Jiangsu Key Laboratory of Marine Bioresources and Environment, HuaiHai Institute of Technology, Cangwu Road 59, Lianyungang, China.
Co-Innovation Center of Jiangsu Marine Bio-industry Technology, HuaiHai Institute of Technology, Cangwu Road 59, Lianyungang, China.
Biotechnol Lett. 2019 Jul;41(6-7):849-857. doi: 10.1007/s10529-019-02686-2. Epub 2019 May 7.
To determine the impact of the N-terminal nonessential domains on the enzymological properties of a pullulanase (BmP) from Bacillus megaterium strain P6.
The domains of BmP were identified by the conserved domain (CD) search online software. BmP was prepared by fermentation with the strain P6 and its N-terminal truncated form (BmNTP) was obtained by heterologous expression. Structure-property relations were analyzed by homology modeling.
BmP showed a domain architecture consisting of CBM41a-CBM41b-X-CBM48-pulA. CBM41a-CBM41b-X was removed in BmNTP. In comparison with BmP, BmNTP was lower in pH stability, specific activity and optimum NaCl concentration, but higher in K value, thermostability, optimum pH and temperature, and activity enhancement by NaCl. Particularly, BmNTP was active over 0-35% (w/v) NaCl concentrations and enhanced 8.7 folds in specific activity (from 17.6 to 170 U/mg) in 10% NaCl. The solvent accessible surface area (SASA) of the catalytic triad was found to be correlated positively to the substrate affinity and negatively to the optimum temperature and activity enhancement by NaCl.
The impact of CBM41a-CBM41b-X on the pullulanase properties was extensive and distinguished from the previous reports. The decrease in SASA may be responsible for the enzymological changes.
确定 N 端非必需结构域对巨大芽孢杆菌菌株 P6 来源的普鲁兰酶（BmP）的酶学性质的影响。
通过在线保守结构域（CD）搜索软件鉴定 BmP 的结构域。通过 P6 菌株发酵制备 BmP，并通过异源表达获得其 N 端截断形式（BmNTP）。通过同源建模分析结构-性质关系。
BmP 表现出由 CBM41a-CBM41b-X-CBM48-pulA 组成的结构域架构。BmNTP 中去除了 CBM41a-CBM41b-X。与 BmP 相比，BmNTP 的 pH 稳定性、比活性和最佳 NaCl 浓度较低，但 K 值、热稳定性、最佳 pH 和温度以及 NaCl 增强活性较高。特别是，BmNTP 在 0-35%（w/v）NaCl 浓度下具有活性，并在 10%NaCl 中比活性提高了 8.7 倍（从 17.6 至 170 U/mg）。发现催化三联体的可及溶剂表面积（SASA）与底物亲和力呈正相关，与最佳温度和 NaCl 增强活性呈负相关。
CBM41a-CBM41b-X 对普鲁兰酶性质的影响广泛，与以往的报道不同。SASA 的减少可能是酶学变化的原因。
