Using the genome of Alteromonas sp.A1-6 as a template, the target gene alg1264 was amplified by polymerase chain reaction (PCR).The recombinant expression vector pET28a-alg1264 was successfully constructed, and the gene alg1264 was heterologously expressed in Escherichia coli BL21(DE3).The enzymatic properties and hydrolysis products of alginate lytic enzyme Alg1264 were systematically investigated.The optimal pH of Alg1264 was 7.0, with relative activity maintained above 77% after incubation in the pH range of 5.0-11.0 for 1 h.Its optimal temperature was 35 ℃, with relative enzyme activity maintained above 86% after incubation at 5-35 ℃ for 30 min.The relative activity of Alg1264 could be kept above 85% when treated with 5 mmol/L of Mg2+ and Ni2+, therefore, the effects of Mg2+ and Ni2+ on it were relatively small, whereas Cu2+ had no significant impact on it.Alg1264 exhibited excellent tolerance to organic reagents.Its relative activity could be maintained above 80% in the presence of 5 mmol/L b-mercaptoethanol, or 20% (v/v) DMSO.Moreover, its relative activity was also maintained above 60% in the presence of high concentrations of 5 mmol/L guanidine hydrochloride, TritonX-100, or 20% (v/v) methanol, ethanol, and isopropanol.The kinetic constants Km and Vmax of Alg1264 for alginate hydrolysis were 4.034 mg/mL and 0.130 μmol/min, respectively.kcat was 52.653 s-1.kcat/Km was 13.052 mL/(mg·s).Alg1264 exhibited a substrate preference for polyguluronic acid (polyG).Liquid chromatograph-mass spectrometer (LC-MS) analysis revealed that Alg1264 degraded alginate, polymannuronic acid (polyM), and polyG into monosaccharides, disaccharides, trisaccharides, and a small number of other oligosaccharides, confirming its dual endo- and exo-cleaving alginate lyase activity.Alg1264 demonstrated excellent stability in organic reagents, strong pH stability, and a preference for polyG, suggesting its potential for industrial-scale alginate oligosaccharides production in the future.
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