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Cloning, secretory expression and characterization of recombinant ?-mannanase from Bacillus circulans NT 6.7.


ABSTRACT: The mannanase gene of B. circulans NT 6.7 was cloned and expressed in an Escherichia coli expression system. The B. circulans NT 6.7 mannanase gene consists of 1,083 nucleotides encoding a 360-amino acid residue long polypeptide, belonging to glycoside hydrolase family 26. The full-length mannanase gene including its native signal sequence was cloned into the vector pET21d and expressed in E. coli BL21 (DE3). ?-Mannanase activities in the culture supernatant and crude cell extract were 37.10 and 515 U per ml, respectively, with most of the activity in the cell extract attributed to the periplasmic fraction. In contrast, expression of mannanase was much lower when using the B. circulans NT 6.7 mannanase gene without its signal sequence. The optimum temperature of recombinant ?-mannanase activity was 50°C and the optimum pH was 6.0. The enzyme was very specific for ?-mannan substrates with a preference for galactomannan. Hydrolysis products of locust bean gum were various mannooligosaccharides including mannohexaose, mannopentaose, mannotetraose, mannotriose and mannobiose, while mannose could not be detected. In conclusion, this expression system is efficient for the secretory production of recombinant ?-mannanase from B. circulans NT 6.7, which shows good characteristics for various applications.

SUBMITTER: Piwpankaew Y 

PROVIDER: S-EPMC4141934 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Cloning, secretory expression and characterization of recombinant β-mannanase from Bacillus circulans NT 6.7.

Piwpankaew Yotthachai Y   Sakulsirirat Supa S   Nitisinprasert Sunee S   Nguyen Thu-Ha TH   Haltrich Dietmar D   Keawsompong Suttipun S  

SpringerPlus 20140813


The mannanase gene of B. circulans NT 6.7 was cloned and expressed in an Escherichia coli expression system. The B. circulans NT 6.7 mannanase gene consists of 1,083 nucleotides encoding a 360-amino acid residue long polypeptide, belonging to glycoside hydrolase family 26. The full-length mannanase gene including its native signal sequence was cloned into the vector pET21d and expressed in E. coli BL21 (DE3). β-Mannanase activities in the culture supernatant and crude cell extract were 37.10 and  ...[more]

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