Naiyu Chi

Characterisation of a chitinase from Pseudoalteromonas sp. DL-6, a marine psychrophilic bacterium

In this study, we isolated a new psychrophilic bacterium, Pseudoalteromonas sp. DL-6 from marine sediments, which grew well on chitin-containing plates at 4°C. One endo-type chitinase gene, chiA, was cloned from the genomic DNA of this bacterium and heterologously expressed in Escherichia coli BL21 (DE3). ChiA showed very high catalytic activity, even at 4°C, and exhibited maximal activity on a chitinous substrate at pH 8.0 and 20°C. Kinetic studies indicated that ChiA has a greater catalytic efficiency on 4-methylumbelliferyl-β-D-N,N,N″-triacetylchitotriose[4-MU(GlcNAc)3] than on 4-methylumbelliferyl-β-D-N,N-diacetylchitobioside[4-MU(GlcNAc)2]. Electrospray ionisation mass spectrometry (ESI-MS) analysis showed that the hydrolysis products of powdered chitin after ChiA digestion consisted of a series of chitin oligomers with different degrees of polymerisation. The ChiA mode of action was also examined using (GlcNAc)2-6 as a substrate, and the results suggested that ChiA is a non-processive endo-type chitinase.

Characterization of a cold-adapted and salt-tolerant exo-chitinase (ChiC) from Pseudoalteromonas sp. DL-6

We have previously reported a non-processive endo-type chitinase, ChiA, from a newly isolated marine psychrophilic bacterium, Pseudoalteromonas sp. DL-6. In this study, a processive exo-type chitinase, ChiC, was cloned from the same bacterium and characterized in detail. ChiC could hydrolyze crystalline chitin into (GlcNAc)2 as the only observed product. It exhibited high catalytic activity even at low temperatures, e.g. close to 0xa0°C, or in the presence of 5xa0M NaCl, suggesting that ChiC was a cold-adapted and highly salt-tolerant chitinase. ChiC could also hydrolyze other substrates, including chitosan and Avicel, indicating its broad substrate specificity. Sequence features indicated that ChiC was a multi-domain protein having a deep substrate-binding groove that was regarded as characteristic of processive exo-chitinases. Enzymatic hydrolysis of chitin by ChiC could be remarkably boosted in the presence of ChiA, suggesting the synergy of ChiC and ChiA. This work provided a new evidence to prove that marine psychrophilic bacteria utilized a synergistic enzyme system to degrade recalcitrant chitin.

Molecular cloning, expression, and biochemical characterization of a novel cold-active α-amylase from Bacillus sp. dsh19-1

A novel gene (ANK58566) encoding a cold-active α-amylase was cloned from marine bacterium Bacillus sp. dsh19-1 (CCTCC AB 2015426), and the protein was expressed in Escherichia coli. The gene had a length of 1302xa0bp and encoded an α-amylase of 433 amino acids with an estimated molecular mass of 50.1xa0kDa. The recombinant α-amylase (AmyD-1) showed maximum activity at 20xa0°C and pH 6.0, and retained about 35.7% of activity at 4xa0°C. The AmyD-1 activity was stimulated by Ca2+ and Na+. However, the chelating agent, EDTA, inactivated the enzyme. Moreover, AmyD-1 displayed extreme salt tolerance, with the highest activity in the presence of 2.0xa0M NaCl and 60.5% of activity in 5.0xa0M NaCl. The Km, Vmax and kcat of AmyD-1 in 2.0xa0M NaCl were 2.8xa0mgxa0ml−1, 21.8xa0mgxa0ml−1 min−1 and 933.5xa0s−1, respectively, at 20xa0°C and pH 6.0 with soluble starch as substrate. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) revealed that the end products of starch hydrolysis by AmyD-1 were glucose, maltose, maltotriose, maltotetraose, and malt oligosaccharides. Thus, AmyD-1 is one of the very few α-amylases that can tolerate low temperatures and high salt concentrations, which makes it to be a potential candidate for research in basic and applied microbiology.