Cloning, identification and characterization of a novel κ-carrageenase from marine bacterium Cellulophaga lytica strain N5-2

Hongli Cui; Yuxin Peng; Bowen Zhao; Yuqing Liu; Fengjia Chen; Haige Wu; Ziang Yao

doi:10.1016/j.ijbiomac.2017.07.071

Cloning, identification and characterization of a novel κ-carrageenase from marine bacterium Cellulophaga lytica strain N5-2

Int J Biol Macromol. 2017 Dec;105(Pt 1):509-515. doi: 10.1016/j.ijbiomac.2017.07.071. Epub 2017 Jul 20.

Authors

Hongli Cui¹, Yuxin Peng¹, Bowen Zhao¹, Yuqing Liu¹, Fengjia Chen¹, Haige Wu¹, Ziang Yao²

Affiliations

¹ School of Life Science and Technology, Dalian University, Dalian 116622, China; Key Laboratory of Marine Biotechnology of Dalian City, Dalian 116622, China; Key Laboratory of Glycolipid Metabolism of Liaoning Province, Dalian 116622, China; School of Life Science and Technology, Dalian University,10 Xuefu Street, Dalian Economic Technological Development Zone, Dalian, Liaoning, 116622, China.
² School of Life Science and Technology, Dalian University, Dalian 116622, China; Key Laboratory of Marine Biotechnology of Dalian City, Dalian 116622, China; Key Laboratory of Glycolipid Metabolism of Liaoning Province, Dalian 116622, China; School of Life Science and Technology, Dalian University,10 Xuefu Street, Dalian Economic Technological Development Zone, Dalian, Liaoning, 116622, China. Electronic address: yaoziang@dlu.edu.cn.

PMID: 28736039
DOI: 10.1016/j.ijbiomac.2017.07.071

Abstract

A novel κ-carrageenase gene (Cly-κ-car) was cloned and heterologously expressed from marine bacterium Cellulophaga lytica strain N5-2. The gene comprised an open reading frame of 1488bp encoding 495 amino acid residues. The deduced protein had a calculated molecular weight of 55.24kDa with an estimated isoelectric point of 9.90. Multiple alignment analysis revealed that Cly-κ-CAR shared identity with κ-carrageenases from Zobellia sp. M-2 (46%), Zobellia galactanivorans (42%) and Rhodopirellula islandica (38%). Recombinant Cly-κ-CAR (R-Cly-κ-CAR) had maximum specific activity of 620.08U/mg at 35°C, pH 7.0, 0.7% κ-carrageenan and in the presence of 0.6% NaCl. It retained >75% of its initial activity after heat treatment below 35°C for 2h. More than 50% of its activity was maintained after incubation at pH 5.0-8.0 and 4°C for 6h. The K_m and V_max values for κ-carrageenan were 0.94mg/ml and 13.42mM/min/mg, respectively. Thin layer chromatographic analysis of the R-Cly-κ-CAR hydrolysis products revealed that the enzyme hydrolyzed κ-carrageenan into neo-κ-carraoctaose and neo-κ-carrahexaose. R-Cly-κ-CAR is a novel κ-carrageenase enzyme and could be a valuable tool to produce high degree of polymerization κ-carrageenan oligosaccharides with various biological activities.

Keywords: Carrageenase; Enzyme properties; Heterologous expression.

MeSH terms

Amino Acid Sequence
Base Sequence
Cloning, Molecular
Computational Biology
Flavobacteriaceae / enzymology*
Glycoside Hydrolases / chemistry
Glycoside Hydrolases / genetics*
Glycoside Hydrolases / metabolism*
Hydrolysis
Protein Multimerization

Substances

Glycoside Hydrolases