Enzyme and Microbial Technology最新文献_第6页

Spectrophotometric assay for the screening of selective enzymes towards DHA and EPA ethyl esters hydrolysis 用于筛选 DHA 和 EPA 乙酯水解选择性酶的分光光度测定法

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-28 DOI: 10.1016/j.enzmictec.2024.110531

Hiram Y. Guerrero-Elias , M. Angeles Camacho-Ruiz , Ruben Espinosa-Salgado , Juan Carlos Mateos-Díaz , Rosa María Camacho-Ruiz , Ali Asaff-Torres , Jorge A. Rodríguez

Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), hold notable significance due to their pharmaceutical relevance. Obtaining PUFAs from diverse sources like vegetables, fish oils, and algae poses challenges due to the mixed fatty acid (FA) composition. Therefore, focusing on particular FAs necessitates purification and resolution processes. To address this, we propose a continuous assay for screening lipases selective for ethyl EPA (E-EPA) or ethyl DHA (E-DHA). Utilizing microplate spectrophotometry, the method enables quantification of liberated fatty acids from ethyl esters (E-EPA or E-DHA). This involves assessing enzyme selectivity by measuring the release of FAs through p-nitrophenolate protonation, either separately for each substrate or in competition with a reference substrate, resorufin acetate. Ten lipases underwent screening, revealing Burkholderia cepacia lipase's (BCL) preference for ethyl DHA hydrolysis (E-EPA/E-DHA = 0.82 ± 0.07 and the lipase selectivity ratio (S) for E-EPA/E-DHA = 0.13 ± 0.04) and Candida antarctica lipase B's (CALB) high specific activity towards both E-EPA and E-DHA (531.14 ± 37.76 and 281.79 ± 2.79 U/mg, respectively) and E-EPA preference (E-EPA/E-DHA = 1.86 ± 0.15 and S E-EPA/E-DHA = 2.59±0.15). Candida rugosa recombinant isoform 4 (rCRLip4) and commercial Candida rugosa lipase (CRL) exhibited significant preference for E-EPA hydrolysis (E-EPA/E-DHA = 2.18 ±0.51 and 2.26 ±0.36, respectively; and S E-EPA/E-DHA = 7.59 ± 1.59 and 7.88 ± 2.13, respectively). Docking analyses of rCRLip4, BCL, and CALB demonstrated no statistically significant differences in activation energies or distances to the catalytic serine; however, they agreed with the experimental results. These findings suggest potential mutagenesis or directed evolution strategies for CALB to enhance E-EPA selectivity, with rCRLip4 emerging as a promising candidate for further investigation. This assay offers a valuable tool for identifying lipases with desired substrate selectivity, with broad implications for pharmaceutical and biotechnological applications.

多不饱和脂肪酸（PUFA），如二十二碳六烯酸（DHA）和二十碳五烯酸（EPA），因其与药物的相关性而具有显著的意义。从蔬菜、鱼油和藻类等不同来源获取 PUFAs 会因混合脂肪酸 (FA) 的组成而面临挑战。因此，要专注于特定的脂肪酸，就必须进行纯化和解析过程。为了解决这个问题，我们提出了一种连续测定法，用于筛选对乙基 EPA（E-EPA）或乙基 DHA（E-DHA）具有选择性的脂肪酶。利用微孔板分光光度法，该方法可对从乙酯（E-EPA 或 E-DHA）中释放出的脂肪酸进行定量。这涉及到通过测量对硝基苯酚质子化释放脂肪酸的情况来评估酶的选择性，既可以针对每种底物分别进行，也可以与参考底物醋酸间苯二酚竞争。十种脂肪酶进行了筛选，发现伯克霍尔德氏菌脂肪酶（BCL）偏好水解 DHA 乙酯（E-EPA/E-DHA = 0.82 ± 0.07，E-EPA/E-DHA 的脂肪酶选择性比（S）= 0.13 ± 0.04），而白色念珠菌脂肪酶（BCL）偏好水解 DHA 乙酯（E-EPA/E-DHA = 0.82 ± 0.07，E-EPA/E-DHA 的脂肪酶选择性比（S）= 0.13 ± 0.04）。E-EPA/E-DHA=0.82±0.07，E-EPA/E-DHA 的脂肪酶选择性比率（S）=0.13±0.04），而白色念珠菌脂肪酶 B（CALB）对 E-EPA 和 E-DHA 均具有较高的特异性活性（分别为 531.14±37.76 和 281.79±2.79 U/mg ）和 E-EPA 偏好（E-EPA/E-DHA=1.86±0.15，S E-EPA/E-DHA =2.59±0.15）。皱褶念珠菌重组异构体 4（rCRLip4）和商用皱褶念珠菌脂肪酶（CRL）表现出明显的 E-EPA 水解偏好（E-EPA/E-DHA = 2.18 ±0.51 和 2.26 ±0.36；S E-EPA/E-DHA = 7.59 ± 1.59 和 7.88 ± 2.13）。rCRLip4、BCL 和 CALB 的对接分析表明，它们的活化能或与催化丝氨酸的距离在统计学上没有显著差异；不过，它们与实验结果一致。这些发现表明，CALB 有可能采用诱变或定向进化策略来提高 E-EPA 的选择性，而 rCRLip4 则有望成为进一步研究的候选对象。这种检测方法为鉴定具有所需底物选择性的脂肪酶提供了一种有价值的工具，对制药和生物技术应用具有广泛的意义。

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引用次数: 0

New insight into acid-resistant enzymes from natural mutations of Escherichia coli Nissle 1917 从大肠杆菌 Nissle 1917 的天然突变中了解耐酸酶的新发现。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-20 DOI: 10.1016/j.enzmictec.2024.110526

Chengfeng Xue, Wan-Wen Ting, Jiun-Jang Juo, I-Son Ng

The probiotic Escherichia coli Nissle 1917 (EcN), known for its superior acid resistance (AR), serves as a promising chassis for live therapeutics due to the effective colonization capabilities. However, the enzymatic activity regarding AR in EcN remains poorly understood. First, we investigated the AR systems of EcN by measuring cell growth under acidic stress and exploring the relationship of mutations to their corresponding enzymatic activities. As a result, the catalytic activity of inducible decarboxylases of GadB, AdiA and CadA, responsible for metabolizing glutamate, arginine, and lysine, exhibited an average 2-fold increase in EcN compared to the reference strain MG1655. Furthermore, we discovered that the glutamate-dependent AR2 system in EcN was meticulously regulated by specific regulons such as GadW. This study not only revealed the physiology of EcN under acidic conditions, but also highlighted that the mutated core enzymes in the AR system of EcN exhibit improved activities.

益生菌大肠埃希氏菌 Nissle 1917（EcN）以其卓越的耐酸性（AR）而闻名，由于其有效的定植能力，它是一种很有前景的活体疗法底盘。然而，人们对 EcN 中 AR 的酶活性仍然知之甚少。首先，我们通过测量酸性胁迫下的细胞生长来研究 EcN 的 AR 系统，并探索突变与其相应酶活性的关系。结果发现，与参考菌株 MG1655 相比，EcN 中负责谷氨酸、精氨酸和赖氨酸代谢的诱导性脱羧酶 GadB、AdiA 和 CadA 的催化活性平均增加了 2 倍。此外，我们还发现 EcN 中依赖谷氨酸的 AR2 系统受到 GadW 等特定调控子的严格调控。这项研究不仅揭示了 EcN 在酸性条件下的生理机理，还突出显示了 EcN AR 系统中突变的核心酶表现出更高的活性。

引用次数: 0

On-site cellulase production by Trichoderma reesei RutC-30 to enhance the enzymatic saccharification of ball-milled corn stover 利用毛霉 RutC-30 现场生产纤维素酶，提高球磨玉米秸秆的酶糖化效果。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-20 DOI: 10.1016/j.enzmictec.2024.110530

Yinghui He , Hui Zhang , Yeledana Huwati , Na Shu , Wei Hu , Xiwen Jia , Kaili Ding , Xueyan Liang , Luoyang Liu , Lujia Han , Weihua Xiao

Cellulases are essential for the enzymatic saccharification of lignocellulose. They play a crucial role in breaking down the structure of lignocellulose to obtain fermentable sugars. In this study, we conducted on-site cellulase production by Trichoderma reesei RutC-30 through submerged fermentation. The effects of carbon source, nitrogen source, KH₂PO₄, and mineral elements on cellulase production were evaluated using the hydrolyzed total sugar concentration of ball-milled corn stover as an indicator. The optimal fermentation medium conditions for cellulase production were determined through orthogonal experimental design analysis. Additionally, by optimizing culture conditions, including inoculation, pH, and bottling volume, we achieved a total sugar concentration of 92.25 g/L. After the optimization, the FPA, CMCA, protein, and total sugar concentration increased by 75.49 %, 18.43 %, 89.71 %, and 17.83 %, respectively. Furthermore, corn stover pretreated by different methods was applied to induce cellulase production. Ball-milled and steam-exploded corn stover was identified as suitable incubation carbon sources with total sugar concentration up to 94.31 g/L. Our work exploits the cellulase induced by lignocellulose and then applies it to lignocellulose, enabling the customization and providing a reference for the production of cellulase with corn stover as an inducer.

纤维素酶对木质纤维素的酶促糖化至关重要。它们在分解木质纤维素结构以获得可发酵糖类方面发挥着至关重要的作用。在本研究中，我们通过浸没发酵法利用毛霉 RutC-30 现场生产纤维素酶。以球磨玉米秸秆的水解总糖浓度为指标，评估了碳源、氮源、KH2PO4 和矿物质元素对纤维素酶生产的影响。通过正交实验设计分析，确定了纤维素酶生产的最佳发酵培养基条件。此外，通过优化接种、pH 值和装瓶量等培养条件，我们获得了 92.25 克/升的总糖浓度。优化后，FPA、CMCA、蛋白质和总糖浓度分别提高了 75.49 %、18.43 %、89.71 % 和 17.83 %。此外，玉米秸秆经不同方法预处理后可诱导纤维素酶的产生。球磨玉米秸秆和蒸汽爆破玉米秸秆被确定为合适的培养碳源，总糖浓度高达 94.31 克/升。我们的工作利用了木质纤维素诱导的纤维素酶，然后将其应用于木质纤维素，实现了定制化，为以玉米秸秆为诱导剂生产纤维素酶提供了参考。

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引用次数: 0

Production of bio-indigo from engineered Pseudomonas putida KT2440 harboring tryptophanase and flavin-containing monooxygenase 利用携带色氨酸酶和含黄素单氧化酶的工程假单胞菌 KT2440 生产生物靛蓝。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-18 DOI: 10.1016/j.enzmictec.2024.110529

Hyun Jin Kim , Suwon Kim , Yeda Lee , Yuni Shin , Suhye Choi , Jinok Oh , Jaeho Jeong , HyunA Park , Jungoh Ahn , Jeong Chan Joo , Kwon-Young Choi , Shashi Kant Bhatia , Yung-Hun Yang

Indigo is a unique blue dye that has been used in the textile industry for centuries and is currently mass-produced commercially through chemical synthesis. However, the use of toxic substrates and reducing agents for chemical synthesis is associated with environmental concerns, necessitating the development of eco-friendly alternatives based on microbial production. In this study, a robust industrial strategy for indigo production was developed using Pseudomonas putida KT2440 as the host strain, which is characterized by its excellent ability to degrade aromatic compounds and high resistance to environmental stress. By introducing the genes tryptophanase (tnaA) and Flavin-containing monooxygenase (FMO), a P. putida HI201 strain was constructed to produce indigo from tryptophan. To enhance the indigo yield, culture conditions, including medium composition, temperature, tryptophan concentration, and shaking speed, were optimized. Under optimal conditions such as TB medium, 15 mM tryptophan, 30°C, 200 rpm, P. putida HI201 biosynthesized 1.31 g/L indigo from tryptophan in a fed-batch fermentation system. The introduction of tnaA and FMO genes also enabled the production of indigo in various P. putida species, and the indigo-producing strain had a blue color, which served as a visual indicator. This study presents a strategy for using P. putida as a host for robust and sustainable microbial production of indigo, highlighting the strain's applicability and efficiency in environment friendly dye synthesis.

靛蓝是一种独特的蓝色染料，几个世纪以来一直用于纺织业，目前通过化学合成进行大规模商业化生产。然而，使用有毒底物和还原剂进行化学合成会引起环境问题，因此有必要开发基于微生物生产的生态友好型替代品。该菌株具有降解芳香族化合物能力强、抗环境胁迫能力强等特点。通过引入色氨酸酶（tnaA）和含黄素单氧化酶（FMO）基因，构建了一株利用色氨酸生产靛蓝的假单胞菌 HI201。为提高靛蓝产量，对培养基成分、温度、色氨酸浓度和振荡速度等培养条件进行了优化。在 TB 培养基、15 mM 色氨酸、30°C、200 rpm 等最佳条件下，P. putida HI201 在饲料批量发酵系统中从色氨酸中生物合成了 1.31 g/L 的靛蓝。引入 tnaA 和 FMO 基因也能在不同种类的 P. putida 中产生靛蓝，而且产生靛蓝的菌株呈蓝色，可作为视觉指标。本研究提出了一种以 P. putida 为宿主进行稳健、可持续的靛蓝微生物生产的策略，凸显了该菌株在环境友好型染料合成中的适用性和高效性。

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引用次数: 0

Targeted metagenomics – Enrichment for enzymes active on sulfated polysaccharides from seaweeds 靶向元基因组学 - 从海藻中富集硫酸化多糖活性酶

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-16 DOI: 10.1016/j.enzmictec.2024.110528

Bjorn Thor Adalsteinsson , Hörður Guðmundsson , Andrius Jasilionis , Morten Schiøtt , Maria Dalgaard Mikkelsen , Elísabet Eik Guðmundsdóttir , Pavithra Sivakumar , Annika Malmgren , Tushar Kaushik , Erik Apelqvist , Signe Vangsgaard , Rébecca Leblay , Ólafur Friðjónsson , Anne S. Meyer , Eva Nordberg Karlsson , Guðmundur Óli Hreggviðsson

Seaweeds (macroalgae) are an attractive resource for diverse microbial- and enzymatic production processes. They are abundant, underutilized, cheap, and rich in carbohydrates, and therefore have the potential to be used as a source of mono- or oligosaccharides, and as substrates for industrial fermentation processes. Many seaweed polysaccharides, including the sulfated polysaccharides ulvan and fucoidan, are however complex and heterogenous in structure, and there are currently few enzymes available to modify them, and understanding of their enzymatic depolymerization remains limited. The present study aimed to identify and characterize robust fucoidanases and ulvan lyases. Metagenomes were obtained from microbial enrichments from an intertidal hot-spring, genes identified that expressed putative fucoidanases and ulvan lyases, and following gene cloning and expression, the respective enzymes were screened for enzymatic activity. Consistent with their origin, the identified protein sequences were considerably divergent from previously characterized enzymes, with a 44 % average maximal sequence identity. In total, the study resulted in the characterization of 10 new fucoidanases (GH107 and GH168 families) and 8 new ulvan lyases (PL24, PL25 and PL40 families). Notably, the new fucoidanases appeared to have functional specificity towards fucoidan containing α-1,3 linked L-fucosyl and several functioned at high temperature. The study contributes a metagenomics-based approach to identify new seaweed polysaccharide degrading enzymes and an increased understanding of the diversity of such enzymes, which may have implications for the realization of biotechnology based valorization of seaweed biomass.

海藻（大型藻类）是一种具有吸引力的资源，可用于多种微生物和酶生产工艺。海藻资源丰富、利用率低、价格便宜且富含碳水化合物，因此有可能被用作单糖或寡糖的来源，以及工业发酵过程的底物。然而，许多海藻多糖（包括硫酸化多糖乌饭子和褐藻糖胶）结构复杂，种类繁多，目前可用来修饰它们的酶很少，人们对其酶解聚合作用的了解仍然有限。本研究旨在鉴定和描述强健的褐藻糖胶酶和乌尔凡酶。研究人员从潮间带温泉的微生物富集物中获得了元基因组，确定了表达假定褐藻糖胶酶和乌尔凡裂解酶的基因，并在基因克隆和表达之后，对相应的酶进行了酶活性筛选。与它们的起源相一致的是，鉴定出的蛋白质序列与以前鉴定出的酶有很大差异，平均最大序列同一性为 44%。这项研究总共鉴定出了 10 种新的褐藻糖胶酶（GH107 和 GH168 家族）和 8 种新的溃疡酶（PL24、PL25 和 PL40 家族）。值得注意的是，新的褐藻糖胶酶似乎对含有α-1,3-L-岩藻糖基的褐藻糖胶具有功能特异性，其中几种还能在高温下发挥作用。这项研究提供了一种基于元基因组学的方法来鉴定新的海藻多糖降解酶，并增加了对此类酶多样性的了解，这可能对实现基于生物技术的海藻生物质增值具有重要意义。

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引用次数: 0

Metabolic engineering of the borneol and camphor degradation pathways in Pseudomonas to produce optically pure bicyclic monoterpenes 假单胞菌降解龙脑和樟脑途径的代谢工程，以生产光学纯双环单萜烯。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-16 DOI: 10.1016/j.enzmictec.2024.110527

Yi-An Hong , Aye Aye Khine , Yu-Wei Lin , Pei-Yun Lee , Wei-Xiang Hong , Anren Hu , Tzenge-Lien Shih , Hao-Ping Chen

Borneol, a medicinally important bicyclic monoterpene, facilitates drug transport across mucous membranes and the blood-brain barrier. Derivatives of borneol and camphor also have numerous biomedical applications. Borneol is currently industrially synthesized via the conversion of turpentine and α-pinene. However, the major product is racemic isoborneol rather than racemic borneol. Both borneol and isoborneol are degraded by the soil bacterium Pseudomonas via a well-established degradation pathway. Two indigenous Pseudomonas strains were used to convert racemic isoborneol to other optically pure bicyclic monoterpenes here. Our results showed that deletion of the camE_2,5 gene alone from the strain TCU-HL1 genome led to the complete loss of borneol and camphor degradation ability. Knockout of both camE_2,5 and bdh1 (TCU-HL1Δbdh1ΔcamE_2,5) restored the degradation capability as the role of Bdh1 was replaced by that of Bdh2. This mutant converted racemic isoborneol into an optically pure bicyclic monoterpene, 2,5-diketocamphane, with a 45 % recovery yield. RT-qPCR results suggested that camE_2,5 expression plays a pivotal role in regulating the borneol/camphor degradation cluster. While (+)-borneol, (–)-borneol and (+)-camphor can be obtained from plants for mass production purposes, (–)-camphor cannot be obtained in the same manner. P. monteilii TCU-CK1 converted racemic isoborneol into (–)-camphor and 3,6-diketocamphane, with 15 % and 10 % recovery yields, respectively. In conclusion, we report the role of camE_2,5 in regulating the borneol/camphor degradation operon and biotransformation methods to produce several optically pure bicyclic monoterpenes.

莰烯醇是一种具有重要药用价值的双环单萜，可促进药物通过粘膜和血脑屏障的转运。莰烯醇和樟脑的衍生物也有许多生物医学应用。莰烯醇目前是通过松节油和 α-蒎烯的转化进行工业合成的。不过，主要产品是外消旋异龙脑，而不是外消旋莰烯。莰烯醇和异龙脑都能被土壤中的假单胞菌通过一种成熟的降解途径降解。两种本地假单胞菌株被用来将外消旋异龙脑转化为其他光学纯双环单萜烯。结果表明，仅从 TCU-HL1 菌株基因组中删除 camE2,5 基因就会导致其完全丧失降解龙脑和樟脑的能力。同时敲除 camE2,5 和 bdh1（TCU-HL1Δbdh1ΔcamE2,5）可恢复降解能力，因为 Bdh1 的作用被 Bdh2 所取代。该突变体将外消旋异龙脑转化为光学纯双环单萜烯，即 2,5-二酮樟脑，回收率为 45%。RT-qPCR 结果表明，camE2,5 的表达在调节龙脑醇/樟脑降解簇中起着关键作用。虽然(+)-冰片醇、(-)-冰片醇和(+)-樟脑可以从植物中获取，用于大规模生产，但(-)-樟脑却不能以同样的方式获取。P. monteilii TCU-CK1 能将外消旋异龙脑转化为(-)-樟脑和 3,6-二酮樟烷，回收率分别为 15 % 和 10 %。总之，我们报告了 camE2,5 在调节龙脑醇/樟脑降解操作子和生物转化方法以生产多种光学纯双环单萜烯中的作用。

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引用次数: 0

Enhanced soluble expression and characterization of human N-acetylglucosaminyltransferase IVa in Escherichia coli 在大肠杆菌中增强人 N-乙酰葡糖胺基转移酶 IVa 的可溶性表达并确定其特性。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-10 DOI: 10.1016/j.enzmictec.2024.110524

Sen-Lin Peng , Yi Ding , Meng-Hai Xiang , Ken Chen , Xiao-Dong Gao , Ning Wang

N-Glycosylation is one of the most important posttranslational modifications of proteins. Nearly the entire surface of cells and almost all secreted proteins in humans are modified with complex-type N-glycans, whose functions are affected by the number of N-glycan branches. N-Acetylglucosaminyltransferase-IVa (GnT-IVa) is a Golgi glycosyltransferase that transfers a GlcNAc to the α-1,3 mannose arm of the biantennary N-glycan GlcNAc2Man3GlcNAc2 to form a β-1,4 GlcNAc branched structure. The soluble expression of mammalian glycosyltransferases in heterologous hosts is often challenging. In the present study, human GnT-IVa (HsGnT-IVa) was cloned as an N-terminal truncated form that was fused with solubility-enhancing tags or signal peptides and overexpressed in Escherichia coli (E. coli). Our results showed that recombinant HsGnT-IVa could be overexpressed in its highest soluble and active form when the first 87 amino acids were removed and was fused with maltose-binding protein (MBP). By optimizing the induction conditions, the expression level of the recombinant protein was increased to yield approximately 540 mg per liter of culture after affinity purification. The purified enzyme exhibited appropriate glycosyltransferase activity, and the K_m value of the acceptor substrate was calculated as 1.1 mM. Characterization of the enzyme revealed that it reached its maximum activity with 5 mM Mn²⁺ at 37 °C in MES/NaOH (pH 7.0). In addition, the effects of key amino acids in the catalytic and lectin domains on enzyme activity were measured. This work offers an efficient approach for the large-scale production of bioactive HsGnT-IVa, which can be used for in vitro synthesis and functional studies of multiantennary complex-type N-glycans.

N-糖基化是蛋白质最重要的翻译后修饰之一。几乎整个细胞表面和人类几乎所有的分泌蛋白都被复合型 N-聚糖修饰过，其功能受 N-聚糖分支数量的影响。N-Acetylglucosaminyltransferase-IVa (GnT-IVa) 是一种高尔基糖基转移酶，它能将一个 GlcNAc 转移到双元 N-聚糖 GlcNAc2Man3GlcNAc2 的 α-1,3 甘露糖臂上，形成一个 β-1,4 GlcNAc 分支结构。哺乳动物糖基转移酶在异源宿主中的可溶性表达通常具有挑战性。本研究克隆了人 GnT-IVa（HsGnT-IVa）的 N 端截短形式，该形式与可溶性增强标签或信号肽融合，并在大肠杆菌（E. coli）中超表达。我们的研究结果表明，当去掉前 87 个氨基酸并与麦芽糖结合蛋白（MBP）融合后，重组的 HsGnT-IVa 可以以可溶性和活性最高的形式过度表达。通过优化诱导条件，重组蛋白的表达水平得以提高，亲和纯化后每升培养物可产生约 540 毫克重组蛋白。纯化后的酶具有适当的糖基转移酶活性，接受底物的 Km 值为 1.1 mM。酶的特性分析表明，在 37 °C、MES/NaOH（pH 7.0）条件下，5 mM Mn2+ 可使酶达到最大活性。此外，还测定了催化结构域和凝集素结构域中的关键氨基酸对酶活性的影响。这项工作为大规模生产具有生物活性的 HsGnT-IVa 提供了一种有效的方法，它可用于体外合成和功能性研究多延复合型 N-聚糖。

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引用次数: 0

Unveiling six novel CALB-like lipases using genome-centric and patent-driven prospection 利用以基因组为中心和专利驱动的探索揭示六种新型 CALB 类脂肪酶

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-09 DOI: 10.1016/j.enzmictec.2024.110525

Priscila Esteves de Faria , Gabriel Stamato Nunes , Gabriela Coelho Brêda , Erika Cristina Gonçalves Aguieiras , Maria Beatriz Santos Mota , Leticia Dobler , Denise Maria Guimarães Freire , Rodrigo Volcan Almeida , Rafael Dias Mesquita

Lipases present biotechnological applications in various industrial sectors due to their ability to perform multiple biochemical reactions. However, the high cost sometimes discourages their potential uses, besides the extensive number of patents involving them. One of the most utilized and researched lipases is Candida antarctica lipase B (CALB), known for its versatility, encompassing enantioselectivity, thermostability, and a wide range of substrates. Therefore, finding new CALB-like lipases is an interesting strategy to enable the implementation of biocatalysts, especially if intellectual property analysis is included. The present study identified and produced six CALB-like enzymes without patent protection, with differences in pocket amino acids and substrate specificity. We conducted genomic searches in almost 7000 Fungal genomes, identifying over 1500 unique CALB homolog candidates. The phylogenetic and intellectual property analysis filtered those results into a few sequences without protection that were very similar to CALB. One cloned lipase had a lower hydrophobicity at the pocket entrance and preferred the C4 p-nitrophenyl ester as substrate. Another had a wider opening and more polar pocket, showing no preference. These results identified new patent-free lipases with conserved essential catalytic elements and diverse substrate specificity due to variations in the catalytic pocket. These enzymes can be the starting point for biocatalyst innovation with potential applications in diverse biotechnological areas.

由于脂肪酶能够进行多种生化反应，因此在各种工业领域都有生物技术应用。然而，除了涉及脂肪酶的大量专利外，高昂的成本有时也阻碍了它们的潜在用途。利用和研究最多的脂肪酶之一是白色念珠菌脂肪酶 B（CALB），它以多功能性著称，包括对映体选择性、热稳定性和广泛的底物范围。因此，寻找新的类 CALB 脂肪酶是实现生物催化剂的一个有趣策略，尤其是在包含知识产权分析的情况下。本研究发现并生产了六种没有专利保护的类 CALB 酶，它们在口袋氨基酸和底物特异性方面存在差异。我们在近 7000 个真菌基因组中进行了基因组搜索，发现了 1500 多个独特的 CALB 同源物候选者。通过系统发育和知识产权分析，我们筛选出了一些与 CALB 非常相似的未受保护序列。其中一种克隆的脂肪酶在口袋入口处的疏水性较低，偏好以 C4 对硝基苯酯为底物。另一种脂肪酶的口袋开口更宽，极性更强，但没有显示出偏好性。这些结果发现了新的无专利脂肪酶，它们具有保守的基本催化元件，但由于催化袋的变化而具有不同的底物特异性。这些酶可以作为生物催化剂创新的起点，在不同的生物技术领域具有潜在的应用价值。

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引用次数: 0

Microbial production of adipic acid from 6-hydroxyhexanoic acid for biocatalytic upcycling of polycaprolactone 微生物利用 6-羟基己酸生产己二酸，用于聚己内酯的生物催化升级再循环

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-05 DOI: 10.1016/j.enzmictec.2024.110521

Yu-Ri Oh , Young-Ah Jang , Gyeong Tae Eom

To valorize waste polycaprolactone (PCL), one of the most widely used biodegradable plastics, into a value-added chemical, we upcycled 6-hydroxyhexanoic acid (6-HHA), the sole monomer of PCL, into adipic acid (AA) using a microbial method. Recombinant Escherichia coli strains expressing chnD (6-HHA dehydrogenase) and chnE (6-oxohexanoic acid dehydrogenase) genes from three bacteria were constructed, and all these strains successfully produced AA from 6-HHA. Among these, the E. coli strain harboring ChnDE genes from Acinetobacter strain SE19 (E. coli [pKK-AcChn]) showed the highest AA-producing ability. To increase the AA production titer, we optimized the culture temperature of this strain in flask culture and performed fed-batch fermentation in a 5 L bioreactor. After the fed-batch fermentation, the AA production titer increased to 15.6 g/L. As 6-HHA is a monomer of PCL, our results provide the groundwork for the development of a biocatalytic upcycling method of PCL.

聚己内酯（PCL）是最广泛使用的生物降解塑料之一，为了将废弃聚己内酯转化为高附加值化学品，我们采用微生物方法将聚己内酯的唯一单体 6-hydroxyhexanoic acid（6-HHA）升级循环为己二酸（AA）。我们构建了表达来自三种细菌的 chnD（6-HHA 脱氢酶）和 chnE（6-氧代己酸脱氢酶）基因的重组大肠杆菌菌株，所有这些菌株都成功地利用 6-HHA 生产出了 AA。其中，含有 SE19 阴沟杆菌 ChnDE 基因的大肠杆菌菌株（E. coli [pKK-AcChn]）的 AA 产率最高。为了提高 AA 的生产滴度，我们优化了该菌株在烧瓶培养中的培养温度，并在 5 L 生物反应器中进行了喂料式批量发酵。批量喂养发酵后，AA 产量滴度增加到 15.6 克/升。由于 6-HHA 是 PCL 的单体，我们的研究结果为开发 PCL 的生物催化上循环方法奠定了基础。

引用次数: 0

Selective transformation of crocin-1 to crocetin-glucosyl esters by β-glucosidase (Lf18920) from Leifsonia sp. ZF2019: Insights from molecular docking and point mutations 来自 Leifsonia sp. ZF2019 的 β-葡萄糖苷酶 (Lf18920) 将 crocin-1 选择性转化为鳄梨素-葡萄糖基酯：分子对接和点突变的启示。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-04 DOI: 10.1016/j.enzmictec.2024.110522

Xi Wang , Chenzhi Zhuhuang , Yi He, Xiaolong Zhang, Yan Wang, Qinxue Ni, Youzuo Zhang, Guangzhi Xu

Crocetin di/mono-glucosyl esters (crocin-4 and crocin-5) are rarely distributed in nature, limiting their potential applications in the food and pharmaceutical industries. In the present study, a novel GH3 family β-glucosidase Lf18920 was identified from Leifsonia sp. ZF2019, which selectively hydrolyzed crocin-1 (crocetin di-gentiobiosyl ester) to crocin-5 and crocin-4, but not to its aglycone, crocetin. Under the optimal condition of 40 °C and pH 6.0 for 120 min, Lf18920 almost completely hydrolyzed crocin-1, yielding 73.50±5.66 % crocin-4 and 16.19±1.38 % crocin-5. Molecular docking and point mutation studies revealed that Lf18920 formed a narrow binding channel that facilitated crocin-1 binding. Five single amino acid variants (D50A, D53A, W274A, G420A, and Q421A) were constructed, all of which showed reduced hydrolytic activity. Mutations at D50 and D53, located distal to the active site, increased binding energy and decreased hydrolytic activity, while mutations at W274, G420, and Q421, proximal to the active site, disrupted hydrolytic function. These findings suggest that the narrow binding channel and specific enzyme-substrate interactions are crucial for Lf18920’s selective hydrolytic activity. Overall, this study is the first to report a β-glucosidase capable of selectively transforming crocin-1 to crocetin di/mono-glucosyl esters, offering potential for synthesizing crocin-4 and crocin-5.

鳄梨素二/单葡萄糖基酯（鳄梨素-4 和鳄梨素-5）在自然界中很少分布，限制了其在食品和制药业中的潜在应用。本研究从 Leifsonia sp. ZF2019 中发现了一种新型 GH3 家族 β-葡萄糖苷酶 Lf18920，它能选择性地将 crocin-1（藏红花素二-胍基酯）水解为 crocin-5 和 crocin-4，但不能水解为其苷元藏红花素。在 40 °C、pH 值为 6.0 的最佳条件下，持续 120 分钟，Lf18920 几乎完全水解了 crocin-1，得到 73.50±5.66 % 的 crocin-4 和 16.19±1.38 % 的 crocin-5。分子对接和点突变研究表明，Lf18920 形成了一个狭窄的结合通道，有利于与 crocin-1 结合。研究人员构建了五个单氨基酸变体（D50A、D53A、W274A、G420A 和 Q421A），所有这些变体的水解活性都有所降低。位于活性位点远端的 D50 和 D53 突变增加了结合能，降低了水解活性，而位于活性位点近端的 W274、G420 和 Q421 突变则破坏了水解功能。这些发现表明，狭窄的结合通道和特定的酶-底物相互作用对 Lf18920 的选择性水解活性至关重要。总之，这项研究首次报道了一种能选择性地将 crocin-1 转化为 crocin 二/单葡萄糖基酯的β-葡萄糖苷酶，为合成 crocin-4 和 crocin-5 提供了潜力。

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引用次数: 0