Solid-state fermentation production and characterization of an alkaline lipase from a newly isolated Burkholderia gladioli strain

The newly isolated Burkholderia gladioli BRM58833 strain was shown to secrete an alkaline lipase highly active and stable in organic solvents. Lipase production was optimized through the cultivation of the strain by solid-state fermentation in wheat bran. The lipase extraction conditions were also optimized. The low-cost extract obtained has shown a high hydrolytic activity of 1096.7 ± 39.3 U·gds−1 (units per gram of dry solids) against pNPP and 374.2 ± 20.4 U·gds−1 against triolein. Proteomic analysis revealed the optimized extract is composed of two esterases and three true lipases, showing a preference for long-chain substrates. The highest activity was obtained at 50 °C and pH 9. However, the extract maintained more than 50% of its maximum activity between pH 8.0 and 10.0 and throughout the whole temperature range evaluated (32–70 °C). The enzymes were inhibited by SDS, EDTA, ZnSO4 and FeCl3 and activated by FeSO4, MgCl2 and BaCl2. The lipases conserved their activity when incubated in solvents as acetonitrile, diethyl ether, n-heptane n-hexane, toluene, methanol and t-butanol. The resistance of these lipases to solvents and expressive thermostability when compared to other lipases, reveal their potential both in hydrolysis reactions and in synthesis of esters.

本研究中新分离得到的唐菖蒲伯克霍尔德菌（Burkholderia gladioli）BRM58833菌株可分泌一种在有机溶剂中兼具高活性与稳定性的碱性脂肪酶。通过以麦麸为基质的固态发酵培养该菌株，本研究优化了脂肪酶的生产工艺，并对脂肪酶的提取条件进行了优化。所获得的低成本粗提物表现出优异的水解活性：对硝基苯基磷酸酯（pNPP）的活性为1096.7 ± 39.3 U·gds⁻¹（单位/克干物质），对三油酸甘油酯（triolein）的活性为374.2 ± 20.4 U·gds⁻¹。蛋白质组学分析显示，该优化后的粗提物包含两种酯酶与三种真性脂肪酶，对长链底物具有底物偏好性。该脂肪酶的最高活性出现在50 ℃、pH 9的条件下。但在pH 8.0至10.0的区间内，以及所测试的全部温度范围（32–70 ℃）中，该粗提物均保留了超过50%的最大活性。该酶类可被十二烷基硫酸钠（SDS）、乙二胺四乙酸（EDTA）、硫酸锌（ZnSO₄）及三氯化铁（FeCl₃）抑制，而可被硫酸亚铁（FeSO₄）、氯化镁（MgCl₂）及氯化钡（BaCl₂）激活。将该脂肪酶在乙腈、乙醚、正庚烷、正己烷、甲苯、甲醇及叔丁醇中孵育后，其活性得以保留。相较于其他脂肪酶，此类脂肪酶兼具有机溶剂耐受性与优异的热稳定性，在水解反应及酯类合成反应中均具有应用潜力。