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Light-mediated biosynthesis of size-tuned silver nanoparticles using Saccharomyces cerevisiae extract. 利用酿酒酵母提取物进行光介导的尺寸调整银纳米粒子的生物合成。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-07-14 DOI: 10.1007/s00449-024-03060-x
Lucia Colleselli, Mira Mutschlechner, Martin Spruck, Florian Albrecht, Oliver I Strube, Pamela Vrabl, Susanne Zeilinger, Harald Schöbel

Bio-based production of silver nanoparticles represents a sustainable alternative to commercially applied physicochemical manufacturing approaches and provides qualitatively highly valuable nanomaterials due to their narrow size dispersity, high stability and biocompatibility with broad application potentials. The intrinsic features of nanoparticles depend on size and shape, whereby the controlled synthesis is a challenging necessity. In the present study, the biosynthesis of size-tuned silver nanoparticles based on cell-free extracts of Saccharomyces cerevisiae DSM 1333 was investigated. Single parameter optimization strategies in phases of cultivation, extraction, and synthesis were performed to modify the nanoparticle scale and yield. Visible light was exploited as a tool in nanoparticle production. The influence of white light on the biosynthesis of silver nanoparticles was determined by using novel LED systems with the exposition of varying irradiation intensities and simultaneous performance of control experiments in the dark. Characterization of the resulting nanomaterials by spectrophotometric analysis, dynamic light scattering, scanning electron microscopy, and energy dispersive X-ray spectroscopy, revealed spherical silver nanoparticles with controlled, light-mediated size shifts in markedly increased quantities. Matching of irradiated and non-irradiated reaction mixtures mirrored the enormous functionality of photon input and the high sensitivity of the biosynthesis process. The silver nanoparticle yields increased by more than 90% with irradiation at 1.0 ± 0.2 mW cm - 2 and the reduction of particle dimensions was achieved with significant shifts of size-specific absorption maxima from 440 to 410 nm, corresponding to particle sizes of 130 nm and 100 nm, respectively. White light emerged as an excellent tool for nano-manufacturing with advantageous effects for modulating unique particle properties.

以生物为基础生产银纳米粒子是商业应用物理化学制造方法的一种可持续替代方法,由于其尺寸分散性小、稳定性高、生物相容性好,具有广泛的应用潜力,因此可提供质量上乘的高价值纳米材料。纳米粒子的固有特性取决于尺寸和形状,因此控制合成是一项具有挑战性的必要条件。本研究考察了基于无细胞萃取的酿酒酵母 DSM 1333 的尺寸调整银纳米粒子的生物合成。研究人员在培养、提取和合成阶段实施了单参数优化策略,以改变纳米粒子的规模和产量。可见光被用作纳米粒子生产的工具。通过使用新型 LED 系统进行不同强度的照射,并同时在黑暗中进行对照实验,确定了白光对银纳米粒子生物合成的影响。通过分光光度分析、动态光散射、扫描电子显微镜和能量色散 X 射线光谱对所产生的纳米材料进行表征,发现球形银纳米粒子的数量明显增加,其大小在光的作用下发生了可控的变化。辐照和非辐照反应混合物的匹配反映了光子输入的巨大功能性和生物合成过程的高灵敏度。在 1.0 ± 0.2 mW cm - 2 的辐照条件下,银纳米颗粒的产量增加了 90% 以上,颗粒尺寸的缩小是通过尺寸特异性吸收最大值从 440 纳米到 410 纳米的显著移动实现的,这分别对应于 130 纳米和 100 纳米的颗粒尺寸。白光是纳米制造的绝佳工具,具有调节颗粒独特性质的优势。
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引用次数: 0
Bioactive nanoparticles derived from marine brown seaweeds and their biological applications: a review. 从海洋褐藻中提取的生物活性纳米粒子及其生物应用:综述。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-06-10 DOI: 10.1007/s00449-024-03036-x
Juhi Puthukulangara Jaison, Balamuralikrishnan Balasubramanian, Jaya Gangwar, Manikantan Pappuswamy, Arun Meyyazhagan, Hesam Kamyab, Kuppusamy Alagesan Paari, Wen-Chao Liu, Mohammad Mahdi Taheri, Kadanthottu Sebastian Joseph

The biosynthesis of novel nanoparticles with varied morphologies, which has good implications for their biological capabilities, has attracted increasing attention in the field of nanotechnology. Bioactive compounds present in the extract of fungi, bacteria, plants and algae are responsible for nanoparticle synthesis. In comparison to other biological resources, brown seaweeds can also be useful to convert metal ions to metal nanoparticles because of the presence of richer bioactive chemicals. Carbohydrates, proteins, polysaccharides, vitamins, enzymes, pigments, and secondary metabolites in brown seaweeds act as natural reducing, capping, and stabilizing agents in the nanoparticle's synthesis. There are around 2000 species of seaweed that dominate marine resources, but only a few have been reported for nanoparticle synthesis. The presence of bioactive chemicals in the biosynthesized metal nanoparticles confers biological activity. The biosynthesized metal and non-metal nanoparticles from brown seaweeds possess different biological activities because of their different physiochemical properties. Compared with terrestrial resources, marine resources are not much explored for nanoparticle synthesis. To confirm their morphology, characterization methods are used, such as absorption spectrophotometer, X-ray diffraction, Fourier transforms infrared spectroscopy, scanning electron microscope, and transmission electron microscopy. This review attempts to include the vital role of brown seaweed in the synthesis of metal and non-metal nanoparticles, as well as the method of synthesis and biological applications such as anticancer, antibacterial, antioxidant, anti-diabetic, and other functions.

生物合成具有不同形态的新型纳米粒子对其生物能力具有良好的影响,因此在纳米技术领域引起了越来越多的关注。真菌、细菌、植物和藻类提取物中的生物活性化合物是合成纳米粒子的主要成分。与其他生物资源相比,褐藻中含有更丰富的生物活性化学物质,因此也可用于将金属离子转化为金属纳米粒子。褐藻中的碳水化合物、蛋白质、多糖、维生素、酶、色素和次生代谢物在纳米粒子的合成过程中起着天然还原剂、封盖剂和稳定剂的作用。在海洋资源中占主导地位的海藻约有 2000 种,但只有少数几种被报道用于纳米粒子的合成。生物合成的金属纳米粒子中含有生物活性化学物质,因此具有生物活性。从棕色海藻中生物合成的金属和非金属纳米粒子因其不同的理化性质而具有不同的生物活性。与陆地资源相比,海洋资源在纳米粒子合成方面的开发并不多。要确认其形态,需要使用吸收分光光度计、X 射线衍射、傅立叶变换红外光谱、扫描电子显微镜和透射电子显微镜等表征方法。本综述试图介绍褐藻在合成金属和非金属纳米粒子中的重要作用、合成方法和生物应用,如抗癌、抗菌、抗氧化、抗糖尿病和其他功能。
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引用次数: 0
The ability of selected fungal strains to produce carboxylesterase enzymes for biodegradation and use of bifenthrin insecticide as carbon source: in vitro and in silico approaches. 选定真菌菌株产生羧基酯酶的能力,以生物降解联苯菊酯杀虫剂并将其用作碳源:体外和硅学方法。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-07-19 DOI: 10.1007/s00449-024-03062-9
Hasnat Mueen, Rafiq Ahmad, Sabaz Ali Khan, Muhammad Shahzad, Ahmed Mahmoud Ismail, Hossam S El-Beltagi, M Jamal Hajjar, Hosny Hamed Kesba

Bifenthrin (BF) is a broad-spectrum type I pyrethroid insecticide that acts on insects by impairing the nervous system and inhibiting ATPase activity, and it has toxic effects on non-target organisms and high persistence in the environment. This study aimed to determine the potential of six different fungi, including Pseudozyma hubeiensis PA, Trichoderma reesei PF, Trichoderma koningiopsis PD, Purpureocillium lilacinum ACE3, Talaromyces pinophilus ACE4, and Aspergillus niger AJ-F3, to degrade BF. Three different concentrations of BF, including 0.1%, 0.2%, and 0.3% w/v, were used in the sensitivity testing that revealed a significant (p ≤ 0.01) impact of BF on fungal growth. Enzymatic assays demonstrated that both intracellular and extracellular carboxylesterases hydrolyzed BF with the enzymatic activity of up to 175 ± 3 U (μmol/min) and 45 ± 1 U, respectively. All tested fungi were capable of utilizing BF as a sole carbon source producing 0.06 ± 0.01 to 0.45 ± 0.01 mg dry biomass per mg BF. Moreover, the presence of PytH was determined in the fungi using bioinformatics tools and was found in A. niger, T. pinophilus, T. reesei, and P. lilacinum. 3D structures of the PytH homologs were predicted using AlphaFold2, and their intermolecular interactions with pyrethroids were determined using MOE. All the homologs interacted with different pyrethroids with a binding energy of lesser than - 10 kcal/mol. Based on the study, it was concluded that the investigated fungi have a greater potential for the biodegradation of BF.

联苯菊酯(BF)是一种广谱 I 型拟除虫菊酯杀虫剂,通过损害神经系统和抑制 ATP 酶活性作用于昆虫,对非靶标生物有毒性作用,在环境中具有高持久性。本研究旨在确定六种不同真菌降解 BF 的潜力,包括湖北假酵母菌 PA、雷氏毛霉菌 PF、科宁拟毛霉菌 PD、紫云英球菌 ACE3、嗜酸塔拉酵母菌 ACE4 和黑曲霉 AJ-F3。灵敏度测试中使用了三种不同浓度的 BF,包括 0.1%、0.2% 和 0.3% w/v,结果显示 BF 对真菌生长有显著影响(p ≤ 0.01)。酶测定表明,细胞内和细胞外的羧基酯酶都能水解 BF,酶活性分别高达 175 ± 3 U(μmol/min)和 45 ± 1 U。所有受试真菌都能利用 BF 作为唯一碳源,每毫克 BF 产生 0.06 ± 0.01 至 0.45 ± 0.01 毫克干生物量。此外,利用生物信息学工具确定了真菌中 PytH 的存在,并在 A. niger、T. pinophilus、T. reesei 和 P. lilacinum 中发现了 PytH。利用 AlphaFold2 预测了 PytH 同源物的三维结构,并利用 MOE 确定了它们与拟除虫菊酯的分子间相互作用。所有同源物与不同除虫菊酯的相互作用结合能均小于 - 10 kcal/mol。根据这项研究得出的结论是,所研究的真菌在生物降解溴化阻燃剂方面具有更大的潜力。
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引用次数: 0
Nutrient removal efficacy and microbial dynamics in constructed wetlands using Fe(III)-mineral substrates for low carbon-nitrogen ratio sewage treatment. 使用铁(III)-矿物基质处理低碳氮比污水的建构湿地中的营养物去除效果和微生物动态。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-07-18 DOI: 10.1007/s00449-024-03063-8
Yu Li, Mengyue Zhang, Liang Li, Wenyuan Gao, Fei Huang, Guanming Lai, Liping Jia, Rui Liu

This study evaluated the roles of two common sources of Fe(III)-minerals-volcanic rock (VR) and synthetic banded iron formations from waste iron tailings (BIF-W)-in vertical flow-constructed wetlands (VFCWs). The evaluation was conducted in the absence of critical environmental factors, including Fe(II), Fe(III), and soil organic matter (SOM), using metagenomic analysis and integrated correlation networks to predict nitrogen removal pathways. Our findings revealed that Fe(III)-minerals enhanced metabolic activities and cellular processes related to carbohydrate decomposition, thereby increasing the average COD removal rates by 10.7% for VR and 5.90% for BIF-W. Notably, VR improved nitrogen removal by 1.70% and 5.40% compared to BIF-W and the control, respectively. Fe(III)-mineral amendment in bioreactors also improved the retention of denitrification and nitrification bacteria (phylum Proteobacteria) and anammox bacteria (phylum Planctomycetes), with increases of 3.60% and 3.20% using VR compared to BIF-W. Metagenomic functional prediction indicated that the nitrogen removal mechanisms in VFCWs with low C/N ratios involve simultaneous partial nitrification, ANAMMOX, and denitrification (SNAD). Network-based analyses and correlation pathways further suggest that the advantages of Fe(III)-minerals are manifested in the enhancement of denitrification microorganisms. Microbial communities may be activated by the functional dissolution of Fe(III)-minerals, which improves the stability of SOM or the conversion of Fe(III)/Fe(II). This study provides new insights into the functional roles of Fe(III)-minerals in VFCWs at the microbial community level, and provides a foundation for developing Fe-based SNAD enhancement technologies.

本研究评估了两种常见的铁(III)矿物来源--火山岩(VR)和废铁尾矿合成带状铁层(BIF-W)--在垂直流构建湿地(VFCWs)中的作用。这项评估是在没有铁(II)、铁(III)和土壤有机质(SOM)等关键环境因素的情况下进行的,利用元基因组分析和综合相关网络来预测脱氮途径。我们的研究结果表明,铁(III)矿物质增强了与碳水化合物分解相关的代谢活动和细胞过程,从而使 VR 和 BIF-W 的平均 COD 去除率分别提高了 10.7% 和 5.90%。值得注意的是,与 BIF-W 和对照组相比,VR 的氮去除率分别提高了 1.70% 和 5.40%。生物反应器中的铁(III)-矿物质添加剂也提高了反硝化和硝化细菌(变形菌门)和氨氧化细菌(拟杆菌门)的存留率,与 BIF-W 相比,VR 的存留率分别提高了 3.60% 和 3.20%。元基因组功能预测表明,低碳氮比 VFCW 的脱氮机制包括同时部分硝化、氨氧化和反硝化(SNAD)。基于网络的分析和相关路径进一步表明,铁(III)-矿物质的优势体现在反硝化微生物的增强上。微生物群落可能因铁(III)-矿物质的功能性溶解而被激活,从而提高了 SOM 的稳定性或铁(III)/铁(II)的转化。这项研究为从微生物群落层面了解 VFCW 中铁(III)-矿物质的功能作用提供了新的视角,并为开发基于铁的 SNAD 增强技术奠定了基础。
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引用次数: 0
Recombinant expression and characterization of the endochitinase Chit36-TA from Trichoderma asperellum in Komagataella phaffii for chitin degradation of black soldier fly exuviae. 在 Komagataella phaffii 中重组表达和鉴定来自毛霉菌 Chit36-TA 的内几丁质酶 Chit36-TA,用于降解黑纹伊蚊的几丁质。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-08-08 DOI: 10.1007/s00449-024-03067-4
Luisa Gebele, Andreas Wilke, Axel Salliou, Laura Schneider, Daniel Heid, Tobias Stadelmann, Corinna Henninger, Uzair Ahmed, Melanie Broszat, Pascale Müller, Georg Dusel, Michał Krzyżaniak, Katrin Ochsenreither, Thomas Eisele

The natural polymer chitin is an abundant source for valuable N-acetylchitooligosaccharides and N-acetylglucosamine applicable in several industries. The endochitinase Chit36-TA from Trichoderma asperellum was recombinantly expressed in Komagataella phaffii for the enzymatic degradation of chitin from unused insect exuviae into N-acetylchitooligosaccharides. Chit36-TA was purified by Ni-NTA affinity chromatography and subsequently biochemically characterized. After deglycosylation, the endochitinase had a molecular weight of 36 kDa. The optimum pH for Chit36-TA was 4.5. The temperature maximum of Chit36-TA was determined to be 50 °C, while it maintained > 93% activity up to 60 °C. The chitinase was thermostable up to 45 °C and exhibited ~ 50% activity after a 15 min incubation at 57 °C. Chit36-TA had a maximum specific enzyme activity of 50 nkat/mg with a Km value of 289 µM with 4-methylumbelliferyl-N,N',N″-triacetyl-β-chitotrioside as substrate. Most tested cations, organic solvents and reagents were well-tolerated by the endochitinase, except for SDS (1 mM), Cu2+ (10 mM) and Mn2+ (10 mM), which had stronger inhibitory effects with residual activities of 3, 41 and 28%, respectively. With a degree of hydrolysis of 32% applying colloidal shrimp chitin (1% (w/v)) and 12% on insect larvae (1% (w/v)) after 24 h, the endochitinase was found to be suitable for the conversion of colloidal chitin as well as chitin from black soldier fly larvae into water-soluble N-acetylchitooligosaccharides. To prove scalability, a bioreactor process was developed in which a 55-fold higher enzyme activity of 49 µkat/l and a tenfold higher protein expression of 1258 mg/l were achieved.

天然聚合物甲壳素是宝贵的 N-乙酰壳寡糖和 N-乙酰葡糖胺的丰富来源,可用于多个行业。在 Komagataella phaffii 中重组表达了来自毛霉的内几丁质酶 Chit36-TA,用于将未使用的昆虫卵壳中的几丁质酶解为 N-乙酰壳寡糖。Chit36-TA 通过 Ni-NTA 亲和层析法纯化,随后进行了生物化学鉴定。脱糖后,内切酶的分子量为 36 kDa。Chit36-TA 的最适 pH 值为 4.5。经测定,Chit36-TA的最高温度为50 °C,而在60 °C时仍能保持大于93%的活性。该几丁质酶的热稳定性可达 45 °C,在 57 °C下培养 15 分钟后显示出约 50%的活性。以 4-甲基伞形酮基-N,N',N″-三乙酰基-β-壳三糖苷为底物时,Chit36-TA 的最大特定酶活性为 50 nkat/mg,Km 值为 289 µM。除了 SDS(1 mM)、Cu2+(10 mM)和 Mn2+(10 mM)具有较强的抑制作用(残留活性分别为 3%、41% 和 28%)外,大多数测试的阳离子、有机溶剂和试剂对内几丁质酶都有很好的耐受性。24 小时后,内切几丁质酶对胶体虾几丁质(1%(w/v))的水解度为 32%,对昆虫幼虫(1%(w/v))的水解度为 12%,因此发现内切几丁质酶适用于将胶体几丁质以及黑翅蝇幼虫的几丁质转化为水溶性 N-乙酰壳寡糖。为了证明其可扩展性,开发了一种生物反应器工艺,其酶活性提高了 55 倍(49 µkat/l),蛋白质表达量提高了 10 倍(1258 mg/l)。
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引用次数: 0
Adsorption of extracellular lipase in a packed-bed reactor: an alternative immobilization approach. 细胞外脂肪酶在填料床反应器中的吸附:另一种固定化方法。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-08-05 DOI: 10.1007/s00449-024-03066-5
Amanda Noli Freitas, Daniela Remonatto, Rodney Helder Miotti Junior, João Francisco Cabral do Nascimento, Adriana Candido da Silva Moura, Valéria de Carvalho Santos Ebinuma, Ariela Veloso de Paula

In light of the growing demand for novel biocatalysts and enzyme production methods, this study aimed to evaluate the potential of Aspergillus tubingensis for producing lipase under submerged culture investigating the influence of culture time and inducer treatment. Moreover, this study also investigated conditions for the immobilization of A. tubingensis lipase by physical adsorption on styrene-divinylbenzene beads (Diaion HP-20), for these conditions to be applied to an alternative immobilization system with a packed-bed reactor. Furthermore, A. tubingensis lipase and its immobilized derivative were characterized in terms of their optimal ranges of pH and temperature. A. tubingensis was shown to be a good producer of lipase, obviating the need for inducer addition. The enzyme extract had a hydrolytic activity of 23 U mL-1 and achieved better performance in the pH range of 7.5 to 9.0 and in the temperature range of 20 to 50 °C. The proposed immobilization system was effective, yielding an immobilized derivative with enhanced hydrolytic activity (35 U g-1), optimum activity over a broader pH range (5.6 to 8.4), and increased tolerance to high temperatures (40 to 60 ℃). This research represents a first step toward lipase production from A. tubingensis under a submerged culture and the development of an alternative immobilization system with a packed-bed reactor. The proposed system holds promise for saving time and resources in future industrial applications.

鉴于对新型生物催化剂和酶生产方法的需求日益增长,本研究旨在评估管曲霉在浸没培养条件下生产脂肪酶的潜力,调查培养时间和诱导剂处理的影响。此外,本研究还调查了管曲霉脂肪酶在苯乙烯-二乙烯基苯珠(Diaion HP-20)上的物理吸附固定条件,以便将这些条件应用于填料床反应器的替代固定系统。此外,还对管氏脂肪酶及其固定化衍生物的最佳 pH 值和温度范围进行了表征。结果表明,管状芽孢杆菌是一种很好的脂肪酶生产者,无需添加诱导剂。该酶提取物的水解活性为 23 U mL-1,在 pH 值为 7.5 至 9.0 和温度为 20 至 50 ℃ 的范围内性能更佳。拟议的固定化系统是有效的,产生的固定化衍生物具有更高的水解活性(35 U g-1),在更宽的 pH 值范围(5.6 至 8.4)内具有最佳活性,对高温(40 至 60 ℃)的耐受性更强。这项研究标志着管氏酵母在浸没培养条件下生产脂肪酶迈出了第一步,并开发出了一种使用填料床反应器的替代固定化系统。拟议的系统有望在未来的工业应用中节省时间和资源。
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引用次数: 0
Biotransformation of ginsenoside compound K using β-glucosidase in deep eutectic solvents. 利用β-葡萄糖苷酶在深共晶溶剂中对人参皂苷化合物K进行生物转化。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-06-27 DOI: 10.1007/s00449-024-03056-7
Yinan Hong, Yue Shi, Yurou Fan, Hong Pan, Xiangyu Yao, Yu Xie, Xiaojun Wang

Ginsenoside compound K (CK) holds significant potential for application in the pharmaceutical industry, which exhibits numerous pharmacological activity such as cardioprotective and antidiabetic. However, the difficult separation technique and limited yield of CK hinder its widespread use. The study investigated the process of converting ginsenoside CK using β-glucosidase. It aimed to determine the specific site where the enzyme binds and the most favorable arrangement of the enzyme. Molecular docking was also employed to determine the interaction between β-glucosidase and ginsenosides, indicating a strong and spontaneous contact force between them. The effectiveness of the conversion process was further improved using a "green" deep eutectic solvent (DES). A univariate experimental design was used to determine the composition of DES and the optimal hydrolysis conditions for β-glucosidase to convert ginsenoside Rb1 into ginsenoside CK. The employment of β-glucosidase enzymatic hydrolysis in the synthesis of rare ginsenoside CK applying the environmentally friendly solvent DES is not only viable and effective but also appropriate for industrial use. The characterization methods confirmed that DES did not disrupt the structure and conformation of β-glucosidase. In ChCl:EG = 2:1 (30%, v/v), pH 5.0 of DES buffer, reaction temperature 50 ℃, enzyme substrate mass ratio 1:1, after 36 h of reaction, the CK yield was 1.24 times that in acetate buffer, which can reach 86.2%. In this study, the process of using β-glucosidase enzymatic hydrolysis and producing rare ginsenoside CK in green solvent DES is feasible, efficient and suitable for industrial production and application.

人参皂苷化合物 K(CK)具有保护心脏和抗糖尿病等多种药理活性,在制药业的应用潜力巨大。然而,人参皂苷化合物 K 的分离技术难度大、产量有限,阻碍了其广泛应用。本研究利用β-葡萄糖苷酶研究了人参皂苷 CK 的转化过程。研究旨在确定酶结合的特定位点以及酶的最有利排列。此外,还采用分子对接法确定了β-葡萄糖苷酶与人参皂苷之间的相互作用,结果表明它们之间存在很强的自发接触力。使用 "绿色 "深共晶溶剂(DES)进一步提高了转化过程的有效性。采用单变量实验设计确定了 DES 的组成以及 β-葡萄糖苷酶将人参皂苷 Rb1 转化为人参皂苷 CK 的最佳水解条件。利用β-葡萄糖苷酶酶解法合成稀有人参皂苷 CK,采用环境友好型溶剂 DES,不仅可行、有效,而且适合工业化应用。表征方法证实,DES 不会破坏 β-葡萄糖苷酶的结构和构象。在 ChCl:EG = 2:1 (30%, v/v)、pH 值为 5.0 的 DES 缓冲液中,反应温度为 50 ℃,酶底物质量比为 1:1,反应 36 h 后,CK 收率是醋酸盐缓冲液的 1.24 倍,可达 86.2%。本研究认为,在绿色溶剂DES中利用β-葡萄糖苷酶酶解生产稀有人参皂苷CK的工艺可行、高效,适合工业化生产和应用。
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引用次数: 0
Cellobionate production from sodium hydroxide pretreated wheat straw by engineered Neurospora crassa HL10. HL10 工程神经孢子菌利用氢氧化钠预处理过的小麦秸秆生产纤维二酸酯。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-07-12 DOI: 10.1007/s00449-024-03061-w
Jiajie Wang, Takao Kasuga, Zhiliang Fan

This study investigated cellobionate production from a lignocellulosic substrate using Neurospora crassa HL10. Utilizing NaOH-pretreated wheat straw as the substrate obviated the need for an exogenous redox mediator addition, as lignin contained in the pretreated wheat served as a natural mediator. The low laccase production by N. crassa HL10 on pretreated wheat straw caused slow cellobionate production, and exogenous laccase addition accelerated the process. Cycloheximide induced substantial laccase production in N. crassa HL10, enabling the strain to yield approximately 57 mM cellobionate from pretreated wheat straw (equivalent to 20 g/L cellulose), shortening the conversion time from 8 to 6 days. About 92% of the cellulose contained in the pretreated wheat straw is converted to cellobionate. In contrast to existing methods requiring pure cellobiose or cellulase enzymes, this process efficiently converts a low-cost feedstock into cellobionate at a high yield without enzyme or redox mediator supplementation.

本研究利用十字花科黑孢子属(Neurospora crassa HL10)研究了木质纤维素基质生产纤维硫酸盐的情况。使用 NaOH 预处理过的小麦秸秆作为底物,无需添加外源氧化还原介质,因为预处理过的小麦中所含的木质素可作为天然介质。在预处理过的小麦秸秆上,N. crassa HL10 的漆酶产量较低,导致胞二酸产生缓慢,而外源漆酶的添加加速了这一过程。环己亚胺诱导 N. crassa HL10 产生大量漆酶,使该菌株能从预处理过的小麦秸秆(相当于 20 克/升纤维素)中产生约 57 毫摩尔的纤维二酸酯,将转化时间从 8 天缩短到 6 天。预处理过的小麦秸秆中所含的纤维素约有 92% 转化为纤维二酸。与需要纯纤维素生物糖或纤维素酶的现有方法相比,该工艺无需补充酶或氧化还原介质,即可高效地将低成本原料转化为高产率的纤维酮酸盐。
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引用次数: 0
Novel differential scanning calorimetry (DSC) application to select polyhydroxyalkanoate (PHA) producers correlating 3-hydroxyhexanoate (3-HHx) monomer with melting enthalpy. 应用新型差示扫描量热法 (DSC) 挑选聚羟基烷酸酯 (PHA) 生产商,将 3-hydroxyhexanoate (3-HHx) 单体与熔化焓相关联。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-08-06 DOI: 10.1007/s00449-024-03054-9
Hee Ju Jung, Byungchan Kim, Tae-Rim Choi, Suk Jin Oh, Suwon Kim, Yeda Lee, Yuni Shin, Suhye Choi, Jinok Oh, So Yeon Park, Young Sik Lee, Young Heon Choi, Yung-Hun Yang

Polyhydroxyalkanoate (PHA) is an environmental alternative to petroleum-based plastics because of its biodegradability. The polymer properties of PHA have been improved by the incorporation of different monomers. Traditionally, the monomer composition of PHA has been analyzed using gas chromatography (GC) and nuclear magnetic resonance (NMR), providing accurate monomer composition. However, sequential analysis of the thermal properties of PHA using differential scanning calorimetry (DSC) remains necessary, providing crucial insights into its thermal characteristics. To shorten the monomer composition and thermal property analysis, we directly applied DSC to the analysis of the obtained PHA film and observed a high correlation (r2 = 0.98) between melting enthalpy and the 3-hydroxyhexanoate (3-HHx) mole fraction in the polymer. A higher 3-HHx fraction resulted in a lower melting enthalpy as 3-HHx provided the polymer with higher flexibility. Based on this, we selected the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) producing strain from Cupriavidus strains that newly screened and transformed with vectors containing P(3HB-co-3HHx) biosynthetic genes, achieving an average error rate below 1.8% between GC and DSC results. Cupriavidus sp. BK2 showed a high 3-HHx mole fraction, up to 10.38 mol%, with T(℃) = 171.5 and ΔH of Tm (J/g) = 48.0, simultaneously detected via DSC. This study is an example of the expansion of DSC for PHA analysis from polymer science to microbial engineering.

聚羟基烷酸酯(PHA)具有生物降解性,是石油基塑料的环保替代品。通过加入不同的单体,PHA 的聚合物特性得到了改善。传统上,PHA 的单体组成是通过气相色谱法(GC)和核磁共振法(NMR)进行分析,从而提供准确的单体组成。然而,使用差示扫描量热仪(DSC)对 PHA 的热特性进行连续分析仍然是必要的,这将为深入了解其热特性提供重要依据。为了缩短单体成分和热特性分析的时间,我们直接使用 DSC 分析所获得的 PHA 薄膜,并观察到熔化焓与聚合物中 3-hydroxyhexanoate (3-HHx) 分子分数之间存在高度相关性(r2 = 0.98)。3-HHx 分数越高,熔化焓越低,因为 3-HHx 使聚合物具有更高的柔韧性。基于这一点,我们从新筛选并用含有 P(3HB-co-3HHx)生物合成基因的载体转化的铜绿微囊藻菌株中选出了生产聚(3-羟基丁酸-co-3-羟基己酸)(P(3HB-co-3HHx))的菌株,使 GC 和 DSC 结果之间的平均误差率低于 1.8%。Cupriavidus sp. BK2 表现出较高的 3-HHx 分子分数,高达 10.38 摩尔%,Tm (℃) = 171.5,Tm 的 ΔH (J/g) = 48.0,同时通过 DSC 检测到。这项研究是将 DSC 用于 PHA 分析从聚合物科学扩展到微生物工程的一个范例。
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引用次数: 0
Protein rational design and modification of erythrose reductase for the improvement of erythritol production in Yarrowia lipolytica. 合理设计和改造赤藓酮糖还原酶,提高脂肪分解亚罗酵母的赤藓糖醇产量。
IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-01 Epub Date: 2024-07-05 DOI: 10.1007/s00449-024-03057-6
Lianggang Huang, Wenjia Wang, Kai Wang, Yurong Li, Junping Zhou, Aiping Pang, Bo Zhang, Zhiqiang Liu, Yuguo Zheng

Erythritol is a natural non-caloric sweetener, which is produced by fermentation and extensively applied in food, medicine and chemical industries. The final step of the erythritol synthesis pathway is involved in erythritol reductase, whose activity and NADPH-dependent become the limiting node of erythritol production efficiency. Herein, we implemented a strategy combining molecular docking and thermal stability screening to construct an ER mutant library. And we successfully obtained a double mutant ERK26N/V295M (ER*) whose catalytic activity was 1.48 times that of wild-type ER. Through structural analysis and MD analysis, we found that the catalytic pocket and the enzyme stability of ER* were both improved. We overexpressed ER* in the engineered strain ΔKU70 to obtain the strain YLE-1. YLE-1 can produce 39.47 g/L of erythritol within 144 h, representing a 35% increase compared to the unmodified strain, and a 10% increase compared to the strain overexpressing wild-type ER. Considering the essentiality of NADPH supply, we further co-expressed ER* with two genes from the oxidative phase of PPP, ZWF1 and GND1. This resulted in the construction of YLE-3, which exhibited a significant increase in production, producing 47.85 g/L of erythritol within 144 h, representing a 63.90% increase compared to the original chassis strain. The productivity and the yield of the engineered strain YLE-3 were 0.33 g/L/h and 0.48 g/g glycerol, respectively. This work provided an ER mutation with excellent performance, and also proved the importance of cofactors in the process of erythritol synthesis, which will promote the industrial production of erythritol by metabolic engineering of Y. lipolytica.

赤藓糖醇是一种天然无热量甜味剂,由发酵法生产,广泛应用于食品、医药和化工行业。赤藓糖醇合成途径的最后一步涉及赤藓糖醇还原酶,其活性和 NADPH 依赖性成为赤藓糖醇生产效率的限制性节点。在此,我们采用分子对接和热稳定性筛选相结合的策略,构建了ER突变体库。我们成功地获得了双突变体ERK26N/V295M(ER*),其催化活性是野生型ER的1.48倍。通过结构分析和 MD 分析,我们发现 ER* 的催化口袋和酶稳定性都得到了改善。我们在工程菌株ΔKU70中过表达了ER*,得到了菌株YLE-1。YLE-1 在 144 小时内可生产 39.47 克/升赤藓糖醇,与未改造菌株相比提高了 35%,与过表达野生型 ER 的菌株相比提高了 10%。考虑到 NADPH 供应的重要性,我们进一步将 ER* 与 PPP 氧化阶段的两个基因 ZWF1 和 GND1 共同表达。结果构建出了 YLE-3,它的产量有了显著提高,在 144 小时内生产了 47.85 克/升赤藓糖醇,与原始基质菌株相比提高了 63.90%。工程菌株 YLE-3 的生产率和产量分别为 0.33 克/升/小时和 0.48 克/克甘油。这项工作提供了一种性能优异的ER突变,同时也证明了辅助因子在赤藓糖醇合成过程中的重要性,这将促进脂溶性酵母菌代谢工程赤藓糖醇的工业化生产。
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Bioprocess and Biosystems Engineering
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