Enzyme and Microbial Technology最新文献_第3页

Pylb-based overexpression of cytochrome P450 in Bacillus subtilis 168

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-20 DOI: 10.1016/j.enzmictec.2025.110587

Thanaporn Wichai , Sarintip Sooksai , Sajee Noitang , Alisa S. Vangnai , Panaya Kotchaplai

Inducer-free expression systems are promising tools for biorefinery because they can reduce the reliance on inducers, reducing production costs and simplifying processes. Owing to their broad range of substrate structures and catalytic reactions, cytochrome P450s are promising biocatalysts to produce value-added compounds. However, unsuitable levels of cytochrome P450 expression could result in cell stress, affecting the efficiency of the biocatalyst. Here, we assessed the potential of Pylb, a reported growth-phase-dependent promoter derived from Bacillus subtilis 168, to develop an inducer-free expression system, especially cytochrome P450 expression, in B. subtilis, a key workhorse strain. Utilizing a green fluorescent protein (GFP) reporter, we observed differential expression patterns under the control of Pylb and the constitutive promoter P43 in recombinant Escherichia coli and B. subtilis. Recombinant B. subtilis cultivated at 37 °C showed 2.8-fold higher bacterial fluorescence compared to cultivation at 30 °C. Codon-optimized engineered P450-BM3, which can convert octane to octanols, was selected as a model cytochrome P450 in this study. In the Pylb-based system, the expression of cytochrome P450 in recombinant B. subtilis can be detected at 24 h and increases over time as shown by the purpald assay. The activity of the overexpressed P450 was confirmed by the conversion of octane to octanols. Within one hour, the resting cells of recombinant B. subtilis produced 0.15 ± 0.04 mM of 1-octanol and 0.31 ± 0.08 mM of 2-octanol. Overall, the inducer-free Pylb-based system developed here is a potential biocatalyst for biorefinery applications.

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引用次数: 0

Unraveling the role of E. coli and calf intestinal alkaline phosphatase in calcium phosphate synthesis

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-14 DOI: 10.1016/j.enzmictec.2025.110586

Maria G. Chernysheva, Georgii S. Mikhailov, Daria-Maria V. Ratova, Ivan V. Mikheev, Gennadii A. Badun, Alexander L. Nikolaev

The enzyme-catalyzed synthesis of calcium phosphate is a promising method for producing calcium-based nanomaterials for biomedical applications. The purpose of this work was to determine the type of phosphate that forms when alkaline phosphatase catalyzes the reaction, and to identify the role of natural biopolymers in calcium phosphate formation. In this research, we analyzed calcium phosphates that were synthesized in the presence of alkaline phosphatase from either E. coli or calf intestinal, analyzed the obtained nanoparticles and compared them by functional composition, elemental ratio, and morphology. Since all syntheses were performed in Tris buffer with the addition of MgCl₂, the final depleted hydroxyapatite incorporated magnesium. It was found that in the first 24 h, the reaction product form is determined by the enzyme source as well as the presence of other biopolymers (in particular, humic acid) in the reaction mixture. Hollow nanospheres of the depleted hydroxyapatite were obtained as a final product for both E. coli and calf-intestinal alkaline phosphatase during a 7-day reaction. When humic acid was added into the reaction mixture, separate spheres of the depleted hydroxyapatite were observed during a 24-h reaction. When Mg ions are present in the reaction mixture as a buffer component, they are evenly incorporated into the structure of the resulting calcium phosphate. The data obtained can be useful in understanding the calcification process of bioobjects and in applying the enzymatic method of calcium phosphate synthesis to biomedical applications.

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引用次数: 0

Isolation of an endophytic yeast for improving the antibacterial activity of water chestnut Jiaosu: Focus on variation of microbial communities 一株内生酵母菌的分离提高荸荠皂素的抑菌活性——以微生物群落的变化为重点。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-13 DOI: 10.1016/j.enzmictec.2025.110584

khadija EI Galai , Wenna Dai , Cheng Qian , Jing Ye , Qin Zhang , Mengdie Gao , Xinyu Yang , Yanbin Li

Recent years have seen an increase in the development of functional Jiaosu products, including eco-friendly Jiaosu and antimicrobial healthcare fermentation products. As a result, research on the antibacterial activity of Jiaosu has attracted attention. In the present study, the endophytic yeast WCF016, which exhibits antibacterial activity against Escherichia coli and Staphylococcus aureus, was isolated from the peel of water chestnut and identified as Candida sake via morphological and phylogenetic analyses based on 26S rDNA D1/D2 region sequencing. Water chestnut Jiaosu with or without WCF016 inoculation exhibited similar flavor and physicochemical properties. However, inoculation significantly enhanced the antibacterial activity of water chestnut Jiaosu, especially in group D (inoculate of both fruit and vegetable enzyme starter and WCF016), which showed the largest diameter in its inhibition zone for both E. coli and S. aureus, reaching 25 ± 0 mm and 24 ± 1.0 mm. Moreover, inoculation with WCF016 influenced the abundance of the microbial community, especially Lactiplantibacillus and Zygoascus, which reached 51.76 % and 24.46 %, respectively, in group B (inoculated WCF016), thereby improving the antibacterial activity and flavor quality of the water chestnut Jiaosu. Notably, final pH, total sugar, and all organic acids effectively promoted fungal diversity and exhibited a positive correlation with most of the fungal genera. These results indicate that conditions conducive to the formation of organic acid-producing microbes and the synthesis of organic acids promote the antibacterial activity of Jiaosu.

近年来，功能性胶素产品的开发有所增加，包括环保胶素和抗菌保健发酵产品。因此，对胶素抗菌活性的研究引起了人们的关注。本研究从荸荠果皮中分离到一株对大肠杆菌和金黄色葡萄球菌具有抗菌活性的内生酵母菌WCF016，通过26S rDNA D1/D2区序列的形态和系统发育分析，鉴定为念珠菌清（Candida sake）。接种WCF016或未接种WCF016的荸荠胶苏具有相似的风味和理化特性。而接种荸荠胶素显著增强了荸荠胶素的抑菌活性，特别是D组（同时接种果蔬酶发酵剂和WCF016），其对大肠杆菌和金黄色葡萄球菌的抑菌区直径最大，分别达到25 ± 0 mm和24 ± 1.0 mm。此外，接种WCF016后，B组（接种WCF016）的微生物群落丰度显著提高，其中乳杆菌和Zygoascus的丰度分别达到51.76 %和24.46 %，从而提高了荸荠角素的抑菌活性和风味品质。最终pH、总糖和所有有机酸均能有效促进真菌多样性，并与大多数真菌属呈正相关。上述结果表明，有利于产有机酸微生物形成和有机酸合成的条件对胶素的抑菌活性有促进作用。

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引用次数: 0

Cell-free biocatalysis for co-production of nicotinamide mononucleotide and ethanol from Saccharomyces cerevisiae and recombinant Escherichia coli 酿酒酵母和重组大肠杆菌协同生产烟酰胺单核苷酸和乙醇的无细胞生物催化研究。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-12 DOI: 10.1016/j.enzmictec.2025.110585

Anoth Maharjan , Mamata Singhvi , Beom Soo Kim

Cell-free enzyme systems have emerged as a promising approach for producing various biometabolites, offering several advantages over traditional whole-cell systems. This study presents an approach to producing nicotinamide mononucleotide (NMN) by combining a Saccharomyces cerevisiae cell-free enzyme with a recombinant Escherichia coli cell-free enzyme. The system leverages the ATP generated by yeast during ethanol fermentation to produce NMN in the presence of nicotinamide (NAM) as a substrate. The optimal cell-free enzyme concentration and substrate concentration were investigated to maximize NMN production. The results showed that combined cell-free enzymes led to increased NMN and ethanol yields, with a maximum production of 1.5 mM NMN (2.7-fold) and ethanol production of 0.45 g/L achieved (1.6-fold) compared to individual cell-free enzymes. Furthermore, the study demonstrated that the protein concentration affected NMN production, with optimal production achieved at 5 g/L. This study demonstrates the potential of integrating multiple metabolic pathways in a single cell-free system, paving the way for the development of more efficient and sustainable bioproduction processes.

无细胞酶系统已经成为生产各种生物代谢物的一种有前途的方法，与传统的全细胞系统相比，它提供了几个优点。本研究提出了一种将酿酒酵母无细胞酶与重组大肠杆菌无细胞酶结合生产烟酰胺单核苷酸（NMN）的方法。该系统利用酵母在乙醇发酵过程中产生的ATP在烟酰胺（NAM）作为底物存在的情况下产生NMN。研究了最佳的无细胞酶浓度和底物浓度，以最大限度地提高NMN的产量。结果表明，与单个无细胞酶相比，组合无细胞酶可提高NMN和乙醇产量，最大产量为1.5 mM NMN（2.7倍），乙醇产量为0.45 g/L（1.6倍）。此外，研究表明，蛋白质浓度影响NMN的产量，最佳产量达到5 g/L。这项研究展示了在一个单一的无细胞系统中整合多种代谢途径的潜力，为开发更有效和可持续的生物生产过程铺平了道路。

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引用次数: 0

Effect of the support alkyl chain nature in the functional properties of the immobilized lipases 载体烷基链性质对固定化脂肪酶功能性质的影响。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-11 DOI: 10.1016/j.enzmictec.2025.110583

Diandra de Andrades , Pedro Abellanas-Perez , Javier Rocha-Martin , Fernando Lopez-Gallego , Andrés R. Alcántara , Maria de Lourdes Teixeira de Moraes Polizeli , Roberto Fernandez-Lafuente

Supports coated with amino-hexyl and amino octyl have been prepared from glyoxyl agarose beads and compared in their performance with octyl-agarose to immobilize lipases A and B from Candida antarctica (CALA and CALB). Immobilization courses were similar using all supports, but enzyme release was more difficult using the amino-alkyl supports suggesting a mixed interfacial activation/ionic exchange immobilization. The enzyme activity and specificity (using p-nitrophenyl propionate, triacetin and both isomers of methyl mandelate) greatly depended on the support. In many instances the enzymes immobilized on the new supports offered higher activities and enantiospecificity in the hydrolysis of both enantiomers of methyl mandelate (mainly using CALB). This was coupled to a lower enzyme stability using the new supports, even in the presence of high ionic strength, suggesting that the amphipathic could be responsible of the enzyme lower stability. Using CALB, it was possible to detect a higher exposition of the enzyme Trp groups to the medium by florescence spectra after its immobilization on the amino-alkyl-supports, correlating to the higher activity and lower stability results.

以乙氧基琼脂糖珠为原料制备了氨基己基和氨基辛基包被载体，并与辛基琼脂糖对南极假丝酵母脂肪酶A和脂肪酶B的固定化性能进行了比较。所有载体的固定化过程相似，但使用氨基烷基载体的酶释放更困难，表明混合界面活化/离子交换固定化。酶的活性和特异性（使用对硝基苯基丙酸酯、三乙酸酯和曼德尔酸甲酯的两种异构体）很大程度上取决于载体。在许多情况下，固定在新载体上的酶在水解曼德尔酸甲酯的两种对映体时具有更高的活性和对映体特异性（主要使用CALB）。这与使用新载体时较低的酶稳定性相结合，即使在存在高离子强度的情况下，这表明两亲性可能是酶稳定性较低的原因。使用CALB，可以通过荧光光谱检测到在氨基烷基载体上固定后，酶Trp基团对培养基的暴露程度较高，这与较高的活性和较低的稳定性结果相关。

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引用次数: 0

Biosynthesis of 2-phenylethanol from styrene using engineered Escherichia coli whole cells 利用工程大肠杆菌全细胞从苯乙烯生物合成2-苯乙醇。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-08 DOI: 10.1016/j.enzmictec.2025.110582

Tianzhen Xiong , Qiuyue Gao , Wei Liu , Wei Li , Guangyan Fan

2-Phenylethanol, an aromatic alcohol with a rose scent, is widely used in the cosmetics, food, and pharmaceutical industries. We designed an efficient multi-enzyme cascade pathway for production of 2-phenylethanol from styrene as the substrate. Initially, 2-phenylethanol was produced by overexpression of styrene monooxygenase A (styA), styrene monooxygenase B (styB), styrene oxide isomerase (SOI), alcohol dehydrogenase (yahK), and glucose dehydrogenase (gdh) in Escherichia coli to give 6.28 mM 2-phenylethanol. Subsequently, plasmids with different copy numbers were employed to balance the expression of pathway enzymes to produce 10.28 mM 2-phenylethanol, resulting in a 63.7 % increase in the final yield. Furthermore, the pH and temperature of the whole-cell conversion reaction were optimized, the optimum pH and temperature are 7.5 and 35℃, respectively. Finally, whole-cell conversion experiment was conducted, and the production of 2-phenylethanol reached 48.17 mM within 10 h. This study provides a theoretical and practical foundation for production of 2-phenylethanol.

2- 苯乙醇是一种具有玫瑰香味的芳香醇，广泛应用于化妆品、食品和制药行业。我们设计了一种以苯乙烯为底物生产 2-苯乙醇的高效多酶级联途径。最初，通过在大肠杆菌中过表达苯乙烯单加氧酶 A（styA）、苯乙烯单加氧酶 B（styB）、苯乙烯氧化异构酶（SOI）、醇脱氢酶（yahK）和葡萄糖脱氢酶（ghdh）来生产 2-苯乙醇，得到 6.28 mM 的 2-苯乙醇。随后，利用不同拷贝数的质粒平衡途径酶的表达，生产出 10.28 毫摩尔的 2-苯乙醇，最终产量增加了 63.7%。此外，还对全细胞转化反应的 pH 值和温度进行了优化，最佳 pH 值和温度分别为 7.5 和 35℃。最后，进行了全细胞转化实验，10 h 内 2-苯基乙醇的产量达到 48.17 mM。这项研究为生产 2-苯基乙醇提供了理论和实践基础。

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引用次数: 0

Analysis of enzyme kinetics of fungal methionine synthases in an optimized colorimetric microscale assay for measuring cobalamin-independent methionine synthase activity 真菌蛋氨酸合成酶动力学的优化比色微标法测定钴胺非依赖性蛋氨酸合成酶活性。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-04 DOI: 10.1016/j.enzmictec.2025.110581

Noël Jung, Tomás Vellozo-Echevarría, Kristian Barrett, Anne S. Meyer

Aspergillus spp. and Rhizopus spp., used in solid-state plant food fermentations, encode cobalamin-independent methionine synthase activity (MetE, EC 2.1.1.14). Here, we examine the enzyme kinetics, reaction activation energies (E_a), thermal robustness, and structural folds of three MetEs from three different food-fermentation relevant fungi, Aspergillus sojae, Rhizopus delemar, and Rhizopus microsporus, and compare them to the MetE from Escherichia coli. We also downscaled and optimized a colorimetric assay to allow direct MetE activity measurements in microplates. The catalytic rates, k_cat, of the three fungal MetE enzymes on the methyl donor (6S)-5-methyl-tetrahydropteroyl-L-glutamate₃ ranged from 1.2 to 3.3 min⁻¹ and K_M values varied from 0.8 to 6.8 µM. The k_cat was lowest for the R. delemar MetE, but this enzyme also had the lowest K_M thus resulting in the highest k_cat/K_M of ∼1.4 min⁻¹ µM⁻¹ among the three fungal enzymes. The k_cat was higher for the E. coli enzyme, 12 min⁻¹, but K_M was 6.4 µM, resulting in k_cat/K_M of ∼1.9 min⁻¹ µM⁻¹. The E_a values of the fungal MetEs ranged from 52 to 97 kJ mole⁻¹ and were higher than that of the E. coli MetE (38.7 kJ mole ⁻¹). The predicted structural folds of the MetEs were very similar. T_m values of the fungal MetEs ranged from 41 to 54 °C, highest for the A. sojae enzyme (54 °C), lowest for the R. delemar (41 °C). At 30 °C, the half-lives of the three fungal enzymes varied significantly, with MetE from A. sojae having the longest (> 600 min, k_D=0), and R. delemar the shortest (17 min). Knowledge of the kinetics of these enzymes is important for understanding methionine synthesis in fungi and a first step in promoting methionine synthesis in fungally fermented plant foods.

曲霉（Aspergillus spp.）和根霉（Rhizopus spp.）用于固态植物食品发酵，编码钴胺不依赖蛋氨酸合成酶活性（MetE, EC 2.1.1.14）。在这里，我们研究了来自大豆曲霉、delemar根霉和小孢子根霉这三种不同的食物发酵相关真菌的三种MetE的酶动力学、反应活化能（Ea）、热稳健性和结构折叠，并将它们与来自大肠杆菌的MetE进行了比较。我们还缩小并优化了比色法，以允许在微孔板上直接测量MetE活性。3种真菌MetE酶对甲基供体(6S)-5-甲基-四氢蝶酰- l- glutamate3的催化速率kcat为1.2 ~ 3.3 min-1， KM值为0.8 ~ 6.8 µM。在3种真菌酶中，R. delemar MetE的kcat最低，但KM也最低，因此kcat/KM最高，为~ 1.4 min-1µM-1。大肠杆菌酶的kcat较高，为12 min-1，但KM为6.4 µM，因此kcat/KM为~ 1.9 min-1µM-1。真菌MetE的Ea值为52 ~ 97 kJ mol -1，高于大肠杆菌MetE的38.7 kJ mol -1。预测的MetEs构造褶皱非常相似。真菌MetEs的Tm值在41 ~ 54°C之间，其中A. sojae酶最高（54°C）， R. delemar最低（41°C）。在30℃时，3种真菌酶的半衰期差异显著，其中黄豆霉的MetE酶的半衰期最长（600 min, kD=0）， delemar酶的半衰期最短（17 min）。了解这些酶的动力学对于理解真菌中蛋氨酸的合成是很重要的，也是促进真菌发酵植物性食品中蛋氨酸合成的第一步。

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引用次数: 0

Engineering glycolytic pathway for improved Lacto-N-neotetraose production in pichia pastoris 改良毕赤酵母生产乳酸-n -新四糖的工程糖酵解途径。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-25 DOI: 10.1016/j.enzmictec.2024.110576

Jiao Yang , Nitesh Kumar Mund , Lirong Yang , Hao Fang

Lacto-N-neotetraose (LNnT) is a primary solid component of human milk oligosaccharides (HMOs) with various promising health effects for infants. LNnT production by GRAS (generally recognized as safe) microorganisms has attracted considerable attention. However, few studies have emphasized Pichia Pastoris as a cell factory for LNnT’s production. Here, we have reported the first-ever synthesis of LNnT employing P. pastoris as the host. Initially, LNnT biosynthetic pathway genes β-1,3-N-acetylglucosaminyltransferase (lgtA) and β-1,4-galactostltransferase (lgtB) along with lactose permease (lac12) and galactose epimerase (gal10) were integrated into the genome of P. pastoris, but only 0.139 g/L LNnT was obtained. Second, the titer of LNnT was improved to 0.162 g/L via up-regulating genes to strengthen the supply of precursors, UDP-GlcNAc (Uridine diphosphate N-acetylglucosamine) and UDP-Gal (Uridine diphosphate galactose), for LNnT biosynthesis. Third, by knocking out critical mediator pfk (6-phosphofructokinase) genes in glycolysis, the major glucose metabolic flux was rewired to the LNnT biosynthesis pathway. As a result, the strain accumulated 0.867 g/L LNnT in YPG medium supplemented with glucose and lactose. Finally, LNnT production was increased to 1.24 g/L in a 3 L bioreactor. The work aimed to explore the potential of P. pastoris as a for LNnT production.

乳-n -新四糖（LNnT）是人乳寡糖（HMOs）的主要固体成分，对婴儿的健康具有多种前景。GRAS（通常被认为是安全的）微生物生产LNnT引起了相当大的关注。然而，很少有研究强调毕赤酵母是生产LNnT的细胞工厂。在这里，我们报道了首次以pastoris为宿主合成LNnT。最初将LNnT生物合成途径基因β-1,3- n -乙酰氨基葡萄糖转移酶（lgtA）和β-1,4-半乳糖转移酶（lgtB）以及乳糖渗透酶（lac12）和半乳糖外聚酶（gal10）整合到P. pastoris基因组中，但LNnT仅为0.139 g/L。其次，通过上调基因将LNnT的滴度提高到0.162 g/L，以加强LNnT生物合成的前体UDP-GlcNAc（二磷酸尿苷n -乙酰氨基葡萄糖）和UDP-Gal（二磷酸尿苷半乳糖）的供应。第三，通过敲除糖酵解中的关键介质pfk（6-磷酸果糖激酶）基因，将主要的葡萄糖代谢通量重新连接到LNnT生物合成途径。结果表明，菌株在添加葡萄糖和乳糖的YPG培养基中积累了0.867 g/L LNnT。最后，在3 L的生物反应器中，LNnT产量提高到1.24 g/L。这项工作旨在探索P. pastoris作为LNnT生产的潜力。

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引用次数: 0

Structures and properties of α-amylase and glucoamylase immobilized by ZIF-8 via one-pot preparation 用 ZIF-8 单锅制备固定α-淀粉酶和葡萄糖淀粉酶的结构和特性。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-25 DOI: 10.1016/j.enzmictec.2024.110579

Yuxin Liu, Qinghua Pan, Zesheng Liang, Jingqiao Li, Rulong Wu

The immobilization of α-amylase and glucoamylase using a metal-organic framework (enzyme@ZIF-8) was prepared in situ through a one-pot method. The morphology, crystal structure, and molecular characteristics of the free enzyme and enzyme@ZIF-8 were characterized. The enzyme@ZIF-8 exhibited the rhombic dodecahedron morphology, with a decrease in particle size. Successful immobilization of α-amylase and glucoamylase within ZIF-8 was confirmed, with 30–40 % loading rate. The immobilization process did not significantly alter the crystal structure of ZIF-8. The changes in secondary structure of enzyme after immobilization resulted in modiﬁcation of catalytic activity of enzyme. The melting enthalpy of enzyme @ZIF-8 increased with the increase of enzyme content. The melting peak temperature of the enzyme immobilized by ZIF-8 increased. The activity of free and immobilized enzymes was influenced by the different time, pH and temperature. At pH 5–8 and temperature 60–80 °C, the activity of the immobilized enzyme was significantly greater than that of the free enzyme. The repeatability of enzyme@ZIF-8 was 61.52 % after three cycles. The kinetic parameters of Michaelis-Menten model for enzymatic reaction were determined by fitting the initial rate of reactions and initial substrate concentration data. The Michaelis-Menten constant (K_M) values of immobilized enzyme were lower than that of free enzyme, indicating the greater affinity between the enzyme and the substrate.

用金属有机骨架（enzyme@ZIF-8）原位固定化α-淀粉酶和葡萄糖淀粉酶。对游离酶和enzyme@ZIF-8的形态、晶体结构和分子特性进行了表征。enzyme@ZIF-8呈菱形十二面体形态，粒径减小。证实了α-淀粉酶和葡萄糖淀粉酶在ZIF-8内的固定成功，负载率为30-40 %。固定过程没有显著改变ZIF-8的晶体结构。固定化后酶的二级结构发生变化，导致酶的催化活性发生改变。酶@ZIF-8的熔化焓随酶含量的增加而增大。ZIF-8固定化酶的熔化峰温度升高。游离酶和固定化酶的活性受时间、pH和温度的影响。在pH 5 ~ 8、温度60 ~ 80℃条件下，固定化酶的活性显著高于游离酶。3个循环后enzyme@ZIF-8的重复性为61.52 %。通过拟合初始反应速率和初始底物浓度，确定了Michaelis-Menten模型的动力学参数。固定化酶的Michaelis-Menten常数（KM）值低于游离酶，说明固定化酶与底物的亲和力较强。

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引用次数: 0

Identification of a bacteria P450 enzyme from B. megaterium H-1 with vitamin D3 C-25 hydroxylation capabilities 具有维生素D3 C-25羟基化能力的巨型芽孢杆菌H-1细菌P450酶的鉴定。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-24 DOI: 10.1016/j.enzmictec.2024.110578

Yulin He , Yina Hou , Hui Li , Fan He , Jingyi Zhou , Xiaomei Zhang , Jingsong Shi , Zhenghong Xu

Calcidiol (25(OH)VD₃) and calcitriol (1α,25(OH)₂VD₃) are active vitamin D₃ with high medicinal value, which can maintain calcium and phosphorus balance and treat vitamin D deficiency. Microbial synthesis is an important method to produce high-value-added compounds. It can produce active vitamin D₃ through the hydroxylation reaction of P450, which can reduce the traditional chemical synthesis steps, and greatly improve the production efficiency and economic benefits. In this work, Bacillus megaterium H-1 was screened for its ability to produce 25(OH)VD₃ and 1α,25(OH)₂VD₃ from vitamin D₃. A new highly inducible vitamin D₃ hydroxylase CYP109E1-H was identified from B. megaterium H-1 through searching for transcripts with cytochrome P450 structural domains, combining the transcriptome sequencing with functional expression in Bacillus subtilis WB600. Biotransformation in recombinant B. subtilis confirmed that CYP109E1-H has C-25 hydroxylase activity towards vitamin D₃. CYP109E1-H is a natural mutant of CYP109E1 with greater stereoselectivity and it is a new vitamin D₃ mono-hydroxylase. The cloning and characterization of the CYP109E1-H gene provide useful information on the structural basis for improving the regional and stereoselectivity of the CYP109E gene.

钙二醇（25(OH)VD3）和骨化三醇（1α,25(OH)2VD3）是具有较高药用价值的活性维生素D3，可维持钙磷平衡，治疗维生素D缺乏症。微生物合成是生产高附加值化合物的重要方法。通过P450的羟基化反应可以生产活性维生素D3，减少了传统的化学合成步骤，大大提高了生产效率和经济效益。本研究筛选了巨芽孢杆菌H-1从维生素D3中产生25(OH)VD3和1α，25(OH)2VD3的能力。通过寻找含有细胞色素P450结构域的转录本，结合转录组测序和枯草芽孢杆菌WB600的功能表达，从巨芽孢杆菌H-1中鉴定出一个新的高诱导性维生素D3羟化酶CYP109E1-H。在重组枯草芽孢杆菌中的生物转化证实了CYP109E1-H对维生素D3具有C-25羟化酶活性。CYP109E1- h是CYP109E1的天然突变体，具有较高的立体选择性，是一种新的维生素D3单羟化酶。CYP109E1-H基因的克隆和鉴定为提高CYP109E基因的区域选择性和立体选择性提供了有益的结构依据。

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