Abstract
The novel β-agarase gene aga575 from the agarolytic bacterium Aquimarina agarilytica ZC1 is composed of 2142 bp, and the encoded protein Aga575 has the highest amino acid sequence homology of only 65.2% with known agarases. Though carrying a domain of glycoside hydrolase family 42 in the C-terminal, Aga575 should belong to glycoside hydrolase family 50 according to the phylogenetic analysis. Gene aga575 was successfully cloned and overexpressed in Escherichia coli Rosetta (DE3) cells. The recombinant protein had the maximal agarase activity at pH 8.0 and 37 °C. The values Km and Vmax toward agarose were 8.4 mg/mL and 52.2 U/mg, respectively. Aga575 hydrolyzed agarose and neoagarooligosaccharides to yield neoagarobiose as the sole product. The agarose hydrolysis pattern of Aga575 indicated that it was an exo-type β-agarase. Random mutagenesis was carried out to obtain two beneficial mutants M1 (R534G) and M2 (S4R-R424G) with higher activities. The results showed that the agarase activity of mutant M1 and M2 reached 162% and 192% of the wild-type agarase Aga575, respectively. Moreover, the activity of the mixed mutant M1/M2 (S4R-R424G-R534G) increased to 227%.
Key points
• Aga575 is a novel exo-type β-agarase degrading agarose to yield neoagarobiose as the sole product.
• Though owning a domain of glycoside hydrolase family GH42, Aga575 should belong to family GH50.
• The agarase activity of one mutant increased to 227% of the wild-type Aga575.
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The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Funding
This work was supported by the Major Project of Talent Team introduction for Guangdong Provincial Laboratory of Southern Marine Science and Engineering (Guangzhou) (GML2019ZD0606), Key Project of Higher Education of Guangdong Province (2016KZDXM011), Funds for PHD researchers of Guangdong Medical University in 2017 (2XB17026), and the Talents Recruitment Grant of Yangfan Plan of Guangdong Province (4YF16003G).
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CD, BL, and YS performed experiments and wrote the manuscript. ZH and BL designed, supervised, and coordinated the research. TP, JL, and MZ contributed to data analysis and manuscript editing. All authors read and approved the manuscript.
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Dong, C., Lin, B., Song, Y. et al. Characterization and activity enhancement of a novel exo-type agarase Aga575 from Aquimarina agarilytica ZC1. Appl Microbiol Biotechnol 105, 8287–8296 (2021). https://doi.org/10.1007/s00253-021-11553-y
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DOI: https://doi.org/10.1007/s00253-021-11553-y