Enzyme and Microbial Technology最新文献_第5页

Study on bio-imprinted Aspergillus niger lipase cross-linked aggregates and catalytic synthesis of Vitamin E succinate 生物印迹黑曲霉脂肪酶交联聚集体及其催化合成维生素E琥珀酸酯的研究。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-23 DOI: 10.1016/j.enzmictec.2025.110766

Junqing Qian, Zhengze Yang, Aomei Huang, Qian Li, Hui Guo

To enhance the esterification and stability of lipase in organic solvents, the bio-imprinted Aspergillus niger lipase combined with cross-linked aggregate immobilization was investigated. The bio-imprinted lipase cross-linked aggregates were applied to the catalytic esterification for the synthesis of Vitamin E succinate in N, N-dimethylformamide (DMF) solution. Lauric acid, serving as a succinic acid analogue, was selected as the bio-imprinting molecule, 0.10 g lauric acid was added to 36 mL of 2.10 mg/mL lipase solution, imprinting 40 mins at pH 8.0, the immobilization yield achieved 91.5 % with cross-linked aggregates by glutaraldehyde. The catalytic activity of the bio-imprinted lipase cross-linked aggregates was significantly enhanced, achieving an esterification yield of 87.4 ± 0.43 % for Vitamin E succinate. Moreover, the bio-imprinted lipase cross-linked aggregates maintained their catalytic activity over five consecutive reaction cycles in DMF. Fluorescence spectroscopy analysis revealed that bio-imprinting promoted the exposure of the lipase active sites, which corresponded with the observed increase in esterification activity. In addition, the mechanism of the substrate analogue-imprinted lipase was characterized. This study provides a theoretical foundation for improving the catalytic esterification performance of lipase as well as a process basis for the enzymatic synthesis of Vitamin E succinate.

为了提高脂肪酶在有机溶剂中的酯化和稳定性，研究了生物印迹黑曲霉脂肪酶与交联聚集体固定化的结合。应用生物印迹脂肪酶交联聚集体在N， N-二甲基甲酰胺（DMF）溶液中催化酯化合成维生素E琥珀酸酯。选择琥珀酸类似物月桂酸作为生物印迹分子，将0.10 月桂酸加入到36 mL 2.10 mg/mL脂肪酶溶液中，在pH 8.0条件下印迹40 min，戊二醛交联聚体的固定化率达到91.5 %。生物印迹脂肪酶交联聚集体的催化活性显著增强，维生素E琥珀酸酯的酯化率为87.4 ± 0.43 %。此外，生物印迹脂肪酶交联聚集体在DMF中连续五个反应循环中保持其催化活性。荧光光谱分析表明，生物印迹促进了脂肪酶活性位点的暴露，这与观察到的酯化活性增加相对应。此外，还对底物类似物印迹脂肪酶的作用机理进行了表征。本研究为提高脂肪酶的催化酯化性能提供了理论基础，也为酶促合成维生素E琥珀酸酯提供了工艺基础。

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引用次数: 0

Microbial-catalyzed biotransformation of Soyasapogenol B by Streptomyces griseus ATCC 13273, and Penicillium griseofulvum CICC 40293 微生物催化灰链霉菌ATCC 13273和灰青霉CICC 40293对大豆皂苷醇B的生物转化

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-17 DOI: 10.1016/j.enzmictec.2025.110765

Richa Raj , Pingping Shen , Xuewa Jiang , Jingling Zhang , Weihang Yao , Wei Wang , Jian Zhang

This study aims to diversify the structure of Soyasapogenol B (1) and generate novel metabolites through microbial-catalyzed biotransformation by two highly efficient microbial strains, Streptomyces griseus ATCC 13273 and Penicillium griseofulvum CICC 40293. Consequently, ten (2−11) unique bioactive metabolites were isolated. Their structures were determined using 1D/2D NMR and HR-ESI-MS data, revealing multiple tailoring reactions, including oxidation, C-C double bond rearrangement, hydroxylation, and dehydrogenation. This highlights the enzymatic ability of these strains to catalyze specific and diverse regioselective modifications on the Soyasapogenol B scaffold. Therefore, this study demonstrates that microbial-catalyzed biotransformation offers a promising approach to increase the chemical diversity of Soyasapogenol B (1), providing a sustainable alternative to chemical synthesis.

本研究旨在通过两种高效微生物菌株——灰霉菌链霉菌ATCC 13273和灰霉菌CICC 40293，使大豆apogenol B(1)的结构多样化，并产生新的代谢物。因此，分离出10（2−11）个独特的生物活性代谢物。利用1D/2D NMR和HR-ESI-MS数据确定了它们的结构，揭示了多种裁剪反应，包括氧化、C-C双键重排、羟基化和脱氢。这突出了这些菌株在大豆皂苷醇B支架上催化特异性和多样化区域选择性修饰的酶促能力。因此，本研究表明，微生物催化的生物转化为增加大豆皂苷醇B(1)的化学多样性提供了一种有希望的方法，为化学合成提供了一种可持续的替代方法。

引用次数: 0

Metabolic engineering of Bacillus subtilis for enhanced p-Coumaric acid production and antimicrobial applications 枯草芽孢杆菌代谢工程提高对香豆酸产量及其抗菌应用。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-13 DOI: 10.1016/j.enzmictec.2025.110762

Junjie Zhang , Guodong Zhang , Wenhu Zhu , Youzhi Li , Yutuo Wei , Xianwei Fan

p-Coumaric acid (p-CA) is widely utilized in the food, pharmaceutical and other industries, and has traditionally been derived from plant extraction or chemical synthesis. However, p-CA synthesized by the safe B. subtilis remains poorly explored. In this study, we first engineered a recombinant B. subtilis strain (PBK) capable of synthesizing p-Coumaric acid, achieving an initial yield of 3.81 mg L⁻¹. A high-yielding strain PBnprE was then developed through promoter substitution, with a yield reaching 60.92 mg L⁻¹, and the yield of PBnprE was further increased to 304.04 mg L⁻¹ by optimizing fermentation conditions and substrates, showing an 80-fold increase over PBK. The optimized fermentation extract of PBnprE displayed increased antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, alongside enhanced DPPH and ABTS scavenging capabilities. Compared to PBK, the optimized extracts showed 4.81-fold higher in DPPH and 3.47-fold in ABTS scavenging, consistent with improved antioxidant properties driven by the increased presence of p-CA. This study first successfully constructed a high-yield p-CA producing engineered strain in B. subtilis, providing a valuable platform for synthesizing other secondary metabolites.

对香豆酸（p-CA）广泛应用于食品、制药等行业，传统上是通过植物提取或化学合成得到的。然而，利用安全的枯草芽孢杆菌合成p-CA的研究还很少。在这项研究中，我们首先设计了一种重组枯草芽孢杆菌菌株（PBK），能够合成对香豆酸，初始产量为3.81 mg L-1。通过启动子置换培养出高产菌株PBnprE，其产量达到60.92 mg L-1，通过优化发酵条件和底物，PBnprE的产量进一步提高到304.04 mg L-1，比PBK提高了80倍。优化后的PBnprE发酵提取物对金黄色葡萄球菌、铜绿假单胞菌和大肠杆菌的抑菌活性增强，对DPPH和ABTS的清除能力增强。与PBK相比，优化后的提取物对DPPH的清除能力提高了4.81倍，对ABTS的清除能力提高了3.47倍，这与p-CA含量的增加对抗氧化性能的提高是一致的。本研究首次在枯草芽孢杆菌中成功构建了高产p-CA工程菌株，为其他次生代谢产物的合成提供了有价值的平台。

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引用次数: 0

Expression and biochemical characterization of wild-type and mutant β-amylases from Priestia flexa in Komagataella phaffii 野生型和突变型柔曲Priestia β-淀粉酶在法菲Komagataella phaffii中的表达及生化特性研究。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-11 DOI: 10.1016/j.enzmictec.2025.110761

Tatiana Leonidovna Gordeeva, Artur Aleksandrovich Tkachenko, Larisa Nikolaevna Borshchevskaya, Natalia Vladimirovna Bulushova, Egor Sergeevich Bobrov, Aleksandr Sergeevich Fedorov, Ekaterina Pavlovna Sakharova, Olga Evgenevna Melkina, Sergey Pavlovich Sineoky

The β-amylases AmyPf1 and AmyPf2 from two distinct Priestia flexa strains were successfully expressed for the first time in Komagataella phaffii. The main biochemical characteristics of the recombinant enzymes were determined. β-Amylases exhibited similar optimal temperature (55 °C) and pH (pH 8.0), as well as high stability at 55 °C and across a broad pH range (4.0–10.0). The thermal inactivation half-lives at 55 °C for both recombinant enzymes were approximately 4 h. The AmyPf2 demonstrated superior specific activity (3836 U•mg⁻¹) and catalytic constant (6367 s⁻¹) compared to AmyPf1 (3411 U•mg⁻¹ and 4224 s⁻¹, respectively). Comparison of the amino acid sequences of the two β-amylases revealed four residue differences. Site-directed mutagenesis introducing AmyPf2-specific substitutions into the AmyPf1 sequence showed that the V180A mutation significantly enhanced specific activity (4009 U•mg⁻¹) and catalytic constant (6656 s⁻¹) of the resulting r-V180A variant. In fed-batch fermentation, the activity of r-V180A reached 5130 U•mL⁻¹. Hydrolysis of corn starch using r-V180A in combination with pullulanase (a starch-debranching enzyme) yielded 87.56 % maltose, indicating that a hydrolysate with high maltose content was obtained. These results highlight the potential of the recombinant r-V180A enzyme for industrial maltose production and underscore the utility of the K. phaffii expression system for efficient production of P. flexa β-amylases.

本文首次成功地表达了两个不同菌株的β-淀粉酶AmyPf1和AmyPf2。测定了重组酶的主要生化特性。β-淀粉酶表现出相似的最佳温度（55°C）和pH (pH 8.0)，在55°C和较宽的pH范围（4.0-10.0）具有较高的稳定性。两种重组酶在55℃下的热失活半衰期约为4 h。与AmyPf1（分别为3411 U•mg-1和4224 s-1）相比，AmyPf2具有更高的比活性（3836 U•mg-1）和催化常数（6367 s-1）。比较两种β-淀粉酶的氨基酸序列，发现4个残基差异。在AmyPf1序列中引入amypf2特异性替换的定点突变表明，V180A突变显著提高了r-V180A变体的比活性（4009 U•mg-1）和催化常数（6656 s-1）。在分批补料发酵中，r-V180A活性达到5130 U•mL-1。用r-V180A与pullulanase（一种淀粉脱支酶）联合水解玉米淀粉，麦芽糖得率为87.56 %，表明得到了麦芽糖含量高的水解产物。这些结果突出了重组r-V180A酶用于工业麦芽糖生产的潜力，并强调了K. phaffii表达系统在高效生产柔性假单抗β-淀粉酶方面的实用性。

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引用次数: 0

An H1N1 virus biosensor based on enzyme activity-gated PER-CRISPR/Cas12a cascade signal amplification 基于酶活性门控PER-CRISPR/Cas12a级联信号扩增的H1N1病毒生物传感器

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-09 DOI: 10.1016/j.enzmictec.2025.110759

Meiyan Xin , Jitong Liu , Hongyu Zhou , Shengjun Bu , Zhuo Hao , He Sun , Jie Lu , Xiangru Feng , Xue Jiang , Qingshuang Wang , Jiayu Wan

The rapid and accurate detection of the H1N1 influenza virus is a key link in epidemic prevention and control. This study innovatively constructed a cascade signal amplification biosensor based on DNA polymerase activity regulation, aiming to achieve ultra-sensitive and highly specific detection of viral nucleic acids. This biosensor has the following significant advantages: (i) Molecular lock-key regulation mechanism: A functional DNA inhibitor is designed to form a complex with Taq DNA polymerase, and the target H1N1 RNA is specifically recognized to release enzyme activity inhibition, converting the target presence information into a PER reaction initiation signal. (ii) Cascade signal amplification system: The single-stranded DNA generated by PER activates Cas12a trans-cleavage activity, achieving a three-level signal amplification of enzyme activity activation → nucleic acid synthesis → CRISPR cleavage. The biosensor exhibits a linear detection range between 1 pM and 1 μM, with a detection limit of 25 fM. Moreover, the platform showed high versatility and could be readily adapted for the detection of other pathogens such as SARS-CoV-2 by simply modifying the nucleic acid sequences of the inhibitor and activator. This study not only provides a new tool for the screening of H1N1 influenza virus, but also offers a novel strategy for the development of next-generation molecular detection technologies suitable for point-of-care diagnostics, indicating considerable application potential.

甲型H1N1流感病毒的快速、准确检测是疫情防控的关键环节。本研究创新性构建了基于DNA聚合酶活性调控的级联信号扩增生物传感器，旨在实现病毒核酸的超灵敏、高特异性检测。该生物传感器具有以下显著优势：(1)分子锁键调控机制：设计功能性DNA抑制剂，与Taq DNA聚合酶形成复合物，特异性识别靶标H1N1 RNA释放酶活性抑制，将靶标存在信息转化为PER反应起始信号。（ii）级联信号扩增系统：PER产生的单链DNA激活Cas12a反式切割活性，实现酶活性激活→核酸合成→CRISPR切割的三级信号扩增。该传感器的线性检测范围为1 pM ~ 1 μM，检测限为25 fM。此外，该平台具有较高的通用性，只需修改抑制剂和激活剂的核酸序列，就可以很容易地用于检测SARS-CoV-2等其他病原体。本研究不仅为H1N1流感病毒的筛查提供了新的工具，而且为开发适用于即时诊断的下一代分子检测技术提供了新的策略，具有相当大的应用潜力。

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引用次数: 0

Anti-biofilm efficiency and substrate specificity of recombinantly produced human paraoxonases 重组人对氧酶的抗生物膜效率和底物特异性。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-08 DOI: 10.1016/j.enzmictec.2025.110760

Manik Goel, Priyamedha Yadav, Fauzia Parween, Sudisha Mukherjee, Rinkoo Devi Gupta

The human paraoxonase (PON) gene family, comprising PON1, PON2, and PON3, has evolved through parallel evolutionary trajectories leading to distinct substrate selectivity, thereby enabling these enzymes to hydrolyze a range of substrates such as lactones, arylesters, and organophosphates. This study explores the evolutionary divergence of PON family members and their varied anti-biofilm activities through in silico molecular docking and in vitro analysis. The huPON1, huPON2, and huPON3 genes were cloned and expressed in the E. coli expression system. These overexpressed proteins formed inclusion bodies, which were further subjected to in vitro refolding and biochemical characterization. The enzymatic studies revealed that huPON1 has unique paraoxonase activity and high arylesterase activity, while huPON2 demonstrated the highest lactonase activity, followed by huPON3 and huPON1. A comparative assessment of the anti-biofilm potential of recombinant huPONs was conducted against Mycobacterium smegmatis, which revealed that all three paraoxonases inhibited biofilm formation; however, huPON2 displayed the highest anti-biofilm activity, followed by huPON3 and huPON1. Additionally, all the huPONs showed a synergistic effect with the conventional TB drug, Rifampicin, in enhancing biofilm disruption. These findings contribute valuable insights into the application of huPONs as a therapeutic tool against drug-resistant bacterial infections.

人类对氧磷酶（PON）基因家族，包括PON1、PON2和PON3，通过平行的进化轨迹进化，导致不同的底物选择性，从而使这些酶能够水解一系列底物，如内酯、芳基酯和有机磷酸盐。本研究通过硅分子对接和体外分析，探讨PON家族成员的进化分化及其不同的抗生物膜活性。克隆了huPON1、huPON2和huPON3基因，并在大肠杆菌表达系统中表达。这些过表达的蛋白形成包涵体，进一步进行体外重折叠和生化表征。酶学研究表明，huPON1具有独特的对氧磷酶活性和较高的芳烯酯酶活性，而huPON2具有最高的内酯酶活性，其次是huPON3和huPON1。对重组huPONs对耻垢分枝杆菌的抗生物膜潜能进行了比较评估，结果表明三种对氧酶均能抑制生物膜的形成；其中，huPON2的抗生物膜活性最高，其次是huPON3和huPON1。此外，所有的huPONs都显示出与传统结核病药物利福平的协同作用，以加强生物膜破坏。这些发现为huPONs作为抗耐药细菌感染的治疗工具的应用提供了有价值的见解。

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引用次数: 0

Enhancing isoprimeverose-producing enzyme in genetically engineered Aspergillus oryzae through impeller shape and pH control for the large-scale fermentation 通过叶轮形状和pH控制来提高转基因米曲霉大规模发酵产异戊糖酶的活性

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-05 DOI: 10.1016/j.enzmictec.2025.110758

Fahmi Baihaqqi , Satoshi Wakai , Filemon Jalu Nusantara Putra , Tomohiro Suzuki , Yoshiro Ikeya , Prihardi Kahar , Yutaro Mori , Akihiko Kondo , Chiaki Ogino

Aspergillus oryzae is a filamentous fungus that serves as a source of polysaccharide-degrading enzymes. We constructed two self-cloning forms of A. oryzae that specifically express isoprimeverose oligoxyloglucanase (IpeA) and endoglucanase. These enzymes are essential for the enzymatic release of the rare sugar isoprimeverose (α-D-xylopyranosyl-(1→6)-D-glucopyranose) from tamarind xyloglucan. Of the two, IpeA is a key enzyme that is currently unavailable commercially. The primary objective of this study was to enhance IpeA production through fermentation engineering. Various fermentation parameters were optimized. These include a source of carbon from either glucose or maltose, pH control, and impeller shapes using either Disk Turbine (DT) or MAXBLEND® (MB) impellers that were tested using a 5-L stirred-tank bioreactor. Cultivation in maltose-supplemented medium resulted in a reduction in carbon catabolite repression compared with that of glucose. Batch fermentation at acidic pH (4.5) using the MB impeller yielded a significantly higher level of biomass and enzyme production with a dry mycelial weight (DMW) yield of ∼19 g/L and IpeA activity of ∼68 U/mL, which compares favorably with ∼14 g/L of DMW and 43 U/mL of IpeA activity under identical pH conditions when using a DT impeller. The findings of this study offer valuable insight for both large-scale and biological production of A. oryzae.

米曲霉是一种丝状真菌，可作为多糖降解酶的来源。构建了特异表达异戊糖寡氧葡聚糖酶（IpeA）和内切葡聚糖酶（endoglucanase）的两种自克隆稻芽孢杆菌（a.m oryzae）。这些酶是罗望子木葡聚糖酶解稀有糖异丙糖（α-D-xylopyranosyl-(1→6)- d -glucopyranose）所必需的。在这两种酶中，IpeA是一种目前无法商业化的关键酶。本研究的主要目的是通过发酵工程提高IpeA的产量。对各种发酵参数进行了优化。这些包括来自葡萄糖或麦麸的碳源，pH控制和叶轮形状，使用圆盘涡轮（DT）或MAXBLEND®（MB）叶轮，使用5升搅拌槽生物反应器进行测试。与葡萄糖相比，在麦芽糖补充培养基中培养导致碳分解代谢抑制减少。在酸性pH（4.5）条件下，使用MB叶轮的批量发酵产生了显著更高的生物量和酶产量，干菌丝重（DMW）产量为~ 19 g/L， IpeA活性为~ 68 U/mL，与相同pH条件下使用DT叶轮的DMW产量为~ 14 g/L， IpeA活性为43 U/mL相比具有优势。本研究结果为米芽孢杆菌的大规模生产和生物生产提供了有价值的见解。

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引用次数: 0

Mechanism of tyrosine 69 in the flavin domain modulating electron transfer efficiency in BVU5 azoreductase 黄素区域酪氨酸69调节BVU5偶氮还原酶电子转移效率的机制

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-09-25 DOI: 10.1016/j.enzmictec.2025.110757

Xuehui Xie , Dongyang Li , Hangmi Zheng , Ziyi Wu , Jiajie Liu , Wanting Li , Songwei Chen , Yiwen Zhang , Daijie Tang , Na Liu , Qingyun Zhang , Ye Chen

The azoreductase BVU5 is a flavoprotein dependent on NAD(P)H/FMN-mediated electron transfer; However, the functional mechanism of key residues within its flavin domain remains unclear. This study identified the conserved residue Tyr⁶⁹ through bioinformatics analysis and constructed Y69F and Y69C mutants. Enzymatic assays demonstrated that mutants exhibited significantly lower decolorization rates than the BVU5 enzyme across 12 dye molecules, including azo, anthraquinone, and triphenylmethane dyes. For example, when decolorizing Reactive Black 5 (RB5) for 2 h: Y69F mutant achieved 60 %-65 % decolorization, Y69C mutant reached 55 %-60 % decolorization, both markedly lower than the 80 %-85 % efficiency of BVU5.The decolorization hierarchy remained azo dyes > triphenylmethane > anthraquinone dyes. Molecular docking revealed that mutations reconfigured FMN-binding patterns. Although Y69F enlarged the substrate-binding pocket, it failed to enhance the degradation efficiency of the bulky dye Chlorazol Black E. This critical contradiction indicates that substrate binding is not the limiting factor. Combined with evidence such as the lighter color of the mutant enzyme solutions, increased A280/A450 ratios, and enzyme dosage experiments, this study confirms that Tyr⁶⁹ is a key residue that sustains electron transfer efficiency by maintaining the FMN-binding conformation, thereby determining the decolorization performance. Consequently, electron transfer efficiency, rather than substrate binding, is the primary mechanism influencing the catalytic function of BVU5.

偶氮还原酶BVU5是一种依赖于NAD(P)H/ fmn介导的电子转移的黄蛋白；然而，其黄素结构域关键残基的功能机制尚不清楚。本研究通过生物信息学分析鉴定了保守残基Tyr69，构建了Y69F和Y69C突变体。酶分析表明，突变体在12种染料分子上的脱色率明显低于BVU5酶，包括偶氮、蒽醌和三苯甲烷染料。例如，当2 h: Y69F突变体对活性黑5 （RB5）脱色时，脱色率为60 %-65 %，Y69C突变体脱色率为55 %-60 %，均明显低于BVU5的80 %-85 %。脱色等级仍为偶氮染料>； 三苯甲烷>； 蒽醌染料。分子对接显示突变重新配置了fmn结合模式。虽然Y69F扩大了底物结合袋，但未能提高对体积较大的染料氯唑黑e的降解效率，这一关键矛盾表明底物结合并不是限制因素。结合突变酶溶液颜色变浅、A280/A450比值增加、酶用量实验等证据，本研究证实Tyr69是通过维持fnm结合构象来维持电子转移效率的关键残基，从而决定了脱色性能。因此，电子转移效率，而不是底物结合，是影响BVU5催化功能的主要机制。

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引用次数: 0

Identification of a thermostable GH6 family cellulase from chaetomium thermophilum exhibiting high cellobiose and ionic liquid tolerance 嗜热毛菌GH6家族耐热纤维素酶的鉴定，具有高纤维二糖和离子液体耐受性

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-09-23 DOI: 10.1016/j.enzmictec.2025.110755

Pooja , Sushant K. Sinha , Supratim Datta

Efficient conversion of lignocellulosic biomass into fermentable sugars requires cellulases that are both thermostable and tolerant to inhibitors such as ionic liquids (ILs). Thermostable enzymes are particularly valuable for industrial applications, as they maintain activity at elevated temperatures for extended periods, improve product yield, and reduce process costs. In this study, we cloned and characterized CtCel6C, a GH6 family cellulase, from the thermophilic fungus Thermochaetoides thermophila (Chaetomium thermophilum). CtCel6C exhibited a specific activity of 28 U/mg on carboxymethyl cellulose (CMC-Na) at 55 °C and pH 6.0, retaining 90 % of its specific activity after 20 h at optimum pH and temperature (55 °C and pH 6.0). CtCel6C demonstrated broad substrate specificity, effectively hydrolyzing oligosaccharides, soluble substrates (CMC and barley β-glucan), and insoluble substrates such as Avicel, consistently producing cellobiose as the sole product and outperforming other GH6 cellobiohydrolases from C. thermophilum. Despite having an open active-site cleft due to a lack of a fifteen-residue stretch in the C-terminal loop, CtCel6C retains the biochemical characteristics of a processive cellobiohydrolase rather than those of an endoglucanase from the GH6 family. The enzyme also exhibited high tolerance to cellobiose, glucose, and to 20 % (v/v) of the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C₂mim][OAc]), in both McIlvaine buffer (pH 6.0) and seawater. The remarkable IL tolerance of CtCel6C makes it a promising candidate for integration into industrial enzyme cocktails for biomass saccharification.

将木质纤维素生物质有效转化为可发酵糖需要既耐热又耐离子液体（ILs）等抑制剂的纤维素酶。耐热酶在工业应用中特别有价值，因为它们在高温下长时间保持活性，提高产品产量，降低工艺成本。本研究从嗜热真菌Thermochaetoides thermophila （Chaetomium thermophilum）中克隆并鉴定了GH6家族纤维素酶CtCel6C。在55℃和pH 6.0条件下，CtCel6C对羧甲基纤维素（CMC-Na）的比活性为28 U/mg，在最佳pH和温度（55℃和pH 6.0）下，20 h后，CtCel6C的比活性仍保持90% %。CtCel6C表现出广泛的底物特异性，可以有效水解低聚糖、可溶性底物（CMC和大麦β-葡聚糖）和不溶性底物（如Avicel），始终如一地产生纤维素二糖作为唯一产物，优于其他来自嗜热c菌的GH6纤维素生物水解酶。尽管由于在c端环中缺乏15个残基延伸而具有开放的活性位点裂缝，但CtCel6C保留了过程纤维素生物水解酶的生化特征，而不是来自GH6家族的内切葡聚糖酶的生化特征。在McIlvaine缓冲液（pH 6.0）和海水中，该酶对纤维素二糖、葡萄糖和20% % （v/v）的离子液体1-乙基-3-甲基咪唑醋酸盐（[C2mim][OAc]）均表现出较高的耐受性。CtCel6C卓越的IL耐受性使其成为整合到生物质糖化工业酶混合物中的有希望的候选者。

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引用次数: 0

Preparation of magnetic xylanase cross-linked aggregates (m-CLXAs) for the hydrolysis of arabinoxylan by mechano-enzymology 机械酶水解阿拉伯木聚糖的磁性木聚糖酶交联聚集体（m-CLXAs）制备。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-09-23 DOI: 10.1016/j.enzmictec.2025.110756

Justin B. Safari , Lebogang Ramatsui , Liam Nisbet , Vincent J. Smith , Rui W.M. Krause , Brett I. Pletschke

The development of a biocatalyst consisting of immobilised xylanases as cross-linked enzyme aggregates (CLEAs) on magnetic nanoparticles (MNPs) as support was undertaken. MNPs were prepared by a coprecipitation reaction of Fe^3 + and Fe²⁺, followed by surface modification with (3-aminopropyl)-trimethoxysilane using a polycondensation reaction. Magnetic CLEAs were prepared via glutaraldehyde cross-linking after precipitation with acetone, and the physicochemical characterisation of the immobilised enzyme was performed at all stages using several techniques (FTIR, PXRD, TGA, and DLS). A one-factor-at-a-time approach (OFAT) was used to investigate the impact of temperature, time, enzyme: MNP ratio, precipitant, and cross-linking agent to determine their effect on the enzyme’s recovered activity. The results demonstrated that all parameters impacted the immobilisation differently, with the optimised conditions determined as 4°C, 12 h, 10 mg/g, 60 % (v/v) acetone, and 200 mM glutaraldehyde, respectively. The immobilisation did not affect the pH and temperature optima of the enzyme, which were 6.0 and 50–70°C, respectively, for both the free and immobilised enzymes. In contrast, the immobilised enzyme could be reused more than ten times to hydrolyse wheat arabinoxylan, without losing 50 % of its initial activity. Values for V_max and K_m only decreased slightly compared to those obtained for the free enzyme. The thermal inactivation parameters showed that the immobilisation procedure did not adversely affect the enzyme's catalytic properties after immobilisation. Finally, we assessed the immobilised enzyme for its ability to catalyse reactions under mechanochemical conditions (grinding and ageing) and found that the free and immobilised enzymes were active during solvent-free and liquid-assisted grinding (LAG).

开发了一种由固定化木聚糖酶作为交联酶聚集体（CLEAs）在磁性纳米颗粒（MNPs）上作为支撑的生物催化剂。采用Fe3 +和Fe2+共沉淀法制备MNPs，再用（3-氨基丙基）-三甲氧基硅烷进行缩聚改性。用丙酮沉淀后，通过戊二醛交联制备了磁性clea，并使用FTIR、PXRD、TGA和DLS等技术对固定化酶进行了各阶段的理化表征。采用单因素一次法（OFAT）研究温度、时间、酶：MNP比、沉淀剂和交联剂对酶恢复活性的影响。结果表明，各参数对固定化效果的影响不同，最佳条件分别为4℃、12 h、10 mg/g、60 % （v/v）丙酮和200 mM戊二醛。固定不影响酶的最佳pH值和温度，游离酶和固定酶的最佳温度分别为6.0°C和50-70°C。相比之下，固定化酶可以重复使用10次以上来水解小麦阿拉伯木聚糖，而不会失去其初始活性的50% %。与游离酶相比，Vmax和Km值仅略有下降。热失活参数表明，固定化过程对固定化后酶的催化性能没有不利影响。最后，我们评估了固定化酶在机械化学条件下（研磨和老化）催化反应的能力，发现游离酶和固定化酶在无溶剂和液体辅助研磨（LAG）期间具有活性。

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