微生物酶催化腈水解反应的研究进展

腈的酶法水解不仅反应条件温各,而且高效性、高选择性,在光学活性羧酸及其衍生物的合成中具有巨大的应用潜力。综述了催化腈水解的酶类、反应类型、反应特性、影响反应的因素及其在工业上的应用前景。     

Virtual screening of carboxylic acid reductases for biocatalytic synthesis of 6-aminocaproic acid and 1,6-hexamethylenediamine

The key precursors for nylon synthesis, that is, 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD), are produced from petroleum-based feedstocks. A sustainable biocatalytic alternative method from bio-based adipic acid has been demonstrated recently. However, the low efficiency and specificity of carboxylic acid reductases (CARs) used in the process hampers its further application. Herein, we describe a highly accurate protein structure prediction-based virtual screening method for the discovery of new CARs, which relies on near attack conformation frequency and the Rosetta Energy Score. Through virtual screening and functional detection, five new CARs were selected, each with a broad substrate scope and the highest activities toward various di- and ω-aminated carboxylic acids. Compared with the reported CARs, KiCAR was highly specific with regard to adipic acid without detectable activity to 6-ACA, indicating a potential for 6-ACA biosynthesis. In addition, MabCAR3 had a lower K_m with regard to 6-ACA than the previously validated CAR MAB4714, resulting in twice conversion in the enzymatic cascade synthesis of HMD. The present work highlights the use of structure-based virtual screening for the rapid discovery of pertinent new biocatalysts.     

Heterogeneous biocatalyst for nitrile and amide transformation based on cells of nitrile-hydrolyzing bacteria and multiwalled carbon nanotubes

A heterogeneous biocatalyst for the biotransformation of nitriles and amides of carboxylic acids in the form of cells of nitrile-hydrolyzing bacteria immobilized on the carrier, was created based on multiwalled carbon nanotubes (MWCNTs). It was shown that bacterial cells form aggregates in contact with powderformed purified or unpurified MWCNTs. The amount of both gram-positive and gram-negative bacteria binding with unpurified MWCNTs was significantly higher than with purified. The nitrile hydratase and amidase activity of bacterial aggregates of purified MWCNTs was preserved to a greater extent as compared to that of unpurified MWCNTs and cells adhered to the surface of the carbonized pyrosealing material with MWCNTs. Both gram-positive Rhodococcus ruber gt1 and gram-negative Alcaligenes faecalis 2 remained viable when cultured in the presence of purified or unpurified MWCNTs. The obtained heterogeneous biocatalyst can be easily separated from the medium by filtration and can be used repeatedly.     

Microbial Isobutyronitrile Utilization under Haloalkaline Conditions

The utilization of isobutyronitrile (iBN) as a C and N source under haloalkaline conditions by microbial communities from soda lake sediments and soda soils was studied. In both cases, a consortium consisting of two different bacterial species capable of the complete degradation and utilization of iBN at pH 10 was selected. The soda lake sediment consortium consisted of a new actinobacterium and a gammaproteobacterium from the genus Marinospirillum. The former was capable of fast hydrolysis of aliphatic nitriles to the corresponding amides and much-slower further hydrolysis of the amides to carboxylic acids. Its partner cannot hydrolyze nitriles but grew rapidly on amides and carboxylic acids, thus acting as a scavenger of products released by the actinobacterium. The soda soil consortium consisted of two Bacillus species (RNA group 1). One of them initiated nitrile hydrolysis, and the other utilized the hydrolysis products isobutyroamide (iBA) and isobutyrate (iB). In contrast to the actinobacterium, the nitrile-hydrolyzing soil Bacillus grew rapidly with hydrolysis products, but it was dependent on vitamins most probably supplied by its product-utilizing partner. All four bacterial strains isolated were moderately salt-tolerant alkaliphiles with a pH range for growth from pH 7.0 to 8.5 up to 10.3 to 10.5. However, both their nitrile hydratase and amidase activities had a near-neutral pH optimum, indicating an intracellular localization of these enzymes. Despite this fact, the study demonstrated a possibility of whole-cell biocatalytic hydrolysis of various nitriles at haloalkaline conditions.     

Construction of an engineered biocatalyst system for the production of medium-chain α,ω-dicarboxylic acids from medium-chain ω-hydroxycarboxylic acids

Medium-chain α,ω-dicarboxylic acids produced from renewable long-chain fatty acids are valuable as precursors in the chemical industry. However, they are difficult to produce biologically at high concentrations. Although improved biocatalyst systems consisting of engineering of Baeyer–Villiger monooxygenases are used in the production of ω-hydroxycarboxylic acids from long-chain fatty acids, the engineering of biocatalysts involved in the production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids has been rarely attempted. Here, we used highly active bacterial enzymes, Micrococcus luteus alcohol dehydrogenase and Archangium violaceum aldehyde dehydrogenase, for the efficient production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids and constructed a biocatalyst with cofactor regeneration system by introducing NAD(P)H flavin oxidoreductase as the NAD(P)H oxidase. The inhibition of the biocatalyst by hydrophobic substrates was attenuated by engineering a biocatalyst system with an adsorbent resin, which allowed us to obtain 196 mM decanedioic, 145 mM undecanedioic, and 114 mM dodecanedioic acid from 200 mM of C10, C11, and C12 hydroxyl saturated carboxylic acids, respectively, and 141 mM undecanedioic acid from 150 mM C11 unsaturated carboxylic acids, with molar conversions of 98%, 97%, 95%, and 94%, respectively. The concentration of undecanedioic acid obtained was approximately 40-fold higher than that in the previously highest results. Our results from this study can be applied for the industrial production of medium-chain α,ω-dicarboxylic acids from renewable long-chain fatty acids.     

Inhibition of the β-class carbonic anhydrases from Mycobacterium tuberculosis with carboxylic acids

The growth of Mycobacterium tuberculosis is strongly inhibited by weak acids although the mechanism by which these compounds act is not completely understood. A series of substituted benzoic acids, nipecotic acid, ortho- and para-coumaric acid, caffeic acid and ferulic acid were investigated as inhibitors of three β-class carbonic anhydrases (CAs, EC 4.2.1.1) from this pathogen, mtCA 1 (Rv1284), mtCA 2 (Rv3588c) and mtCA 3 (Rv3273). All three enzymes were inhibited with efficacies between the submicromolar to the micromolar one, depending on the scaffold present in the carboxylic acid. mtCA 3 was the isoform mostly inhibited by these compounds (K_Is in the range of 0.11–0.97 µM); followed by mtCA 2 (K_Is in the range of 0.59–8.10 µM), whereas against mtCA 1, these carboxylic acids showed inhibition constants in the range of 2.25–7.13 µM. This class of relatively underexplored β-CA inhibitors warrant further in vivo studies, as they may have the potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug or extensive multi-drug resistance.     

Development of a radiolabeled ATP assay for carboxylic acid:CoA Ligases and its use in the characterization of the xenobiotic carboxylic acid:CoA ligases of bovine liver mitochondria

A radiolabeled ATP assay was developed for measuring carboxylic acid:CoA ligase activity. The assay was designed to measure the formation of [γ-³³P]pyrophosphate from [γ-³³P]ATP in the course of the reaction. The assay was linear with protein concentration, and rates as low as 1 pmol/min were measurable. Rates determined with this assay were in agreement with rates determined with [14C]carboxylic acids. The assay was used to characterize the substrate specificity of the XL-I, XL-II, and XL-III ligases from bovine liver mitochondria. Forty carboxylic acids were tested for activity. The enzymes differed in their substrate specificities with XL-I and XL-II being the most similar and XL-III having the broadest specificity. This study has uncovered 19 new carboxylic acids that are substrates for these enzymes. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 151–155, 1998     

代谢工程改造解脂耶氏酵母合成羧酸的研究进展

解脂耶氏酵母是一种具有独特生理代谢特征的非常规酵母.它具有可以利用多种廉价碳源、低pH值耐受性好、分泌能力强等优点,因此非常适合用于各种工业产品的微生物发酵.目前,解脂耶氏酵母已被证实具有高效生产多种(同源或异源)有机羧酸的能力.本文对近年来利用代谢工程及合成生物学技术改造解脂耶氏酵母生产羧酸的实例进行了总结,并重点介...     

A plate method for screening of bacteria capable of degrading aliphatic nitriles

A novel indicator plate method was developed for screening of aliphatic-nitrile-degrading bacteria. Isolated bacteria were tested for utilization of acetonitrile as sole source of carbon and nitrogen with the release of ammonia. The released ammonia causes increase of the pH of the medium. Phenol red indicator is used for detection of ammonia based on colour change of the indicator dye from red to pink. The liberation of ammonia from aliphatic-nitrile-utilizing bacteria is also studied in plates containing other indicators such as bromothymol blue and phenolphthalein. The usefulness of the indicator plate is demonstrated for bacteria that degrade certain aliphatic nitriles. Bacteria degrading nitriles as a nitrogen source can also be isolated with a medium containing additional carbon source. This plate method would be useful in isolation and screening of bacteria for degradation of aliphatic nitriles and also for production of nitrile-hydrolyzing enzymes.     

Electromicrobial regeneration of pyridine nucleotides and other preparative redox transformations with Clostridium thermoaceticum

Clostridium thermoaceticum contains interesting enzymes suitable for redox reactions. Various AMAPOR (artificial-mediator-accepting pyridine-nucleotide oxidoreductase) activities were used for electromicrobial pyridine nucleotide regeneration. The combination of AMAPOR with commercially available pyridine-nucleotide-dependent oxidoreductases led to (S)-glutamate, (2R,3S)-isocitrate, (2S,3R)-isocitrate, 6-phosphogluconate and ribulose 5-phosphate. The redox equivalents were provided by electrochemically regenerated artificial mediators. Methylviologen or cobalt sepulchrate were used for NAD(P)H regeneration, whereas carboxamidomethylviologen (CAV) or anthraquinone sulphonates (AQ-S) were suitable for NAD(P)⁺ regeneration. With resting cells of C. thermoaceticum productivity numbers {mmol product/[biocatalyst (kg dry weight) × time (h)]} of about 30 000 for NADPH, 7000 for NADH and 14 000 for NADP⁺ regeneration could be reached. The cycle number for NADPH regeneration was up to 4300, that for NADP⁺ regeneration was at least 1600. An aldehyde and an alcohol oxidoreductase were used to reduce non-activated carboxylic acids to the alcohols and to dehydrogenate primary alcohols to the aldehydes or carboxylates. The electromicrobial reduction of 6-chloropyridine 3-carboxylate to the corresponding alcohol was compared with the reduction by CO as electron donor.The application of phenothiazine-dye-type mediators (thionine, methylene blue) converted primary alcohols to the aldehydes with productivity numbers up to 1400 in the presence of hydrazine as aldehyde scavenger. With CAV or AQ-S, alcohols were dehydrogenated to carboxylic acids with productivity numbers of almost 1700.     

Enantioselective kinetic resolution of phenylalkyl carboxylic acids using metagenome‐derived esterases

Enantiomerically pure β-arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities. A library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter plate-based screening using a pH-indicator assay. Out of these, 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity. The most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions. Thus, both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields.     

A convenient method for the synthesis of norstatine derivatives from N-Fmoc or N-Boc amino acids

Summary A new and convenient method for synthesizing norstatine derivatives has been developed. The key step is the preparation of propargylic alcohols from N-Fmoc or N-Boc amino aldehydes, followed by transformation to carboxylic acids after protection of the alcohols. The N-Boc-protected derivatives are coupled to a dipeptide sequence to afford peptide mimetics (norstatines).     

Di(1‐naphthyl) methanol ester of carboxylic acids for absolute stereochemical determination

The absolute stereochemistry of chiral carboxylic acids is determined as a di(1‐naphthyl)methanol ester derivative. Computational scoring of conformations favoring either P or M helicity of the naphthyl groups, capable of exciton‐coupled circular dichroic coupling, leads to a predicted stereochemistry for the derivatized carboxylic acids.     

A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids

Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs) as key components of crucial nutrients, soaps, industrial chemicals, and fuels. However, our ability to control the composition of microbially synthesized FFAs is still limited, particularly, for producing medium-chain fatty acids (MCFAs). This is mainly due to the lack of high-throughput approaches for FFA analysis to engineer enzymes with desirable product specificity. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. In particular, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and a higher m/z peak ratio at 730 and 758 was used as an indication for improved MCFA production. This colony-based method can be performed at a rate of ~2 s per sample, representing a substantial improvement over gas chromatography-MS (typically >30 min per sample) as the gold standard method for FFA detection. To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified nine missense mutations that resulted in improved MCFA production relative to the wild-type strain. Colony-based MALDI-ToF MS screening provides an effective approach for engineering microbial fatty acid compositions in a high-throughput manner.     

A new irreversible enzyme-aided esterification method in organic solvents

A new irreversible esterification method for carboxylic acids catalyzed by a lipase from Candida antarctica (Novozyme 435) in organic solvents has been developed. The water produced during the process is chemically destroyed by a corresponding ester of acetoacetate, which acts as a sacrificial substrate in this reaction. The flavour esters isobutyl acetate, methyl butyrate, ethyl butyrate and benzyl butyrate were synthesized either in small scale (0.05 mol) or large scale (1 mol). The yields range from 82 to 92% within 24 h at 52°C. Optimal molar ratios of reactants were 1:1:1 (carboxylic acid:alcohol:acetoacetate).     

一种3-氰基吡啶水解酶产生菌的高通量筛选策略及应用

利用改进的羟肟酸铁分光光度比色法建立了一种简单、快速、高通量的腈水解酶筛选方法.应用该方法从土壤中筛选获得1株具有3-氰基吡啶水解酶活性的菌株CCZU10 -1,经16S rDNA序列分析,鉴定该菌为红球菌属Rhodococcus sp.;同时确定了最适反应温度、pH和金属离子添加剂分别为30℃、7.0和Ca2+ (0.1 mmol/L).在最适催化反应条件下,催化转化50 mmol/L烟腈36 h,烟酸的产率可达到93.5％.     

Biocatalytic reduction of short‐chain carboxylic acids into their corresponding alcohols with syngas fermentation

Short‐chain carboxylic acids generated by various mixed‐ or pure‐culture fermentation processes have been considered valuable precursors for production of bioalcohols. While conversion of carboxylic acids into alcohols is routinely performed with catalytic hydrogenation or with strong chemical reducing agents, here, a biological conversion route was explored. The potential of carboxydotrophic bacteria, such as Clostridium ljungdahlii and Clostridium ragsdalei, as biocatalysts for conversion of short‐chain carboxylic acids into alcohols, using syngas as a source of electrons and energy is demonstrated. Acetic acid, propionic acid, n‐butyric acid, isobutyric acid, n‐valeric acid, and n‐caproic acid were converted into their corresponding alcohols. Furthermore, biomass yields and fermentation stoichiometry from the experimental data were modeled to determine how much metabolic energy C. ljungdahlii generated during syngas fermentation. An ATP yield of 0.4–0.5 mol of ATP per mol CO consumed was calculated in the presence of hydrogen. The ratio of protons pumped across the cell membrane versus electrons transferred from ferredoxin to NAD⁺ via the Rnf complex is suggested to be 1.0. Based on these results, we provide suggestions how n‐butyric acid to n‐butanol conversion via syngas fermentation can be further improved. Biotechnol. Bioeng. 2013; 110: 1066–1077. © 2012 Wiley Periodicals, Inc.     

The Comparative Analysis of Carboxylic Acid Composition of Four Iris Species from Ukraine

The present article reports results of analysis of carboxylic acids in leaves of Iris species from Ukraine using a gas chromatography (GC) method with mass spectrometric (MS) detection (GC/MS). Carboxylic acids play significant roles in contemporary society as evidenced by multiple applications in fields of medicine, agriculture, pharmacy, food, and other industries. Study of natural plant products as a source of organic acids is of particular interest. Carboxylic acid composition of leaves of Iris hungarica Waldst. & Kit., Iris germanica L., Iris pallida Lam., and Iris variegate L. was studied for the first time applying GC/MS method. The mass spectrums of compounds were matched with NIST and WILEY Libraries. The GC/MS analysis revealed the presence of 26 common acids in the plant raw materials studied. The short-chain carboxylic acids, such as citric (1337.5–12364.4 mg/kg), malic (50.8–4558.0 mg/kg) and oxalic (1199.0–3435.2 mg/kg) acids were contained in significantly high quantity in all samples. Ferulic, p-coumaric and vanillic acids were the most abundant among phenolic acids. α-Linolenic acid was dominant in the leaves of I. germanica (869.5 mg/kg), I. pallida (753.3 mg/kg), and I. variegate (250.3 mg/kg) among polyunsaturated fatty acids, however, linoleic acid prevailed in the plant raw material of I. hungarica (1150.7 mg/kg). Since the leaves of Iris species studied contain carboxylic acids with diverse pharmacological activity, extracts of these raw materials are perspective for development food supplements and medicines.     

Characterization of Plasmodium phosphatidylserine decarboxylase expressed in yeast and application for inhibitor screening

Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparum PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodium PSD in yeast as a tool for screening a library of anti‐malarials. One of these compounds is 7‐chloro‐N‐(4‐ethoxyphenyl)‐4‐quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4‐quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity