Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

Cuiping Ma,Xinzhi Lu,Chao Shi,Jingbao Li,Yuchao Gu,Yiming Ma,Yan Chu,Feng Han,Qianhong Gong,Wengong Yu

doi:10.1074/jbc.m607888200

Cuiping Ma, Xinzhi Lu + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m607888200

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Abstract

Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type beta-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites +1 to +8 and 4 sugar units toward the non-reducing end spanning subsites -4 to -1, and enzymatic cleavage taking place between subsites -1 and +1. In addition, 1H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.

Highlights

EXPERIMENTAL PROCEDURESAll chemicals used were of analytical grade or higher. Enzymes were purchased from TaKaRa (Dalian, China)
Rides from Gigartinales and Solieriales, differ from agars in the D-configuration of the [134]-linked galactose residues and in the density of ester-sulfate substituents per digalactose repeating unit
Isolation and Identification of Strain CY24—Based on sole carbon source culture and clearing zone screening on agar plates, 50 agarase-producing bacterial strains were obtained from seawater

Summary

EXPERIMENTAL PROCEDURES

All chemicals used were of analytical grade or higher. Enzymes were purchased from TaKaRa (Dalian, China). The E. coli BL21 (DE3) cells carrying the agarase gene (pET-24a-agaB) were grown at 37 °C in LB medium containing 30 ␮g/ml kanamycin sulfate until the cell density reached 0.5 A600. At this point a final concentration of 0.1 mM isopropylthio-␤-galactopyranoside was added. The elution fractions with agarase activity were subjected to purification through a phenyl-Sepharose high performance column (Amersham Biosciences) equilibrated with 20 mM sodium phosphate buffer (pH 7.5) containing 2 M NaCl. The column was washed with equilibration buffer, and the protein was eluted using 20 mM phosphate buffer (pH 6.5) directly. To identify the structure of final hydrolysis products, oligosaccharides with different degree of polymerization (DP) were purified from a hydrolytic mixture of agarose by AgaB. The degradation product was subjected to gradient (18 –25%) discontinuous PAGE using a vertical slab gel electrophoresis system (Amersham Biosciences)

RESULTS

The kinetic parameters of AgaB on different oligosaccharides

DISCUSSION