Expression, Fermentation And Characterization Of Protopectinase In Pichia Pastoris

The enzyme that catalyzes water-soluble pectin substance from protopectin is protopectinase. In 1978, the first study on protopectinase from yeast was reported by Sakai and Okushina, and then characterization and applications of protopectinases from Yeast, Bacillus and Aspergillus were also done. So far, researches on microbial protopectinases and applications of those have been not reported in China, and protopectinase expressed in Pichia pastoris have been not reported in the world. In this paper it was investigated that the isolation of Bacillus subtilis XZ2 producing protopectinase, protopectinase expression in Pichia pastoris, optimization of fermentation parameters, purification of protopectinase and characterization of protopectinase. The main results are as followed:1. B. subtilis XZ2 producing protopectinase was isolated from many Bacilli and the protopectinase gene were amplified by polymerase chain reaction (PCR) with the genomic DNA of B. subtilis XZ2 as template and sequenced. The sequence of protopectinase gene has a length of 1200bp and encodes 399 amino acids. The protopectinase from B.subtilis XZ2 has high homology of 99.6% and 99.5% to that from Genbank(Accession: x74880) in nucleotides and amino acids respectively. Fours nucleotides were replaced and only 2 amino acid replacements was observed in the protopectinase from B. subtilis XZ2. No replacernent was observed in the two conserved region。2. Protopectinase gene and plasmid pPICZaA digested by XhoI and XbaI were ligated and transformed in E.coli DH5a to construct the recombinant plasmid pPICZαA-PPase. The recombinant plasmid linearized with BstXI was mixed the competent cells of P.pastoris X-33 and transformed with the conditions of 1.5 kV, 25μF, and 200Ω. The transformants were screened by YPD containing 100,200,400, 800 and 1000μg zeocin /ml and identified by the Pichia pastoris genomic DNA PCR, the final recombinant X-33/pPICZaA-PPase was gained。3. It was investigated that induction medium, the length of induction, methanol concentration, cell optical density, pH, the additive of PTM1, rotary speed and high cell density fermentation can affected the expression level of protopectinase. The results showed that protopectinase can reach a good expression with BMGY/mBMMY, pH6.0, 1.0ï¼…methanol, cell optical density of 6 to 8, 0.006ï¼…PTM1, 240 r/min and induction of 96 h. After 210 h cultivation in fermentator the protopectinase activity reached the highest level of 240.2 U/ml, protein concentration was 3.21 g/L, wet cell weight was 192 g/L and cell optical density gained 251 by control the soluble oxygen of 20ï¼…in the culture.5. The crude protopectinase solution from shaking flask was purified by ammonium sulfate, Sephadex G75 and Sepharose Fast Flow and the specific activity increased to 219.27 U/mg,752.48 U/mg,1948.64 U/mg respectively, and the purification reached 2.13-fold purification, 7.31-fold purification and 18.91-fold purification, the molecular weight of the purified protopectinase was 43.17 kDa by SDS-PAGE electrophoresis。6. Studies on characterization of the purified protopectinase showed optimum pH 7.0 and temperature of 50℃. Stable pH range varied from 3-10 and thermostability was below 50℃. The protopectinase activity was strongly inhibited by 1 mM Hg²⁺, Cu²⁺, Ba²⁺, Mn²⁺, and EDTA completely, but strongly activated by 0.2 mM Ca²⁺ to about 2.5 folds, Fe²⁺, Mg²⁺ had little effects on protopectinase. Michaelis constant Km and Vmax were 0.851, 0.445μmol/min, respectively.