Inspecting the genome sequence and agarases of Microbulbifer pacificus LD25 from a saltwater hot spring

https://doi.org/10.1016/j.jbiosc.2018.10.001Get rights and content

Neoagaro-oligosaccharides prepared by agar hydrolysis have various application fields, including the pharmaceutical, cosmetic, and food industries. In this study, an agarolytic strain was isolated from a saltwater hot spring and identified as Microbulbifer pacificus LD25 by 16S rRNA. The whole genome sequence of M. pacificus LD25 was obtained. It had a size of 4.27 Mb and comprised 3062 predicted genes in 37 contigs with a G+C content of 58.0%. Six agarases were annotated and classified into three families, namely, GH16 (AgaL1), GH86 (AgaL2, AgaL3), and GH50 (AgaL4, AgaL5, AgaL6), which shared 75–96% identities with unpublished hypothetical proteins and agarases. AgaL1, AgaL4, and AgaL6 can be successfully expressed and purified in Escherichia coli. AgaL1 and AgaL4 displayed a significantly agarolytic capability, whereas AgaL6 exhibited a rarely detectable enzymatic activity. The optimal temperature and pH required for the activity of AgaL1 and AgaL4 was 50°C and 60°C, respectively, at pH 7. The specific activities of AgaL1 and AgaL4 were achieved at 16.8 and 9.6 U per mg of protein. Both agarases were significantly inhibited in the presence of EDTA, MgO, ZnCl2, and H2O2. However, AgaL1 was resistant to 0.1% SDS and AgaL4 was slightly activated by CaCl2. Substrate hydrolysis detected by LC-MS/MS analysis indicated that neoagarobiose was the main product during AgaL1 and AgaL4 catalysis. Furthermore, AgaL4 was thermostable and retained over 93% of its relative activity after pre-incubation at 70°C for 180 min. Consequently, M. pacificus LD25 has a potential for agarase production in E. coli and industrial applications.

Section snippets

Bacterial growth and 16S rRNA analysis

The bacterial LD25 strain was isolated and purified from Zhaori Spring, Green Island, Taiwan, a world-class saltwater hot spring with temperature of approximately 60–70°C. LD25 was cultivated in marine agar medium (BCRC Medium 61) at 30°C for 3 days. The agarase, protease, gelatinase, esterase, cellulose, and xylanase, activities of LD25 were determined by agar plate method. To observe the clear zone of enzymatic activities, the agar media were utilized as follows. (i) Agarase: Observation of

Identification of LD25 by 16S rRNA

LD25 was isolated and purified from a saltwater hot spring in Green Island, Taiwan. Several enzymatic activities including agarase, amylase, cellulase, esterase, gelatinase, protease, and xylanase were determined by agar plate and broth method. The results revealed that LD25 exhibited significant activities in agarase, cellulase, and gelatinase, followed by xylanase, amylase, esterase, and protease.

To realize the LD25 strain, 16S rRNA sequencing and phylogeny were conducted (Fig. S1). The 16S

Discussion

Several studies on the analysis of Microbulbifer species including the characterizations of agarase, chitinase, esterase, alginate lyase, amylase, and carrageenase have been published 14, 25, 26, 27, 28. In this study, M. pacificus LD25 was isolated from a saltwater hot spring and exhibited various enzymatic activities. The draft genome sequence of M. pacificus LD25 was carried out and submitted to the NCBI database for further inspection of its functional proteins. Compared with the different

Acknowledgements

This work was supported by the Educational and Scientific Research Program for Young Scholar Sponsored by Educational Department of Fujian Province (JAT170715), Fujian, China, the Doctor Startup Fund Project of Xiamen Medical Colledge (K2016-31), Fujian, China, and Systematic Management and Industrialized Application of Bioresources (4/4) (107-EC-17-A-22-0525), Ministry of Economic Affairs, Taiwan.

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