Enzyme and Microbial Technology最新文献_第10页

Efficient isoprimeverose production using an enzyme cocktail from engineered Aspergillus oryzae and yeast-assisted purification 利用工程米曲霉和酵母辅助纯化的鸡尾酒酶高效生产异戊糖

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-06-19 DOI: 10.1016/j.enzmictec.2025.110698

Satoshi Wakai , Nanami Nakashima , Hiroko Tsutsumi , Yoji Hata , Fahmi Baihaqqi , Akihiko Kondo , Chiaki Ogino

Aspergillus oryzae is a filamentous fungus that possesses various types of carbohydrate -degrading enzymes. Among these, isoprimeverose-producing enzyme (IpeA), acts on a key component of the plant cell wall structure, xyloglucan, to catalyze the release of isoprimeverose — a rare disaccharide that is expected to possess valuable prebiotics properties. Despite these expectations, however, a process for the effective production of isoprimeverose from the xyloglucan still requires further development for commercial-level application. A complicating factor for the lack of such a valuable process is that plant-derived xyloglucan is often modified with other sugars such as galactose and arabinose. Therefore, the effective production of isoprimeverose requires a cooperative form of degradation that must utilize different enzymes. In this study, we genetically engineered two A. oryzae strains — one produces IpeA and the other produces endoglucanase. The two strains were cultivated separately, and an enzyme cocktail was prepared using their respective culture supernatants. This enzyme cocktail successfully produced isoprimeverose from tamarind xyloglucan and tamarind seed gum. Approximately 14 g/L of isoprimeverose was obtained, which corresponds to a theoretical conversion rate of over 90 %. Although glucose and galactose remained in the reaction solution after enzymatic degradation, these by-products could be easily removed via treatment with Saccharomyces cerevisiae. Our developed process, which mimics traditional Japanese sake fermentation using A. oryzae and S. cerevisiae, has enabled efficient production of isoprimeverose.

米曲霉是一种丝状真菌，具有多种类型的碳水化合物降解酶。其中，异基戊糖生成酶（IpeA）作用于植物细胞壁结构的关键成分木葡聚糖，催化异基戊糖的释放，这是一种罕见的双糖，有望具有宝贵的益生元特性。然而，尽管有这些期望，从木葡聚糖中有效生产异戊糖的工艺仍需要进一步开发以实现商业应用。缺乏这样一个有价值的过程的一个复杂因素是，植物衍生的木葡聚糖经常被其他糖修饰，如半乳糖和阿拉伯糖。因此，异戊糖的有效生产需要一种协同降解形式，必须利用不同的酶。在这项研究中，我们对两株稻谷曲霉进行了基因工程改造，一株产生IpeA，另一株产生内切葡聚糖酶。分别培养两株菌，用各自的培养上清配制酶鸡尾酒。该酶混合物成功地从罗望子木葡聚糖和罗望子籽胶中生产出了异丙糖。得到的异戊糖约为14 g/L，理论转化率为90 %以上。虽然葡萄糖和半乳糖在酶降解后仍留在反应溶液中，但这些副产物可以很容易地通过酿酒酵母处理去除。我们开发的工艺，模仿传统的日本清酒发酵，使用米曲霉和酿酒酵母，使异戊糖的高效生产成为可能。

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引用次数: 0

Comparison of novel α-glucosidases in glycoside hydrolase family 97 isolated from Bacteroides thetaiotaomicron 拟杆菌糖苷水解酶家族97中新型α-葡萄糖苷酶的比较

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-06-18 DOI: 10.1016/j.enzmictec.2025.110696

Dam-Seul Ko , Hyun-Mo Jeong , Yu-Jeong Shin, Da-Woon Jeong, Na-Ri Kim, Jae-Hoon Shim

In this study, three genes encoding novel Glycoside Hydrolase (GH) 97 enzymes were cloned from Bacteroides thetaiotaomicron and expressed in Escherichia coli. The recombinant enzymes (Bt_4581, Bt_0683, Bt_3163) were purified using Ni-NTA affinity chromatography and subsequently characterized. All three enzymes released glucose from the non-reducing ends of oligosaccharides and displayed metal ion dependency. Among them, Bt_4581 hydrolyzed a wide range of α-glycosidic linkages, while Bt_0683 and Bt_3163 showed narrower substrate specificity. Amino acid sequence analysis indicated that Bt_4581 and Bt_0683 belong to Group 1, whereas Bt_3163 is part of Group 3. Kinetic studies revealed that Bt_4581 preferred maltooligosaccharides with an odd number of glucosyl units. In contrast, Bt_3163 exhibited a preference for α-pNPG, confirming it as the first characterized α-glucosidase in Group 3 of the GH 97 family.

本研究从拟杆菌（Bacteroides thetaiotaomicron）中克隆了3个编码糖苷水解酶（GH） 97酶的基因，并在大肠杆菌中表达。重组酶（Bt_4581、Bt_0683、Bt_3163）采用Ni-NTA亲和层析纯化，并进行了结构表征。这三种酶都从低聚糖的非还原端释放葡萄糖，并表现出金属离子依赖性。其中，Bt_4581水解α-糖苷键的范围较广，而Bt_0683和Bt_3163的底物特异性较窄。氨基酸序列分析表明，Bt_4581和Bt_0683属于第1族，Bt_3163属于第3族。动力学研究表明，Bt_4581倾向于选择具有奇数个葡萄糖基单位的低聚麦芽糖。相比之下，Bt_3163表现出对α-pNPG的偏好，证实了它是GH 97家族第3组中第一个被鉴定的α-葡萄糖苷酶。

引用次数: 0

Arazyme prevents skin aging through regulation of matrix metalloproteinase and collagen synthesis Arazyme通过调节基质金属蛋白酶和胶原合成来防止皮肤老化

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-06-18 DOI: 10.1016/j.enzmictec.2025.110695

Jong-Hoon Kim , Hwa Lee , Kwang-Hee Son , Tae-Sook Jeong , Ho-Yong Park

Arazyme, an enzyme derived from Serratia proteamaculans, has demonstrated efficacy in enhancing skin barrier function in studies involving skin cell treatments and topical application on animal skin. The objective of this study was to assess the anti-wrinkle and anti-aging effects of Arazyme in skin keratinocytes and fibroblasts subjected to ultraviolet B (UVB) radiation and oxidative stress. Keratinocytes (HaCaT cells) and fibroblasts (CCD-986sk) were exposed to UVB (15 mJ/cm²) radiation or oxidative stress induced by 2 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), followed by treatment with Arazyme (0.1–0.5 μM) for 24 h. The effects of Arazyme were compared to those of individual treatments with papain, trypsin, or retinol, which served as reference compounds. Key parameters examined included the expression of matrix metalloproteinases (MMP-1, MMP-3, and MMP-13), collagen synthesis, and cellular senescence markers (LMNB1, p16, p21, and p53). Additionally, the impact of Arazyme on cellular signaling pathways, including ERK, JNK, and NF-κB, was assessed. Arazyme significantly suppressed UVB-induced expression of MMP-1, MMP-3, and MMP-13 in a dose-dependent manner in HaCaT cells compared to other treatments. In UVB-exposed fibroblasts, Arazyme reduced both mRNA and protein levels of MMPs, while also enhancing procollagen concentration and collagen gene expression. Furthermore, Arazyme inhibited the activation of ERK, JNK, and NF-κB signaling pathways in keratinocytes. In AAPH-stimulated HaCaT cells, Arazyme significantly attenuated the expression of senescence-related markers, including LMNB1, p16, p21, and p53, and decreased the proportion of senescence-positive cells in fibroblasts. Our in vitro findings suggest that Arazyme may help attenuate UVB- and oxidative stress-induced markers of skin aging, indicating its potential as a candidate for further investigation in anti-aging skincare research.

在涉及皮肤细胞治疗和动物皮肤局部应用的研究中，Arazyme是一种源自蛋白沙雷氏菌的酶，已证明具有增强皮肤屏障功能的功效。本研究的目的是评估Arazyme在紫外线B （UVB）辐射和氧化应激下皮肤角质形成细胞和成纤维细胞中的抗皱和抗衰老作用。将角质形成细胞（HaCaT细胞）和成纤维细胞（CCD-986sk）暴露于UVB （15 mJ/cm²）辐射或2 mM 2,2 ' -偶氮（2-氨基丙烷）二盐酸（AAPH）诱导的氧化应激下，然后用Arazyme（0.1-0.5 μM）处理24 h。将Arazyme的效果与作为参比化合物的木瓜蛋白酶、胰蛋白酶或视黄醇单独处理的效果进行了比较。检测的关键参数包括基质金属蛋白酶（MMP-1、MMP-3和MMP-13）、胶原合成和细胞衰老标志物（LMNB1、p16、p21和p53）的表达。此外，我们还评估了Arazyme对细胞信号通路的影响，包括ERK、JNK和NF-κB。与其他处理相比，Arazyme以剂量依赖的方式显著抑制uvb诱导的HaCaT细胞中MMP-1、MMP-3和MMP-13的表达。在uvb暴露的成纤维细胞中，Arazyme降低MMPs mRNA和蛋白水平，同时提高前胶原浓度和胶原基因表达。此外，Arazyme抑制角化细胞中ERK、JNK和NF-κB信号通路的激活。在aaph刺激的HaCaT细胞中，Arazyme显著降低了衰老相关标志物LMNB1、p16、p21和p53的表达，降低了成纤维细胞中衰老阳性细胞的比例。我们的体外研究结果表明，Arazyme可能有助于减弱UVB和氧化应激诱导的皮肤衰老标志物，这表明它有潜力在抗衰老护肤研究中进一步研究。

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引用次数: 0

Semi-rational engineering of 3-Ketosteroid-Δ1-dehydrogenase boosts catalytic efficiency and robustness for steroid bioconversion 半合理工程的3-酮类固醇-Δ1-dehydrogenase提高催化效率和稳健性类固醇生物转化

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-06-11 DOI: 10.1016/j.enzmictec.2025.110692

Zijuan Tao , Yusong Zhang , Yanmei Dai , Changshun Huang , Liangli Luo , Junhao Yue , Jing'e Yue , Hanbing Shi , Zhimin Ou

Steroid drugs play a pivotal role in clinical therapeutics, with androsta-1,4-diene-3,17-dione (ADD) serving as a critical intermediate whose efficient biosynthesis relies on the catalytic activity of 3-ketosteroid-Δ1-dehydrogenase (KstD). Building upon the previously engineered KstD₂^ep variant, this study employed semi-rational design strategies to enhance KstD₂’s high-substrate-loading adaptability. Key residues (V332, L334, G534) were systematically identified through homology modeling and molecular docking, followed by constructing a combinatorial mutant library. Through alanine scanning and iterative screening of single/multiple-site mutations, the optimal mutant KstD₂^ep (V332E/L334T/G534V) demonstrated a 1.1-fold enhancement in catalytic efficiency compared to the KstD₂^ep. Molecular dynamics simulations confirmed significantly enhanced structural stability in the mutant. Whole-cell catalytic optimization revealed expanded operational tolerance to temperature fluctuations, pH variations, and co-solvent exposure. Implementing high-density fermentation coupled with fed-batch substrate supplementation, the process achieved a 1.5-fold increase in substrate conversion efficiency, yielding 117.75 g/L ADD. These advancements position the engineered variant as a high-potential candidate for scalable steroid biotransformation, addressing key barriers to enzymatic stability and process efficiency for enzyme-driven biosynthesis of steroidal pharmaceutical intermediates.

甾体药物在临床治疗中发挥着关键作用，androsta-1,4-二烯-3,17-二酮（ADD）作为关键中间体，其有效的生物合成依赖于3-酮类固醇-Δ1-dehydrogenase （KstD）的催化活性。在先前设计的KstD2ep变体的基础上，本研究采用半合理的设计策略来增强KstD2的高底物负载适应性。通过同源建模和分子对接，系统鉴定关键残基V332、L334、G534，构建组合突变文库。通过丙氨酸扫描和单/多位点突变的迭代筛选，最优突变体KstD2ep （V332E/L334T/G534V）的催化效率比KstD2ep提高了1.1倍。分子动力学模拟证实突变体的结构稳定性显著增强。全细胞催化优化揭示了对温度波动、pH变化和共溶剂暴露的扩展操作耐受性。通过高密度发酵加上补料批底物的补充，该工艺将底物转化效率提高了1.5倍，产生117.75 g/L ADD。这些进展使该工程变体成为可扩展类固醇生物转化的高潜力候选物，解决了酶驱动类固醇药物中间体生物合成的酶稳定性和工艺效率的关键障碍。

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引用次数: 0

Expanding fluorescence imaging tools in Staphylococcus aureus: Optimized expression of red fluorescent proteins 金黄色葡萄球菌扩展荧光成像工具：红色荧光蛋白的优化表达

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-06-11 DOI: 10.1016/j.enzmictec.2025.110694

Fuminori Kato

Fluorescent proteins (FPs) are widely used as molecular imaging tools for visualizing protein localization, gene expression, and bacterial labeling. However, most commercially available FPs are optimized for expression in human cells or Escherichia coli, and often exhibit poor expression in other bacterial species due to differences in codon usage preference, which critically affects translation efficiency. Staphylococcus aureus, a clinically important Gram-positive bacterium, presents additional challenges for heterologous protein expression due to codon usage bias and gene regulatory mechanisms. While our previous study has developed expression vectors that enable robust expression of GFP and its color variants in S. aureus, expression of red fluorescent proteins (RFPs) remain limited. In this study, I improved the expression of red fluorescent proteins (RFPs)—mCherry, mOrange2, E2-Crimson—and the photoconvertible protein Dendra2 in S. aureus. Codon optimization was performed based on S. aureus codon usage preferences, and RNA secondary structures at the 5′ region were minimized to enhance translation efficiency. The fully optimized mCherry(Sa2) exhibited strong fluorescence at both colony and single-cell levels. Similarly, mOrange2(Sa), E2-Crimson(Sa), and Dendra2(Sa) showed robust expression following host-adapted codon design, and Dendra2(Sa) retained its photoconvertible functionality. These results demonstrate that codon usage adjustment and RNA structure optimization are effective strategies for achieving high-level expression of diverse fluorescent proteins in S. aureus and provide valuable insights into optimizing heterologous protein expression in non-model bacterial systems.

荧光蛋白（FPs）被广泛用作可视化蛋白定位、基因表达和细菌标记的分子成像工具。然而，大多数市售的FPs都是针对人类细胞或大肠杆菌的表达进行了优化，并且由于密码子使用偏好的差异，在其他细菌物种中往往表现不佳，这严重影响了翻译效率。金黄色葡萄球菌是临床重要的革兰氏阳性细菌，由于密码子使用偏差和基因调控机制，对异源蛋白表达提出了额外的挑战。虽然我们之前的研究已经开发了能够在金黄色葡萄球菌中鲁棒表达GFP及其颜色变体的表达载体，但红色荧光蛋白（rfp）的表达仍然有限。在本研究中，我提高了金黄色葡萄球菌中红色荧光蛋白(rfp) -mCherry, mOrange2， e2 - crimson和光转化蛋白Dendra2的表达。根据金黄色葡萄球菌密码子的使用偏好进行密码子优化，最小化5′区的RNA二级结构以提高翻译效率。充分优化的mCherry（Sa2）在集落和单细胞水平上均表现出较强的荧光。同样，mOrange2（Sa）、E2-Crimson（Sa）和Dendra2（Sa）在宿主适应密码子设计后表现出稳健的表达，Dendra2（Sa）保留了其光转换功能。这些结果表明，密码子使用调整和RNA结构优化是实现多种荧光蛋白在金黄色葡萄球菌中高水平表达的有效策略，并为优化非模式细菌系统中的外源蛋白表达提供了有价值的见解。

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引用次数: 0

Characterization of a GH10 family thermophilic, alkali- and salt-tolerant xylanase from Xinjiang salt lake 新疆盐湖GH10家族嗜热耐碱耐盐木聚糖酶的研究

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-06-07 DOI: 10.1016/j.enzmictec.2025.110693

Rong-Huan Song , Dan Zhu , Zhong-qiao Yang , Jian-Ling Li , Zheng-Feng Yang , Zhi-Hua Lv , Kai-Qing Xie , Li-Quan Yang , A. Zhou-Cun , Peng Sang , Yi-Rui Yin

Xylanase is extensively employed in the food, feed, and paper sectors, with those derived from extreme environments offering distinct advantages. This study identified a novel xylanase gene (designated xynaes) through metagenomic analysis of samples from Aiting Lake, Xinjiang, China. Cloned and expressed in Escherichia coli after PCR amplification. The recombinant protein was purified using Ni-NTA affinity chromatography XynAES demonstrated optimal activity at pH 8.0 and 65 °C, its half-life (T_1/2) was 120 min. XynAES preserved over 80 % residual activity after 12 h in pH 6.0–9.0 buffer. Its activity was enhanced to 132 % and 135 % in the presence of 1 mM Mg²⁺ and Zn²⁺, respectively. Additionally, XynAES maintained over 60 % relative activity in 0–3.0 M NaCl and its Km and Vmax of XynAES were determined to be 3.23 mg/mL and 72.46 μmol/min/mg, respectively. It is worth noting that the main products of XynAES enzymatic hydrolysis of xylan are xylose disaccharides and xylose tetrasaccharides, and XynAES shows obvious activity against the pre-treated wheat bran. In summary, XynAES is a thermophilic, alkali-tolerant, and salt-resistant xylanase, signifying its potential applications in the feed, food baking, paper manufacturing, and prebiotic production industries.

木聚糖酶广泛应用于食品、饲料和造纸行业，在极端环境中产生的木聚糖酶具有明显的优势。本研究通过对新疆艾亭湖样品的宏基因组分析，鉴定出一个新的木聚糖酶基因（称为xynaes）。经PCR扩增克隆并在大肠杆菌中表达。重组蛋白经Ni-NTA亲和层析纯化得到，XynAES在pH 8.0和65℃条件下活性最佳，半衰期（T1/2）为120 min。在pH 6.0-9.0的缓冲液中，xnaes在12 h后保留了80% %的残留活性。在1 mM Mg2+和Zn2+存在下，其活性分别提高到132 %和135 %。在0 ~ 3.0 M NaCl中，XynAES的相对活性保持在60% %以上，其Km和Vmax分别为3.23 mg/mL和72.46 μmol/min/mg。值得注意的是，XynAES酶解木聚糖的主要产物是木糖二糖和木糖四糖，并且XynAES对预处理后的麦麸具有明显的活性。综上所述，XynAES是一种耐热、耐碱、耐盐的木聚糖酶，在饲料、食品烘焙、造纸和益生元生产等行业具有潜在的应用前景。

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引用次数: 0

Influence of a rumen ciliate-derived xylanase on the gut microbiota composition: A potential enzyme for prebiotic applications 瘤胃纤毛虫衍生的木聚糖酶对肠道菌群组成的影响：一种潜在的益生元应用酶

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-05-31 DOI: 10.1016/j.enzmictec.2025.110683

Weide Su , Huiying Luo , Xiaolian Chen , Gaoxiang Ai , Qipeng Wei , Zhiheng Zou , Chuanhui Xu , Jiang Chen , Pingwen Xiong , Wenjing Song , Qiongli Song

A xylanase derived from rumen ciliates designated as XynC, was successfully expressed and purified. The enzyme's catalytic properties, including its optimal pH (5.5) and temperature (40 °C), were determined, demonstrating high stability under mildly acidic and moderate thermal conditions. The substrate specificity of XynC was investigated by analyzing its hydrolysis pattern with beechwood xylan, where it preferentially degraded higher xylo-oligosaccharides (xylotetrose (X4), xylopentose (X5), xylohexose (X6)) while showing no activity on smaller oligosaccharides like xylobiose (X2) and xylotriose (X3). The in vivo effects of XynC on gut microbiota were evaluated by administering the enzyme to mice, followed by analysis of fecal 16S rRNA to assess shifts in gut bacterial populations. The results revealed dose-dependent modulation of gut microbiota composition, with a significant increase in Lactobacillus abundance observed in the medium- and high-dose groups, while no notable change was detected in the low-dose group. These dose-related microbial responses suggest that the efficacy of XynC in promoting probiotic growth is dependent on administration level. These findings suggest that XynC selectively promotes the growth of beneficial probiotics in a dosage-sensitive manner, supporting its potential as a functional feed additive for enhancing gut health and nutrient utilization in livestock.

从瘤胃纤毛中成功表达并纯化了一种木聚糖酶，命名为XynC。测定了该酶的催化性能，包括其最佳pH值（5.5）和温度（40°C），在轻度酸性和中等热条件下表现出高稳定性。通过分析XynC与山毛榉木聚糖的水解模式，研究了其底物特异性，其中XynC优先降解高级低聚木糖（xylotetrose （X4）、xylopentose （X5）、xylohexose (X6)），而对低聚木糖（xylobeose （X2）和xylotriose (X3)）没有活性。通过给小鼠注射XynC酶，然后分析粪便16S rRNA来评估肠道细菌群的变化，从而评估XynC对肠道微生物群的体内影响。结果显示了肠道微生物群组成的剂量依赖性调节，在中剂量组和高剂量组中观察到乳酸杆菌丰度显著增加，而在低剂量组中没有发现显着变化。这些剂量相关的微生物反应表明，XynC促进益生菌生长的功效取决于给药水平。这些结果表明，XynC以剂量敏感的方式选择性地促进有益益生菌的生长，支持其作为增强牲畜肠道健康和营养物质利用的功能性饲料添加剂的潜力。

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引用次数: 0

Mutational and structural analysis of ribose 5-phosphate isomerase B from Acetivibrio thermocellus: relationship between transformation efficiency and substrate binding pocket conformation 热细胞活动弧菌核糖5-磷酸异构酶B的突变和结构分析：转化效率与底物结合袋构象的关系

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-05-29 DOI: 10.1016/j.enzmictec.2025.110671

Shitong Dong , Yuanwei Wang , Han Zhao , Yingshuai Wu , Ruiming Wang , Chunling Ma

D-allose is a rare hexose sugar with a variety of potential application in food, medicine and other fields. Ribose 5-phosphate isomerase（RPI） plays a pivotal role in the synthesis of D-allose. AtRpiB from Acetivibrio thermocellus can convert D-psicose to D-allose; however, improvements in its stability, optimal temperature, and conversion efficiency are necessary. This study aimed to investigate the catalytic properties, stability, and substrate-binding affinity of AtRpiB using D-psicose as the substrate. Using various strategies, 10 amino acid residues were selected to construct a library of 82 single mutants. Nine single mutants showed high conversion rates of D-allose. Furthermore, 27 double mutants were constructed by combining nine successful single mutation sites. Notably, the mutants R109W/R132Q, S39I/R109F, and S39V/R109F showed a 1.39-fold increase in enzyme activity at 40 ºC, 1.58-fold increase at 60 ºC, and 1.9-fold increase at 80 ºC, respectively. The S39V/R109F mutant converted 100 g/L D-psicose to 38.21 g/L D-allose after 18 hours at 40°C, the highest reported conversion rate for RpiB in D-allose production to date. Analysis of enzymatic characteristics, structure, and molecular dynamics simulations revealed that changes in the amino acid composition and conformational adjustments in loops 3, 9, and 10 of AtRpiB significantly affected the entry and exit of substrates and products into the active pocket, conversion efficiency, and enzyme stability.

d -醛糖是一种罕见的己糖，在食品、医药等领域具有多种潜在的应用前景。核糖5-磷酸异构酶（RPI）在D-allose的合成中起关键作用。来自热细胞活动弧菌的AtRpiB可以将D-psicose转化为D-allose；然而，它的稳定性，最佳温度和转换效率的改进是必要的。本研究旨在以D-psicose为底物考察AtRpiB的催化性能、稳定性和底物结合亲和力。采用不同的策略，选取10个氨基酸残基构建了82个单突变体文库。9个单突变体表现出较高的D-allose转化率。通过9个成功的单突变位点组合，构建了27个双突变体。值得注意的是，突变体R109W/R132Q、S39I/R109F和S39V/R109F在40℃、60℃和80℃下的酶活性分别提高了1.39倍、1.58倍和1.9倍。在40°C下，S39V/R109F突变体在18 小时后将100 g/L D-psicose转化为38.21 g/L D-allose，这是迄今为止报道的RpiB在D-allose生产中的最高转化率。酶学特性、结构分析和分子动力学模拟表明，AtRpiB的氨基酸组成和3、9、10环构象调整的变化显著影响底物和产物进入活性口袋的进出、转化效率和酶的稳定性。

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引用次数: 0

Nicotinic acid production from 3-methylpyridine by E. coli whole-cell biocatalyst 大肠杆菌全细胞生物催化剂从3-甲基吡啶生产烟酸

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-05-27 DOI: 10.1016/j.enzmictec.2025.110682

Sineenat Sripattanakul , Piyasiri Chueakwon , Le Thi Thuy Trinh , Rung-Yi Lai

Nicotinic acid, a bioactive form of niacin (vitamin B3), is an essential nutrient involved in various metabolic pathways. Its deficiency can lead to severe health issues, emphasizing the need for effective production methods. Traditional chemical methods for synthesizing nicotinic acid often require harsh reaction conditions and produce environmentally hazardous byproducts. This has prompted increasing interest in more sustainable biocatalytic alternatives. In this study, we investigated the bioconversion of 3-methylpyridine (3-MP) to nicotinic acid using resting cells of recombinant Escherichia coli expressing enzymes from Pseudomonas putida mt-2. Through systematic optimization, E. coli MG1655 RARE cells co-overexpressing xylene monooxygenase (XMO: XylM and its reductase XylA) and benzyl alcohol dehydrogenase (XylB) achieved efficient biotransformation of 9 mM 3-MP to 8.28 ± 0.35 mM nicotinic acid within 12 hours, corresponding to a yield of 92.0 % ± 3.9 %. This work presents a streamlined biocatalytic route for nicotinic acid synthesis and offers a foundation for further metabolic engineering of microbial production systems.

烟酸是烟酸（维生素B3）的一种生物活性形式，是参与多种代谢途径的必需营养素。它的缺乏可导致严重的健康问题，强调需要有效的生产方法。传统的合成烟酸的化学方法往往需要苛刻的反应条件，并产生对环境有害的副产物。这促使人们对更可持续的生物催化剂替代品越来越感兴趣。在这项研究中，我们研究了利用表达恶臭假单胞菌mt-2酶的重组大肠杆菌静息细胞将3-甲基吡啶（3-MP）转化为烟酸。通过系统优化,MG1655罕见的大肠杆菌细胞co-overexpressing二甲苯单氧酶(XMO: XylM及其还原酶XylA)和苄醇脱氢酶(XylB)实现高效的生物转化9 8.28 mM它 ±0.35  mM烟酸 12小时内,对应的收益率92.0 % ±3.9  %。本研究为烟酸合成提供了一种流线型的生物催化途径，为微生物生产系统的进一步代谢工程奠定了基础。

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引用次数: 0

Leveraging Se-nanoparticle@Candida tropicalis for efficient di-carboxylic acid production and methodological advances in selective product separation 利用Se-nanoparticle@Candida热带草高效二羧酸生产和选择性产品分离的方法进步

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-05-26 DOI: 10.1016/j.enzmictec.2025.110681

Pragya Gupta, Prakash C. Sahoo, Srikanth Sandipam, Manoj Kumar, Ravi Prakash Gupta, Rajesh Badhe, Umish Srivastva, Alok Sharma

This study investigates a bio-inorganic hybrid of selenium nanoparticles (SeNP@C. tropicalis) with an in-house developed mutant of Candida tropicalis (Castellani) Berkhout for enhanced bioconversion of crude dodecane (C₁₂H₂₆) into di-carboxylic acids (DCAs) under challenging conditions. The presence of SeNP@C. tropicalis improves NAD+ /NADH regeneration by enhancing enzymatic activity, thereby optimizing the metabolic transformation process. The activation of low-reactivity alkanes to DCAs, facilitated by cytochrome P450 monooxygenase, NADPH, and alcohol dehydrogenase (ADH), presents a sustainable approach for biorefineries when paired with effective downstream processing. At a 2 L scale, the SeNP@C. tropicalis hybrid achieved a 40.87 % increase in DCA production compared to the control. High-resolution transmission electron microscopy (HRTEM) confirmed the deposition of well-defined, spherical SeNP@C. tropicalis on the yeast surface, and X-ray diffraction (XRD) spectra validated the crystallinity of the nanoparticles, along with Confocal imaging. For downstream processing, four methods were evaluated, with vacuum distillation followed by crystallization achieving the highest DCA purity (>75 %) and recovery (>84 %). This research highlights the potential of SeNP@C. tropicalis as an effective catalyst for DCA production, offering new opportunities for advancing bioconversion technologies and enhancing the sustainability of biorefineries.

本研究研究了硒纳米粒子的生物无机杂化（SeNP@C）。利用内部开发的热带假丝酵母（Castellani） Berkhout突变体，在具有挑战性的条件下增强粗十二烷（C₁₂H₂₆）向二羧酸（DCAs）的生物转化。SeNP@C的存在。热带草通过增强酶活性促进NAD+ /NADH再生，从而优化代谢转化过程。细胞色素P450单加氧酶、NADPH和乙醇脱氢酶（ADH）促进了低反应性烷烃对DCAs的活化，当与有效的下游加工相结合时，为生物炼制提供了一种可持续的方法。在2 L的比例下，SeNP@C。与对照相比，热带稻杂交种的DCA产量提高了40.87 %。高分辨率透射电子显微镜（HRTEM）证实了沉积明确的球形SeNP@C。通过共聚焦成像和x射线衍射（XRD）光谱验证了纳米颗粒的结晶度。对于下游加工，评估了四种方法，其中真空蒸馏后结晶可获得最高的DCA纯度（>75 %）和回收率（>84 %）。这项研究突出了SeNP@C的潜力。热带植物是DCA生产的有效催化剂，为推进生物转化技术和提高生物精炼厂的可持续性提供了新的机会。

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引用次数: 0