Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene (agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K m and V max for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and (13)C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type β-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.

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An agarase gene (agaH71) was identified from Pseudoalteromonas hodoensis, an agar utilizing marine bacterium. The nucleotide sequence revealed that AgaH71 had significant homology to glycosyl hydrolase (GH) 16 agarases. agaH71 encodes a primary translation product (32.7 kDa) of 290 amino acids, including a 21-amino-acid signal peptide. The entire AgaH71 was expressed in a fused protein with glutathione-S-transferase (GST) at its N-terminal (GST-AgaH71) in Escherichia coli. Purified GST-AgaH71 had an apparent molecular weight of 59 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was consistent with the calculated molecular weight (58.7 kDa). Agarase activity of the purified protein was confirmed by zymogram assay. GST-AgaH71 could hydrolyze p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for GST-AgaH71 were 6.0 and 45 °C, respectively. GST-AgaH71 retained more than 95 and 90 % of its initial activity at 40 and 45 °C after heat treatment for 60 min, respectively. The K m and V max for agarose were 28.33 mg/ml and 88.25 U/mg, respectively. GST-AgaH71 did not require metal ions for its activity, but severe inhibition by divalent metal ions was observed. Thin-layer chromatography (TLC) analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectrometry of the GST-AgaH71 hydrolysis products revealed that GST-AgaH71 is an endo-type β-agarase that hydrolyzes agarose into predominantly neoagarotetraose and small proportions of neoagarobiose and neoagarohexaose.

A newly isolated bacterial strain, Bacillus sp. MX47, was actively producing extracellular xylanase only in xylan-containing medium. The xylanase was purified from the culture broth by two chromatographic steps. The xylanase had an apparent molecular weight of 26.4 kDa with an NH(2)-terminal sequence (Gln-Gly-Gly-Asn-Phe) distinct from that of reported proteins, implying it is a novel enzyme. The optimum pH and temperature for xylanase activity were 8.0 and 40 °C, respectively. The enzyme activity was severely inhibited by many divalent metal ions and EDTA at 5 mM. The xylanase was highly specific to beechwood and oat spelt xylan, however, not active on carboxymethyl cellulose (CMC), avicel, pectin, and starch. Analysis of the xylan hydrolysis products by Bacillus sp. MX47 xylanase indicated that it is an endo-β-1,4-xylanase. It hydrolyzed xylan to xylobiose as the end product. The K(m) and V(max) values toward beechwood xylan were 3.24 mg ml(-1) and 58.21 μmol min(-1) mg(-1) protein, respectively.