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Abstract Endoglucanase is one of the most important cellulose enzyme, which cooperates with cellobihydrolase and β-1,4-glucosidase to translate cellulose to glucose completely. Our objectives were to express an endoglucanase gene in Pichia pastoris and find out expression conditions for high-level expression of neutral endoglucanase. According to the sequence of endoglucanase gene (GenBank Accession No.DQ782954) from Bacillus subtilis, a pair of primers was designed and synthesized, and a high-fidelity polymerase (Pfu DNA polymerase) was used for amplifying the mature endoglucanase expression gene. A 1.4kb fragment, which does not contain signal peptide sequence obtained, and was ligated to expression vector pPIC9k. pPIC-End was constructed and transformed to Pichia pastoris GS115. Three transformants named GS115-pPIC-EndⅠ, GS115-pPIC-EndⅦ, GS115-pPIC-EndⅧ were obtained through MD,MM plate screening and enzymatic activity test. The expression conditions containing the initial cell density, pH and methanol concentration were studied in shaking culture. Optimal endoglucanase production was observed when the initial cell density OD600was 5, and the optimal methanol concentration was 0.5%-1%. The endoglucanase production increased obviously when provided adequate aeration, whereas it did not seem to vary when cells were induced at pH4-8. Under the above expression condition, the endoglucanase activity of the three transformants was 860.7U, 760.3U, 786.2U, 13.5 times, 11.9 times and 12.3 times compared with the endoglucanase activity of the original strain (63.78U) respectively. The enzyme maintained over 80% of the original enzyme activity after incubated at 65℃ for 30 min, and the SDS-PAGE showed that The molecular weight is about 79.82KDa.
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Received: 08 August 2000
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Corresponding Authors:
Zhen-Fang WU
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