Cloning and characterisation of a novel neoagarotetraose-forming-β-agarase, AgWH50A from Agarivorans gilvus WH0801

Nan Liu; Xiangzhao Mao; Zongjun Du; Bozhong Mu; Dongzhi Wei

doi:10.1016/j.carres.2014.02.019

Cloning and characterisation of a novel neoagarotetraose-forming-β-agarase, AgWH50A from Agarivorans gilvus WH0801

Carbohydr Res. 2014 Mar 31:388:147-51. doi: 10.1016/j.carres.2014.02.019. Epub 2014 Feb 26.

Authors

Nan Liu¹, Xiangzhao Mao², Zongjun Du³, Bozhong Mu⁴, Dongzhi Wei⁵

Affiliations

¹ State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
² College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China. Electronic address: xzhmao@ouc.edu.cn.
³ College of Marine Science, Shandong University at Weihai, Weihai 264209, China.
⁴ Laboratory for Advanced Materials and Institute of Applied Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
⁵ State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. Electronic address: dzhwei@ecust.edu.cn.

PMID: 24642364
DOI: 10.1016/j.carres.2014.02.019

Abstract

AgWH50A, a novel β-agarase, was cloned from Agarivorans gilvus WH0801 by degenerate and nested PCR. It consists of 942 amino acids (105 kDa), including a 21-amino acid signal peptide. AgWH50A shares the highest amino acid sequence homology with AgaD02 from Agarivorans sp. QM38 (53%). The recombinant agarase gene was expressed in Escherichia coli and purified by affinity chromatography. Maximum enzymatic activity (Km 5.97 mg/mL and Vmax 0.781 U/mg) was observed at pH 6.0 and 30 °C. Using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry, Fourier transform-nuclear magnetic resonance spectrometry and thin-layer chromatography, we analysed the hydrolysis products and concluded that AgWH50A is a neoagarotetraose-forming β-agarase, which can cleave agarose into neoagarotetraose. This novel agarase has potential applications in the industrial production of neoagarotetraose and provides a new agarose hydrolysis model for future research.

Keywords: Agarivorans gilvus; GH50; Neoagarotetraose; β-Agarase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alteromonadaceae / chemistry*
Alteromonadaceae / enzymology
Amino Acid Sequence
Bacterial Proteins / chemistry*
Bacterial Proteins / genetics
Cloning, Molecular
Escherichia coli / enzymology
Escherichia coli / genetics
Galactosides / chemistry*
Glycoside Hydrolases / chemistry*
Glycoside Hydrolases / genetics
Hydrogen-Ion Concentration
Hydrolysis
Kinetics
Molecular Sequence Data
Molecular Weight
Oligosaccharides / chemistry*
Open Reading Frames
Phylogeny
Protein Sorting Signals
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Sepharose / chemistry*

Substances

Bacterial Proteins
Galactosides
Oligosaccharides
Protein Sorting Signals
Recombinant Proteins
neoagarotetraose
Sepharose
Glycoside Hydrolases
agarase