Overexpression of Keratinase Candidate from Bacillus velezensis LPL061 in Escherichia coli BL21(DE3)

IF 3.9 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology Pub Date : 2026-03-01 Epub Date: 2025-11-22 DOI:10.1016/j.enzmictec.2025.110789

Desi Sagita , Tirza Jeli Ping , Aluicia Anita Artarini , Rachmat Mauluddin , Edwin Setiawan , Anto Budiharjo , Catur Riani

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Abstract

Proteases have diverse industrial applications including keratinase, which degrade keratin-rich substrates, such as hair, nails, feathers, and skin. This study aimed to express a protease of Bacillus velezensis LPL061 (WP_003155195.1) in Escherichia coli BL21(DE3) and to evaluate its keratinolytic as a new source of keratinase. Two gene constructs were designed, one containing the propeptide domain (kerfull) and one without it (kerhalf). Expression was induced with 0.05 mM IPTG at 20°C for 20 h. KerFull was partially purified using sequential ultrafiltration. SDS-PAGE analysis showed that KerHalf as a 32.2 kDa protein, while KerFull appeared as a 38 kDa fusion and a 28.5 kDa mature form. QTOF-MS confirmed the amino acid sequence. Only KerFull exhibited proteolytic and keratinolytic, highlighting the importance of the I9 domain for proper folding and enzyme activation. The partially purified mature KerFull protein (P50 fraction) retained activity despite low yield. KerFull showed a broad pH stability (6−11) with optimum at pH 9 and active over a wide temperature range (37–70℃), with an optimum at 60℃. Protein remained stable at 20–40℃ and pH 8. Specific activity reached 16.67 U/mg in the crude and 34.14 U/mg of P50 fraction. When combined with DTT, KerFull effectively degraded chicken feather barbules within 4 h at 37 °C. These findings suggest that one of the proteases from B. velezensis LPL061 (WP_003155195.1) is a robust keratinase candidate, offering broad operational pH and temperature, and efficient keratin degradation even at low concentrations, making it a promising candidate for industrial keratinase applications.

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韦氏芽孢杆菌候选角化酶LPL061在大肠杆菌BL21（DE3）中的过表达

蛋白酶有多种工业应用，包括角化酶，它能降解富含角蛋白的底物，如头发、指甲、羽毛和皮肤。本研究目的是在大肠杆菌BL21（DE3）中表达一种名为velezensis Bacillus LPL061 （WP_003155195.1）的蛋白酶，并评价其角化酶作为角化酶的新来源。设计了两个基因结构，一个包含前肽结构域（kerfull），一个不包含前肽结构域（kerhalf）。0.05 mM IPTG在20℃下诱导表达20 h。采用序贯超滤法对KerFull进行部分纯化。SDS-PAGE分析显示KerHalf为32.2 kDa蛋白，而KerFull为38 kDa融合蛋白和28.5 kDa成熟蛋白。QTOF-MS证实了氨基酸序列。只有KerFull表现出蛋白水解和角蛋白水解，这突出了I9结构域对正确折叠和酶激活的重要性。部分纯化的成熟KerFull蛋白（P50部分）尽管产量低，但仍保持活性。KerFull具有较宽的pH稳定性（6−11），其中pH值为9时的稳定性最佳；在较宽的温度范围（37 ~ 70℃）内具有活性，60℃时的稳定性最佳。蛋白在20 ~ 40℃、pH值8条件下保持稳定。粗馏分比活性为16.67 U/mg， P50馏分比活性为34.14 U/mg。当KerFull与DTT联合使用时，在37℃下，在4 h内可有效降解鸡毛小管。这些发现表明，velezensis中的一种蛋白酶LPL061 （WP_003155195.1）是一种强大的角化酶候选物，具有广泛的工作pH和温度，即使在低浓度下也能有效降解角化酶，使其成为工业角化酶应用的有希望的候选物。

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来源期刊

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.