Four alginate lyase genes were cloned and sequenced from the genomic DNAs of deep-sea bacteria, namely members of Vibrio and Agarivorans. Three of them were from Vibrio sp. JAM-A9m, which encoded alginate lyases, A9mT, A9mC, and A9mL. A9mT was composed of 286 amino acids and 57% homologous to AlxM of Photobacterium sp. A9mC (221 amino acids) and A9mL (522 amino acids) had the highest degree of similarity to two individual alginate lyases of Vibrio splendidus with 74% and 84% identity, respectively. The other gene for alginate lyase, A1mU, was shotgun cloned from Agarivorans sp. JAM-A1m. A1mU (286 amino acids) showed the highest homology to AlyVOA of Vibrio sp. with 76% identity. All alginate lyases belong to polysaccharide lyase family 7, although, they do not show significant similarity to one another with 14% to 58% identity. Among the above lyases, the recombinant A9mT was purified to homogeneity and characterized. The molecular mass of A9mT was around 28 kDa. The enzyme was remarkably salt activated and showed the highest thermal stability in the presence of NaCl. A9mT favorably degraded mannuronate polymer in alginate. We discussed substrate specificities of family 7 alginate lyases based on their conserved amino acid sequences.

A high-alkaline, salt-activated alginate lyase is produced by Agarivorans sp. JAM-A1m from a deep-sea sediment off Cape Nomamisaki on Kyushu Island, Japan. Purified to homogeneity, as judged by SDS-PAGE, the enzyme (A1m) had a molecular mass of approximately 31 kDa. The optimal pH was around 10 in glycine-NaOH buffer, and the activity was increased to 1.8 times by adding 0.2 M NaCl. However, when the optimal pH in the presence of 0.2 M NaCl was shifted to pH 9.0, the activity was more than 10 times compared with that at pH 9 in the absence of NaCl. A1m showed the optimal temperature at around 30 degrees C and was stable to incubation between pH 6 and 9. The enzyme degraded favorably mannuronate-guluronate and guluronate-rich fragments in alginate. Shotgun cloning and sequencing of the gene for A1m revealed a 930-bp open reading frame, which encoded a mature enzyme of 289 amino acids (32,295 Da) belonging to polysaccharide lyase family 7. The deduced amino acid sequence showed the highest similarity to that of a Klebsiella enzyme, with only 54% identity.

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721(T). The enzyme activity increased around 4-fold by the addition of 0.2-2.0 M NaCl in 0.1 M glycine-NaOH buffer (pH 9.0). KCl, Na2SO4, NaBr, NaNO3, CH3COONa, LiCl, NH4NO3, and NH4Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7-9.4 and 60 degrees C with 0.2 M NaCl, but pH 6.5-7 and 50 degrees C without NaCl; enzyme activity increased approximately 6-fold at 60 degrees C with 0.2 M NaCl compared to that at 50 degrees C without NaCl in 0.1 M glycine-NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe2+ and Hg2+ ions), regardless whether NaCl was present or not.

A new alkaline protease (AcpII) was purified from a culture of the deep-sea bacterium Alkalimonas collagenimarina AC40(T). AcpII degraded collagen three times faster than it degraded casein. The optimal pH was 8.5-9, and the optimal temperature was 45 degrees C for the degradation of collagen. AcpII was completely inhibited by phenylmethylsulfonyl fluoride and partially by EDTA. Cloning and sequencing the gene for AcpII revealed a 2,283-bp open reading frame encoding a protein of 760 amino acids. AcpII comprises a prepropeptide, a catalytic domain that includes a protease-associated domain (PA domain), and tandem repeat prepeptidase C-terminal domains. To elucidate the role of the PA domain of AcpII, we constructed genes for two enzyme derivatives that possessed the catalytic domains with or without the PA domain and expressed them in Escherichia coli. The derivative without the PA domain showed increased specific activities toward all proteinaceous substrates tested, including gelatin, casein, and collagen, compared with those of the derivative with the PA domain.

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