Cloning, Expression And Characterization Of A Polygalacturonase In Pichia Pastoris From The Penicillium Sp.

Pectiases are a kind of enzymes that can hydrolyze pectin, taking up to a quarter of enzymes used globally, and they are widely used in food, feed, textile and paper industry. Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain with the introduction of water across the oxygen bridge, and the viscosity of the pectin are decreased rapidly. They are the most extensively studied among the family of pectinolytic enzymes. Polygalacturonase are widely distributed among bacteria, fungi and many plants. As a result, the study of polygalacturonase is of profound theoretical and practical significance.A Penicillium sp. FJ2, which produced polygalacturonase and characterized by 18 S rDNA, has been selected in this research.Polygalacturonases belong to glycoside hydrolases family 28 (GH 28),and some conserved regions existed in this family. Depending on this phenomenon, some protein sequences were downloaded from NCBI and were aligned by ClustalW2. Two conserved regions were selected and the corresponding primers were designed to amplified the sequences located between the conserved regions. Through sequencing and designing nested primers, the whole gene were obtained by thermal asymmetric interlaced PCR (TAIL-PCR). The localization and number of intrones were confirmed by GENSCAN and NCBI Blastx. The whole cDNA sequence was obtained by overlap- PCR and the signal peptide was predicted by SignalP 3.0. Through the above work, a polygalacturonase gene without signal peptide coding sequence pgp1 was cloned. The length of the polygalacturonase pgp1 gene and its cDNA are 1225 bp and 1104 bp, respectively. The cDNA of pgp1 encode 367 amino acids and a termination codon, first 18-residue amino acids is signal peptide. This gene was cloned into a expression vector pPIC9 and overexpressed in Pichia pastoris GS115 with the maximum polygalacturonase activity of 700 U/mL. The recombinant PGP1 exhibited activity towards polygalacturonic acid was optimally active at pH 5.0 and 38℃, and remained 90% relative activity at pH 4.0～6.0. The Km and Vmax values for polygalacturonic acid were 1.172±0.169 mg/ml and 0.061±0.002 mg/min/mL, respectively. The metal ions Na+,K+ and EDTA had no effect on the activity of PGP1, but SDS and CTAB strongly inhibited PGP1 activity. On the contary, Triton could enhanced PGP1 activity. Mg²⁺ (10 mmol/L) would decrease 50% of the activity of PGP1 and the ions Ca²⁺, Co²⁺, Mn²⁺, Pb²⁺ and Zn²⁺ (10 mmol/L) would completely inhibited PGP1 activity.