Enzyme and Microbial Technology最新文献_第2页

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

Lignin-based monophenolic model compounds in L-tyrosine derivative synthesis via tyrosine phenol lyase 通过酪氨酸苯酚裂解酶合成 L-酪氨酸衍生物中以木质素为基础的单酚模型化合物。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-03 DOI: 10.1016/j.enzmictec.2024.110519

Idamaria Romakkaniemi, Johanna Panula-Perälä, Juha Ahola, Marja Mikola, Juha Tanskanen

Tyrosine phenol lyase (TPL) synthesises L-tyrosine derivatives from monophenols, pyruvate and ammonia. Production of such high-value aromatic chemicals from biomass-derived raw materials is of great interest. In this study, six monophenols (guaiacol, phenol, o-cresol, m-cresol, catechol and syringol) were chosen based on the structure of lignin and were studied as substrates in the enzymatic reaction. Single monophenol reactions (SMR) and binary monophenol reactions (BMR) with guaiacol were carried out. TPL-M379V was found to be selective towards guaiacol (84.5 % conv.). The highest single activity was measured towards phenol (93.9 % conv.). However, the enzyme preferred guaiacol over phenol in the BMRs. Syringol was found to be inert in the reaction, whereas catechol had an inhibitory effect on the enzymatic reaction, in addition to causing degradation of all the substrates in the medium. Doubling the guaiacol concentration in the SMR did not significantly increase the production of 3-O-methyldopa (conv. 45.9 %). However, in the binary reaction systems the total monophenol conversions were higher with guaiacol and phenol (total 62.4 %) or o-cresol (total 57.1 %). This indicates possible substrate/product specific inhibition. The study provides new data on activity, selectivity and inhibitory effects of monophenols in the synthetic reaction catalysed by TPL-M379V, especially in mixed-substrate reactions.

酪氨酸酚裂解酶（TPL）可从单酚、丙酮酸和氨中合成 L-酪氨酸衍生物。从生物质衍生原料中生产此类高价值芳香化学品具有重大意义。本研究根据木质素的结构选择了六种单酚（愈创木酚、苯酚、邻甲酚、间甲酚、儿茶酚和丁香酚）作为酶反应的底物进行研究。进行了与愈创木酚的单酚反应（SMR）和二元单酚反应（BMR）。研究发现，TPL-M379V 对愈创木酚具有选择性（84.5% 的转化率）。测得对苯酚的单一活性最高（93.9 % conv.）。然而，在 BMRs 中，该酶偏好愈创木酚而非苯酚。在反应中发现丁香酚是惰性的，而儿茶酚除了会导致培养基中所有底物的降解外，还会对酶反应产生抑制作用。将 SMR 中愈创木酚的浓度增加一倍并不能显著提高 3-O-甲基多巴的产量（conv. 45.9 %）。不过，在二元反应体系中，愈创木酚和苯酚（总转化率 62.4%）或邻甲酚（总转化率 57.1%）的单酚总转化率更高。这表明可能存在底物/产物特异性抑制。这项研究为 TPL-M379V 催化合成反应，特别是混合底物反应中单苯酚的活性、选择性和抑制作用提供了新的数据。

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引用次数: 0

Protein engineering of an alkaline protease from Bacillus licheniformis (BLAP) for efficient and specific chiral resolution of the racemic ethyl tetrahydrofuroate 地衣芽孢杆菌碱性蛋白酶（BLAP）的蛋白质工程，用于高效、特异性手性解析外消旋四氢糠酸乙酯。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-03 DOI: 10.1016/j.enzmictec.2024.110523

Xinjun Yu , Yichao Li , Zhaoxia Qian, Litian Wei, Jing Xie, Meijun Tong, Yinjun Zhang

Enzymatic resolution of ethyl tetrahydrofuroate to produce (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid is a green biomanufacturing strategy. However, enzymatic activity and selectivity are still limiting factors of their industrial applications and development. In previous study, we incidentally found that a Bacillus licheniformis alkaline protease (BLAP), not a lipase, could specifically resolve ethyl tetrahydrofuroate to produce (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid. In this study, the point-saturation-mutation libraries based on the seven amino acid sites (L105, I113, P114, L115, V309, Y310, and M326) were constructed and screened using the molecular docking technology. It was found that activity of the mutant BLAP^Y310E reached 182.78 U/mL with high stereoselectivity, 3.14 times higher than that of the wild-type BLAP. Further simulated mutation analysis showed that the Y310E mutation increased the distance from the substrate ligand to the binding pocket from 2.3 Å to 4.5 Å, reducing steric hindrance to the active center. Under the optimal conditions and after 3.5 h of reaction catalyzed by BLAP^Y310E, 200 mM ethyl tetrahydrofuroate was converted to (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid with the ee values of 99.9 % and 68.63 %, respectively. The enantiomeric ratio of BLAP^Y310E was 105.5, which was 30.23 times higher than that of BLAP. This study advances the comprehension of protease activity and selectivity mechanisms in resolving ester substances and lays a robust foundation for the industrial production of the optically pure (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid via biological enzymatic methods.

酶解四氢糠酸乙酯生成(S)-2-四氢糠酸乙酯和(R)-2-四氢糠酸是一种绿色生物制造策略。然而，酶的活性和选择性仍是其工业应用和发展的限制因素。在之前的研究中，我们偶然发现地衣芽孢杆菌碱性蛋白酶（BLAP）而非脂肪酶能特异性地分解四氢糠酸乙酯，生成(S)-2-四氢糠酸乙酯和(R)-2-四氢糠酸。本研究构建了基于七个氨基酸位点（L105、I113、P114、L115、V309、Y310 和 M326）的点饱和突变库，并利用分子对接技术进行了筛选。结果发现，突变体BLAPY310E的活性达到182.78 U/mL，具有很高的立体选择性，是野生型BLAP的3.14倍。进一步的模拟突变分析表明，Y310E突变使底物配体到结合口袋的距离从2.3埃增加到4.5埃，减少了对活性中心的立体阻碍。在最佳条件下，BLAPY310E催化反应3.5 h后，200 mM四氢糠酸乙酯被转化为(S)-2-四氢糠酸乙酯和(R)-2-四氢糠酸，ee值分别为99.9 %和68.63 %。BLAPY310E 的对映体比率为 105.5，是 BLAP 的 30.23 倍。这项研究加深了人们对蛋白酶解析酯类物质的活性和选择性机制的理解，为通过生物酶解方法工业化生产光学纯的(S)-2-乙基四氢呋喃酸和(R)-2-四氢呋喃酸奠定了坚实的基础。

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引用次数: 0

Characterization of lycopene β-cyclase from Dunaliella bardawil for enhanced β-carotene production and salt tolerance 巴达维杜莎藻番茄红素β-环化酶的表征，以提高β-胡萝卜素产量和耐盐性。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-10-01 DOI: 10.1016/j.enzmictec.2024.110520

Yu-Chen Xie , Zhi-Wei Ye , Jv-Liang Dai , Hao-Hong Chen , Jian-Guo Jiang

Dunaliella can accumulate more β-carotene (10 % or even more of the dry weight of cells) than any other species. Lycopene β-cyclase (LcyB) is the key enzyme in the catalysis of lycopene to β-carotene. In the present research, we used Escherichia coli BL21 (DE3) as host to construct two different types of engineering bacteria, one expressing the D. bardawil LcyB and the other expressing the orthologue Erwinia uredovora crtY. The catalytic ability of LcyB and CrtY were evaluated by comparing the β-carotene yields of the two E. coli BL21(DE3) strains, whose salt tolerance was simultaneously compared by cultivated them under different NaCl concentrations (1 %, 2 %, and 4 %). We also interfered with the LcyB gene to investigate the effect of LcyB in D. bardawil. Results displayed that the β-carotene yield of the LcyB-transformant significantly increased by about 48 % compared with the crtY-transformant. Additionally, LcyB was verified to be able to enhance the salt tolerance of E. coli BL21 (DE3). It is concluded that D. bardawil LcyB not only has better catalytic ability but also is able to confer salt tolerance to cells. Interfering D. bardawil LcyB induced the low expression of LcyB and the changes of growth and carotenoids metabolism in D. bardawil.

杜纳利藻积累的 β-胡萝卜素（占细胞干重的 10%，甚至更高）比任何其他物种都多。番茄红素β-环化酶（LcyB）是将番茄红素催化成β-胡萝卜素的关键酶。在本研究中，我们以大肠杆菌 BL21 (DE3) 为宿主，构建了两种不同类型的工程菌，一种表达 D. bardawil LcyB，另一种表达 Erwinia uredovora crtY 的直向同源物。通过比较两株大肠杆菌 BL21（DE3）的β-胡萝卜素产量，评估了 LcyB 和 CrtY 的催化能力。我们还干扰了 LcyB 基因，以研究 LcyB 对 D. bardawil 的影响。结果显示，与 crtY 转化株相比，LcyB 转化株的β-胡萝卜素产量显著增加了约 48%。此外，还验证了 LcyB 能够增强大肠杆菌 BL21 (DE3) 的耐盐性。结论是，D. bardawil LcyB 不仅具有更好的催化能力，还能赋予细胞耐盐性。干扰 D. bardawil LcyB 会导致 LcyB 的低表达以及 D. bardawil 生长和类胡萝卜素代谢的变化。

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引用次数: 0

Action pattern of Sulfolobus O-α-glycoligase for synthesis of highly water soluble resveratrol 3,4′-α-diglucoside 硫醇杆菌 O-α-糖苷酶合成高水溶性白藜芦醇 3,4′-α- 二糖苷的作用模式

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-09-24 DOI: 10.1016/j.enzmictec.2024.110518

Hee-Won Ahn , Jetendra Kumar Roy , Jaeick Lee , Mi-Jin Lee , Sang-Ho Yoo , Young-Wan Kim

This study presents the enzymatic synthesis of resveratrol-3,4′-O-α-diglucoside (RDG) using a hyperactive O-α-glycoligase (MalA-D416R/Q450S) and α-glucopyranosyl fluoride as the donor substrate. The transglycosylation rate for resveratrol by MalA-D416R/Q450S was maximized in 100 mM Tris-HCl (pH 9.5) containing 20 % DMSO at 45°C. Because the pK_a of the 4′-OH group of resveratrol is lower than that of the 3-OH group, the 4′-OH group is more nucleophilic at the alkaline pH, leading to a preference for glycosylation at the 4′-OH site rather than the 3-OH site. This preference makes resveratrol 3-O-α-glucoside (R3G) as the more efficient acceptor than resveratrol 4′-O-α-glucoside (R4′G), resulting in negligible production of resveratrol 3-O-α-glucoside (R3G) due to its complete consumption in the second transglycosylation reaction when using a 2:1 ratio of donor to acceptor substrates. From a preparative scale reaction, R4′G and RDG were isolated with yields of 41.2 % and 43.3 %, respectively. The water solubility of RDG exceeded 1.67 M, which represents more than a 9,800-fold improvement compared to resveratrol. In a hydrolysis experiment using intestinal α-glycosidase from rat, the α-glucosides of resveratrol (R4′G and RDG) were completely deglycosylated to the aglycone.

本研究采用超活性 O-α-糖苷酶（MalA-D416R/Q450S）和α-吡喃葡萄糖酰氟作为供体底物，酶法合成了白藜芦醇-3,4′-O-α-二葡萄糖苷（RDG）。MalA-D416R/Q450S 在含有 20% DMSO 的 100 mM Tris-HCl（pH 9.5）、45°C 条件下对白藜芦醇的转糖基化率达到最大。由于白藜芦醇的 4′-OH基团的 pKa 低于 3-OH基团，因此 4′-OH基团在碱性 pH 值下更具亲核性，导致糖基化更倾向于 4′-OH位点而不是 3-OH位点。这种偏好使白藜芦醇 3-O-α-葡萄糖苷（R3G）成为比白藜芦醇 4′-O-α-葡萄糖苷（R4′G）更有效的受体，导致白藜芦醇 3-O-α-葡萄糖苷（R3G）的产量微乎其微，因为当供体和受体底物的比例为 2:1 时，它在第二次转糖基化反应中完全消耗掉了。在制备规模的反应中，分离出了 R4′G 和 RDG，产量分别为 41.2 % 和 43.3 %。RDG 的水溶性超过 1.67 M，与白藜芦醇相比提高了 9,800 多倍。在使用大鼠肠道α-糖苷酶进行的水解实验中，白藜芦醇的α-葡萄糖苷（R4′G 和 RDG）被完全脱糖为苷元。

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引用次数: 0

Changes in ficin specificity by different substrate proteins promoted by enzyme immobilization 酶固定化促进不同底物蛋白改变菲辛的特异性

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-09-21 DOI: 10.1016/j.enzmictec.2024.110517

Alex D. Gonzalez-Vasquez , El Siar Hocine , Marcela Urzúa , Javier Rocha-Martin , Roberto Fernandez-Lafuente

Ficin extract has been immobilized using different supports: glyoxyl and Aspartic/1,6 hexamethylenediamine (Asp/HA) agarose beads. The latter was later submitted to glutaraldehyde modification to get covalent immobilization. The activities of these 3 kinds of biocatalysts were compared utilizing 4 different substrates, casein, hemoglobin and bovine serum albumin and benzoyl-arginine-p-nitroanilide at pH 7 and 5. Using glyoxyl-agarose, the effect of enzyme-support reaction time on the activity versus the four substrates at both pH values was studied. Reaction time has been shown to distort the enzyme due to an increase in the number of covalent support-enzyme bonds. Surprisingly, for all the substrates and conditions the prolongation of the enzyme-support reaction did not imply a decrease in enzyme activity. Using the Asp/HA supports (with different amount of HA) differences in the effect on enzyme activity versus the different substrates are much more significant, while with some substrates the immobilization produced a decrease in enzyme activity, with in other cases the activity increased. These different effects are even increased after glutaraldehyde treatment. That way, the conformational changes induced by the biocatalyst immobilization or the chemical modification fully altered the enzyme protein specificity. This may also have some implications when following enzyme inactivation.

Ficin 提取物使用不同的支持物进行固定：乙醛基和天冬氨酸/1,6-己二胺（Asp/HA）琼脂糖珠。后者后来经过戊二醛修饰，实现了共价固定。利用 4 种不同的底物（酪蛋白、血红蛋白、牛血清白蛋白和苯甲酰精氨酸对硝基苯胺），在 pH 值为 7 和 5 的条件下比较了这 3 种生物催化剂的活性。利用乙醛基琼脂糖，研究了在两种 pH 值下酶支持反应时间对四种底物活性的影响。事实证明，反应时间会使酶变形，因为共价支持键-酶键的数量会增加。令人惊讶的是，在所有底物和条件下，酶与支持物反应时间的延长并不意味着酶活性的降低。使用 Asp/HA 支持物（HA 含量不同）时，不同底物对酶活性的影响差异要大得多。这些不同的影响在戊二醛处理后甚至会加剧。因此，生物催化剂固定化或化学修饰引起的构象变化完全改变了酶蛋白的特异性。这也可能对酶失活后产生一些影响。

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引用次数: 0

Poly(3-hydroxybutyrate) production for food packaging from biomass derived carbohydrates by cupriavidus necator DSM 545 利用坏死葡萄球菌 DSM 545 从生物质中提取的碳水化合物生产用于食品包装的聚（3-羟基丁酸）乙酸酯

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-09-17 DOI: 10.1016/j.enzmictec.2024.110516

Gianfrancesco Russo , Paola Scocca , Mattia Gelosia , Giacomo Fabbrizi , Tommaso Giannoni , Stefania Urbani , Sonia Esposto , Andrea Nicolini

The extensive utilization of conventional plastics has resulted in a concerning surge in waste. A potential solution lies in biodegradable polymers mostly derived from renewable sources. Cupriavidus necator DSM 545 is a microorganism capable, under stress conditions, of intracellularly accumulating Poly(3-hydroxybutyrate) (PHB), a bio-polyester. This study aimed to identify optimal conditions to maximize the intracellular accumulation of PHB and its global production using natural media obtained by processing lignocellulosic residues of cardoon, a low-cost feedstock. An intracellular PHB accumulation was observed in all of the tested media, indicating a metabolic stress induced by the lack of macronutrients. Increasing C/N ratios led to a significant decrease in cellular biomass and PHB production. Furthermore C. necator DSM 545 was incapable of consuming more than 25 g/L of supplied monosaccharides. Surprisingly, in the samples supplied with 60 % of the pentose-rich liquid fraction, complete consumption of xylose was observed. This result was also confirmed by subsequent tests using Medium 1 growth media containing xylose as the sole carbon source. Using a diluted medium with a C/N ratio of 5, a PHB production of 5.84 g/L and intracellular PHB accumulation of 77 % w/w were respectively achieved. Finally, comparative shelf-life tests conducted against conventional pre-packaging materials in PP suggested that PHB films performed similarly in preserve ready-to-eat products.

传统塑料的广泛使用导致废物激增，令人担忧。生物可降解聚合物是一种潜在的解决方案，这种聚合物大多来自可再生来源。坏死葡萄球菌（Cupriavidus necator）DSM 545 是一种微生物，在压力条件下能够在细胞内积累生物聚酯聚（3-羟基丁酸）（PHB）。本研究旨在确定最佳条件，以最大限度地提高 PHB 的细胞内积累，并利用加工低成本原料--芒果的木质纤维素残渣所获得的天然培养基生产 PHB。在所有测试的培养基中都观察到了细胞内 PHB 的积累，这表明缺乏大量营养物质会导致新陈代谢压力。提高 C/N 比会导致细胞生物量和 PHB 产量显著下降。此外，C. necator DSM 545 无法消耗超过 25 克/升的单糖。令人惊讶的是，在富含 60% 戊糖液体组分的样品中，木糖被完全消耗。随后使用含木糖作为唯一碳源的 Medium 1 生长培养基进行的测试也证实了这一结果。使用 C/N 比为 5 的稀释培养基，PHB 产量分别达到 5.84 克/升和 77 % w/w 的胞内 PHB 积累。最后，与传统的聚丙烯预包装材料进行的货架期比较测试表明，PHB 薄膜在保存即食产品方面的表现类似。

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引用次数: 0

Characterization of Runella zeae D-mannose 2-epimerase and its expression in Bacillus subtilis for D-mannose production from D-glucose Runella zeae D-甘露糖 2-酰亚胺酶的特征及其在枯草芽孢杆菌中的表达，以利用 D-葡萄糖生产 D-甘露糖

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-09-05 DOI: 10.1016/j.enzmictec.2024.110506

Yuhan Wei , Wei Xu , Wenli Zhang , Penka Petrova , Kaloyan Petrov , Dawei Ni , Wanmeng Mu

D-Mannose 2-epimerase (MEase) catalyzes the bioconversion between D-glucose and D-mannose. It is an important potential biocatalyst for large-scale production of D-mannose, a functional monosaccharide used in pharmaceutical and food industries. In this study, a new microbial MEase was characterized from Runella zeae DSM 19591. The enzyme was purified by one-step nickel-affinity chromatography and determined to be a dimeric protein with two identical subunits of approximately 86.1 kDa by gel filtration. The enzyme showed the highest activity at pH 8.0 and 40 °C, with a specific activity of 2.99 U/mg on D-glucose and 3.71 U/mg on D-mannose. The melting temperature (T_m) was 49.4 °C and the half-life was 115.14 and 3.23 h at 35 and 40 °C, respectively. The purified enzyme (1 U/mL) produced 115.7 g/L of D-mannose from 500 g/L of D-glucose for 48 h, with a conversion ratio of 23.14 %. It was successfully expressed in Bacillus subtilis WB600 via pP43NMK as the vector. The highest fermentation activity was 10.58 U/mL after fed-batch cultivation for 28 h, and the whole cells of recombinant B. subtilis produced 114.0 g/L of D-mannose from 500 g/L of D-glucose, with a conversion ratio of 22.8 %.

D-Mannose 2-epimerase (MEase) 催化 D-葡萄糖和 D-甘露糖之间的生物转化。它是大规模生产 D-甘露糖（一种用于制药和食品工业的功能性单糖）的重要潜在生物催化剂。本研究对 Runella zeae DSM 19591 的一种新型微生物 MEase 进行了鉴定。通过一步镍亲和层析法纯化了该酶，并通过凝胶过滤确定其为二聚体蛋白，含有两个相同的亚基，分子量约为 86.1 kDa。该酶在 pH 值为 8.0、温度为 40 ℃ 时活性最高，对 D-葡萄糖的比活度为 2.99 U/mg ，对 D-甘露糖的比活度为 3.71 U/mg 。熔融温度（Tm）为 49.4 °C，在 35 °C和 40 °C时的半衰期分别为 115.14 和 3.23 h。纯化酶（1 U/mL）从 500 g/L D-葡萄糖中产生 115.7 g/L D-甘露糖，持续 48 h，转化率为 23.14 %。该酶以 pP43NMK 为载体在枯草芽孢杆菌 WB600 中成功表达。重组枯草芽孢杆菌全细胞从 500 g/L 的 D-葡萄糖中产生 114.0 g/L 的 D-甘露糖，转化率为 22.8 %。

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引用次数: 0