Lee Hyo-Jung, Lee Yong-Suk, Choi Yong-Lark
Department of Biotechnology, Dong-A University, Busan, 49315 Republic of Korea.
Biotechnol Biofuels. 2018 Nov 8;11:303. doi: 10.1186/s13068-018-1299-1. eCollection 2018.
The ability to use organic solvents in enzyme reactions offers a number of industrially useful advantages. However, most enzymes are almost completely inactive in the presence of organic solvents. Thus, organic solvent-tolerant enzymes have potential applications in industrial processes.
A chitinase gene from DAU221 () was cloned and expressed in BL21 (DE3). The molecular weight of the expressed MtCh509 protein was approximately 60 kDa, and it was purified by His-tag affinity chromatography. Enzymatic assays showed that the optimum temperature for MtCh509 chitinase activity was 55 °C, and the enzyme was stable for 2 h at up to 50 °C. The optimum pH for MtCh509 activity was in the sub-acidic range, at pH 4.6 and 5.0. The activity of MtCh509 was maintained in presence of 1 M salt, gradually decreasing at higher concentrations, with residual activity (20%) detected after incubation in 5 M salt. Some organic solvents (benzene, DMSO, hexane, isoamyl alcohol, isopropyl alcohol, and toluene; 10-20%, v/v) increased the reactivity of MtCh509 relative to the aqueous system. When using NAG, as a substrate, MtCh509 produced NAG as the major product, as well as NAG, demonstrating that MtCh509 has transglycosylation activity. The and values for MtCh509 using colloidal chitin as a substrate were 9.275 mg/mL and 20.4 U/mg, respectively. Thus, MtCh509 could be used in extreme industrial conditions.
The results of the hydrolysate analysis and the observed increase in enzyme activity in the presence of organic solvents show that MtCh509 has industrially attractive advantages. This is the first report on an organic solvent-tolerant and transglycosylating chitinase from species.
在酶促反应中使用有机溶剂具有许多工业上有用的优势。然而，大多数酶在有机溶剂存在下几乎完全失活。因此，耐有机溶剂的酶在工业过程中具有潜在的应用价值。
从DAU221（）中克隆了几丁质酶基因，并在BL21（DE3）中表达。表达的MtCh509蛋白的分子量约为60 kDa，并通过His标签亲和层析进行纯化。酶活性测定表明，MtCh509几丁质酶活性的最适温度为55℃，该酶在高达50℃的温度下可稳定2小时。MtCh509活性的最适pH值在亚酸性范围内，为pH 4.6和5.0。MtCh509的活性在1 M盐存在下保持，在较高浓度下逐渐降低，在5 M盐中孵育后仍检测到残余活性（20%）。一些有机溶剂（苯、二甲基亚砜、己烷、异戊醇、异丙醇和甲苯；10 - 20%，v/v）相对于水相体系提高了MtCh509的反应活性。当使用NAG作为底物时，MtCh509产生NAG作为主要产物，以及NAG，表明MtCh509具有转糖基化活性。以胶体几丁质为底物时，MtCh509的和值分别为9.275 mg/mL和20.4 U/mg。因此，MtCh509可用于极端工业条件。
水解产物分析结果以及在有机溶剂存在下观察到的酶活性增加表明，MtCh509具有工业上有吸引力的优势。这是关于来自物种的耐有机溶剂且具有转糖基化活性的几丁质酶的首次报道。
