ACS Agricultural Science & Technology
·
2023
·
IF 2.5
Liang Jin,Xiao-Di Zhao,Jun-Ao Liao,Li-Jun Fu,Yi Lin
Cry toxins produced by Bacillus thuringiensis (Bt) are toxic to Lepidoptera, Coleoptera, and Diptera, but display very low activity against aphids. Recently, Cry41-related toxin was found to show moderate toxicity against Myzus persicae with the 50% lethal concentration (LC50) of 32.7 μg/mL, and its mode of action was proposed to be the acceleration of the apoptosis of aphid cells by enhancing Cathepsin B activity. This study focused on constructing Cry41-related mutants against M. persicae based on its interaction with Cathepsin B. First, eight key interacting residues in Cry41-related toxin were identified using alanine scanning and site-directed saturation mutagenesis. Subsequently, the positive mutant Cry41-7M protein (mutations of Gly48, Ile59, Lys364, Gln367, Gln377, Tyr378, and Ser400 to Tyr, Ala, Arg, Lys, Lys, Lys, and Ala in Cry41-related toxin, respectively) and the negative mutant Cry41-6A protein (mutations of Gly48, Lys364, Gln367, Gln377, and Tyr378 to Ala and mutation of Pro453 to Glu in Cry41-related toxin) were constructed, expressed, and purified. We then found that Cry41-7M protein performed slightly stronger than Cry41-related toxin in enhancing the enzymatic activity of Cathepsin B, whereas Cry41-6A protein did not affect Cathepsin B activity. A further bioassay showed that the mortality caused by Cry41-7M protein (LC50 = 19.144 μg/mL) was marginally higher than that of Cry41-related toxin, while Cry41-6A protein caused a decreased mortality (LC50 = 42.478 μg/mL). These results are expected to open new avenues for improving Cry aphidicidal activity.
研究领域:
昆虫学
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分子生物学
·
生物技术
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产品关联
DOI
高影响因子
ACS Synthetic Biology
·
2022
·
IF 5.249
Amreesh Parvez,Yuvaraj Ravikumar,Renu Bisht,Junhua Yun,Yang Wang,Sabapathy Poorna Chandrika,Hossain M. Zabed,Xianghui Qi
Biosynthesis of 1,3-propanediol (1,3-PD) by 1,3-propanediol oxidoreductase (PDOR) is often limited by the stability issues. To address this issue, the goal of the present study was to engineer the Clostridium butyricum PDOR dimeric interface. The interface exists between the chains and plays a role in the synthesis of 1,3-PD, which is hindered by the increased temperature and pH. Herein, we engineered PDOR by HotSpot Wizard 3.0 and molecular dynamics simulations, improving its thermal stability, pH tolerance, and catalytic properties with respect to the wild-type PDOR activity at 37 °C. Compared to the activity of the wild-type PDOR, the N298C mutant showed 0.5-fold greater activity at pH 8.0, while the P299E mutant showed significantly increased activity of over five fold at pH 4.0. Further structural comparisons between the wild-type and P299E mutant revealed that the extraordinary stability of the P299E mutant could be due to the formation of additional hydrogen bonds and salt bridges. The N298C mutant also exhibits thermal stability at a broad range of temperature at pH 8 with respect to wild-type PDOR and other mutants. The molecular dynamics simulations revealed that stability profiles of P299E mutants at pH 4.0 are attributed to identical root mean square deviation values and stable conformations in the motif region present in the dimer interface of the enzyme. These findings suggest that the dimer interface motifs are essential for the compactness and stability of the PDOR enzyme; therefore, engineering the PDOR using a structure-guided approach could aid in improving its activity and stability under various physiological conditions (pH and temperature).
研究领域:
生物化学
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酶工程
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
PLANT SCIENCE
·
2015
·
IF 3.607
Chao Huang,Qing-Bo Yu,Xin-Bo Yuan,Zi-Ran Li,Jing Wang,Lin-Shan Ye,Ling Xu,Zhong-Nan Yang
The fructokinase-like protein2 (FLN2) is a component of the PEP complex. FLN2 knockout mutants displayed a delayed greening phenotype on sucrose-containing medium. Our previous work indicated that partial PEP activity is essential for its greening phenotype. In this study, we further report that sufficient Rubisco accumulation is critical for fln2-4 greening. Sugar serves many important functions, such as an energy source and signaling molecule. Through pharmacological experiments using a sugar analog and sugar signaling inhibitor, we demonstrate that sugar serves as energy to support the fln2-4 greening. Seed-reserve and photosynthetic CO 2 -fixation are the primary energy sources for early seedling growth . No obvious differences were observed in the seed-reserve of the wild-type and fln2-4 by comparing their seed size and dark-germination, indicating that the defective carbon fixation may account for the energy deficit in fln2-4 during its early seedling growth. The Rubisco content was low in fln2-4 , but it rapidly accumulated during the greening of fln2-4 . Expression of a nuclear-encoded rbcL gene facilitates Rubisco accumulation and partially complements the mutant defects. These results suggest that the Rubisco accumulation is critical for fln2-4 greening. In summary, the rapid Rubisco accumulation that depends on sufficient PEP activity is important for normal seedling growth.
研究领域:
植物学
·
分子生物学
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DOI
高影响因子
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2022
·Vol. 38
·期 5
·1-8
·
IF 4.253
Li Yudi,Chen Guiying,Ge Fanglan,Dang Ting,Ren Yao,Zeng Bei,Li Wei
γ-aminobutyric acid (GABA) has various physiological functions and is widely used in medicine, food, and other fields. Glutamate decarboxylase (GAD) is a key enzyme that catalyzes the decarboxylation of L-glutamate to synthesize GABA. However, the industrial application of microorganism-derived GAD is limited by its rapid loss of enzymatic activity with pH approaching neutrality. In this study, a novel glutamate decarboxylase, GAD MSM, from Mycobacterium smegmatis was overexpressed and purified. On the basis of homologous modeling and substrate molecular docking, several GAD MSM mutants were constructed, and their enzymatic properties were analyzed. The results showed that the optimal pH of wild-type GAD MSM is 5.4; at pH 6.2, 22.8% enzymatic activity was retained. The T211I replacement in GAD and C-terminal deletion mutant GAD MSMΔC showed relatively high catalytic activity in a pH range of 5.0–7.0. The V max and K m values of GAD MSMΔC were 14.69 and 5.70, respectively, at pH 5.5, and 9.87 and 6.17, respectively, at pH 7.0. Compared with the wild-type GAD, GAD MSMΔC maintained higher affinity and enzymatic activity of the substrate, maintaining 78.5% of the highest enzymatic activity even at pH 7.0, which is the highest reported activity retention for GAD under neutral pH condition. Therefore, GAD MSMΔC can be used for the transformation of high-yielding strains and industrial production of GABA.
研究领域:
微生物学
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酶工程
·
生物化学
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产品关联
DOI
PubMed
高影响因子
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2025
·Vol. 41
·期 3
·1-11
·
IF 4.2
Liu Ming-Yu,Wang Yu-Yue,Li Han-Zhi,Luo Xue-Mei,Feng Jia-Xun,Zhao Shuai
Nitriles exhibit acute cytotoxicity to human and animal cells. Nitrilase is a green biocatalyst that can directly convert nitrile into nontoxic carboxylic acids and ammonia. However, the nitrilases capable of degrading 3-butenenitrile and 4-pentenenitrile derived from glucosinolate present in rapeseed meals are still limited. This study expressed the nit rilase 2 gene from bacterium Paraburkholderia g raminis ( PgNIT2 ) in Escherichia coli . The purified recombinant nitrilase rPgNIT2 showed specific activities of 5.94 U/mg and 6.66 U/mg when using 3-butenenitrile and 4-pentenenitrile as substrates at the optimal pH 7.0 and temperature 45 °C. The substrate binding pocket of PgNIT2 comprising 12 amino acids was required for rPgNIT2 activity. Remarkably, the specific activity of mutant enzyme A190I increased by 68.3% and 57.3%, respectively, against 3-butenenitrile and 4-pentenenitrile compared with the wild-type enzyme. Furthermore, the A190I showed improved thermostability and hydrolysis ability towards 3-butenenitrile and 4-pentenenitrile compared with rPgNIT2 but shared the same optimal pH and temperature. These results provided theoretical guidance for engineering of bacterial nitrilases used for rapeseed meal detoxification.
研究领域:
生物化学
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Plants-Basel
·
2024
·Vol. 13
·期 2
·315
·
IF 4
Sumei Li,Min Yin,Peng Wang,Lulu Gao,Fenni Lv,Rutong Yang,Ya Li,Qing Wang,Linfang Li,Yongdong Liu,Shuan Wang
Background: The yellow-leafgl1mutant ofLagerstroemia indicaexhibits an altered phenylpropanoid metabolism pathway compared to wild-type (WT). However, details on the metabolites associated with leaf color variation, including color-specific metabolites with bioactive constituents, are not fully understood. Methods: Chemical and metabolomics approaches were used to compare metabolite composition and antioxidant capacity between thegl1mutant and WT leaves. Results: The mutant exhibited an irregular xylem structure with a significantly lower phenolic polymer lignin content and higher soluble phenolic compounds. Untargeted metabolomics analysis identified phenolic compounds, particularly lignans, as key differential metabolites betweengl1and WT, with a significant increase in the mutant. The neolignan derivative balanophonin-4-O-D-glu was identified as a characteristic metabolite in thegl1mutant. The soluble phenolic compounds of thegl1mutant exhibited higher FRAP, ABTS, DPPH, and hydroxyl radical scavenging activity than in WT. Correlation analysis showed a positive relationship between antioxidant capacity and phenolic compounds inL. indica. Conclusions: Metabolites associated with leaf color variation in theL. indicayellow-leafgl1mutant demonstrated high antioxidant capacity, particularly in scavenging hydroxyl radicals.
研究领域:
植物学
·
代谢组学
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遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Synthetic Biology
·
2024
·
IF 3.7
Jialing Wang,Jie Chen,Kemin Lv,Zhen Gao,Jiahuang Li,Bin Wu,Bingfang He,Gerhard Schenk
Processive endoglucanases have generated significant interest due to their bifunctionality in the degradation of cellulose and low product inhibition. However, enhancing their catalytic efficiency through engineering remains a formidable challenge. To address this bottleneck, our engineering efforts targeted loop regions located in the substrate channel of processive endoglucanase EG5C-1. Guided by a comparative analysis of characteristic structural features of the substrate channels in cellobiohydrolase, endoglucanase, and processive endoglucanase, a highly active triple mutant CM6 (N105H/T205S/D233L) was generated that had a 5.1- and 4.7-fold increase in catalytic efficiency toward soluble substrate carboxymethyl cellulose-Na and insoluble substrate phosphoric acid-swollen cellulose (PASC), compared with wild-type EG5C-1. Furthermore, this mutant exhibited greater processivity compared to EG5C-1. Molecular dynamics simulations unveiled that the mutations in the loop regions reshaped the substrate channel, leading to a deeper cleft, resembling the closed channel configuration of cellobiohydrolases. The increased compactness of the substrate channel induced changes in the substrate binding mode and substrate deformation, thereby enhancing both binding affinity and catalytic efficiency. Moreover, metadynamics simulations demonstrated that the processive velocity of cellulose chain through the binding channel in mutant CM6 surpassed that observed in EG5C-1.
研究领域:
生物化学
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酶工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2025
·
IF 3.3
He Ying,Huang Ao,Liu Yun
Phospholipase D (PLD) is a valuable enzyme in industrial processes for converting phosphatidylcholine (PC) to phosphatidylserine (PS). In this study, a strain of Bacillus cereus was isolated from soil and identified through 16S rRNA sequencing. To enhance PLD activity, various mutagenesis strategies—including chemical treatment, irradiation, and their combinations—were employed, resulting in four high-activity positive mutants (C-7, I-12, CI 7–12, and IC 13–14). Among these, the CI 7–12 mutant exhibited a significantly higher enzymatic activity, showing a 3.12-fold increase (312.2%) compared to the wild-type strain. Fermentation conditions were optimied using response surface methodology (RSM), achieving a PLD activity of 35 U/mL. The enzyme demonstrated stability over a wide temperature range (30–60 °C) and pH range (6–10), with a half-life of 128 days. Kinetic analysis revealed a V max of 20.04 μmol/h and a K m of 7.13 μmol/mL, indicating efficient activity. In bioconversion experiments, the PLD-enriched fermentation broth catalyzed the conversion of PC to PS, achieving a 53.0% conversion rate and a 92.3% selectivity for PS in a two-phase system. These findings expand the potential sources of PLD and underscore its applicability for PS production in biotechnological applications.
研究领域:
微生物学
·
生物化学
·
工业生物技术
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2025
·
IF 6.2
Zihao Yin,Wanqing Wei,Wei Song,Jian Wen,Guipeng Hu,Xiaomin Li,Cong Gao,Jia Liu,Jing Wu
Epoxides, a class of ethers with a three-membered ring structure, are widely used in the textile, pharmaceutical, and packaging industries. Chemoenzymatic epoxidation presents a promising method for synthesizing epoxides. However, its epoxidation efficiency is hindered by low chemoselective perhydrolysis, which is caused by the hydrolysis side reaction in the aqueous phase. In this study, a chemoenzymatic epoxidation process in the aqueous phase was developed by utilizing an acyltransferase from P. litoralis (PlAcT) for its chemoselective perhydrolysis. Crystal structure analysis, molecular dynamics simulations, and quantum mechanics calculations, along with site-specific mutagenesis, revealed that the selectivity of perhydrolysis is due to a lower energy barrier in the acyl transfer step compared to that in hydrolysis. Furthermore, the mutant PlAcTM3–2 exhibited a 7.6-fold improvement in solvent stability and a 1.3-fold increase in perhydrolysis activity compared to the wild type, achieved by reshaping interface interactions. As a result, the engineered strain Y07, harboring PlAcTM3–2, successfully synthesized compounds 3a–3n with conversions ranging from 11–99%, and the titers of compounds α-pinene oxide(3i), β-pinene oxide(3j), 3-carene oxide(3k), and limonene dioxide(3l–3) reached 55.8, 16.7, 75.2, and 21.4 g/L, respectively. These results demonstrate a sustainable method for chemoenzymatic epoxidation in the aqueous phase.
研究领域:
生物化学
·
酶工程
·
绿色化学
✓ 已验证
DOI
PubMed
高影响因子
PLANT JOURNAL
·
2025
·Vol. 122
·期 1
·e70127
·
IF 5.7
Junjie Liu,Yuanyuan Huang,Huan Du,Jing Tian,Fan Zhu,Jianguo Zhang,Qing Zhang,Xiaolin Wang,Liangfa Ge
SUMMARY The microbial community residing on the phyllosphere is influenced by many factors, including the host plant's genotype as well as its secondary metabolites. Anthocyanins are a group of flavonoids renowned for their antioxidative properties and are widely distributed across plant tissues. However, the potential impact of anthocyanins on plant-associated microbial communities remains unknown. In the model legume Medicago truncatula , we isolated a mutant named purple leaves ( pl ) that produces purple leaves at a young stage due to over-accumulated anthocyanins. Through sequencing 16S rRNA amplicons of phyllosphere microbes in the pl mutant, we show that anthocyanins significantly enhance the abundance of endophytic lactic acid bacteria within plant leaves. Further in vitro study revealed that anthocyanins derived from pl can significantly promote the growth of lactic acid bacteria under anaerobic conditions. The accumulated anthocyanins in pl leaves reduced reactive oxygen species (ROS), thereby creating a favorable environment for the growth of facultative anaerobic lactic acid bacteria and resultantly increasing the abundance of phyllosphere lactic acid bacteria. Our findings elucidate the role of anthocyanins in modulating the community structure of phyllosphere microbiota in M. truncatula and provide new insights into the relationship between plant secondary metabolites and phyllosphere microbiota.
研究领域:
植物学
·
微生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOCHEMISTRY
·
2016
·
IF 2.876
Zhenqiu Li,Ruiping Gao,Qinggang Hao,Huifang Zhao,Longbin Cheng,Fang He,Li Liu,Xiuhua Liu,Wayne K. W. Chou,Huajie Zhu,David E. Cane
The T296V mutant of amorpha-4,11-diene synthase catalyzes the abortive conversion of the natural substrate (E,E)-farnesyl diphosphate mainly into the acyclic product (E)-β-farnesene (88%) instead of the natural bicyclic sesquiterpene amorphadiene (7%). Incubation of the T296V mutant with (3R,6E)-nerolidyl diphosphate resulted in cyclization to amorphadiene. Analysis of additional mutants of amino acid residue 296 and in vitro assays with the intermediate analogue (2Z,6E)-farnesyl diphosphate as well as (3S,6E)-nerolidyl diphosphate demonstrated that the T296V mutant can no longer catalyze the allylic rearrangement of farnesyl diphosphate to the normal intermediate (3R,6E)-nerolidyl diphosphate, while retaining the ability to cyclize (3R,6E)-nerolidyl diphosphate to amorphadiene. The T296A mutant predominantly retained amorphadiene synthase activity, indicating that neither the hydroxyl nor the methyl group of the Thr296 side chain is required for cyclase activity.
研究领域:
植物生物学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
iScience
·
2024
·
IF 4.6
Jiawei Wang,Xingyu Ouyang,Shiyu Meng,Jiayi Li,Liangxu Liu,Chaofeng Li,Hengrun Li,Haotian Zheng,Chao Liao,Yi-Lei Zhao,Jun Ni
Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of Th Ado, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes.
研究领域:
生物化学
·
生物技术
·
酶学
·
可持续化学
✓ 已验证
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）