PROTEIN EXPRESSION AND PURIFICATION
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2015
·
IF 1.695
Jian-yong Zheng,Jian Wang,Sha-sha Zhou,Xiao-jun Li,Xiang-xian Ying,Zhao Wang
Esterases (EC 3.1.1.X) have been used as biocatalysts due to their good stability, high chemo-, regio- and stereoselectivity. In our previous studies, Bacillus megaterium WZ009 harboring esterase displayed the unique capability to convert ( S )-4-Chloro-3-hydroxyethylbutyrate (CHBE) in the racemate to ( S )-3-hydroxy-γ-butyrolactone (HL) through stereoselective hydrolysis, dechlorination, and lactonization. The remaining ( R )-CHBE and formed ( S )-HL could be obtained in a one-pot enzymatic reaction. An esterase from B. megaterium WZ009 was purified and was found to have 466 encoded amino acids and an apparent molecular mass of 55 kDa. The purified esterase exhibited maximal activity at a temperature of 25 °C and at a pH of 11.5 towards 100 mM CHBE. When the stereoselective biocatalysis of rac -CHBE was performed using the recombinant Escherichia coli BL21 (DH3) cells harboring the esterase, the catalytic activity increased by 20-fold compared with the original strain B. megaterium WZ009. With the addition of activated carbon (62 g/L) in the reaction system, the conversion was increased from 39% to 45% at a substrate concentration of 750 mM. Another remarkable advantage is that both of the obtained residual ( R )-CHBE and the formed ( S )-HL had high optical purities ( e.e. s > 99.9%, e.e. p > 99.9%), thereby making this esterase a usable biocatalyst for industrial application.
研究领域:
生物化学
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分子生物学
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工业酶学
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产品关联
DOI
PubMed
高影响因子
PEDOSPHERE
·
2024
·
IF 5.2
Yang LIU,Lihua ZHU,Junyuan ZHANG,Zhuo WEI,Xuhan HUANG,Christian E.W. STEINBERG,Hao QIU,Martina G. VIJVER,Jing ZHAO,Willie J.G.M. PEIJNENBURG
Due to the complex composition of biochar and the potential interactions between the components that jointly make up biochar, previous studies have always shown controversial results regarding impacts of biochar on soil phosphorus (P) solubilization. Using a nested experimental design, this study investigated the effects of different components of biochar, i.e. whole biochar (BC), dissolved biochar (DBC) and washed biochar (WBC) on the microbial-mediated P solubilizing process. The growth of Bacillus megaterium , acid/alkaline phosphatase activity, differential metabolites of bacteria and metabolic pathways were measured in treatments with biochar pyrolyzed at 200, 300, 400, 500 and 600 °C. Results showed that BC, DBC and WBC all negatively affected the growth and alkaline phosphatase activity of B. megaterium , and BC500/WBC500 and BC400/WBC400 had the greatest impacts. The negative effects of high-temperature biochar on cell proliferation were caused by persistent free radicals and included down-regulation of histidine metabolism by BC400 and interference in nucleotide metabolism by BC600. The impacts of low-temperature biochar on cell proliferation and alkaline phosphatase activity were highly related to the metal content. Using the independent action model, antagonistic effects were found between DBC and WBC on inhibiting B. megaterium growth, while these biochars had synergistic effects on inhibition of alkaline phosphatase activity. Overall, this study provides insights on how biochar adversely modulates microbial-mediated P solubilization.
研究领域:
环境科学
·
微生物学
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DOI
高影响因子
Molecular Catalysis
·
2023
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IF 4.6
Nan Jiang,Xiaotong Du,Liangyu Zheng
In this study, a series of chiral lactams were successfully prepared by one-pot two-step reactions. It included a transamination reaction catalyzed by a ω -transaminase from Bacillus megaterium ( ω -TA Bm ), and subsequent spontaneous cyclisation. The different amine donors were firstly screened with ethyl levulinate as an amine acceptor, and both the excellent conversion and enantiomeric excess ( e.e. p ) of >99% could be achieved with 4-bromophenylethylamine as an amine donor, however, the long reaction time of 24 h was unfavorable for its application. After the amino acid residue Thr120 situated in the small pocket was mutated to Ser, the T120S variant could exhibit 10.6-fold higher activity comparing to that of wild type, and achieve the reaction with both conversion and e.e. p of >99% within 3 h. The T120S variant could also be applied to prepare the other chiral γ- and δ-lactams with excellent conversion and e.e. p . Furthermore, the inclusion bodies of ω -TA Bm and its variants could be aggregated as “native-like structures” to continuously prepare the chiral lactam for 18 h. The aim of the study is to provide a simply and efficiently biocatalytic route for preparing chiral lactams.
研究领域:
生物化学
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酶工程
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有机合成
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产品关联
DOI
高影响因子
Nanomaterials
·
2024
·Vol. 14
·期 6
·512
·
IF 4.4
Yuan Chen,Jiaxing Wang,Daidi Chen,Boxi Wang,Jinchuan Wu,Rongrong Liu,Qingxin Li
Many studies have been conducted on the microbial reduction of Pd (II) to palladium nanoparticles (Pd-NPs) due to the environmental friendliness, low cost, and the decreased toxicity of Pd (II) ions. In this study, we investigate the reduction mechanism of Pd (II) byBacillus megateriumY-4 through proteomics. The data are available via ProteomeXchange with identifier PXD049711. Our results revealed thatB. megateriumY-4 may use the endogenous electron donor (NAD(P)H) generated by nirB, tdh, and fabG and reductase to reduce Pd (II) to Pd-NPs. The expression levels of fabG, tdh, gudB, and rocG that generate NAD(P)H were further increased, and the number of reduced Pd-NPs was further increased with the exogenous electron donor sodium formate. Endogenous electron mediators such as quinones and flavins inB. megateriumY-4 can further enhance Pd (II) reduction. The findings provided invaluable information regarding the reduction mechanism of Pd (II) byB. megateriumY-4 at the proteome level.
研究领域:
微生物学
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环境科学
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产品关联
DOI
PubMed
高影响因子
ACS ES&T Engineering
·
2025
·
IF 6.7
Zulv Huang,Xipeng Wei,Lijuan Zhang,Tao Chen,Yi Huang,Zhangwei Yang,Wanqing Zeng,Hongxing Chen,Lingtian Xie,Bo Yan
Microbial reduction of pentavalent vanadium [V(V)] to less toxic tetravalent vanadium [V(IV)] is associated with the biogeochemical cycling of sulfur. However, the capability of elemental sulfur-reducing bacteria (S0RB) to reduce V(V) in the presence of elemental sulfur (S0) remains largely unexplored. Herein, we systematically investigated the V(V) reduction capabilities and underlying mechanisms of Bacillus megaterium R01, a S0RB strain isolated from the stone coal mining region. Under sulfur-depleted conditions, B. megaterium R01 demonstrated limited reduction capacity (<54.3% removal efficiency for 200 mg/L V(V)). In contrast, S0 supplementation enabled near-complete V(V) reduction (97.6 ± 2.1%) even at elevated concentrations (400 mg/L), achieving a relatively low carbon consumption rate (TOC/V(V) of ∼0.6). Sulfhydryl (SH) compounds, reaching 424.2 ± 9.9 μmol/L within 5 days, were likely crucial for reducing V(V) to V(IV). This mechanism differs from that of other V-reducing bacteria. The transcriptome analysis revealed that the up-regulation of genes associated with sulfur metabolism, cysteine, and methionine metabolism in B. megaterium R01 is likely to contribute to its enhanced resistance toward V(V). Additionally, the biosynthesis of intracellular BSH in B. megaterium R01, and the extracellular SH groups (e.g., Trx and TrxR encode enzymes) play an irreplaceable role in reducing V(V) by B. megaterium R01. This study enhances our understanding of V(V) reduction in sulfur-rich environments and suggests a promising approach for remediating V(V)-contaminated groundwater, sediments, and soils.
研究领域:
环境微生物学
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污染修复
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生物地球化学
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产品关联
DOI
高影响因子
FOOD CHEMISTRY
·
2021
·
IF 7.514
Jinghui Zhou,Changwei Liu,Shimin Zhao,Ya Liu,Sheng Zhang,Qiang Zhao,Fen Wang,Gang Xu,Jianan Huang,Zhonghua Liu
Theaflavin-3,3ʹ-digallate (TFDG) in black tea possesses several health benefits. However, low TFDG yields limit its application. Herein, tyrosinases from Bacillus megaterium (Bmtyrc) were used to synthesize TFDG. To improve the catalytic efficiency of tyrosinase, a directed evolution strategy and a high-throughput screening method was employed. Compared with the wild type, mutant Bmtyrc-3 (N205D/D166E/D167G/F197W) showed 6.46 and 4.91-folds higher specific activity and 51.97- and 1.95-folds higher Vmax values towards epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), respectively. Moreover, Bmtyrc-3 displayed significantly enhanced catalytic efficiencies, and the space–time yield of TFDG was 35.35 g L -1 d -1 . Bmtyrc-3 presents a broader substrate binding area, caused by a mutation (N205D) encompassing the active site. Changes in the potential of the substrate binding site and hydrogen bonds, and the electrostatic effect on the protein surface resulted in an increased activity of the substrates EGCG and ECG.
研究领域:
生物技术
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生物化学
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酶学
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食品科学
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DOI
PubMed
高影响因子
Journal of Cleaner Production
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2024
·
IF 9.7
Linya Yi,Shuyan Chen,Yuancai Chen
Bio-remediation are low-cost and environmentally friendly, but microbial enzymes tend to lose their activity when treated antibiotic contaminants due to its high toxicity. In this study, Bacillus megaterium Y-4 was demonstrated to in situ synthesize Pd 0 @cells, and detoxify oxytetracycline (OTC) through enhancing electron transport chain via Pd 0 -dependent F O F 1 -ATPase (proton pumps). Toxicity analyses showed that increasing Pd 0 loading significantly reduced acute and chronic bio-toxicity as well as ecological risk of OTC effluent, as evidenced by the promoting cellular response to oxidative stress with increasing enzymes GSH-Px and IDH activities (4.24 and 1.20-fold). Electrochemical (I-t, LSV, DPV and EDC) showed that the incorporated bio-Pd 0 mainly promoted FDH/Hase-based short chain (gradually took precedence over CoQ-based long chain), diverting OTC extracellular degradation to markedly detoxify OTC to cell. Inhibition (DCCD and TBA) assays and structural equation modelling indicated that bio-Pd 0 upregulated ATP and NADH synthesis. It also acted as transmembrane F O F 1 -ATPase proton pump, which enabled the backward of protons out of the restriction of PMF , eventually maximizing the energy utilization. This study sheds new insights into the detoxification of antibiotics by bio-nanoparticles through proton transmembrane transfer/redistribution.
研究领域:
微生物学
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环境科学
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产品关联
DOI
高影响因子
SCIENCE OF THE TOTAL ENVIRONMENT
·
2017
·
IF 4.9
Nai-Xian Feng,Jiao Yu,Ce-Hui Mo,Hai-Ming Zhao,Yan-Wen Li,Bing-Xiao Wu,Quan-Ying Cai,Hui Li,Dong-Mei Zhou,Ming-Hung Wong
Phthalic acid esters (PAEs) are a group of recalcitrant and hazardous organic compounds that pose a great threat to both ecosystem and human beings. A novel endophytic strain YJB3 that could utilize a wide range of PAEs as the sole carbon and energy sources for cell growth was isolated from Canna indica root tissue. It was identified as Bacillus megaterium based on morphological characteristics and 16S rDNA sequence homology analysis. The degradation capability of the strain YJB3 was investigated by incubation in mineral salt medium containing di-n-butyl-phthalate (DBP), one of important PAEs under different environmental conditions, showing 82.5% of the DBP removal in 5 days of incubation under the optimum conditions (acetate 1.2 g·L − 1 , inocula 1.8%, and temperature 34.2 °C) achieved by two-step sequential optimization technologies. The DBP metabolites including mono-butyl phthalate (MBP), phthalic acid (PA), protocatechuic acid (PCA), etc. were determined by GC–MS. The PCA catabolic genes responsible for the aromatic ring cleavage of PCA in the strain YJB3 were excavated by whole-genome sequencing. Thus, a degradation pathway of DBP by the strain YJB3 was proposed that MBP was formed, followed by PA, and then the intermediates were further utilized till complete degradation. To our knowledge, this is the first study to show the biodegradation of PAEs using endophyte. The results in the present study suggest that the strain YJB3 is greatly promising to act as a competent inoculum in removal of PAEs in both soils and crops.
研究领域:
环境微生物学
·
生物修复
·
基因组学
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产品关联
DOI
PubMed
高影响因子
BIOTECHNOLOGY LETTERS
·
2017
·Vol. 39
·期 8
·1175-1181
·
IF 1.73
Zhou Youzhi,Liu Ke,Zhang Jinsong,Chu Jianlin,He Bingfang
Objective To improve the stability of β-galactosidase from Bacillus megaterium YZ08 (BMG) in aqueous hydrophilic solvents and promote its application in the galactosylation of natural products. Results The addition of 5 mM Mg 2+ significantly enhanced the stability of BMG in aqueous hydrophilic solvents, and the half-lives of BMG in these solutions reached 56 min to 208 h, while they were only 7 min to 5.9 h without addition of Mg 2+ . Studies on the kinetic parameters in buffer solution and 30% dimethyl sulfoxide (DMSO) indicated that the affinity of BMG to 2-nitrophenyl-β- d -galactopyranoside and its catalytic efficiency ( κ
cat / K
m ) increased with the addition of Mg 2+ . Furthermore, the addition of Mg 2+ facilitated galactosylation reactions in 30% DMSO and increased product conversions by 24–41% due to the reversal of the thermodynamic equilibrium of hydrolysis. Conclusion A convenient approach was established to improve the stability of BMG in aqueous hydrophilic solvents.
研究领域:
酶学
·
生物催化
·
生物化学
·
有机化学
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产品关联
DOI
PubMed
高影响因子
CHEMOSPHERE
·
2019
·
IF 5.108
Yuan Chen,Keqiang Hu,Yuancai Chen
In this study, we screened a new aerobic bacterium ( Bacillus megaterium Y-4) that can efficiently reduce Pd(II) with different electron donors. The best electron donor was sodium formate and the best reduction of Pd(II) were by log growth phase cells. The high removal capacity of Pd(II) (1658.3 mg/g) was obtained with 30 mg/L dry cell weight and 50 mg/L Pd (II) in the presence of 5 mM sodium formate. The removal amount of Pd(II) increased with initial Pd(II) concentrations ranging from 50 to 200 mg/L with 100 mg/L Pd(II) being completely removed by 148 mg/L dry cell weight in 6 h. The cell wall, periplasmic space and intracellular contents of B . megaterium Y-4 contains different kinds of enzymes for reducing Pd(II). In addition, the activity of extracellular and periplasmic enzymes was more sensitive to temperature than intracellular enzymes . XRD and XPS analysis revealed that the enzyme for reducing Pd(II) in B. megaterium Y-4 can tolerate a broad range of temperatures (20–60 °C) and pH (2.0–7.0) but was sensitive to oxygen. TEM analysis showed that biogenic palladium nanoparticles (Pd-NPs) were mainly distributed evenly in the periplasmic space of the live cells and were released from cells into aqueous solution, which reduced the toxicity of Pd(II), allowing Pd-NP recovery without cell destruction. B. megaterium Y-4 is a potential bacterium for efficient treatment and reclamation of Pd(II) pollution and formation of Pd-NPs.
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DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2022
·
IF 3.705
Rui Pei,Xiaoli Fu,Libing Tian,Shu-Feng Zhou,Wei Jiang
In recent years, with the increasing number of patients with depression, the efficient synthesis of the first-line antidepressant drug duloxetine intermediate ( S-N,N- dimethyl-3-hydroxy-3-(2-thienyl)−1-propanamine, S -DHTP) has attracted great attention. The wild-type AKR3–2–9 from Bacillus megaterium YC4-R4 exhibits high application potential of catalyzing N,N -dimethyl-3-keto-3-(2-thienyl)−1-propanamine (DKTP) to prepare S -DHTP, but there is still much room for improvement. In this work, rational design was carried out to enhance the catalytic potential of AKR3–2–9. Notably, compared to the wild-type AKR3–2–9, three mutants (Ile189Val, Asn256Asp, and Ile189Val + Asn256Asp) were obtained, and their catalytic efficiencies were increased by 1.3 times, 2.3 times, and 1.31 times, respectively. Besides, the thermal stability and organic solvent resistance were improved. More importantly, when the concentration of the substrate DKTP was 0.5 g/L, the catalytic yields of Ile189Val, Asn256Asp and Ile189Val + Asn256Asp were increased by 1.45 times, 1.86 times, and 2.05 times, respectively. Besides, the corresponding optical purities of the three mutants were 92.7 %, 94.3 % and 93.8 %. The above results indicated that the rational design of the AKR of Bacillus megaterium YC4-R4 enhanced its potential for biocatalytic preparation of S -DHTP.
研究领域:
生物化学
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药学
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酶学
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生物催化
✓ 已验证
DOI
PubMed
高影响因子
Journal of Environmental Chemical Engineering
·
2025
·
IF 7.2
Yating Jia,Jing Lu,Yongfen Long,Bin Hou,Yuancai Chen
Microbially synthesized palladium nanoparticles (bio-Pd 0 ) hold great promise for catalysis, but the catalytic activity of bio-Pd 0 relies heavily on its subcellular localization. Electron donors could affect enzymatic reduction efficiency of Pd(II) and control the subcellular location of bio-Pd 0 , yet the underlying mechanisms were unclear. Here, the regulatory mechanism of electron donors on Pd(II) reduction pathways in the non-model bacterium B. megaterium Y-4 was comprehensively investigated by multiple methodologies including cell fractionation, DPV/CV, XPS, FTIR, TEM, proteomics. Key findings revealed that membrane-bound hydrogenases drove periplasmic Pd 0 synthesis via biohydrogen, while the new one-electron extracellular transfer (EET) channel consisting of membrane-attached cytochromes ( ccsB ) and multi-heme cytochromes-bound flavin mediated the extracellular Pd(II) reduction. Notably, electron donors could regulate Pd(II) reduction routes, thereby altering the subcellular distribution of bio-Pd 0 . In lactate-added systems, the higher expression of these proteins related to EET processes (NADH dehydrogenase, membrane-attached cytochrome c, heme-based electron transfer proteins and flavodoxin) promoted extracellular Pd 0 formation (65.4 %). Whereas, in formate-added systems, hydrogenase-driven periplasmic Pd 0 synthesis (64.0 %) was facilitated due to more active formate dehydrogenase, hydrogenase and NADPH-reducing hydrogenase. Moreover, synergistical metabolism of formate and lactate balanced both pathways and maximized the apparent Pd(II) reduction efficiency (98.13 %). Proteomic analysis confirmed formate-specific energy rewiring and compensatory energy via Na⁺-translocating Rnf complex and V-ATPase overexpression. These findings significantly advance our understanding of microbial metal reduction by delineating a true dual-pathway strategy in a Gram-positive bacterium, highlighting the mechanistic diversity beyond model Gram-negative systems and offering new insights for harnessing non-model organisms in biocatalysis and nanoparticle synthesis.
研究领域:
微生物生物技术
·
纳米生物技术
·
环境微生物学
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蛋白质组学
·
生物无机化学
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）