A novel GH16 beta-agarase isolated from a marine bacterium, Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris

• Published in: International journal of biological macromolecules Vol. 119; pp. 1164 - 1170
• Main Authors: Li, Ren-Kuan, Chen, Zeng, Ying, Xi-Juan, Ng, Tzi Bun, Ye, Xiu-Yun
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2018
• Subjects: Alteromonadaceae - enzymology; Alteromonadaceae - genetics; Amino Acid Sequence; Beta-agarase; Cloning, Molecular; Expression; Gene Expression; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; Hydrolysis; Microbulbifer sp; Phylogeny; Pichia - genetics; Pichia pastoris; Beta-agarase; Pichia pastoris; Microbulbifer sp; Expression
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2018.08.053

An agar-degrading bacterium, strain BN3, was isolated from a coastal soil sample collected in Taiwan Strait, China and identified to be a novel species of the genus Microbulbifer. The gene (N3-1) encoding for a novel β-agarase from the isolate was cloned and sequenced. It encoded a mature protein with 274 amino acids and a calculated molecular mass of 34.3 kDa. The deduced amino acid sequence manifested sequence similarity (61–84% identity) to characterized β-agarases in the glycoside hydrolase family 16. The recombinant agarase was hyper-produced extracellularly using Pichia pastoris as the host. After induction in a shake flask for 96 h, the yield of recombinant N3–1 protein reached 0.406 mg/mL, and the enzyme activity attained 502.1 U/mL. The enzyme purified by ion exchange chromatography displayed a specific activity of 6447 U/mg at pH 6.0 and 50 °C. The optimal pH and temperature for agarase activity were approximately 6 and 50 °C, respectively. The pattern of agarose hydrolysis showed that the enzyme was an endo-type β-agarase, capable of hydrolyzing agarose and Gracilaria lemaneiformis, with neoagarobiose and neoagarotetraose as the final main products.