关键词: 纤维二糖酶, 黑曲霉, 里氏木霉, 纤维素酶, 胞外分泌

Abstract:

Cellobiase (CB）is an important component in the cellulase system.At present, the cellulase preparation produced by Trichoderma reesei is poor in cellobiase and restricts the conversion of cellobiose to glucose.In this study, the CB gene of 2526bp from Aspergillus niger was cloned by the reverse transcription PCR method, and was inserted between the strong promoter Pcbh1 (including secreting signal peptide sequence）and the terminator Tcbh1 from T.reesei to construct a recombinant plasmid pHB9 including the hygromycin B resistance marker.The plasmid pHB9 was transformed into the conidia of T.reesei by using the PEG-CaCl₂ method to obtain 387 positive transformants.Ten fast growth transformants were isolated by the rescreening culture plates using cellobiose as sole carbon source.It was confirmed that the CB gene from A.niger was integrated into the chromosomal DNA ofT.reesei by the PCR test.Cellulase production by the recombinant transformants was performed in shaking flasks, and five high CB activity transformants were obtained.After 48h fermentation, the CB activity in culture broth could reach 5.3 IU·ml^-1, which was 106 times that of the host strain.It demonstrated that CB gene from A.niger was efficiently expressed and the enzyme was successfully secreted.

Key words: 纤维二糖酶, 黑曲霉, 里氏木霉, 纤维素酶, 胞外分泌