Extracellular expression of a novel \u03b2-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber

Qian Su,Haijin Mou,Min Yang,Yuan Yu,Tianyi Jin,Li Li

doi:10.1186/s13568-017-0525-8

Abstract

A novel endo-type β-agarase was cloned from an agar-degrading bacterium, Microbulbifer sp. Q7 (CGMCC No. 14061), that was isolated from sea cucumber gut. The agarase-encoding gene, ID2563, consisted of 1800 bp that encoded a 599-residue protein with a signal peptide of 19 amino acids. Sequence analysis suggested that the agarase belongs to the GH16 family. The agarase was expressed in Escherichia coli with a total activity of 4.99 U/mL in fermentation medium. The extracellular enzyme activity accounted for 65.73% of the total activity, which indicated that the agarase can be extracellularly secreted using the wild-type signal peptide from Microbulbifer sp. Q7. The agarase exhibited maximal activity at approximately 40 °C and pH 6.0. It was stable between pH 6.0 and pH 9.0, which was a much wider range than most of the reported agarases. The agarase was sensitive to some metal ions (Cu2+, Zn2+ and Fe3+), but was resistant to urea and SDS. The agarase hydrolyzed β-1,4-glycosidic linkages of agarose, primarily yielding neoagarotetraose and neoagarohexaose as the final products. These indicate that this recombinant agarase can be an effective tool for the preparing functional neoagaro-oligosaccharides.

Highlights

Agarose consists of alternating 3-O-linked β-d-galactose and 4-O-linked 3,6-anhydro-α-l-galactose units (Duckworth and Yaphe 1971)
Sequence analyses of the ID2563 gene A single open-reading frame composed of 1800 bp was obtained directly from the genomic DNA of Microbulbifer sp
The SignalP 4.1 server suggested the cleavage site of the signal peptide was likely between Ala19 and Ala20

Summary

Introduction

Agarose consists of alternating 3-O-linked β-d-galactose and 4-O-linked 3,6-anhydro-α-l-galactose units (Duckworth and Yaphe 1971). Oligosaccharides prepared from agarose exhibit various biological and physiological functions, such as antioxidant (Wang et al 2004), and antiinflammatory activities (Yun et al 2013), moisturizing and whitening effects on melanoma cells (Kobayashi et al 1997), inhibition of bacterial growth, and inhibition of starch degradation (Giordano et al 2006). Owing to these properties of agar-oligosaccharides, their preparation and potential applications have attracted widespread attention.

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Results

Conclusion