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Cloning, Expression, and Characterization of Endo-β-1,4-Endoglucanase and Putative Mannase from Sulfolobus

Author: ZhuZuo
Tutor: LiangYunXiang
School: Huazhong Agricultural University
Course: Microbiology
Keywords: Solfataricus Mannanase Endo- beta -1 , 4 - glucanase DNS method Expression
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 30
Quote: 0
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Abstract


β-mannanase and endo-p-1, 4 - glucanase important industrial enzymes, widely used in the feed industry, paper industry, beer industry and other fields. In order to meet the requirements of the production of the high activity of the industrial enzyme heat acid is increasing. The main purpose of the wood research from extremophiles to mining potential industrial value thermophilic enzyme, try the Iceland solfataricus protein expression system, to explore the feasibility of these thermophilic enzymes in industrial applications. Following results obtained in this study: (1) p-1 with and without a signal peptide coding sequence of the inner cut from Iceland solfataricus REY15A (S. islandicus Rey15A) were amplified by PCR using 4 - endo glucanase gene (Sis eng), and cloned into the expression of solfataricus the carrier pZC2, to construct the recombinant expression vector pZC2-eng-YS and pZC2-eng-WS, and converted to S.islandicus E233S (△ pyrEF △ lacS). Recombinant strains by D-arabinose induction, cell disruption supernatant was purified by Ni-NTA column, the recombinant protein. SDS-PAGE results showed that the molecular weight of the glucanase (ENG-W) with a signal peptide of 41 ku, molecular weight (ENG-SP) with a signal peptide of 43 ku. Enzymatic properties analysis showed no activity of the former; latter enzyme activity was 103.4 U / L, the optimum reaction temperature of 90 ° C, the optimum pH was 4.0, heat resistance and experimental results show that the enzyme activity and stability after 60 min incubation at 90 ℃ In more than 40% of the highest enzyme activity; metal ion experimental results show that the final concentration of 1mmol / L Mn2 recombinant enzyme promoting effect, its enzymatic activity increased by about 59%, the final concentration of 1mmol / L of Ca2 its its enzyme activity decreased to 43%, the strongest inhibition. (2) the use of PCR technology mannan endo gene from the sulfur mine solfataricus (Sulfolobus solfataricus P2) amplified and cloned into E. coli expression vector pET-30α solfataricus expression vector pZC2 recombinant expression vector pET-30α-3007,, and pZC2-3007, and was transformed into E. coli Rosetta and bacteria S.islandicus E233S (△ pyrEF △ lacS), respectively. IPTG induction of recombinant strain E. coli Rosetta/pET-30α-3007 is, by SDS-PAGE analysis showed that most of the target protein form of inclusion bodies and the molecular size of 70 ku, in line with forecasts; over after its inclusion body refolding The Ni-NTA column purification to obtain the target protein 3007-R. The recombinant strains the S. islandicus E233S/pZC2-3007 after D-arabinose induction, cell disruption supernatant by Ni-NTA column purification of recombinant proteins 3007-S. The enzymatic properties analysis showed that the recombinant proteins 3007-R and 3007-S are no mannan activity, experimental results confirmed that under the conditions of this experiment Sso3007 NCBI forecast activity of mannan. Mass spectrum analysis of recombinant protein 3007-S, and results showed that the gene sequences are all expression.

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