Cloning,Expression Of Penicillium Aculeatum Dextranase In Picha Pastoris And Study On Enzymatic Propertoes

Dextranase(?-1,6-D-glucan-6-glucanohydrolase;E.C.3.2.1.11)is a hydrolase that degrades polymer dextran to low molecular weight polysaccharides.Currently,the enzyme and its products play increasingly important roles in the food,medical and chemical industries.In this study,the dextranase gene(dex)has been cloned from Penicillium aculeatum F1001,and a recombinant Pichia pastoris that efficiently expresses exogenous dextranase was constructed.Recombinant dextranase was obtained and its properties were analyzed.Firstly,the Penicillium aculeatum was cultured for dextranase high expression and the gene(dex)was amplified by reverse transcription PCR from the genome of Penicillium aculeatum.Then recombinant E.coli was constructed by TA ligation.Sequencing the recombinant plasmid revealed that the sequence of dex consists of 1866 base pairs and encodes a protein with 622 amino acid residues.Based on codon bias of Pichia pastoris synonymous mutations was carried out,a new gene sequence was obtained as opt-dex.The GC content of dex reduced from 49.4% to 38.3%,and codon preference index adjust from 0.65 to 0.92.The opt-dex and dex were cloned into expression plasmid p PICZ?A,and transformed into E.coli to obtain the recombinant plasmid opt-dex-p PICZ?A and dex-p PICZ?A,respectively.Both were electro-transformed into Pichia pastoris X33.Depending on the size of transparent zone,several dextranase-producing recombinant opt-dexp PICZ?A/X33 and dex-p PICZ?A/X33 were selected on radiantly Zeocin resistant plates and blue-dextran T-2000 specific plates.The expression of opt-dexp PICZ?A/X33 was significantly higher than that of dex-p PICZ?A/X33.The fermentation conditions for the preliminary optimized enzyme production were: incubation temperature 25?,initial p H5.0,methanol addition(per 24 h)1%(V/V),Tween-80 0.4% and sorbitol addition(per 24 h)0.5%,50 m L medium was added to the 500 m L shake-flask bottle to maintain oxygen.The final recombinant dextranase activity reached 240.74 U/m L.The affinity chromatography was used to purify the recombinant dextranase.SDS-PAGE showed that the size of the recombinant dextranase was about 65 k Da.The optimal catalytic temperature of the recombinant dextranase is 35?,and the optimal catalytic p H is 5.0,which are consistent with the original Penicillium aculeatum dextranse.The effet of metal ions on recombinant detranase is a little different from that of original dextranse from Penicillium aculeatum.Both of them showed the same substrate specificity and product property.