MBio Labs at Monaghan Mushrooms Ireland Ultd, Tyholland, Monaghan, Ireland.
Chemical Sciences Department, University of Limerick, Castletroy, Limerick, Ireland.
Extremophiles. 2023 Apr 18;27(2):10. doi: 10.1007/s00792-023-01293-4.
An acid-active exo/endo-chitinase; comprising a GH18 catalytic domain and substrate insertion domain; originating from the thermophilic filamentous fungus Rasamsonia emersonii, was expressed in Pichia pastoris. In silico analysis including phylogenetic analysis, and recombinant production, purification, biochemical characterisation, and industrial application testing, was carried out. The expressed protein was identified by SDS-PAGE as a smear from 56.3 to 125.1 kDa, which sharpens into bands at 46.0 kDa, 48.4 kDa and a smear above 60 kDa when treated with PNGase F. The acid-active chitinase was primarily a chitobiosidase but displayed some endo-chitinase and acetyl-glucosamidase activity. The enzyme was optimally active at 50 °C, and markedly low pH of 2.8. As far as the authors are aware, this is the lowest pH optima reported for any fungal chitinase. The acid-active chitinase likely plays a role in chitin degradation for cell uptake in its native environment, perhaps in conjunction with a chitin deacetylase. Comparative studies with other R. emersonii chitinases indicate that they may play a synergistic role in this. The acid-active chitinase displayed some efficacy against non-treated substrates; fungal chitin and chitin from shrimp. Thus, it may be suited to industrial chitin hydrolysis reactions for extraction of glucosamine and chitobiose at low pH.
一种酸活性的外切/内切几丁质酶；包含 GH18 催化结构域和底物插入结构域；源自嗜热丝状真菌拉沙姆森尼 emersonii，在巴斯德毕赤酵母中表达。进行了包括系统发育分析、重组生产、纯化、生化特性和工业应用测试在内的计算分析。通过 SDS-PAGE 鉴定表达的蛋白为 56.3 至 125.1 kDa 的弥散带，经 PNGase F 处理后，该蛋白变为 46.0 kDa、48.4 kDa 和高于 60 kDa 的弥散带。该酸活性几丁质酶主要是几丁质寡糖酶，但显示出一些内切几丁质酶和乙酰葡萄糖胺酶活性。该酶在 50°C 时具有最佳活性，在 pH 值为 2.8 时活性明显较低。据作者所知，这是报道的任何真菌几丁质酶的最低 pH 最佳值。在其天然环境中，酸活性几丁质酶可能在细胞摄取过程中发挥几丁质降解作用，也许与几丁质脱乙酰酶协同作用。与其他拉沙姆森尼 emersonii 几丁质酶的比较研究表明，它们可能在这方面发挥协同作用。该酸活性几丁质酶对未经处理的底物（真菌几丁质和虾壳几丁质）具有一定的功效。因此，它可能适合在低 pH 值下用于工业几丁质水解反应，以提取氨基葡萄糖和壳二糖。
