Biochemical Engineering Journal最新文献_第6页

Long lasting degradation of all alkanes in soil by Pseudomonas activated after Fenton pre-oxidation 经 Fenton 预氧化处理的假单胞菌对土壤中所有烷烃的长效降解作用

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-10-02 DOI: 10.1016/j.bej.2024.109511

Jinlan Xu , Yikai Li , Mengzhen Gao , Jianan Dai , Huan Li , Jiayi Wang

This study investigated the function and mechanism of Fenton pre-oxidation on the long lasting degradation of all alkanes in soil contaminated by petroleum. The findings demonstrated that the biological removal amount of all alkanes in the respiratory regulation group reached 4083.46 mg/kg, which was twice that of the non-regulation group, and the removal amount gradually increased in the four stages of bioremediation. In addition, the removal amount of all alkanes in the non-regulated group did not change much and showed a downward trend, indicating that long lasting degradation of all alkanes could be achieved by the respiratory regulation group, and the biodegradation cycle was saved by 251 days compared with the non-regulated group. Furthermore, the total number of bacteria in the respiratory regulation group (6.73 log CFU/g) was significantly higher than that in the non-regulation group (2.25 log CFU/g). Pseudomonas became the dominant genus in the respiratory regulation group with an average relative abundance of 32.17 %. In the respiratory regulation group, a large amount of ammonia nitrogen (1703.62 mg/kg) was consumed during the degradation process, which stimulated the tricarboxylic acid cycle respiratory metabolism process of Pseudomonas and accelerated the hydrocarbon conversion. This may be the reason why the long lasting degradation of all alkanes in soil could be achieved by the respiratory regulation group.

本研究探讨了 Fenton 预氧化作用对石油污染土壤中所有烷烃的长效降解作用及其机理。研究结果表明，呼吸调节组对所有烷烃的生物去除量达到 4083.46 mg/kg，是非调节组的两倍，并且在生物修复的四个阶段中去除量逐渐增加。此外，非调节组对所有烷烃的去除量变化不大，呈下降趋势，这表明呼吸调节组可实现对所有烷烃的长效降解，生物降解周期比非调节组节省了 251 天。此外，呼吸调节组的细菌总数（6.73 log CFU/g）明显高于非调节组（2.25 log CFU/g）。假单胞菌成为呼吸调节组的优势菌属，平均相对丰度为 32.17%。呼吸调节组在降解过程中消耗了大量氨氮（1703.62 mg/kg），刺激了假单胞菌的三羧酸循环呼吸代谢过程，加速了碳氢化合物的转化。这可能就是呼吸调节组能够长期降解土壤中所有烷烃的原因。

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引用次数: 0

Natural biocide-assisted ultrasonic disinfection of wastewater effluent following a response surface methodology approach 采用响应面方法对废水进行天然杀菌剂辅助超声波消毒

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-10-01 DOI: 10.1016/j.bej.2024.109517

Arkadeep Mukherjee, Young-Ho Ahn

The increasing prevalence of multidrug-resistant (MDR) bacteria in wastewater poses a significant threat to public health and the environment, necessitating more effective and sustainable disinfection methods. Ozonation and chlorination frequently fall short of eliminating these bacteria and can create toxic byproducts. This study introduces a novel disinfection strategy that combines ultrasonication with tea tree oil to target MDR bacteria in residential wastewater treatment systems, aiming to provide an eco-friendly, efficient, and scalable solution. The method harnesses tea tree oil's natural biocidal properties alongside the physical effects of ultrasonication, particularly acoustic cavitation, to enhance bacterial inactivation. Temperature, biocide dosage, and ultrasonication power were the three main factors that were optimized using response surface methodology. The system achieved a 2.2–2.4 log CFU/mL reduction of total bacteria in secondary effluent within 30 min and complete disinfection of modified effluent inoculated with high-strength MDR bacteria (6-log CFU/mL) in 50 min. Optimal conditions were 698.4 Watt power, 1.234 µl/mL tea tree oil, and 20.64 °C. Nucleic acid release and respiratory chain dehydrogenase inhibition indicated bacterial cell membrane rupture. Regrowth tests showed long-term effectiveness, with no bacterial colonies after three days. Using a natural biocide, the hybrid technique reduces operational costs and time, thus having commercial and environmental benefits. The capacity to remove MDR bacteria makes it an attractive contender for large-scale wastewater treatment.

废水中的耐多药（MDR）细菌日益增多，对公共卫生和环境构成了严重威胁，因此需要采用更有效、更可持续的消毒方法。臭氧消毒和氯化消毒往往无法消除这些细菌，而且会产生有毒的副产品。本研究介绍了一种新颖的消毒策略，它将超声波与茶树油相结合，针对住宅污水处理系统中的 MDR 细菌，旨在提供一种环保、高效和可扩展的解决方案。该方法利用茶树油的天然杀菌特性和超声波的物理效应，尤其是声空化效应，来提高细菌灭活效果。温度、杀菌剂用量和超声功率是利用响应面方法进行优化的三个主要因素。该系统可在 30 分钟内使二级污水中的细菌总数减少 2.2-2.4 log CFU/mL，并在 50 分钟内完全消毒接种了高强度 MDR 细菌（6-log CFU/mL）的改良污水。最佳条件为 698.4 瓦功率、1.234 微升/毫升茶树油和 20.64 °C。核酸释放和呼吸链脱氢酶抑制表明细菌细胞膜破裂。重新生长测试表明，该方法长期有效，三天后就没有细菌菌落了。混合技术使用天然杀菌剂，降低了操作成本，缩短了操作时间，因此具有商业和环境效益。该技术能够去除 MDR 细菌，因此成为大规模废水处理的有力竞争者。

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引用次数: 0

Fermentative production of 3-hydroxypropionic acid by using metabolically engineered Klebsiella pneumoniae strains 利用代谢工程肺炎克雷伯菌株发酵生产 3-羟基丙酸

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-10-01 DOI: 10.1016/j.bej.2024.109516

Chan Woo Song, Mina Kwon, Jong Myoung Park, Hyohak Song

3-Hydroxypropionic acid (3-HP) is an industrially important platform chemical for super-absorbent or biodegradable polymers. Its production via biological methods is expected to be more competitive than chemical methods. Klebsiella pneumoniae is the most promising host due to its innate capabilities for 3-HP and vitamin-B12 production, ease of culture, and ease of engineering. In this study, step-by-step metabolic engineering and fermentation technologies were used to enhance the production of 3-HP. K. pneumoniae-derived ydcW gene was overexpressed using a plasmid after screening candidate genes. Major competing pathways encoded by dhaT, yqhD, ldhA, glpK, poxB, and pta-ackA were blocked. Additionally, it was demonstrated that simultaneous reinforcement of two native aldehyde dehydrogenase encoded by the ydcW gene preferring NADPH and the puuC gene preferring NADH, synergistically improved 3-HP production. Additional reinforcement of the acs gene to reduce acetate accumulation resulted in 93.7 g/L of 3-HP with a yield of 0.42 g/g·glycerol over a 72-h fed-batch fermentation. This performance is deemed sufficient for industrial applications.

3-羟基丙酸（3-HP）是一种重要的工业平台化学品，可用于制造超吸收或生物降解聚合物。与化学方法相比，通过生物方法生产 3-HP 预计更具竞争力。肺炎克雷伯菌是最有前途的宿主，因为它具有生产 3-HP 和维生素-B12 的先天能力，易于培养和工程化。本研究采用逐步代谢工程和发酵技术来提高 3-HP 的产量。在筛选候选基因后，利用质粒过量表达了肺炎双球菌衍生的ydcW基因。由 dhaT、yqhD、ldhA、glpK、poxB 和 pta-ackA 编码的主要竞争途径被阻断。此外，研究还表明，同时加强由偏好 NADPH 的 ydcW 基因和偏好 NADH 的 puuC 基因编码的两种原生醛脱氢酶，可协同提高 3-HP 的产量。在 72 小时的喂料批次发酵过程中，通过进一步强化 acs 基因以减少乙酸酯的积累，3-HP 的产量为 93.7 克/升，甘油产量为 0.42 克/克。这一性能足以满足工业应用的需要。

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引用次数: 0

Enhanced S-adenosyl-L-methionine synthesis in Saccharomyces cerevisiae using metabolic engineering strategies 利用代谢工程策略提高酿酒酵母中 S-腺苷-L-蛋氨酸的合成能力

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-09-28 DOI: 10.1016/j.bej.2024.109504

Yuanshan Wang , Jinhao Wang , Zuoyu Huang , Liangzhuang Tan , Yuehan Zhang , Zhongce Hu , Zhiqiang Liu , Yuguo Zheng

S-adenosyl-L-methionine (SAM) plays pivotal roles in various physiological processes. With its increasing application in the treatment of diseases such as liver disease, depression, osteoarthritis and Alzheimer's, interest in SAM production aroused. Currently, Saccharomyces cerevisiae is the main industrial producer of SAM. With the surge in demand for SAM, improving the SAM biosynthesis is of importance. In this study, a multimodule engineering strategy was employed to improving SAM production: 1) Enhancing the gene expression of the sulfur assimilation pathway; 2) Strengthening the metabolic flux of the SAM synthesis pathway; 3) Weakening the SAM degradation pathway; 4) Increasing ATP supply. The resulting engineered mutant SC06 (S. cerevisiae CEN.PK2–1CΔgal80∷T_cyc1-sam2-P_gal1-P_gal10-met14-T_adh1, Δlsc2∷T_cyc1-hom6-P_gal1-P_gal10-met6-T_adh1, Δsah1Δmls1) displayed the highest SAM titer of 240.86 mg/L, which was 10.22-fold increase compared with the original strain. With optimized conditions, the SAM titer of mutant SC06 in shake flask fermentation reached 473.02 mg/L with a specific yield of 127.18 mg/g dry cell weight (DCW). In a 5 L fermenter with fed-batch fermentation, the maximal SAM yield of mutant SC06 reached 1.25 g/L with a specific yield of 166.67 mg/g DCW after 58 h cultivation. Therefore, the established metabolic engineering strategies displayed promising efficiency in improving the SAM productivity of S. cerevisiae CEN.PK2–1C, which may provide a useful tool for the improvement of SAM-producing strains.

S- 腺苷-L-蛋氨酸（SAM）在各种生理过程中发挥着关键作用。随着 SAM 在肝病、抑郁症、骨关节炎和老年痴呆症等疾病治疗中的应用日益广泛，人们对 SAM 的生产产生了浓厚的兴趣。目前，酿酒酵母是 SAM 的主要工业生产者。随着对 SAM 需求的激增，改善 SAM 的生物合成具有重要意义。本研究采用了多模块工程策略来提高 SAM 的产量：1) 增强硫同化途径的基因表达；2) 加强 SAM 合成途径的代谢通量；3) 削弱 SAM 降解途径；4) 增加 ATP 供应。结果发现，SC06（S. cerevisiae CEN.PK2-1CΔgal80∷Tcyc1-sam2-Pgal1-Pgal10-met14-Tadh1，Δlsc2∷Tcyc1-hom6-Pgal1-Pgal10-met6-Tadh1，Δsah1Δmls1）的SAM滴度最高，为240.86 mg/L，比原菌株提高了10.22倍。在优化条件下，突变体 SC06 在摇瓶发酵中的 SAM 滴度达到 473.02 mg/L，比产量为 127.18 mg/g（干细胞重量）。在 5 升的发酵罐中进行分批进行发酵，经过 58 小时的培养，突变体 SC06 的 SAM 产量达到 1.25 克/升，比产量为 166.67 毫克/克干细胞重量（DCW）。因此，已建立的代谢工程策略在提高 S. cerevisiae CEN.PK2-1C 的 SAM 产率方面表现出了良好的效率，可为 SAM 生产菌株的改良提供有用的工具。

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引用次数: 0

Advanced oxidation enhanced microbial electrolysis cell coupled with anaerobic digestion: A novel approach to coal gasification wastewater treatment 高级氧化强化微生物电解池与厌氧消化相结合：煤气化废水处理的新方法

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-09-27 DOI: 10.1016/j.bej.2024.109512

Yajie Li , Ou Wang , Yuyao Zhang , Weikang Kong , Nana Cheng , Salma Tabassum , Hongbo Liu

Coal gasification wastewater (CGW) contains a range of refractory and toxic organic pollutants with low biodegradability and significant biological toxicity. This study synthesized a hemin-graphene (H-graphene) catalyst for permonosulfate (PMS) activation. The MEC-AD (Microbial electrolysis cell- Anaerobic digestion) reactor (K1) was designated as the control group. The experimental groups were the MEC-AD reactor (K2) with PMS and H-graphene, and the MEC-AD reactor (K3) with advanced oxidation as a synthetic CGW pretreatment. The results demonstrated that the COD removal rates of K1, K2 and K3 reactors were 76.7 %, 79.5 % and 87.4 %, while the total phenol removal rates were 74.1 %, 90.1 % and 100 %, respectively. Quinoline and indole were removed at rates greater than 90 % in the microbial electrolytic cell reactors K2 and K3, and 100 % in the K3 reactor. In K2 and K3, there was a considerable decrease in the abundance of unclassified _ f _ Alcaligenaceae, Arenimonas and unclassified _ f _ Gracilibacteraceae as compared to K1. The abundance of unclassified _ p _ Zixibacteria, Candidatus _ Caldatribacterium, unclassified _ c _ JS1 and JGI-0000079-D21, which are responsible for promoting the anaerobic degradation of long-chain fatty acids and anaerobic fermentation of acid production increased.

煤气化废水（CGW）中含有一系列难降解的有毒有机污染物，生物降解性低，生物毒性大。本研究合成了一种用于过硫酸盐（PMS）活化的氨基石墨烯（H-graphene）催化剂。MEC-AD（微生物电解池-厌氧消化）反应器（K1）被指定为对照组。实验组为添加了 PMS 和 H-石墨烯的 MEC-AD 反应器（K2），以及添加了高级氧化作为合成 CGW 预处理的 MEC-AD 反应器（K3）。结果表明，K1、K2 和 K3 反应器的 COD 去除率分别为 76.7%、79.5% 和 87.4%，总酚去除率分别为 74.1%、90.1% 和 100%。在微生物电解池反应器 K2 和 K3 中，喹啉和吲哚的去除率超过 90%，在 K3 反应器中超过 100%。与 K1 反应器相比，K2 和 K3 反应器中未分类的 _ f _ Alcaligenaceae、Arenimonas 和未分类的 _ f _ Gracilibacteraceae 的数量大幅减少。负责促进长链脂肪酸厌氧降解和厌氧发酵产酸的未分类 _ p _ Zixibacteria、Candidatus _ Caldatribacterium、未分类 _ c _ JS1 和 JGI-0000079-D21 的数量有所增加。

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引用次数: 0

Cutting-edge computational approaches in enzyme design and activity enhancement 酶设计和活性增强的前沿计算方法

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-09-26 DOI: 10.1016/j.bej.2024.109510

Ruobin Sun, Dan Wu, Pengcheng Chen, Pu Zheng

Enzyme activity is crucial in biocatalysis, making methods to enhance enzyme performance a major focus of research. Computational design provides an efficient approach to boosting enzyme activity, thereby expanding its applications across various fields. This review highlights three main computational methods: molecular dynamics simulations, Rosetta, and machine learning, and explores recent advances in their use for rapidly enhancing enzyme activity in enzyme engineering. These techniques provide a novel perspective on enzyme activity optimization, significantly reducing the complexity of traditional screening processes. By integrating these advanced computational approaches, high-activity enzymes can be designed more rapidly, accelerating progress in protein engineering and synthetic biology.

酶活性在生物催化中至关重要，因此提高酶性能的方法成为研究的重点。计算设计提供了提高酶活性的有效方法，从而将其应用扩展到各个领域。本综述重点介绍了三种主要计算方法：分子动力学模拟、Rosetta 和机器学习，并探讨了在酶工程中使用这些方法快速提高酶活性的最新进展。这些技术为酶活性优化提供了新的视角，大大降低了传统筛选过程的复杂性。通过整合这些先进的计算方法，可以更快地设计出高活性酶，加快蛋白质工程和合成生物学的进展。

引用次数: 0

Recombinant beta-galactosidase derived from Enterobacter cloacae Zjut HJ2001 for efficient biotransformation of galactooligosaccharides 用于高效生物转化半乳寡糖的重组β-半乳糖苷酶，来源于泄殖腔肠杆菌 Zjut HJ2001

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-09-25 DOI: 10.1016/j.bej.2024.109514

Guangxue Wang, Jing Jiang , Lihong Liu, Jin Huang

Galactooligosaccharides (GOS), as a type of prebiotics, have excellent physical and chemical properties, and can be widely used in the pharmaceutical and food fields. Recently, the microbial β-galactosidases have gained widespread attentions in industrial GOS production. However, most β-galactosidases from microorganisms have low transgalactosylation activity, resulting in poor GOS yield of enzymatic transformation from lactose. In this paper, a brand new β-galactosidase derived from Enterobacter cloacae Zjut HJ2001 was screened out from soil, and successfully overexpressed, characterized, and mutated by combinatorial alanine-scanning and site-saturation mutagenesis. Compared to the yield of 51.73 % obtained by wild-type β-galactosidase with lactose concentration of 380 g/L, the obtained mutant β-gal-H542V achieved a higher GOS yield of 67.08 %, which was the highest in the reported literature. These results suggested that the developed mutagenesis strategy could improve the transgalactosylation efficiency, and the mutant β-gal-H542V could be regarded as a prospective biocatalyst for GOS industrial manufacturing.

半乳寡糖（GOS）作为一种益生元，具有优良的物理和化学特性，可广泛应用于医药和食品领域。近年来，微生物 β-半乳糖苷酶在工业化生产 GOS 方面受到广泛关注。然而，大多数微生物β-半乳糖苷酶的转半乳糖基化活性较低，导致从乳糖酶解转化的 GOS 产率较低。本文从土壤中筛选出了一种全新的β-半乳糖苷酶，并通过丙氨酸扫描和位点饱和突变的组合方法对其进行了成功的过表达、表征和突变。在乳糖浓度为 380 g/L 的条件下，野生型 β-半乳糖苷酶的产率为 51.73%，与之相比，所获得的突变体 β-gal-H542V 的 GOS 产率高达 67.08%，为文献报道的最高产率。这些结果表明，所开发的诱变策略可以提高转半乳糖基化的效率，突变体β-gal-H542V可被视为一种有望用于GOS工业生产的生物催化剂。

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引用次数: 0

Copper and zinc sulfides bioleaching by an extremely thermophilic archaeon in high NaCl concentration 高浓度氯化钠条件下极嗜热古生物对硫化铜和硫化锌的生物浸出作用

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-09-24 DOI: 10.1016/j.bej.2024.109509

Flávio Luiz Martins , Yago Costa Roberto , Versiane Albis Leão

Chloride bioleaching has received attention of several mineral processing industries, particularly in countries where there is scarcity of freshwater and only chloride-containing waters can be used. Therefore, the present work investigated the effect of NaCl (1.0 mol L⁻¹) on the bioleaching of three sulfide minerals: chalcopyrite, bornite, and sphalerite by the thermophilic archaea Sulfolobus acidocaldarius. Chalcopyrite dissolution was only 25 % in the biotic experiment in the absence of chloride, but reached 90 % in the presence of both microorganisms and chloride, while less than 60 % extraction was observed in the abiotic experiment with chloride. In the experiments of bornite bioleaching, 86 % and 77 % of copper were extracted in the biotic and abiotic tests with chloride, respectively. In the absence of NaCl, the biotic and abiotic experiments presented similar copper dissolution (∼35 %). Finally, bioleaching experiments carried out with sphalerite showed zinc extractions below 35 % in all conditions tested. The main contribution from the archaea was its ability to produce low concentrations of ferric ion, which was partially precipitated as jarosite, resulting in low redox potential values (< 450 mV vs. Ag/AgCl), and efficiently bioleached bornite and chalcopyrite. Furthermore, XRD and SEM-EDS analyses demonstrated that sphalerite was practically not leached while bornite was transformed into new copper sulfide phases (CuS and Cu₃FeS₄). Jarosite and elemental sulfur were products of chalcopyrite and bornite bioleaching in the presence of chloride.

氯化物生物浸出已受到多个矿物加工行业的关注，尤其是在淡水匮乏且只能使用含氯化物水的国家。因此，本研究调查了 NaCl（1.0 mol L-1）对嗜热古菌 Sulfolobus acidocaldarius 对黄铜矿、辉锑矿和闪锌矿这三种硫化矿物进行生物浸出的影响。在无氯化物的生物实验中，黄铜矿的溶解度仅为 25%，但在微生物和氯化物同时存在的情况下，溶解度达到了 90%，而在有氯化物的非生物实验中，黄铜矿的提取率不到 60%。在波来石生物浸出实验中，在有氯化物的生物和非生物试验中，铜的萃取率分别为 86% 和 77%。在没有氯化钠的情况下，生物实验和非生物实验的铜溶解度相似（35%）。最后，用闪锌矿进行的生物浸出实验表明，在所有测试条件下，锌的提取率都低于 35%。古细菌的主要贡献在于其产生低浓度铁离子的能力，铁离子部分沉淀为绿泥石，导致低氧化还原电位值（< 450 mV vs. Ag/AgCl），并有效地生物浸蚀了辉铜矿和黄铜矿。此外，XRD 和 SEM-EDS 分析表明，闪锌矿几乎没有被浸出，而辉铜矿则转变成了新的硫化铜相（CuS 和 Cu3FeS4）。箭石和元素硫是黄铜矿和辉铜矿在氯化物存在下生物浸出的产物。

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引用次数: 0

Assessment of in situ product recovery techniques to enhance 2-phenylethanol production by Acinetobacter soli ANG344B 评估原位产品回收技术，以提高固氮醋酸杆菌 ANG344B 的 2-苯基乙醇产量

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-09-23 DOI: 10.1016/j.bej.2024.109508

Ana R.S. Bernardino , Cristiana A.V. Torres , João G. Crespo , Maria A.M. Reis

The 2-phenylethanol (2-PE) production process by the newly isolated Acinetobacter soli ANG344B is limited by product toxicity. To overcome this limitation and enhance 2-PE production process, various alternatives based in in situ product removal (ISPR) approaches were evaluated. The approaches selected for assessment were gas stripping using the air supplied to the bioreactor, liquid-liquid extraction and adsorption. Adsorption was found to be the most promising approach to increase 2-PE production. Amberlite XAD 4 was chosen from the different adsorbents tested since it has high affinity for 2-PE, being able to adsorb 205.8 ± 8.1 mg_2-PE/g_{dry resin}. In a batch cultivation process, in presence of 3 % (dry w/v) of Amberlite XAD 4, A. soli ANG344B was able to produce 6.99 ± 0.06 g/L of 2-PE with a volumetric productivity of 0.17 ± 0.00 g/L.h, which represents an improvement of 3.3-fold. To the best of our knowledge, this is the highest 2-PE production reported for a wild-type bacteria. These findings highlight the potential of Acinetobacter soli ANG344B as 2-PE producer, contributing to the development of natural 2-PE production process.

新分离出的固态醋酸乙烯杆菌（Acinetobacter soli ANG344B）生产 2-苯基乙醇（2-PE）的过程受到了产物毒性的限制。为了克服这一局限性并改进 2-PE 生产工艺，我们评估了基于原位产物去除（ISPR）方法的各种替代品。选定用于评估的方法包括利用提供给生物反应器的空气进行气提、液液萃取和吸附。吸附法被认为是最有希望提高 2-PE 产量的方法。从测试的不同吸附剂中选择了 Amberlite XAD 4，因为它对 2-PE 有很高的亲和力，能够吸附 205.8 ± 8.1 mg2-PE/gdry 树脂。在批量培养过程中，在含有 3 %（干重/体积）Amberlite XAD 4 的情况下，A. soli ANG344B 能够生产 6.99 ± 0.06 g/L 的 2-PE，体积生产率为 0.17 ± 0.00 g/L.h，提高了 3.3 倍。据我们所知，这是野生型细菌的最高 2-PE 产量。这些发现凸显了单歧杆菌 ANG344B 作为 2-PE 生产者的潜力，有助于天然 2-PE 生产工艺的发展。

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引用次数: 0

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases 多糖合成糖基转移酶的无细胞表达和生化鉴定

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal

Pub Date : 2024-09-23 DOI: 10.1016/j.bej.2024.109507

Dharanidaran Jayachandran , Amar D. Parvate , Jory T. Brookreson , James E. Evans , Shishir P.S. Chundawat

Polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs) that play critical structural, metabolic, or functional roles in cells. Though GT-2 family has the maximum number of deposited gene sequences, the biochemistry is poorly understood due to enzyme production challenges. Here, we developed a cell-free expression (CFE) protocol to produce two GT-2 family representative cellulose synthase (PttCesA8 from Populus tremula x tremuloides) and hyaluronan synthase (SeHAS from Streptococcus equisimilis). The CFE products were obtained as reconstituted proteoliposomes directly at high yields and short processing times compared to other approaches. Enzyme expression was confirmed using SDS-PAGE and immunoblotting, while integration of GTs into lipid layers was observed using cryogenic electron microscopy. Both GTs were catalytically active with Michalis-Menten kinetic constants, K_m for PttCesA8, was 295.8 µM, and SeHAS was 321.51 µM (toward UDP N-Acetyl Glucosamine) and 207.88 µM (toward UDP Glucuronic Acid), respectively, and with UDP inhibiting both GTs. Mutation of conserved residues in SeHAS also confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, CFE methods enable expression of polysaccharide-synthesizing enzymes as proteoliposomes at high yields with relative ease for rapid biochemical and structural characterization studies.

纤维素和透明质酸等多糖是由膜结合的 2 族糖基转移酶（GTs）合成的，它们在细胞中发挥着关键的结构、代谢或功能作用。虽然GT-2家族拥有最多的基因序列，但由于酶的生产面临挑战，人们对其生物化学还知之甚少。在此，我们开发了一种无细胞表达（CFE）方案，用于生产两种 GT-2 家族的代表性纤维素合成酶（来自杨树 x tremuloides 的 PttCesA8）和透明质酸合成酶（来自马氏链球菌的 SeHAS）。与其他方法相比，CFE 产物以重组蛋白脂质体的形式直接获得，产量高，处理时间短。使用 SDS-PAGE 和免疫印迹法确认了酶的表达，同时使用低温电子显微镜观察了 GTs 与脂质层的整合。两种 GT 都具有催化活性，其 Michalis-Menten 动力常数为：PttCesA8 的 Km 为 295.8 µM，SeHAS 为 321.51 µM（对 UDP N-Acetyl 葡萄糖胺）和 207.88 µM（对 UDP 葡萄糖酸），UDP 对两种 GT 都有抑制作用。对 SeHAS 中保守残基的突变也证实了赖氨酸-139、谷氨酰胺-248 和苏氨酸-283 残基在透明质酸生物合成中的重要性。总之，CFE 方法能以蛋白脂质体的形式高产表达多糖合成酶，而且相对容易进行快速生化和结构表征研究。

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