Clonging Of ?-glucosidase Gene From Penicillium Oxalicum And Expression In Pichia Pastoris

In this experiment,through the extraction of Penicillium oxalicum SJ1,the research utilizes PCR&RT-PCR technologies for amplification and obtains the DNA and cDNA sequence of P-glucosidase BGL1 and BGL2.The result of sequencing and bioinformatics analysis showed that the bgl1 has the length of 2963 bp,contains 5 intors and 6 exons,and ecods 861 amino acids,while the bgl2 has the length of 1778 bp,contains 3 intors and 4 exons,and encods 463 amino acids.After the CDS sequence of BGL1 was connected with pPIC9K,the recombinant expression vector pPIC9K-bgll was constructed.The linearized expression plasmid was transformed into Pichia Pastoris GS115 by the electric shock.After filtered by G418 plate(0.25 mg/mL)?color-display plate(0.25%esculin and 0.1%ammonium ferric citrate),verified by template PCR and PCR,obtain the yeast engineering strain of GS115-pPIC9K-bgl1.The recombinant strain was induced by 1.5%methanol,the activity of ?-glucosidase was detected by pNPG method.The maximum enzyme activitie of hydrolytic pNPG is 0.297 U/mL.The results of SDS-PAGE electrophoresis showed that expression product was about 97 KDa.With the method of response surface methodology,the best optiumum fermentation medium for inducing yeast powder 12.01 g/L?ammonia sulfate 16.89 g/L?sorbitol 23.41 g/L?methanol 1.5%,and the culture medium can improve the expression level of 19.47%.The crude enzyme was purified by lyophilization,ammonium sulfate fractionation,dialysis and Sephadex G 100 chromatography,and the purified BGL1 enzyme solution was obtained.As showed in analysis result,the best reaction time of BGL1 was 30 minutes.the optimum reaction temperature was,the optimum pH was 6,the optimum storage temperature was low,and the optimum PH was 6.Ba2+ ions can promote the activity of BGL1 enzyme,and Cu2+ ions can inhibit the activity of BGL1 enzyme.