp and 626 bp, considered as the same gene fragment after comparing them. BLAST in the NCBI and was found similar with Aspergillus terreus NIH2624 gene sequence of unknown proteins. Illustrate HBZ003 CBH gene sequence was different from the reported, It is speculated that CBH of HBZ003 is CBH Ⅱ type of cellobiohydrolase.