Optimization Of Fermentation Medium For Protopectinase Production Of Penicillium Sp. Y702and Cloning And Expression Of An Endo-arabinanase

Protopectinase is an enzyme that solubilizes protopectin forming highly polymerized soluble pectin. A protopectinase-producing strain, Penicillium sp. Y702, was isolated from persimmon garden. The fermentation conditions for maximum activity of protopectinases (PPase) in flask was optimized by using Plackett-Burman design and Response Surface methodology. The optimum medium was composed of NaNO32g/1, MgSO4·7H2O0.3g/l, K2HPO4·3H2O0.15g/1, KCl0.5g/1, MgSO4·7H2O0.5g/l, FeSO4·7H2O0.01g/1, CaCl20.01g/1and MnCl2·4H2O0.01g/l, and maximum activity of PPase reached205U/ml, which was about200%higher than before and consistent with the maximum predicted value.Endo-arabinanase belonging to B-type PPase seems to attack the hair regions of protopectins. We succeed in cloning an endo-arabinanase (AbnC) gene by overlap-PCR from genomic DNA of Penicillium sp. Y702, which was expressed in Pichia pastor is. The coding sequence containing963nucleotides encodes for320amino acids, including a signal peptide of21amino acids. Three catalytic residues (Asp34, Asp148, and Glu199) of AbnC belonging to GH family43are highly conserved, and are deduced as active sites.The recombinant protein was further purified by ion exchange chromatography and resulted in7.94-fold enzymatic activity improvement. The molecular weight was determined as35kDa by SDS-PAGE. The optimum pH and temperature of recombinant AbnC were5.0and50℃. The activity of AbnC was highly stable at pH5.0-7.0and45℃or below, which were different from those of the recombinant AbnC expressed in Escherichia coli. The Km and Vmsx values with linear arabinan as the substrate were24.8±4.7mg/ml and88.5±5.6U/mg, respectively, and enzymatic activity was20U/mg. Besides, TLC and HPLC analysis verified that the recombinant enzyme (AbnC) is an endo-arabinanase, which yielded a major product of arabinobiose and arabinotriose. Through51fermentor the resulting AbnC activity reached up to1.03U/ml and was300%higher than that of shake flask. The synergistic action of AbnC with ABFs resulted in1.9mg release of pectic substance of apple protopectin, indicating that the synergy of enzymes can exert their hydrolysis better, which has certain guiding significance to the development and utilization of the natural plant polysaccharides.