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.
Keywords: Agarase; Exo-type; GH50; Neoagarobiose; Random mutagenesis.
© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.