ORGANIC & BIOMOLECULAR CHEMISTRY
·
2015
·Vol. 13
·期 26
·7323-7330
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IF 3.562
Kelu Yan,Daoshan Yang,Wei Wei,Jing Zhao,Yuanyuan Shuai,Laijin Tian,Hua Wang
A novel, efficient, and catalyst-free strategy has been initially developed for the construction of thioesters via the direct radical oxidative decarboxylation of α-keto acids with thiols, and the corresponding target products were obtained in moderate to good yields. It offers an alternative approach for the synthesis of useful diverse thioesters.
研究领域:
有机化学
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合成化学
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DOI
PubMed
高影响因子
ADVANCED SYNTHESIS & CATALYSIS
·
2018
·Vol. 361
·期 4
·803-812
·
IF 5.123
Xinjian Yin,Yayun Liu,Lijun Meng,Haisheng Zhou,Jianping Wu,Lirong Yang
Glutamate dehydrogenases (GluDHs) are promising biocatalysts for the synthesis of chiral α-amino acids by asymmetric reductive amination of α-keto acids. However, their strict substrate specificity limits their applications. To address this problem, we developed a molecular engineering method for GluDHs that enhances the asymmetric reductive amination of bulky α-keto acids. Based on rational design, a “cave” located in the active site pocket of Pp GluDH (GluDH from Pseudomonas putida ), which plays an essential role in substrate recognition, was tailored to facilitate the accepting of bulky substrates. Two mutants (A167G and V378A) were discovered to have significantly enhanced catalytic activity toward 2-oxo-4-[(hydroxy)(methyl)phosphinyl]butyric acid (PPO) and several other bulky substrates. This molecular engineering method was then applied to ten other GluDHs from different sources and with different properties. All engineered GluDHs acquired substantial improvements in PPO-oriented catalytic activity. The most efficient mutant of NADP + (nicotinamide adenine dinucleotide phosphate)-specific GluDHs showed up to 1820-fold increased activity and the specific activity reached 111.02 U/mg-protein. The NAD + (nicotinamide adenine dinucleotide)-specific GluDHs, which have no detectable wild type activity toward PPO, acquired a considerable level of activity (1.90–29.48 U/mg-protein). In batch production of L-phosphinothricin, these “cave-tailored” GluDHs exhibited markedly improved catalytic efficiencies compared with their wild types and ee values of >99%. The space-time yields (STY) varied from 818.16 to 1482.96 g ⋅ L −1 ⋅ d −1 , suggesting potential practical applications of these mutants.
研究领域:
生物化学
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酶工程
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合成生物学
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DOI
高影响因子
FOOD CHEMISTRY
·
2017
·
IF 4.529
Biying Wang,Yajun Bai,Taiping Fan,Xiaohui Zheng,Yujie Cai
Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291 kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40–50 °C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg 2+ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. K m and k cat values for phenyl pyruvate were 0.62 mM and 77.38 s −1 , respectively, and the k cat / K m value was 124.81 mM −1 s −1 . The enzyme properties suggest it may act effectively under cheese ripening conditions.
研究领域:
生物化学
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食品科学
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微生物学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Lijun Yang,Tiantian Zhou,Zhiyi Zhang,Wei Qi,Mengfan Wang
The enzymatic oxidation of amino acids using amino acid oxidases (AAOs) as biocatalysts for α-keto acid synthesis has garnered increasing attention due to its environmental friendliness and exceptional reaction efficiency. However, the by-product H 2 O 2 inevitably causes the decarboxylation of α-keto acids and subsequently deactivates the enzyme. Herein, we used a simple in-situ biomineralization method to immobilize Pt NPs with catalase-like activity and L-amino acid oxidase (LAAO) in to a hydrogen-bonded organic framework ( TW HOF) under a mild condition, and the synthesized LAAO-Pt@ TW HOF cascade catalytic system was applied to synthesis of α-keto acid. Compared with free LAAO, the LAAO-Pt@ TW HOF exhibited a 3.3-fold activity, well reusability and complete conversion (100 %) of L-tryptophan to indole-3-pyruvic acid was achieved. This work offers a potential application of HOF materials in constructing high-efficiency enzyme-metal coupled catalyst for cascade reactions.
研究领域:
生物化学
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材料科学
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催化反应
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产品关联
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2022
·
IF 3.705
Jinbin Liu,Kaipeng Wang,Mian Wang,Huaxiang Deng,Xiaodong Chen,Yueling Shang,Xiaochen Liu,Xiaohong Yu
An engineered Escherichia coli was constructed by co-expressing L -amino acid deaminase, α-keto acid decarboxylase, alcohol dehydrogenase , and glucose dehydrogenase through two plasmids for tyrosol production. The activity of the rate-limiting enzyme L -amino acid deaminase from Cosenzaea myxofaciens (CmAAD) toward tyrosine was improved by structure-guided modification. The enzyme activity of triple mutant CmAAD V438G/K147V/R151E toward tyrosine was ~5.12-fold higher than that of the wild-type CmAAD. Secondly, the plasmid copy numbers and the gene orders were optimized to improve the titer of tyrosol. Finally, the recombinant strain CS-6 transformed 10 mM tyrosine into 9.56 ± 0.64 mM tyrosol at 45 ℃, and the space-time yield reached 0.478 mM·L −1 ·h −1 . This study proposes a novel idea for the efficient and natural production of tyrosol, which has great potential for industrial application.
研究领域:
生物技术
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酶学
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微生物学
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产品关联
DOI
PubMed
高影响因子
PROTEIN EXPRESSION AND PURIFICATION
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2024
·
IF 1.4
Junzhang Chang,Yuxin Zhang,Zhiwei Li,Yunfeng Ma,Xueqin Hu,Jingwen Yang,Hongbin Zhang
Chiral amino acids and their deamination products, α-keto acids, have important applications in food, medicine, and fine chemicals. In this study, two l -amino acid deaminase genes from Proteus mirabilis , PM473 of type Ⅰ and PM471 of type Ⅱ were cloned and expressed in Escherichia coli respectively, expected to achieve the chiral separation of amino acids . Extensive substrate preference testing showed that both deaminases had catalytic effects on the d -amino acid component of the D, l -amino acids, and PM473 has a wider catalytic range for amino acids. When D, L-Cys was used as the substrate, all L-Cys components and 75.1 % of D-Cys were converted to mercapto pyruvate , and the remaining D-Cys was a single chiral enantiomer . Molecular docking analysis showed that the interaction between the substrate and the key residues affected the stereoselectivity of enzymes . The compatibility of hydrophobicity between the binding pocket and substrate may be the basic factor that affects the substrate selectivity. This work provides an alternative method for the production of α-keto acids and the resolution of chiral amino acids.
研究领域:
生物化学
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生物催化
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酶学
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药物化学
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DOI
PubMed
高影响因子
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
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2024
·
IF 3.1
Chi Zhang,Haipiao Huang,Changhui Li,Lifang Wei,Jingru Wu,Rui Wang,Shiying Huang,Qiugu Chen,Pingli Mo,Yinghao Yin,Jianping Chen
The dysregulated levels of branched chain amino acids (BCAA) contribute to renal fibrosis in chronic kidney disease (CKD), yet specific analysis of BCAA contents and how they are regulated still remain unclear. It is therefore of great scientific interest to understand BCAA catabolism in CKD and develop a sensitive method for simultaneous determination of individual BCAA and their metabolites branched chain α-ketoacids (BCKA). In this work, the important role of BCAA metabolism that drives renal fibrosis in the process of CKD was first revealed by using transcriptomics. The key target genes controlling BCAA metabolism were then validated, that is, mRNA levels of BCKDHA and BCKDHB , the regulating rate-limiting enzymes during BCAA metabolism were abnormally reduced by quantitative PCR (qPCR), and a similar drop-off trend of protein expression of BCKDH, HIBCH and MCCC2 that are closely related to BCAA metabolism was also confirmed by western blotting. Furthermore, we established a novel strategy that simultaneously determines 6 individual BCAA and BCKA in serum and tissue. The method based on dansylhydrazine derivatization and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-QQQ-MS) achieved to simultaneously determine the contents of BCAA and BCKA, which is efficient and stable. Compared with normal rats, levels of BCAA including leucine, isoleucine and valine in serum and kidney of CKD rats was decreased, while BCKA including α-ketoisocaproic acid, α-ketomethylvaleric acid and α-ketoisovaleric acid was increased. Together, these findings revealed the abnormality of BCAA metabolism in driving the course of kidney fibrosis and CKD. Our current study sheds new light on changes in BCAA metabolism during CKD, and may facilitate development of drugs to treat CKD and renal fibrosis.
研究领域:
肾病学
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代谢组学
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分子生物学
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DOI
PubMed
高影响因子
Biotechnology Journal
·
2018
·Vol. 14
·期 3
·1800253
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IF 3.507
Junping Zhou,Yaling Wang,Jiajie Chen,Meijuan Xu,Taowei Yang,Junxian Zheng,Xian Zhang,Zhiming Rao
Unnatural amino acids (UAAs) play a key role in modern medicinal chemistry such as small molecules and peptide-based drugs with fast-growing markets. Low efficiency for natural enzymes including leucine dehydrogenase (LeuDH, EC1.4.1.9) are one major challenge for UAA production. Here, rational engineering of LeuDH from Bacillus cereus with a structure-based design approach is studied. The results achieve higher enzymatic activity and stability toward α-keto acid reduction by improving the hydrophobic and rigidity of enzymatic substrate entrance tunnel. High catalytic efficiency for variant E116V is associated with the presence of more hydrophobic tunnels that allows easy substrate diffusion, which is confirmed in absorbance spectroscopy study. For variant T45M/E116V, melting temperature and half-lives of thermal inactivation at 60 °C is 62.8 °C and 29.2 h, respectively, much higher than 48.4 °C and 3.4 h of wild type. Structural analysis indicates that an additional hydrogen bond in β5 fold is formed in variant T45M, which results in a more rigid β5 fold leading to better stability. Furthermore, asymmetric synthesis of α-amino acids with coenzyme regeneration reveals higher productivities for variant T45M/E116V. This study indicates the importance of substrate entrance tunnel for enzymatic activities and stability, the engineered LeuDH would better serve UAA production. Graphical Low yield rates for natural enzymes including leucine dehydrogenase (LeuDH, EC1.4.1.9) are one major challenge to the production of unnatural amino acids in industrial scale. Here, the catalytic activity and stability of Bc LeuDH toward α-keto acid reduction are remarkably improved via rational engineering of substrate entrance tunnel based on a structure-based design approach. This work will better serve the production of especially unnatural α-amino acids.
研究领域:
生物化学
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酶工程
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药物化学
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生物催化
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
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2023
·
IF 5.4
Kaihao Liu,Peng Yang,Xudong Zhang,Di Zhang,Liu Wu,Lan Zhang,Huan Zhang,Guoliang Li,Ruren Li,Liangyan Rong
Microbial interactions play an important role in regulating the metabolic function of fermented food communities, especially the production of key flavor compounds. However, little is known about specific molecular mechanisms that regulate the production of key flavor compounds through microbial interactions. Here, we designed a synthetic consortium containing Debaryomyces hansenii D1, Staphylococcus xylosus S1, and Pediococcus pentosaceus PP1 to explore the mechanism of the microbial interactions underlying the branched-chain aldehydes production. In this consortium, firstly, D. hansenii secreted amino acids that promoted the growth of P. pentosaceus and S. xylosus . Specifically, D. hansenii D1 secreted alanine , aspartate, glutamate, glutamine, glycine, phenylalanine , serine , and threonine , which were the primary nutrients for bacterial growth. P. pentosaceus PP1 utilized all these eight amino acids through cross-feeding, whereas S. xylosus S1 did not utilize aspartate and serine. Furthermore, D. hansenii D1 promoted the production of branched-chain aldehydes from S. xylosus and P. pentosaceus through cross-feeding of α-keto acids (intermediate metabolites). Thus, the accumulation of 2-methyl-butanal was promoted in all co-culture. Overall, this work revealed the mechanism by which D. hansenii and bacteria cross-feed to produce branched-chain aldehydes in fermented sausages .
研究领域:
食品科学
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微生物学
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代谢工程
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产品关联
DOI
PubMed
高影响因子
JACS Au
·
2024
·
IF 8.5
Yaoyun Wu,Yaozhong Cui,Wei Song,Wanqing Wei,Zhizhen He,Jinyang Tao,Dejing Yin,Xiulai Chen,Cong Gao,Jia Liu,Liming Liu,Jing Wu
l-Amino acid oxidase (LAAO) is an important biocatalyst used for synthesizing α-keto acids. LAAO from Rhodococcus opacus (RoLAAO) has a broad substrate spectrum; however, its low total turnover number limits its industrial use. To overcome this, we aimed to employ crystal structure-guided density functional theory calculations and molecular dynamic simulations to investigate the catalytic mechanism. Two key steps were identified: S → [TS1] in step 1 and Int1 → [TS2] in step 2. We reprogrammed the transition states [TS1] and [TS2] to reduce the identified energy barrier and obtain a RoLAAO variant capable of catalyzing 19 kinds of l-amino acids to the corresponding high-value α-keto acids with a high total turnover number, yield (≥95.1 g/L), conversion rate (≥95%), and space-time yields ≥142.7 g/L/d in 12–24 h, in a 5 L reactor. Our results indicated the promising potential of the developed RoLAAO variant for use in the industrial production of α-keto acids while providing a potential catalytic-mechanism-guided protein design strategy to achieve the desired physical and catalytic properties of enzymes.
研究领域:
生物催化
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酶工程
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计算化学
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蛋白质工程
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DOI
PubMed
高影响因子
Nature Catalysis
·
2023
·Vol. 6
·期 10
·906-915
·
IF 37.8
Li Mengyang,Wu Yongmeng,Zhao Bo-Hang,Cheng Chuanqi,Zhao Jinghui,Liu Cuibo,Zhang Bin
Amino acids have wide application in the food and pharmaceutical industries. Current biotic and chemical syntheses suffer from low efficiency, complex purification operations and high energy consumption. Here we report a sustainable electrocatalytic synthesis of alanine from NO and pyruvic acid over oxide-derived Ag with low-coordination sites under ambient conditions. Mechanistic studies reveal a cascade NO → NH 2 OH → pyruvate oxime → alanine pathway. The quick pyruvate oxime formation and slow pyruvate oxime reduction steps cause various side reactions, leading to low alanine production. Then, a spatially decoupled two-pot electrosynthesis system using flow reactors loaded with oxide-derived Ag is designed for pyruvate oxime formation and reduction reactions. This decoupled system delivers 3.85 g of easily purified alanine with a total Faradaic efficiency of 70% and a purity of >98% at 100 mA cm −2 . Further techno-economic analysis demonstrates the potential. This method is suitable for solar-energy-driven alanine electrosynthesis from polylactic acid wastes and for the fabrication of other amino acids.
研究领域:
化学工程
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电化学
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可持续化学
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生物技术
✓ 已验证
DOI
高影响因子
ACS Applied Materials & Interfaces
·
2021
·
IF 9.229
Zhenfu Wang,Yang Liu,Xiaoyan Dong,Yan Sun
Chemoenzymatic catalysis combining the traits of chemical and enzymatic catalysis provides tremendous possibilities for the design of biosynthetic pathways utilizing inorganic catalysts and enzymes. However, the efficiency of chemoenzymatic catalysis is usually governed by the synergy and compatibility of the two catalysts. Here, we report for the first time the catalase-like activity of cobalt phosphate nanocrystals (CoPs). By a one-pot biomimetic mineralization with CoPs and l-amino acid oxidase (LAAO) under a mild condition, we have fabricated a hybrid nanobiocatalyst, LAAO@CoPs, for the chemoenzymatic synthesis of α-keto acid. The as-fabricated nanobiocatalyst with directly contacted catalytic sites of the enzyme and nanozyme maximizes the substrate channeling effects for in situ chemical decomposition of the oxidative intermediate, H2O2, during the enzymatic oxidation of l-tryptophan (l-Trp), thus minimizing the H2O2 accumulation and byproduct generation. Benefiting from the superiority of LAAO@CoPs, complete conversion (100.0%) of l-Trp to indole pyruvic acid is achieved, over two times higher than the yield of the free LAAO system (47.6%). Meanwhile, LAAO@CoPs show high stabilities against heat and proteolytic treatments. This work offers a new design approach for constructing a high-performance nanobiocatalyst for cascade reactions, especially for those systems with toxic or reactive intermediates.
研究领域:
纳米技术
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生物催化
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化学生物学
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酶学
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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）