1. Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum
- Author
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Xun Li, Hao Shi, Liangliang Wang, Huaihai Ding, Yu Zhang, Fei Wang, and Yingjuan Huang
- Subjects
Arabinose ,Glycoside Hydrolases ,Molecular Sequence Data ,Biology ,Polysaccharide ,medicine.disease_cause ,Arabinan ,Chromatography, Affinity ,Substrate Specificity ,Thermotoga thermarum ,Gram-Negative Anaerobic Straight, Curved, and Helical Rods ,Hydrolysis ,chemistry.chemical_compound ,Affinity chromatography ,Polysaccharides ,Escherichia coli ,medicine ,Endo-arabinanase ,Glycoside hydrolase ,Amino Acid Sequence ,Thermostability ,chemistry.chemical_classification ,Temperature ,Substrate (chemistry) ,Gene Expression Regulation, Bacterial ,Hydrogen-Ion Concentration ,Recombinant Proteins ,Protein Structure, Tertiary ,Kinetics ,Biochemistry ,chemistry ,Research Article ,Biotechnology - Abstract
Arabinan is an important plant polysaccharide degraded mainly by two hydrolytic enzymes, endo-arabinanase and α-L-arabinofuranosidase. In this study, the characterization and application in arabinan degradation of an endo-arabinanase from Thermotoga thermarum were investigated. The recombinant endo-arabinanase was expressed in Escherichia coli BL21 (DE3) and purified by heat treatment followed by purification on a nickel affinity column chromatography. The purified endo-arabinanase exhibited optimal activity at pH 6.5 and 75°C and its residual activity retained more than 80% of its initial activity after being incubated at 80°C for 2 h. The results showed that the endo-arabinanase was very effective for arabinan degradation at higher temperature. When linear arabinan was used as the substrate, the apparent K m and V max values were determined to be 12.3 ± 0.15 mg ml−1 and 1,052.1 ± 12.7 μmol ml−1 min−1, respectively (at pH 6.5, 75°C), and the calculated k cat value was 349.3 ± 4.2 s−1. This work provides a useful endo-arabinanase with high thermostability andcatalytic efficiency, and these characteristics exhibit a great potential for enzymatic conversion of arabinan.
- Published
- 2014