以Alteromonas sp.A1-6的基因组为模板,PCR扩增目的基因alg1264,成功构建重组表达载体pET28a-alg1264,实现了alg1264的克隆表达,并对其编码的褐藻胶裂解酶Alg1264的酶学性质和水解产物开展了系统研究。Alg1264的最适pH值为7.0,在pH 5.0~11.0条件下保温1 h后相对酶活力仍能保持在77%以上;最适温度为35 ℃,在5~35 ℃保温30 min后相对酶活力仍能保持在86%以上;Mg2+、Ni2+对Alg1264的影响比较小,在5 mmol/L的浓度影响下,其活力维持在85%以上,Cu2+对Alg1264活力基本没影响。Alg1264对有机试剂有良好的耐受性,在5 mmol/L β-巯基乙醇或者体积分数20%的二甲基亚砜的处理下,Alg1264的相对酶活力可以维持在80%以上;在5 mmol/L盐酸胍、TritonX-100或者体积分数20%的甲醇、乙醇以及异丙醇存在下,Alg1264的相对酶活力也能维持在60%以上。Alg1264对底物褐藻胶的最大反应速率Vmax为0.130 μmol/min,米氏常数Km为4.034 mg/mL,kcat为52.653 s-1,kcat/Km为13.052 mL/(mg·s)。Alg1264对聚β-D-古罗糖醛酸(polyG)具有底物偏好性;液质分析结果显示该酶降解褐藻胶,聚甘露糖醛酸(polyM),polyG生成的终产物主要为单糖、二糖、三糖和少量其他寡糖,故Alg1264是一种同时具有内切和外切酶活力的聚古罗糖醛酸裂解酶。Alg1264具有较好的有机溶剂耐受性、pH稳定性,同时具有明显的聚古罗糖醛酸偏好性,该酶具备的独特性质可能使其在褐藻寡糖制备方面具有巨大的应用潜力。
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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