Cloning and heterologous expression of a novel halo/alkali-stable multi-domain xylanase (XylM) from a marine bacterium Marinimicrobium sp. strain LS-A18.

Halophilic bacteria are good bioresources for halotolerant alkaline enzymes. A multi-domain high-molecular-weight endo-β-1,4-xylanase gene, xylM, was cloned from a halophilic marine bacterium Marinimicrobium sp. LS-A18. XylM is different from any of the functionally reported xylanases. It has a glycosyl hydrolase (GH) 43 domain, a GH10 domain, and two serine-rich linkers, representing a novel family. The gene, encoding 1022 residues, was cloned and heterologously expressed in Escherichia coli BL21(DE3) cells. Purified XylM was proved to be a xylanase. It showed diminished activity without salt and showed activity with a broad NaCl range from 0.2 to 25% (w/v). NaCl can increase the optimal temperature from 30 °C (0% NaCl) to 50 °C (10% NaCl). The purified XylM was active between pH 6.0 and 10.0 and was optimally active at pH 7.0. The xylanase activities were basically unchanged at a NaCl concentration range from 10 to 20% or pH from 7 to 10 after 24 h incubation. The apparent K and V values of XylM for xylan were 2.76 mg/mL and 60.0 U/mg, respectively. The GH10 domain of this enzyme, XylM, was expressed and characterized. XylM also showed xylanase activity and maintained halo-stable property. The apparent K and V values of XylM for xylan were 1.60 mg/mL and 130.1 U/mg, respectively. Other domains of XylM showed no xylanase activity. In summary, XylM is a halo-tolerant and alkali-stable endoxylanase which is a suitable candidate for xylan biodegradation in high-salt and alkali conditions. To our knowledge, this is the first report of a multidomain high-molecular-weight xylanase.