Cloning, Expression, And Characterization Of Endo-β-1,4-Endoglucanase And Putative Mannase From Sulfolobus

(3-mannanase and endo-1,4-β-glucanase, as important enzymes, have been widely used in many fields such as animal feed industry, pulp and paper industry and beer industry. In order to meet the need in industrial production, the exploitation of thermoacidophilic industrial enzymes with high activity is increasing. The main purpose of this study is to find thermophilic endoglucanase and mannase from extremophilic microorganisms, which have potential industrial value. The author tried to use Sulfolobus islandicus protein expression system to explore these thermophilic enzymes for potential industrial applications.(1) Endo-β-1,4-glucanase gene (with or without signal peptide coding sequence) was amplified from Sulfolobus islandicus REY15A genomic DNA and inserted into the Sulfolobus expression vector pZC2, generating recombinant plasmid pZC2-eng-YS and pZC2-eng-WS respectively. Then they were electro-transformed into the host strain S. islandicus E233S(△pyrEF△lacS). After induced by D-arabinose and purified with the Ni2+-nitrilotriacetate column, two sharp protein bands revealled on the SDS-PAGE and their protein molecular weight were about 43ku and 41ku respectively. The following analysis showed the recombinant protein (ENG-W) without signal peptide does not have endo-β-1,4-glucanase activity. However, the activity of recombinant protein (ENG-SP) with signal peptide was 103.4U/L. The optimal temperature for the recombinant protein (ENG-SP) was 90℃and the optimal pH was 4.0. Further research indicated that after incubating at 90℃for an hour, ENG-SP still had 40% of the highest activity. Metal ions 1mmol/L Mn2+could increase the activity of ENG-SP by 59%, while 1mmol/L Ca+ could inhibit the activity of ENG-SP by 43%.(2) Sso3007 gene was amplified from Sulfolobus solfataricus P2 genomic DNA, inserted into the Escherichia coli expression vector pET-30a and the Sulfolobus expression vector pZC2, generating recombinant plasmid pET-30a-3007 and pZC2-3007 respectively. Then they were transformed into the host strain E.coli Rosetta and S. islandicus E233S(△pyrEF△lacS) respectively. The recombinant strain E.coli Rosetta/pET-30a-3007 was induced by IPTG, SDS-PAGE analysis showed that most of the target protein was in inclusion body, and the protein molecular weight was 70ku, which was consistent with forecasts. Moreovr, S. islandicus E233S/pZC2-3007 was induced by D-arabinose and purified with the Ni2+-nitrilotriacetate column. The target protein was 3007-S. The following analysis showed 3007-R and 3007-S did not have mannanase activity. The result of Mass spectrometric analysis about recombinant protein 3007-S showed Sso3007 were correctly expressed.