Mass production and characterization of an endoglucanase from Coleoptera insect (Monochamus saltuarius) in yeast Kluyveromyces lactis

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2024-07-04 DOI:10.1016/j.pep.2024.106540

Hyunjun Ko , Yong Chul Park

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Abstract

To harness the diverse industrial applications of cellulase, including its use in the food, pulp, textile, agriculture, and biofuel sectors, this study focused on the high-yield production of a bioactive insect-derived endoglucanase, Monochamus saltuarius glycoside hydrolase family 5 (MsGHF5). MsGHF5 was introduced into the genome of Kluyveromyces lactis to maintain expression stability, and mass production of the enzyme was induced using fed-batch fermentation. After 40 h of cultivation, recombinant MsGHF5 was successfully produced in the culture broth, with a yield of 29,000 U/L, upon galactose induction. The optimal conditions for the activity of purified MsGHF5 were determined to be a pH of 5 and a temperature of 35 °C, with the presence of ferrous ions enhancing the enzymatic activity by up to 1.5-fold. Notably, the activity of MsGHF5 produced in K. lactis was significantly higher than that produced in Escherichia coli, suggesting that glycosylation is crucial for the functional performance of the enzyme. This study highlights the potential use of K. lactis as a host for the production of bioactive MsGHF5, thus paving the way for its application in various industrial sectors.

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鞘翅目昆虫（Monochamus saltuarius）内切葡聚糖酶在乳酸克鲁伊酵母（Kluyveromyces lactis）中的批量生产和特性分析。

为了利用纤维素酶的多种工业应用，包括其在食品、纸浆、纺织品、农业和生物燃料领域的应用，本研究重点研究了一种生物活性昆虫内切葡聚糖酶--Monochamus saltuarius GH Family 5（MsGHF5）的高产生产。为了保持表达的稳定性，MsGHF5 被导入到乳酸克鲁维酵母菌（Kluyveromyces lactis）的基因组中，并通过饲料批量发酵诱导该酶的大规模生产。经过40小时的培养，在半乳糖诱导下，重组MsGHF5成功地在培养液中产生，产量为29,000 U/L。纯化的MsGHF5活性的最佳条件被确定为pH值为5，温度为35 °C，亚铁离子的存在可使酶活性提高1.5倍。值得注意的是，在乳酸菌中产生的MsGHF5的活性明显高于在大肠杆菌中产生的活性，这表明糖基化对酶的功能表现至关重要。这项研究强调了利用乳酸菌作为宿主生产具有生物活性的MsGHF5的潜力，从而为其在各种工业领域的应用铺平了道路。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.