Cloning And Expression Of Pel9A Encoding A Thermostable Pectate Lyase

Pectate lyase can be used for the separation of flavor oil and carotenoid, the fermentation of coffee and tea, the disposing of waste water and the distilling of base oil. It was also found recently that the pectate lyase could be used for the purification of plant virus, bleaching of paper pulp and the bio-refining of textiles etc.The pel9A gene encoding pectate lyase was amplified by polymerase chain reaction using Clostridium stercorarium chromosomal DNA as a template, and then it was cloned into pET28a to recombine a expression plasmid pPel9A, finally the plasmid pPel9A was transformed into E.coli BL21 and expressed under the control of T7lac promoter at 37℃with 0.8mmol/L IPTG as inducer for 5 hours. SDS-PAGE analysis revealed that the recombinant strain bearing the plasmid pPel9A could express the target protein (130kD), with the molecular weight as the same of expected pectate lyase protein. The specific activity of pectate lyase was 15U/mg crude protein.The conditions for E. coli BL21(pPel9A) cell growth in flask were optimized. The optimized medium is composed of: glucose 10 g/L, (NH4)2SO4 2 g/L, peptone 2 g/L, citric acid 1 g/L, KH2PO4 12.5 g/L, MgSO4·7H2O 1.0 g/L, NaCl 2 g/L, trace elements solution (TES) 5 mL/L. 2.696 g/L of dry cell weight was obtained. The effect of dissolved oxygen (DO) concentration and the feeding of carbon source for the growth of E. coli BL21(pPel9A) were studied at a 5.0L fermentor. When the DO was controlled above 30%, the initial glucose concentration was 10g/L and 10g/L of glucose was feeded at log phase of cell growth, dry cell weight reached 6.388 g/L after 8h incubation, and activity of pectate lyase was 35 U/mL cell culture.The recombinant pectate lyase was purified with HiTrap Chelating Column and Superdex 200 Column. The partial purified recombinant pectate lyase was characterized. The optimum pH was found to be pH7.0 when the enzyme activity was assayed in Brittion-Robinson's universal buffer solution at various pHs. The enzyme was stable in the range of pH 6.5 to 8.0, when incubated in the same buffer solution at various pHs at 4℃for 12 hrs. The enzyme was optimally active at 65℃and stable in the ranged of 40 ~ 75℃under heat treatment for 10 min. The enzyme activity was completely inhibited by 0.1 mmol/L Fe2+, and was partly inhibited by Mg2+, K+, Co2+, Ni2+ and Na+. The enzyme was activated throughout a range of CaCl2 concentration from 0.01 to 0.2 mmol/L, and the optimal activity was observed at 0.05mmol/L of CaCl2.