Enzyme and Microbial Technology最新文献_第2页

Microbial-enzyme co-fermentation of low-grade tobacco: Metagenomics and metabolomic insights into flavor formation 低品位烟草的微生物-酶共发酵：元基因组学和代谢组学对风味形成的见解

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-11 DOI: 10.1016/j.enzmictec.2025.110803

Ming Shu , Huijie Xue , Yang Yang , Xiao Zhang , Shitou Li , Tengfei Bian , Kailong Yuan , Chunping Xu

Microbial-enzyme co-fermentation effectively enhances the quality of low-grade tobacco leaves quality, but the underlying mechanisms of flavor formation remain unclear. This study investigated the dynamics and relationships of microbial communities and volatile aroma metabolites during low-grade tobacco leaves fermentation through metagenomics and headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Results showed that during microbial-enzyme co-fermentation, the tobacco leaves fermented for four days (D4) exhibited the highest levels of total sugars and reducing sugars, the peak total content of aroma metabolites, and the best sensory quality. Pseudomonadota, Bacillota, and Ascomycota were dominant microorganisms during fermentation. During the initial stage (D1–D4), Saccharomyces was the dominant genus, which was subsequently displaced by Pantoea at D5. This microbial succession coincided with a decline in sensory quality, indicating its crucial role in shaping flavor evolution during co-fermentation. During microbial-enzyme co-fermentation process, a total of 46 volatile metabolites were detected in low-grade tobacco leaves. Among them, seven esters with high variable important in projection values and strong microbial correlations were identified as characteristic aroma metabolites, including ethyl phenylacetate, benzyl acetate, phenylethyl acetate, ethyl myristate, ethyl palmitate, ethyl oleate, and methyl linolenate. Gene function annotation revealed carbohydrate metabolism was the most abundant, followed by amino acid metabolism. Spearman correlation analysis elucidated the formation mechanism of characteristic ester metabolites. Specifically, short-chain esters correlated with glycerolipid and amino acid metabolism, while long-chain esters linked to glycolysis and fatty-acid biosynthetic pathways.

微生物-酶共发酵可有效提高低质烟叶品质，但其风味形成机制尚不清楚。本研究采用宏基因组学、顶空固相微萃取-气相色谱-质谱联用技术研究了低度烟叶发酵过程中微生物群落与挥发性香气代谢物的动态变化及其相互关系。结果表明，发酵4 d （D4）烟叶总糖和还原糖含量最高，香气代谢产物总含量最高，感官品质最佳。假单胞菌、杆状杆菌和子囊菌是发酵过程中的优势菌群。在初始阶段（D1-D4）， Saccharomyces是优势属，随后在D5被Pantoea取代。这种微生物演替与感官品质的下降相吻合，表明其在共发酵过程中形成风味演变的关键作用。在微生物-酶共发酵过程中，低品位烟叶共检测到46种挥发性代谢物。其中，7种具有高投影值重要变量和强微生物相关性的酯类被鉴定为特征香气代谢物，包括苯基乙酸乙酯、乙酸苄酯、乙酸苯乙酯、肉豆酸乙酯、棕榈酸乙酯、油酸乙酯和亚麻酸甲酯。基因功能注释显示碳水化合物代谢最丰富，其次是氨基酸代谢。Spearman相关分析阐明了特征性酯代谢产物的形成机制。具体来说，短链酯类与甘油脂和氨基酸代谢相关，而长链酯类与糖酵解和脂肪酸生物合成途径相关。

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引用次数: 0

A unique highly efficient, thermostable and multi-substrate specific galactanase (AtGH53) from Acetivibrio thermocellus cleaving both β (1,4)- and β (1,6)- linked galactans 一种独特的高效、耐热和多底物特异性半乳糖酶（AtGH53），来自热细胞活动弧菌，可切割β(1,4)-和β(1,6)-连接的半乳糖

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-08 DOI: 10.1016/j.enzmictec.2025.110801

Shreya Biswas , Ardhendu Mandal , Carlos M.G.A. Fontes , Arun Goyal

The gene encoding endo-β-1,4-galactanase, AtGH53 from Acetivibrio thermocellus was cloned, expressed and the encoded soluble protein was biochemically characterized. Purified AtGH53 showed molecular mass of approximately, 36 kDa, an optimum temperature 70°C and half-life of 15 h at 70°C. AtGH53 displayed stability in acidic and alkaline pH ranges, with an optimum pH 7.5. AtGH53 activity increased over 30 % by Ni or Co²⁺ ions. AtGH53 exhibited broad substrate specificity, displaying the highest activity with potato pectic-galactan with V_max of 1432 U.mg⁻¹ and K_M of 1.2 mg.mL⁻¹ . TLC and HPLC analyses of potato galactan hydrolysis by AtGH53 showed initially the endo-lytic cleaving property and later shifting to exo-lytic mode. This was confirmed by the release of β-1,4-linked galacto-oligosaccharides of higher degrees of polymerization (DP>3) from potato galactan in first 2 h, followed by accumulation of galactobiose and galactose up to 24 h. In contrast, hydrolysis of larch-arabinogalactan by AtGH53 (specific activity, 148.6 U.mg⁻¹) resulted in the release of β-1,6-galactobiose from branches of the polymer. This indicated that AtGH53 also exhibits lower efficiency in hydrolyzing β-1,6-galactan of arabinogalactan via an exo-mode of action. The broad substrate specificity, pH stability and thermostability of AtGH53 make it a versatile enzyme for biotechnological applications.

克隆、表达了热细胞活动弧菌内切-β-1,4-半乳糖酶（endo-β-1,4-半乳糖酶）编码基因AtGH53，并对编码的可溶性蛋白进行了生化表征。纯化后的AtGH53分子量约为36 kDa，最适温度为70℃，70℃时半衰期为15 h。AtGH53在酸性和碱性范围内均表现出稳定性，最适pH值为7.5。Ni 或Co2+离子使AtGH53活性增加30% %以上。AtGH53具有广泛的底物特异性，对马铃薯果胶半乳的活性最高，Vmax为1432 U。mg¹ 和KM 1.2 mg. ml⁻¹ 。AtGH53对马铃薯半乳氨酸水解的TLC和HPLC分析显示，半乳氨酸最初具有内解裂解性质，随后转变为外解模式。马铃薯半乳糖在前2 h内释放出较高聚合度的β-1,4-连接半乳糖低聚糖（DP>3），随后在24 h内积累半乳糖和半乳糖，证实了这一点。相比之下，AtGH53水解落叶松阿拉伯半乳聚糖(比活性，148.6 U。Mg−1)导致β-1,6-半乳糖二糖从聚合物的分支中释放。这表明AtGH53通过外显作用模式水解阿拉伯半乳聚糖中的β-1,6-半乳聚糖的效率也较低。AtGH53广泛的底物特异性、pH稳定性和热稳定性使其成为生物技术应用的多功能酶。

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引用次数: 0

Peach gum-derived immobilization of Klebsiella sp. cells combined with nanoparticle addition for robust enhancement of biohydrogen production 桃胶源克雷伯氏菌细胞的固定化与纳米颗粒的结合增强了生物制氢的能力。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-08 DOI: 10.1016/j.enzmictec.2025.110800

Qin Zhang , Qianzi Cheng , Juanjuan Cao , Cancan Zhang , Bo Wu , Yanbin Li , Sejabodile Precious Moanokeng

This study developed a peach-gum-derived immobilization of Klebsiella sp. cells combined with nanoparticles (NPs) to enhance biohydrogen production. The optimal ratio of peach gum (PG) to sodium alginate was determined to be 1.5:1 for effective immobilization of Klebsiella sp. cells, which was further improved by the addition of magnetite NPs and NiFe₂O₄ NPs (NFNPs). The results showed that the Im + NFNP 20 mg/L treatment was optimal, achieving the highest cumulative hydrogen production of 5996.35 ± 0.29 mL/L at 120 h, which was 1.89 times higher than the control treatment. Notably, this treatment resulted in the maximum Y_H2/S (128.42 ± 2.05 mL/g) and the highest peak number of viable cells ((2.91 ± 0.03) * 10⁹ cfu/mL), as well as enhanced peak activities of hydrogenase and formate-hydrogen lyase. Furthermore, the combination of immobilization and optimum NFNP addition noticeably promoted the expressions of several hydrogenase and formate-hydrogen lyase related genes and altered the distribution of soluble metabolites, which enhanced the hydrogenase- and formate-hydrogen lyase-catalyzed pathways. Thus, we propose a mechanism in which PG-derived immobilization facilitates the entry of NPs into Klebsiella sp. cells, and elucidate the molecular basis for the efficient cellular uptake and distribution of NFNPs in Klebsiella sp. cells.

本研究开发了一种由桃胶衍生的克雷伯氏菌细胞与纳米颗粒（NPs）结合的固定化方法，以提高生物氢的产生。确定桃胶（PG）与海藻酸钠的最佳固定比为1.5:1，通过添加磁铁矿NPs和NiFe2O4 NPs （NFNPs）进一步提高固定化克雷伯氏菌细胞的效果。结果表明，Im + NFNP 20 mg/L处理效果最佳，在120 h时累计产氢量最高，为5996.35 ± 0.29 mL/L，是对照处理的1.89倍。其中，YH2/S最高（128.42 ± 2.05 mL/g），活细胞峰值数最高（2.91 ± 0.03）* 109 cfu/mL)，氢化酶和甲酸解氢酶活性峰值增强。此外，固定化和最佳NFNP添加的结合显著促进了几个氢化酶和甲酸氢裂解酶相关基因的表达，改变了可溶性代谢产物的分布，从而增强了氢化酶和甲酸氢裂解酶催化的途径。因此，我们提出了一种pg衍生的固定化促进NPs进入克雷伯氏菌细胞的机制，并阐明了NFNPs在克雷伯氏菌细胞中有效摄取和分布的分子基础。

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引用次数: 0

Mechanistic investigation of the enhanced catalytic activity of B. sub lipase A mutant I157V and its application in biodiesel production b亚脂肪酶A突变体I157V催化活性增强机理研究及其在生物柴油生产中的应用

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-03 DOI: 10.1016/j.enzmictec.2025.110798

Zhong Ni, Jia Zhao, Huimin Zhou, Huayun Jia, E-Bin Gao, ZhongJian Guo, Zhaoyang Hu, Shangshang Ma, Yong Chen

Optimizing lipases for the transesterification of long-chain fatty acid esters plays a vital role in enhancing biodiesel yields, especially from recycled cooking oils. In this study, we applied error-prone PCR to create a diverse mutagenesis library based on Bacillus subtilis lipase A(BSLA). The I157V mutant, selected through 96-well high-throughput screening, exhibited over sixfold greater catalytic efficiency (k_cat/K_m) than the wild-type enzyme. Insights from molecular docking and dynamic simulations pointed to the mutation’s effect on aligning substrates more effectively within the enzyme’s catalytic center. This alignment minimized non-productive binding modes and reinforced the enzyme-substrate interactions, which in turn boosted catalytic output. In biodiesel production using waste cooking oil as feedstock, the I157V mutant achieved a methyl ester yield of 87 % (w/w) after 10 h, representing an ∼45 % increase compared to 60 % (w/w) for the wild type. These findings not only advance enzyme engineering for industrial biocatalysis but also underline the potential of targeted mutations in supporting sustainable energy solutions.

优化脂肪酶对长链脂肪酸酯酯的酯交换反应对提高生物柴油的产量，特别是从再生食用油中提取生物柴油具有重要作用。本研究以枯草芽孢杆菌脂肪酶a （Bacillus subtilis lipase a， BSLA）为基础，应用易出错PCR技术建立了一个多样化的诱变文库。通过96孔高通量筛选筛选出的I157V突变体的催化效率（kcat/Km）是野生型酶的6倍以上。来自分子对接和动态模拟的见解指出，突变对酶的催化中心内的底物更有效地排列产生了影响。这种排列最小化了非生产性结合模式，加强了酶与底物的相互作用，从而提高了催化产量。在以废食用油为原料的生物柴油生产中，I157V突变体在10 h后的甲酯产率为87 % (w/w)，比野生型的60 % （w/w）提高了~ 45 %。这些发现不仅推动了工业生物催化的酶工程，而且强调了靶向突变在支持可持续能源解决方案方面的潜力。

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引用次数: 0

Dithionite-supported biohydrogenation of muconic acid to adipic acid by lyophilized Escherichia coli cells expressing recombinant enoate reductase 表达重组乙酸还原酶的冻干大肠杆菌细胞在二硫代酸支持下将粘液酸生物加氢为己二酸。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-02 DOI: 10.1016/j.enzmictec.2025.110797

Khorcheska A. Batyrova , Anna N. Khusnutdinova , Alexander F. Yakunin

Adipic acid is an important six-carbon dicarboxylic acid with numerous industrial applications in polymers (nylon) and the food industry. Traditional manufacturing of adipic acid relies on petroleum feedstocks and involves energy-intensive chemical processes with negative environmental impacts. Consequently, alternative synthesis methods are being developed, including the hydrogenation of biobased muconic acid to adipic acid via chemical catalysis or enzymatic reduction with 2-enoate reductases. This study revealed that purified full-length 2-enoate reductase ERBC from Heyndrickxia (Bacillus) coagulans can reduce the three muconic acid isomers (cis,cis, cis,trans, trans,trans) using NADH as a reductant. Titration of the purified ERBC with different chemical reductants showed that its redox cofactors (FMN, FAD, and [4Fe-4S]) can also be reduced by dithionite and Ti(III)-citrate. However, only dithionite and NADH supported the biohydrogenation of trans-cinnamic acid and cis,cis-muconic acid. The individually expressed and purified large domain of ERBC also catalyzed muconic acid reduction with these reductants, but exhibited lower activity and produced only 2-hexenedioic acid as the product. Efficient conversion of muconic acid to adipic acid was demonstrated using lyophilized E. coli cells expressing full-length ERBC as the catalyst, with dithionite acting as both a reductant and an oxygen scavenger. The use of lyophilized recombinant Escherichia coli cells with dithionite for ERBC-mediated biohydrogenation of muconic acid eliminates the need for protein purification and costly natural cofactors (NAD(P)H), as well as enhances ERBC tolerance to high substrate concentrations and creates anaerobic conditions for ERBC activity. This approach shows promise for biobased adipic acid production and other applications of 2-enoate reductases.

己二酸是一种重要的六碳二羧酸，在聚合物（尼龙）和食品工业中有着广泛的工业应用。传统的己二酸制造依赖于石油原料，涉及能源密集型的化学过程，对环境有负面影响。因此，人们正在开发其他合成方法，包括通过化学催化或用2-烯酸还原酶将生物基粘膜酸加氢成己二酸。本研究发现，从海因德里克氏杆菌（芽孢杆菌）凝固物中纯化的全长2-烯酸还原酶ERBC可以用NADH作为还原剂还原三种粘膜酸异构体（顺式、顺式、顺式、反式、反式、反式）。用不同的化学还原剂对纯化的ERBC进行滴定，结果表明其氧化还原辅助因子（FMN、FAD和[4Fe-4S]）也可以被二亚硝酸盐和柠檬酸钛还原。然而，只有二亚硝酸盐和NADH支持反式肉桂酸和顺式、顺式粘膜酸的生物加氢。单独表达纯化的ERBC大结构域也能催化这些还原剂还原黏液酸，但活性较低，产物仅为2-己烯二酸。用表达全长ERBC的冻干大肠杆菌细胞作为催化剂，二亚硝酸盐同时作为还原剂和氧气清除剂，证明了粘液酸向己二酸的有效转化。利用冻干重组大肠杆菌细胞和二亚硝酸盐进行ERBC介导的粘膜酸生物加氢，消除了蛋白质纯化和昂贵的天然辅助因子（NAD(P)H）的需要，同时增强了ERBC对高底物浓度的耐受性，并为ERBC活性创造了厌氧条件。这种方法显示了生物基己二酸生产和2-烯酸还原酶的其他应用前景。

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引用次数: 0

Effect of lipopolysaccharide on ganglioside expression in human induced pluripotent stem cell-derived kidney organoids. 脂多糖对人诱导多能干细胞衍生肾类器官中神经节苷脂表达的影响。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-01 Epub Date: 2025-08-06 DOI: 10.1016/j.enzmictec.2025.110730

Jae-Sung Ryu, Jin Ok Yu, Ki Kwan Kim, Eun-Jeong Jeong, Min Young Kim, Hyo Gyeong Yun, Eun Bin Song, Ji-Su Kim, Young-Woock Noh, Young-Kug Choo

An organoid is a self-organizing, three-dimensional (3D), stem cell-derived structure that closely mimics the structural, cellular, and functional properties of specific organs or tissues. Organoids are widely utilized for assessing drug efficacy, safety, and industrial chemical toxicity. The purpose of this study was to generate a kidney organoid from human induced pluripotent stem cells (iPSCs) and establish a sepsis-associated acute kidney injury (SA-AKI) model by treatment with lipopolysaccharide (LPS). We further analyzed changes in ganglioside expression following LPS treatment in kidney organoids. As a result, we observed that the expression of kidney-specific markers was significantly increased during differentiation. Next, we confirmed that the levels of inflammation-related markers and reactive oxygen species (ROS) were significantly increased, whereas mitochondrial membrane potential (MMPΨ) was significantly reduced in LPS-treated kidney organoids. Interestingly, ganglioside GM3, GM2, GD3, and GD1a expression, as well as their biosynthesis, was notably decreased in LPS-treated kidney organoids. These findings suggest that gangliosides play critical roles in inflammation and may contribute to the pathophysiology of SA-AKI, highlighting the potential of kidney organoids as a valuable model system for studying kidney injury and associated inflammatory responses.

类器官是一种自组织的三维（3D）干细胞衍生的结构，它密切模仿特定器官或组织的结构、细胞和功能特性。类器官被广泛用于评估药物的有效性、安全性和工业化学毒性。本研究的目的是利用人诱导多能干细胞（iPSCs）生成肾类器官，并通过脂多糖（LPS）处理建立脓毒症相关急性肾损伤（SA-AKI）模型。我们进一步分析了脂多糖处理后肾类器官中神经节苷脂表达的变化。结果，我们观察到在分化过程中肾脏特异性标志物的表达显著增加。接下来，我们证实了炎症相关标志物和活性氧（ROS）水平显著增加，而在lps处理的肾类器官中，线粒体膜电位（MMPΨ）显著降低。有趣的是，神经节苷脂GM3、GM2、GD3和GD1a的表达及其生物合成在lps处理的肾类器官中显著降低。这些发现表明神经节苷在炎症中起关键作用，并可能参与SA-AKI的病理生理，突出了肾类器官作为研究肾损伤和相关炎症反应的有价值的模型系统的潜力。

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引用次数: 0

Modulating dioxygenase and hydroperoxide isomerase activities in Burkholderia thailandensis lipoxygenase. 调节泰国伯克霍尔德菌脂加氧酶的双加氧酶和氢过氧化物异构酶活性。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-01 Epub Date: 2025-07-08 DOI: 10.1016/j.enzmictec.2025.110709

Ruth Chrisnasari, Roelant Hilgers, Guanna Li, Jean-Paul Vincken, Willem J H van Berkel, Marie Hennebelle, Tom A Ewing

Lipoxygenases (LOXs) are enzymes that catalyze the regioselective dioxygenation of polyunsaturated fatty acids (PUFAs), leading to the formation of fatty acid hydroperoxides (FAHPs). In addition to dioxygenase activity, some eukaryotic LOXs exhibit hydroperoxide isomerase (HPI) activity under specific conditions, resulting in the production of structurally diverse compounds such as epoxy alcohols and ketones. Until now, the presence of HPI activity in bacterial LOXs has not been documented. In this study, we investigated the HPI activity of LOX from Burkholderia thailandensis (Bt-LOX) and examined the effects of reaction conditions on its catalytic profile using three different C18 PUFA substrates. The results demonstrated that Bt-LOX exhibits significant HPI activity, especially at high enzyme concentrations, with ketone formation showing strong substrate dependence. Oxygen level was identified as a critical factor in directing the catalytic performance of Bt-LOX: HPI activity was inhibited under O₂-saturated conditions and enhanced under O₂-limited conditions. These findings establish Bt-LOX as the first bacterial LOX reported to exhibit pronounced HPI activity, and highlights its expanded potential for biocatalytic applications.

脂氧合酶（LOXs）是催化多不饱和脂肪酸（PUFAs）的区域选择性双氧作用，导致脂肪酸氢过氧化物（FAHPs）形成的酶。除了双加氧酶活性外，一些真核lox在特定条件下还表现出氢过氧化物异构酶（HPI）活性，从而产生结构多样的化合物，如环氧醇和酮类。到目前为止，HPI活性在细菌LOXs中的存在尚未被证实。在这项研究中，我们研究了泰国伯克霍尔德氏菌（Bt-LOX）的HPI活性，并研究了三种不同的C18 PUFA底物对其催化性能的影响。结果表明，Bt-LOX表现出显著的HPI活性，特别是在高酶浓度下，酮的形成表现出强烈的底物依赖性。氧水平被认为是指导Bt-LOX催化性能的关键因素：HPI活性在O₂饱和条件下被抑制，而在O₂限制条件下被增强。这些发现确定了Bt-LOX是第一个报道具有明显HPI活性的细菌LOX，并突出了其在生物催化应用方面的扩展潜力。

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引用次数: 0

Overexpression of Keratinase Candidate from Bacillus velezensis LPL061 in Escherichia coli BL21(DE3) 韦氏芽孢杆菌候选角化酶LPL061在大肠杆菌BL21（DE3）中的过表达

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub 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

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.

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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引用次数: 0

PhcK mediates transport of a β-5-type lignin-derived dimer in Sphingobium lignivorans SYK-6 PhcK介导木质素鞘SYK-6中β-5型木质素衍生二聚体的转运

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-11-22 DOI: 10.1016/j.enzmictec.2025.110784

Mitsuru Kawazoe , Masaya Fujita , Shojiro Hishiyama , Naofumi Kamimura , Eiji Masai

Lignin is a major component of plant cell walls; however, its heterogeneous and complex structure has hindered its efficient utilization. Recently, strategies that combine chemical pretreatment with microbial conversion to produce valuable chemicals have attracted attention. To develop an ideal microbial platform for this purpose, it is essential to elucidate the complete bacterial catabolic system for lignin-derived aromatic compounds. Here, we identified an inner membrane transporter gene involved in the uptake of a metabolite of dehydrodiconiferyl alcohol (DCA), a lignin-derived β-5 dimer, in Sphingobium lignivorans SYK-6. SLG_12820 (phcK) was found to encode a major facilitator superfamily transporter belonging to the endosomal spinster family and is regulated by PhcR, a transcriptional regulator of DCA catabolism genes. Through mutant analysis and a specific uptake assay based on PhcR effector recognition, we demonstrated that PhcK functions as an inner membrane transporter that specifically imports the DCA metabolite, 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)acrylic acid.

木质素是植物细胞壁的主要成分；然而，其异质复杂的结构阻碍了其高效利用。近年来，将化学预处理与微生物转化相结合以生产有价值化学品的策略引起了人们的关注。为了开发一个理想的微生物平台，有必要阐明木质素衍生芳香族化合物的完整细菌分解代谢系统。在这里，我们发现了一个参与木质素衍生的β-5二聚体脱氢二硝基醇（DCA）代谢物摄取的内膜转运基因。SLG_12820 （phcK）编码一个属于内体spinster家族的主要促进体超家族转运蛋白，并受DCA分解代谢基因的转录调节因子PhcR调控。通过突变体分析和基于PhcR效应识别的特异性摄取实验，我们证明PhcK作为一种内膜转运体，特异性地进口DCA代谢物3-（2-（4-羟基-3-甲氧基苯基）-3-（羟甲基）-7-甲氧基-2,3-二氢苯并呋喃-5-基）丙烯酸。

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引用次数: 0

Structure and functional role of the SLHs-CBM54 tandem in bacterial multimodular glycoside hydrolases SLHs-CBM54串联在细菌多模糖苷水解酶中的结构和功能作用

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-11-21 DOI: 10.1016/j.enzmictec.2025.110787

Oksana V. Berezina , Alina I. Selimzyanova , Kirill V. Gordeev , Wolfgang H. Schwarz , Natalia A. Lunina

Carbohydrate-binding modules (CBMs) enhance enzymatic degradation of polysaccharides by improving substrate binding. CBM54, a family of carbohydrate-binding modules, is found in surface-displayed multimodular glycoside hydrolases (MGHs) of gram-positive bacteria. These modules bind insoluble polysaccharides, including chitosan, chitin, xylan, cellulose, and fungal cell wall β-glucans. In MGHs, the CBM54 module is invariably positioned downstream of three S-layer homology modules (SLHs), which anchor the enzyme to the bacterial cell surface. The SLHs-CBM54 tandem promotes bacterial adherence to substrates, concentrating hydrolytic enzymes at the interface and facilitating efficient uptake of soluble degradation products. CBM54 contains a cleavage site that divides it into two structurally distinct parts, which remain associated via hydrogen bonding. The processing of CBM54 promotes bacterial detachment from the substrate and their migration toward new nutrient sources. Beyond MGHs, the SLHs-CBM54 tandem occurs in many other bacterial proteins with uncharacterized functions. SLH modules may self-assemble into 2D arrays on synthetic supports, enabling their use as matrices for protein nucleation and crystal growth. The SLHs-CBM54 tandem fusion with functional modules offers an innovative approach to surface modification, with broad biomedical and biotechnological applications. Due to its versatile architecture, this system holds promise for antigen display, vaccine development, and enhanced polysaccharide degradation processes.

碳水化合物结合模块（CBMs）通过改善底物结合来促进多糖的酶降解。CBM54是一个碳水化合物结合模块家族，存在于革兰氏阳性细菌的表面显示的多模块糖苷水解酶（MGHs）中。这些模块结合不溶性多糖，包括壳聚糖、几丁质、木聚糖、纤维素和真菌细胞壁β-葡聚糖。在MGHs中，CBM54模块总是位于三个s层同源模块（SLHs）的下游，SLHs将酶固定在细菌细胞表面。SLHs-CBM54串联促进细菌粘附在底物上，在界面上集中水解酶，促进可溶性降解产物的有效吸收。CBM54含有一个裂解位点，将其分成两个结构不同的部分，它们通过氢键保持联系。CBM54的处理促进了细菌脱离基质并向新的营养源迁移。除了MGHs外，SLHs-CBM54串联还存在于许多其他功能未知的细菌蛋白中。SLH模块可以在合成支架上自组装成二维阵列，使其能够用作蛋白质成核和晶体生长的基质。SLHs-CBM54串联融合功能模块提供了一种创新的表面改性方法，具有广泛的生物医学和生物技术应用。由于其多功能结构，该系统有望用于抗原展示，疫苗开发和增强多糖降解过程。

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引用次数: 0