College of Arts and Sciences of Beijing Union University, Beijing 100191, China.
Appl Biochem Biotechnol. 2012 Aug;167(8):2241-56. doi: 10.1007/s12010-012-9758-9. Epub 2012 Jun 14.
Alkaline pectin lyase (PNL) shows potential as a biological control agent against several plant diseases. We isolated and characterized a new Bacillus clausii strain that can produce 4,180 U/g of PNL using sugar beet pulp as a carbon source and inducer. The PNL was purified to apparent homogeneity using ultrafiltration, ammonium sulfate fractionation, DEAE Sepharose Fast Flow, and Sephadex G-75 gel filtration. The purified PNL was found to be a monomeric protein with a molecular weight of 35 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It demonstrated optimal activity with K(m) of 0.87 mg/ml at pH 10.0 and 60 °C. The enzyme is stable in the pH range of 8.0-10.0 and temperature ≤40 °C. Ca(2+) was found to stimulate the enzymatic activity of the PNL by up to 410 %. Mass spectrometric results gave 38 % match coverage with pectate lyase from B. clausii KSM-K16 (gi|56961845). The PNL was found to elicit disease resistance in cucumber seedlings, suggesting that it may have applications in biocontrol and sustainable agriculture.
碱性果胶裂解酶（PNL）在防治多种植物病害方面具有应用潜力。本研究从糖用甜菜渣中分离到一株能够产生 4180 U/g PNL 的新解淀粉芽孢杆菌（Bacillus clausii），并对其进行了鉴定。采用超滤、硫酸铵分级沉淀、DEAE Sepharose Fast Flow 柱层析和 Sephadex G-75 凝胶过滤对 PNL 进行了分离纯化，得到了电泳纯的 PNL。SDS-PAGE 结果显示，PNL 为单体蛋白，分子量为 35 kDa。最适反应条件为 pH 10.0、60℃，K(m)值为 0.87 mg/ml。该酶在 pH 8.0-10.0 和温度≤40℃的范围内稳定。Ca(2+)可使 PNL 的酶活提高 410%。质谱鉴定结果表明，该酶与解淀粉芽孢杆菌 KSM-K16 中的果胶裂解酶（gi|56961845）有 38%的序列相似性。PNL 可诱导黄瓜幼苗产生抗病性，表明其在生物防治和可持续农业中有应用潜力。
