PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY
·
2021
·
IF 3.963
Yanan Sun,Haiping Shi,Chenxin Mao,Jianyan Wu,Chuanqing Zhang
The occurrence of bakanae disease of rice caused by the fungus Fusarium fujikuroi in Zhejiang Province has become increasingly aggravated in recent years, concomitant with the development of resistance to the widely applied fungicides , prochloraz and phenamacril. In this study, the activity of a novel succinate dehydrogenase inhibitor (SDHI) fungicide, penflufen, against different fungi was evaluated in addition to the potential of penflufen in controlling F. fujikuroi infections. Penflufen exhibited good bioactivity against F. fujikuroi , but weak activity against Fusarium spp. and other investigated plant-pathogenic fungi including Colletotrichum spp. In addition to inhibiting mycelial growth , penflufen effectively inhibited F. fujikuroi conidium production. For germination, penflufen could effectively inhibit that of small conidia, but only delay the germination of large conidia. In addition, the sensitivity to penflufen among 100 F. fujikuroi isolates that were collected in areas that were never exposed to SDHIs was determined based on mycelium growth. Sensitivities surprisingly exhibited bimodal distributions, indicating the presence of natural resistance. Cross-resistance was not observed between penflufen in F. fujikuroi and two fungicides that have been extensively applied in field including prochloraz (a DMI) and phenamacril (a 2-cyanoacrylate fungicide), nor with the three SDHIs, fluopyram, benzovindiflupyr, and pydiflumetofen. Additional analysis identified five different point mutations in SDH-A (i.e., at residues 46, 225, 283, 430, and 586) of naturally resistant isolates. These results inform the potential application of the new SDHI fungicide penflufen for managing crop diseases and understanding possible resistance mechanisms among pathogens .
研究领域:
植物病理学
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真菌学
·
农药科学
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DOI
PubMed
高影响因子
PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY
·
2024
·
IF 4.2
Xuan Zhao,Mengsong Chen,Hanxin Mao,Chuanqing Zhang,Jianyan Wu
Gray mold caused by Botrytis spp. is a common disease on various hosts, including strawberries. In this study, we obtained 59 Botrytis isolates from strawberries from greenhouses in Zhejiang Province, China. Identification of the sampled isolates at species level was performed by a duplex PCR assay method, the result showed that, in Zhejiang, gray mold on strawberry fruits is caused by a complex of Botrytis groups including B. cinerea group N (47.5 %) and B. cinerea group S (52.5 %). The sensitivities of all Botrytis isolates to pyrimethanil were determined based on discriminatory dose method on L-asparagine-based agar medium plate. Our results showed that the isolates obtained from the greenhouses with continuous use of pyrimethanil developed severe resistance to pyrimethanil, and the resistance frequencies of B. cinerea group N and group S isolates were 89.3 % and 41.9 %, respectively. By sequencing, four different resistance-related point mutations were identified in 38 Botrytis isolates: Bcpos 5 L412F (16 isolates, 42.1 %), Bcpos 5 L412V (14 isolates, 36.8 %), Bcmdl 1 E407K (2 isolates, 5.3 %), and Bcpos 5 L412S ⸱ Bcmdl 1 E407K (1 isolate, 2.6 %). The exogenous addition of methionine could not completely alter the resistance of Botrytis isolates to pyrimethanil. In this study, the pyrimethanil resistance in Botrytis isolates was steadily inherited, and compared to the pyrimethanil-sensitive isolates, the resistant mutants exhibited good fitness in sporulation capacity, spore germination rate, and virulence on strawberries. In conclusion, our results provided a description of the genetic structure of Botrytis groups complex on strawberry fruits and reminded growers to focus on the stable pyrimethanil resistance in Botrytis isolates, caused by point mutations in BcPos5 and BcMdl1.
研究领域:
植物病理学
·
分子遗传学
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产品关联
DOI
PubMed
高影响因子
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY
·
2023
·
IF 6.8
Jiali Li,Linglong Dai,Yu Feng,Zhenxiao Cao,Yuting Ding,Hao Xu,An Xu,Hua Du
Flame retardants (FRs) have raised public concerns because of their environmental persistence and negative impacts on human health. Recent evidence has revealed that many FRs exhibit reproductive toxicities and transgenerational impacts, whereas the toxic effects of FRs on germ cells remain barely explored. Here we investigated the multigenerational effects of three flame retardants (TBBPA, TCEP and TCPP) on germ cell development in Caenorhabditis elegans , and examined the germ cell mutagenicity of these FRs by using whole genome sequencing. Parental exposure to three FRs markedly increased germ cell apoptosis, and impeded oogenesis in F1-F6 offspring. In addition, the double-increased mutation frequencies observed in progeny genomes uncover the mutagenic actions of FRs on germ cells. Analysis of mutation spectra revealed that these FRs predominantly induced point mutations at A:T base pairs, whereas both small and large indels were almost unaffected. These results revealed the long-term effects of FRs on development and genomic stability of germ cells, which may pose risks to environmental organisms and human reproductive health. Taken together, our findings suggest that germ cell mutagenicity should be carefully examined for the environmental risk assessment of FRs and other emerging pollutants.
研究领域:
环境科学
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遗传学
·
毒理学
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产品关联
DOI
PubMed
高影响因子
Molecular Catalysis
·
2022
·
IF 5.089
Jiacheng Zhang,Jieyu Zhou,Guochao Xu,Ye Ni
Kp ADH is a promising biocatalyst for the synthesis of chiral bulky-bulky alcohols. Substrate specificity toward diaryl ketone substrates with different substituents at phenyl group was investigated. The results reveal that the para -position of diaryl ketones plays important role in manipulating the stereoselectivity of Kp ADH. Two vital residues, E214 and T215, were identified through single-point mutagenesis, and saturation mutagenesis was performed to evaluate their contributions to the stereoselectivity. Furthermore, stereodivergent evolution of Kp ADH was achieved through combinatorial mutagenesis. Among them, E214I/T215S/S237A (ISA) and F161V/S196G/E214G (VGG) exhibited e.e. values of >99% ( S ) and 99% ( R ) toward [4-(trifluoromethyl)phenyl]-2-pyridinylmethanone ( 5a ), and e.e. values of >99% ( S ) and 98% ( R ) toward (4-methylphenyl)-2-pyridinylmethanone ( 6a ), respectively. Kinetic characterization and interaction analysis further prove that the eliminated side-chain collision and enhanced electrostatic interactions are mainly responsible for the increased catalytic efficiency and stereoselectivity. This study provides beneficial mutants with complementary stereoselectivity toward diaryl ketones with para -substituents and also provides guidance for the engineering of homologous alcohol dehydrogenases toward bulky-bulky ketones.
研究领域:
生物化学
·
酶工程
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DOI
高影响因子
CHEMBIOCHEM
·
2017
·Vol. 19
·期 5
·521-526
·
IF 2.847
Zheng Zhou,Min Li,Jian-He Xu,Zhi-Jun Zhang
Graphical Reduced to clear : The nitrile reductase from Pectobacterium carotovorum is able to catalyze the reduction of nitriles to primary amines with the consumption of two molecules of NADPH. A single mutation at the entrance of the substrate channel increases both the activity and stability of the nitrile reductase. Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g prot −1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3- d ]pyrimidin-4-one (preQ 0 ) to 2-amino-5-aminomethylpyrrolo[2,3- d ]pyrimidin-4-one (preQ 1 ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.
研究领域:
生物化学
·
合成生物学
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DOI
高影响因子
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
·
2015
·
IF 2.128
Hui Fei,Gang Xu,Jian-Ping Wu,Li-Rong Yang
DERA SEP (2-deoxy- d -ribose-5-phosphate aldolase cloned from Staphylococcus epidermidis ) shows higher catalytic activity toward 2-deoxy- d -ribose-5-phosphate (DRP) than that of DERA ECO (2-deoxy-ribose aldolase cloned from Escherichia coli ). However, the acetaldehyde tolerance of DERA SEP is still at low level, which limits its use in large-scale applications. Although the mechanism responsible for enzyme inactivation in the presence of solvents is unclear, earlier work indicated the positive correlation between the tolerance of an enzyme for organic solvents and the rigidity of its structure. The acetaldehyde tolerance of DERA SEP was improved by site-directed mutagenesis on the basis of enhancing the rigidity of the protein structure. After calculating the virtual mutation energy (stability) of the enzyme and experimental verification, variant 10 mutant DERA SEP with three amino acid substitutions (T120C, G174I, and G213C) was harvested after three rounds of site-directed mutagenesis. It showed surprisingly high tolerance to acetaldehyde, with almost 70.5% residual catalytic activity after exposure to 300 mM acetaldehyde for 2 h, while the wild-type DERA SEP retained only 11.3% activity under the same condition. Because the rigidity of the protein was increased after incorporating the mutations, the specific activity of the variant 10 was only half that of DERA SEP . Thus, the acetaldehyde tolerance of DERA SEP was efficiently increased by enhancing the rigidity of the structure. These results suggested that the inactivation of DERA by exposure to acetaldehyde may result from protein structural damage.
研究领域:
生物化学
·
酶工程
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产品关联
DOI
高影响因子
Biomolecules
·
2024
·Vol. 14
·期 9
·1188
·
IF 4.8
Jiao Ma,Zhongke Sun,Zifu Ni,Yanli Qi,Qianhui Sun,Yuansen Hu,Chengwei Li
This study identified a salt-tolerant GH11 xylanase, Xyn st , which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xyn st and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xyn st in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan ( K m = 0.30 mg mL −1 for Xyn st and 0.18 mg mL −1 for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency ( K cat / K m = 15483.33 mL mg −1 s −1 ). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xyn st did. The conversion efficiencies of Xyn st and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries.
研究领域:
生物化学
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
Systems Microbiology and Biomanufacturing
·
2021
·Vol. 2
·期 3
·473-486
·
IF 0
Wu Aiai,Bai Yajun,Fan TaiPing,Zheng Xiaohui,Cai Yujie
l -Lactate dehydrogenases can reduce alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum. l -Lactate dehydrogenase gene (LF- l -LDH0845) with reducing activity towards 3,4-dihydroxyphenylpyruvate and phenylpyruvate was obtained from Lactobacillus fermentum JN248. To change the substrate specificity of LDH0845 and improve its catalytic activity towards large substrates, site-directed mutation of Tyr221 was performed by analyzing the amino acids in the active center. Kinetic parameters show that the k cat values of Y221F mutant on 3,4-dihydroxyphenylpyruvate, 4-methyl-2-oxopentanoate, and glyoxylate are 1.21 s −1 , 1.35 s −1 , and 0.72 s −1 , respectively, which are 420%, 150% and 130% of the wild-type LDH0845. This study shows that the mutations of Y221 can significantly change the substrate specificity of LDH0845, making it become a potential tool enzyme for the reduction of alpha-keto carboxylic acids with large functional groups.
研究领域:
生物化学
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分子生物学
·
酶工程
✓ 已验证
DOI
WEED SCIENCE
·
2024
·
IF 2.1
Yu Liu,Kerong Shi,Haitao Gao,Shaojing Yin,Liyao Dong,Zhike Feng
Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is increasingly infesting imidazolinone-tolerant (IMI-T) rice fields in China, imazamox resistance of E. crus-galli has become the major concern for weed management in IMI-T rice fields. In this study, the susceptible population JLGY-3 (S) and the suspected resistant population JHXY-2 (R) collected from IMI-T rice fields were used as research subjects. When treated with imazamox, JHXY-2 (R) population showed a high level of herbicide resistance with a resistance index (RI) of 31.2. JHXY-2 (R) was cross-resistant to all five acetolactate synthase (ALS) inhibitors from different chemical families, but sensitive to herbicides inhibiting acetyl-CoA carboxylase (ACCase). In order to understand the reason why JHXY-2 (R) was resistant to imazamox, we performed experiments to characterize potential TSR and NTSR mechanisms. A trp-574-leu amino acid mutation in ALS and low imazamox ALS sensitivity were the main mechanism underlying imazamox resistance in this JHXY-2 (R) population. There was no significant difference in ALS gene expression and ALS protein abundance between R and S populations. High-performance liquid chromatography-tandem mass spectrometry analysis showed enhanced metabolism of imazamox in JHXY-2 (R), which was in contrast to the results of pretreatment with a metabolic enzyme inhibitor. Treatments with the P450/GST inhibitors did not alter the resistance level of JHXY-2 (R) against imazamox. To further clarify the NTSR mechanism of JHXY-2 (R), transcriptome sequencing showed that there was almost no significant difference in the expression of P450 and GST metabolic enzyme genes between R and S populations, and only GST-U1 showed a significant induction in R population. In conclusion, amino acid mutation and higher enzyme activity of ALS are the main causes of imazamox resistance in JHXY-2 (R). However, given the differences in imazamox residues in the leaves of the E. crus-galli, there may still be undetectable NTSR that are causing imazamox resistance in the R population.
研究领域:
植物学
·
遗传学
·
农业科学
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产品关联
DOI
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2015
·Vol. 16
·期 4
·7273-7288
·
IF 2.862
Dongming Lan,Qian Wang,Jinxin Xu,Pengfei Zhou,Bo Yang,Yonghua Wang
Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase fromMalassezia globosais a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273–278 and the 3104 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.Keywords:mono- and diacylglycerol lipase;thermostability;substrate selectivity;site-directed mutagenesis
研究领域:
生物化学
·
蛋白质工程
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Lingzhi Zhang,Rui Zhou,Dekai Liu,Meinan Zhu,Guangya Zhang,Lijuan Zhang,Shu-Feng Zhou,Wei Jiang
Given their outstanding efficiency and selectivity, enzymes are integral in various domains such as drug synthesis, the food industry, and environmental management. However, the inherent instability of natural enzymes limits their widespread industrial application. In this study, we underscore the efficacy of enhancing protein thermal stability through comprehensive protein design strategies, encompassing elements such as the free energy of protein folding, internal forces within proteins, and the overall structural design. We also demonstrate the efficiency and precision of combinatorial screening in the thermal stability design of aldo-keto reductase (AKR7-2-1). In our research, three single-point mutations and five combinatorial mutations were strategically introduced into AKR7-2-1, using multiple computational techniques. Notably, the E12I/S235I mutant showed a significant increase of 25.4 °C in its melting temperature ( Tm ). Furthermore, the optimal mutant, E12V/S235I, maintained 80 % of its activity while realizing a 16.8 °C elevation in Tm . Remarkably, its half-life at 50 °C was increased to twenty times that of the wild type. Structural analysis indicates that this enhanced thermal stability primarily arises from reduced oscillation in the loop region and increased internal hydrogen bonding. The promising results achieved with AKR7-2-1 demonstrate that our strategy could serve as a valuable reference for enhancing the thermal stability of other industrial enzymes.
研究领域:
生物化学
·
分子生物学
·
工业酶工程
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DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2015
·
IF 6.409
Jingqi Tian,Qian Liu,Jinle Shi,Jianming Hu,Abdullah M. Asiri,Xuping Sun,Yuquan He
Considerable recent attention has been paid to homogeneous fluorescent DNA detection with the use of nanostructures as a universal “quencher”, but it still remains a great challenge to develop such nanosensor with the benefits of low cost, high speed, sensitivity, and selectivity . In this work, we report the use of iron-based metal–organic framework nanorods as a high-efficient sensing platform for fluorescent DNA detection. It only takes about 4 min to complete the whole “mix-and-detect” process with a low detection limit of 10 pM and a strong discrimination of single point mutation . Control experiments reveal the remarkable sensing behavior is a consequence of the synergies of the metal center and organic linker. This work elucidates how composition control of nanostructures can significantly impact their sensing properties, enabling new opportunities for the rational design of functional materials for analytical applications.
研究领域:
生物化学
·
纳米技术
·
分析化学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）