Abstract

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.

摘要

新分离的剑兰伯克霍尔德菌BRM58833 菌株被证明可以分泌一种碱性脂肪酶，在有机溶剂中具有高活性和稳定性。通过在麦麸中通过固态发酵培养菌株来优化脂肪酶的生产。脂肪酶提取条件也进行了优化。所获得的低成本提取物对p NPP 具有 1096.7 ± 39.3 U·gds ^-1（单位/克干固体）的高水解活性和 374.2 ± 20.4 U·gds ^-1对抗三油精。蛋白质组学分析显示优化的提取物由两种酯酶和三种真正的脂肪酶组成，显示出对长链底物的偏好。在 50 °C 和 pH 9 时获得最高活性。然而，在 pH 8.0 和 10.0 之间以及在整个评估温度范围 (32–70 °C) 内，提取物保持其最大活性的 50% 以上。这些酶被SDS、EDTA、ZnSO ₄和FeCl ₃抑制并被FeSO ₄、MgCl ₂和BaCl ₂激活。当在溶剂如乙腈、乙醚、正庚烷、正己烷、甲苯、甲醇和t-丁醇。与其他脂肪酶相比，这些脂肪酶对溶剂的耐受性和显着的热稳定性揭示了它们在水解反应和酯合成中的潜力。