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Study on Agarase Production from Marine Strains HZ105 and ZC1

Author: LinBoKun
Tutor: HuZhong
School: Shantou University
Course: Biochemistry and Molecular Biology
Keywords: identification agarase purification cloning expression MS/MS
CLC: Q93
Type: Master's thesis
Year: 2008
Downloads: 36
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Abstract

Agarase is a kind of enzyme which can degrade agar or agarose into agaro-oligosaccharides. Production of agaro-oligosaccharides from agar has become one of the important applications of agarase, because of that more and more bioactivities of agaro-oligosaccharides have been discovered. Besides, agarase can be used to reclaim DNA from agarose gel, degrade the cell walls of marine algae for the preparation of protoplasts and select signal sequences. Up to date, most of the agarases studied are produced by marine bacteria.The sea near Shantou city is an important region of seaweed aquaculture in south China. This study reported two agar-degrading marine bacteria (strain HZ105 and strain ZC1) which were respectively isolated from marine sediments and lavers collected from the sea near Shantou. Identification and optimization of culture conditions of the two bacteria were described. And three extracellular agarases from strain HZ105 were purified and then analyzed by tandem mass spectrometry. One agarase gene of strain HZ105 was cloned. All the work laid good groundwork for high-level production of agarase and the study of new bioactivities of agaro-oligosaccharides.Based on the analysis of 16S rRNA gene sequence and phenotypic and biochemical analysis, strain HZ105 should be classified as belonging to the genus Agarivorans, named Agarivorans sp.HZ105. According to the analysis of 16S rRNA gene sequence, the major cellular fatty acids, carbon-utilizing, DNA G+C mol% and phenotypic and biochemical analysis, strain ZC1 should be assigned to a novel species in the family Flavobacteriaceae as the type strain.Strain HZ105 showed good growth and good production of agarase at NaCl concentration range from 1.0 % to 3.0 %, at pH range from 7.0 to 10.0 which indicating the possible production of alkaline extracellular agarases from strain HZ105. The crude agarase solution produced by strain HZ105 could keep 90.9% of agarase activity after being stored at 25℃for two days. Strain ZC1 showed good growth and good production of agarase at 2-3%NaCl, at pH 7-8. As assessed by Biolog GN microplates, strain ZC1 was only able to metabolize 11 of 95 carbon sources, like D-Galactose, a-D-Glucose, Maltose, Dextrin, Glycogen. And strain ZC1’s production of agarase was not agar-inducible.Three extracellular agarases of strain HZ105 were purified using the method of reclaiming protein from the polyacrylamide gel after PAGE, the molecular masses of which were 54kDa, 58kDa and 105kDa respectively estimated by SDS-PAGE. The three extracellular agarases matched threeβ-agarases belonged to glycosyl hydrolase family 50 by the analysis of tandem mass spectrometry.Based on the result of tandem mass spectrometry, the gene of the 105kDa extracellular agarase of strain HZ105 was cloned using PCR method, the coding sequence of which was 2931bp. After submitting this gene sequence to the CDD database on the wensite of NCBI, no conserved domain was found which suggested the presence of a new catalytic domain.The expression of this gene was studied in E.coli strain using vector pET-32a(+) for the construction of recombinant.

Study on Agarase Production from Marine Strains HZ105 and ZC1

Abstract

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