Research On Alginate Lyase And Agarase Produced By Agarivorans Albus YKW-34

A marine bacterium strain YKW-34 that degrades the cell wall of some seaweed including Laminaria japonica and Gelidium amansii was isolated from the gut of a turban shell. This strain was identified as Agarivorans albus based on 16S rRNA gene sequence analysis. This paper describes the optimization of production, purification, and characterization of an alginate lyase and an agarase, as well as gene cloning and expression of an agarase from this strain. The effects of medium composition and culture condition on the production of alginate lyase by A. albus YKW-34 were investigated using batch shake flasks. No alginate lyase was produced in the marine broth medium. After optimization, the activity of the alginate lyase reached 5 U/ml. The optimal production conditions for alginate lyase by A. albus YKW-34 were: inoculum volume, 10%; inoculum age, 12 h; initial pH of the medium, 8.0; culture temperature, 25°C; carbon source, 0.5% Laminaria powder; nitrogen source, 0.1% KNO3. Alginate could induce alginate lyase production, but not as efficient as did Laminaria powder. The addition of fucoidan, cellulose, and glucose had negative effect on the production of alginate lyase. Other kinds of nitrogen sources such as yeast extract, beef extract, and peptone affected positively the growth of the microorganism, but negatively the alginate lyase production. In addition, the optimal harvest time was 48 h based on the time course of alginate lyase production.An alginate lyase with high specific enzyme activity was purified from A. albus YKW-34 by in order of ion exchange, hydrophobic, and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. It was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for the activity of the alginate lyase were pH 7.0 and 40°C, respectively. It was stable over pH 7.0–10.0 and at temperature below 50°C. The enzyme had substrate specificity for both poly-guluronate and poly-mannuronate units. The kcat/Km value for alginate (heterotype) was 1.7×106 s-1M-1. The enzyme activity was completely lost by dialysis and restored by addition of Na+ or K+. The optimal activity exhibited in 0.1 M of Na+ or K+. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT), and chelating reagents (EGTA and EDTA).Effects of medium composition and culture conditions on agarase production by A. albus YKW-34 were investigated in shake flasks. Effects of carbon and nitrogen sources and culture temperature on agarase production were evaluated by one-factor-at-a-time design. Agar, yeast extract, and 25°C were found to be most suitable for agarase production. The most important nutritional components and culture conditions influencing agarase production were selected by Plackett-Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effect on agarase production. The optimum levels of these variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy resulted in the enhancement of agarase production from 0.23 U/ml to 0.87 U/ml. The activity staining of crude agarase preparation after electrophoresis revealed the presence of an agarase with molecular mass of 50 kDa.An extracellularβ-agarase, AgaA34, was purified from A. albus YKW-34 by ion exchange and gel filtration chromatographies to homogeneity with a recovery of 30% and a fold of 10. AgaA34 was composed of a single polypeptide chain with the molecular mass of 50 kDa. N-terminal amino acid sequencing revealed a sequence of ASLVTSFEEA, which exhibited a high similarity (90%) with those of agarases from glycoside hydrolase family 50. The pH and temperature optima of AgaA34 were pH 8.0 and 40°C, respectively. It was stable over pH 6.0–11.0 and at temperature below 50°C. Hydrolysis of agarose by AgaA34 produced neoagarobiose (75 mol%) and neoagarotetraose (25 mol%), whose structures were identified by MALDI-TOF MS and 13C NMR. AgaA34 cleaved both neoagarohexaose and neoagarotetraose into neoagarobiose. The kcat/Km values for agarose and neoagarotetraose were 4.04×103 and 8.1×102 s–1M–1, respectively. AgaA34 was resistant to denaturing reagents (SDS and urea). Metal ions were not required for its activity, while reducing reagents (β-Me and DTT) increased its activity by 30%.Aβ-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium Agarivorans albus YKW-34. The nucleotide sequence of agaB34 consisted of 1362 bp and encoded a protein of 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a glycoside hydrolase family 16 (GH-16) domain and a carbohydrate-binding module (CBM), showed 37–86% identity to those of known agarases in glycoside hydrolase family 16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli DH5αas a host. The purified rAgaB34 was aβ-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, while it could not further degrade NA4. The maximal activity of rAgaB34 was observed at 30°C and pH 7.0. It was stable over pH 5.0–9.0 and at temperature up to 50°C. Its specific activity and kcat/Km value for agarose were 242 U/mg and 1.7×106 s-1M-1, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT,β-Me), and denaturing reagents (SDS and urea).