Screening of microbial esterases for asymmetric hydrolysis of 2-ethylhexyl butyrate

Summary A pH indicator agar plate method was used to screen for esterase activities for hydrolysis of 2-ethylhexyl butyrate. Seven hundred and fifty-seven selected microbial cultures, including 325 bacteria, and 432 yeasts and actinomycetes from the ARS Culture, Collection, were screened. Among them, 62 cultures hydrolyzed 2-ethylhexyl butyrate. Of these strains only 17 showed lipase activity on a rhodamine B lipase screen. The reaction products, 2-ethyl-1-hexanol andn-butyric acid were confirmed by gas-liquid chromatography (GC) and GC/MS analyses. The yield of 2-ethyl-1-hexanol varied depending on the strains of the microorganisms, with the highest yield at 79.1% by a strain ofPseudomonas myxogenes Product analyses with a cyclodextrin GC chiral column showed that two strains ofPseudomonas produced, greater than 80% enantiomeric excess of S(+)-2-ethyl-1-hexanol.     

热解纤维素菌属F32中乙酰木聚糖酯酶Axe7的表征

【目的】从嗜热厌氧微生物热解纤维素菌属F32(Caldicellulosiruptor sp.F32)菌株中鉴定出可水解木聚糖侧链乙酰基团的脂酶。【方法】通过基因组序列注释、比对以及蛋白结构预测的方法,发现一个潜在的脂酶7家族的(CE-7)乙酰木聚糖脂酶Axe7。利用基因克隆、质粒构建以及在大肠杆菌中表达目标蛋白并纯化等实验方法,获得了该酶的重组蛋白。【结果】以4-甲基乙酸伞形酯(4-Methylumbelliferyl-acetate)作为底物时,该酶的最适反应p H在6.5-7.0之间,最适反应温度为85°C,在最适的温度和p H条件下,Axe7活性半衰期(Half-life)超过4 h。在不同金属离子(1.5 mmol/L)存在下,Axe7活性可保持为最适反应酶活的(66.3±4.6)%-(95.7±2.3)%之间,说明金属离子对其酶活有一定的影响。通过测定酶动力学发现Axe7的Km和kcat值分别为0.39 mmol/L和66.95 s-1。【结论】从高温厌氧微生物中发现并表征一个热稳定性良好的乙酰木聚糖脂酶,为木质纤维素的高温糖化和生物炼制提供了一个可工业化的潜在选择。     

Partial characterization of neuropathy target esterase and related phenyl valerate esterases from bovine adrenal medulla

The mechanism by which organo-phosphorus-induced delayed polyneuropathy is induced relates to the specific inhibition and subsequent modification (“aging”) of a protein known as neuropathy target esterase (NTE), operatively defined as paraoxon-resistant and mipafox-sensi-tive phenyl valerate (PV) esterase activity. This protein has fundamentally been investigated in hen brain, the latter being the habitually employed OPIDP study model. In the present article, a partial characterization is made of the NTE and other related PV esterases in the bovine adrenal medulla and brain; NTE sensitivity to the neurotoxic or-ganophosphorus compound mipafox is investigated, and its subcellular distribution is studied. The NTE activity of the adrenal medulla was found to be the highest of those among the tissues studied to date (5000 ± 1400 mU/g tissue; ± SD, n = 12). This activity represented 93% of the PV esterase activity resistant to 40 μm paraoxon in the par-ticulate fraction of the adrenal medulla and approximately 50% of total PV esterase activity. In the bovine brain, these proportions were 72 and 26%, respectively, i.e., similar to those described in hen brain. The mipafox inhibition curve of PV esterase activity resistant to 40μM paraoxon in the particulate fraction of the adrenal medulla suggests that NTE activity fundamentally comprises a mipafox-sensitive component with an I ₅₀ of 6.39 μM at 30 minutes, which is similar to the value reported in hen brain. NTE activity in the bovine adrenal medulla is almost exclusively limited to the particulate fraction, the microsomal fraction, plasma membrane, and chromaffin granule-enriched fractions being the highest in terms of specific activity. On the contrary, the mitochondria-enriched fraction was very poor in such activity. In bovine brain, most NTE activity was likewise limited to the particulate fraction.     

Selectivity of lipases and esterases towards phenol esters

The selectivity of 28 lipases and esterases in the hydrolysis of butanoates of o-, m- or p-substituted phenols was investigated in a microtiterplate format. The phenols released during enzyme-catalyzed hydrolysis were converted in situ with Gibbs’ reagent to form a blue indophenol complex, which was quantified spectrophotometrically at 600 nm. Substantial differences in rates were found, which exhibits that the type and position of the substituent at the alkyl group has a strong influence on the selectivity of the enzymes. For various enzymes, the p-nitro derivative was the best substrate, whereas for other enzymes the m-Cl-derivative was preferentially hydrolyzed. Analysis of the data using the Hammett equation showed that sometimes the observed changes followed a predictable trend, but in several cases the result is very unexpected.     

Purification and partial characterisation of a 1.57 kDa thermostable esterase from Bacillus stearothermophilus

The molecular mass of esterases usually falls in the range of 20–160 kDa, although an esterase of 5.7 kDa from Candida lipolytica has been described. Three other enzymes smaller than 10 kDa have been reported, all of which were more thermostable than their higher molecular mass counterparts. This paper describes the purification of an extracellular esterase hydrolysing fluorescein dibutyrate from Bacillus stearothermophilus NCIMB 13335. The esterase had a molecular mass of 1.57 kDa when analysed by SDS-PAGE, gel filtration and MALDI-TOF spectrometry. This enzyme retained more than 90% of its activity after incubation at 90°C for 2 h.     

微生物酯酶研究进展

酯酶自发现以来,逐渐被开发利用于医药、化工、食品等领域,其中动植物来源酯酶工业化应用较少,微生物作为天然的酶资源库,是新型酯酶的主要来源之一。然而,大量新型微生物酯酶由于活性低、稳定性差等原因难以达到工业应用的要求;同时酯酶的筛选、活性评价方法仍存在通用性低、成本高的问题,一定程度上阻碍了新型微生物酯酶挖掘和改造。据此,本文总结了近年来微生物酯酶分类与发现、结构与催化特性、改造和优化以及应用等领域的研究新进展,以期促进酯酶的挖掘和工业化应用。     

Est-7, a set of genes controlling green tissue esterases in wheat and related species

Summary Analysis using isoelectric focusing of Chinese Spring wheat genetic stocks revealed a set of coleoptile and leaf esterase loci, designated Est-7, on the long arms of the group 2 chromosomes. A survey of 38 other hexaploid genotypes revealed onyl a single variant, at Est-D7. Homoeoloci were found on chromosome (arm) 2HL of Hordeum vulgare, 2RL of Secale cereale, 2R ^m of S. montanum, 2U of Aegilops umbellulata, 2E of Agropyron elongation and 2V of Dasypyrum villosa.     

Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus

A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts l- or d-glutamate to d- or l-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K _m and k_cat values of the overexpressed A. pyrophilus glutamate racemase for the racemization of l-glutamate to the d-form and the conversion of d-glutamate to the l-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06 s⁻¹ or 0.50 ± 0.07 mM and 0.25 ± 0.01 s⁻¹, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min. Received: September 11, 1998 / Accepted: January 12, 1999     

Initial degradation of dimethylphthalate by esterases from Bacillus species

Dimethylphthalate (DMP), one of the phthalate esters, is used in the manufacture of plasticizers, insect repellents, and synthetic fibers, and contributes to environmental pollution. In the present study, we report a novel bacterium belonging to the Bacillus sp., which has the ability to utilize DMP as the sole source of carbon. The esterases from the cell-free extract of the Bacillus de-esterified DMP. Native polyacrylamide gel electrophoresis showed the presence of four isoesterases designated Et1--4. The isoesterases Et-4 and Et-1 showed a higher preference towards DMP hydrolysis as compared with Et-2 and 3. A megaplasmid of about 60 kb was detected in this bacterium. The ability of this bacterium to utilize DMP as the sole source of carbon was lost upon plasmid curing. The isoesterases Et-1--4 were absent in the cell-free extracts of the cured bacterium. The results from our studies clearly demonstrate that de-esterification is the initial step in the degradation of DMP and the genes for these esterases seem to be harbored on the plasmid in this bacterium.     

The genetic control of grain esterases in hexaploid wheat

Summary Analysis of grain esterase isozymes in Chinese Spring aneuploid genotypes by IEF confirmed that genes on the long arms of chromosomes 3A, 3B and 3D (Est-5) control the production of 19 isozymes. Allelic variants have been found for the isozyme pattern controlled by each chromosome. Segregational data involving null alleles and complex phenotypic differences indicate that the wheat grain esterases are encoded by three compound and probably homoeoallelic loci, each capable of producing at least six different isozymes. In a sample of 138 hexaploid genotypes, seven alleles were distinguished.     

A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in biocatalysis

An esterase from Bacillus subtilis DSM402 (BS2) was cloned and functionally expressed in E. coli. The enzyme is active up to 50 degrees C, and the V(max) (1449 mM/min) and K(M) values (119 mM) were determined using p-nitrophenyl acetate as substrate. BS2 belongs to the few hydrolases that can act on tertiary alcohols and was therefore used to resolve racemic acetates of selected tertiary alcohols, but also to selectively remove the tert-butyl ester protecting group from peptides. In addition, the enzyme shows promiscuous amidase activity.     

Enhancement of cheese flavors with microbial esterases

The characteristic flavor of hard Italian cheeses is associated with the presence of fatty acids, particularly butyric acid, liberated from milk fat during the ripening process. To ensure proper development and control of flavor, animal pregastric esterases or lipases are routinely added to the milk before coagulation of the curd. Such esterases are also used to generate flavor in enzyme modified cheese and other dairy products. Esterases from microbial sources have been investigated as agents to enhance flavor in cheese. We have found that an esterase from Mucor miehei exhibits the type of lipolytic activity needed for this application. Romano and fontina cheeses of excellent quality have been prepared by the use of this esterase. It has also been used successfully in the preparation of enzyme modified cheese, and, in turn, processed American cheese.     

Identification of genes encoding microbial glucuronoyl esterases

One type of covalent linkages connecting lignin and hemicellulose in plant cell walls is the ester linkage between 4-O-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. An enzyme that could hydrolyze such linkages, named glucuronoyl esterase, occurs in the cellulolytic system of the wood-rotting fungus Schizophyllum commune. Here we report partial amino acid sequences of the enzyme and the results of subsequent search for homologous genes in sequenced genomes. The homologous genes of unknown functions were found in genomes of several filamentous fungi and one bacterium. The gene corresponding to the cip2 gene of Hypocrea jecorina (Trichoderma reesei), known to be up-regulated under conditions of induction of cellulolytic and hemicellulolytic enzymes, was over-expressed in H. jecorina. The product of the cip2 gene was purified to homogeneity and shown to exhibit glucuronoyl esterase activity.     

Functional classification of the microbial feruloyl esterases

Feruloyl esterases have potential uses over a broad range of applications in the agri-food industries. In recent years, the number of microbial feruloyl esterase activities reported has increased and, in parallel, even more related protein sequences may be discerned in the growing genome databases. Based on substrate utilisation data and supported by primary sequence identity, four sub-classes have been characterised and termed type-A, B, C and D. The proposed sub-classification scheme is discussed in terms of the evolutionary relationships existing between carbohydrate esterases.     

Molecular cloning and characterisation of a novel xanthine oxidase from Cellulosimicrobium cellulans ATCC21606

Xanthine oxidase (XOD) catalyses the oxidation of hypoxanthine into xanthine and xanthine into uric acid. The enzyme plays a key role in the purine metabolic pathway. Despite the presence of different XODs in prokaryotes, the functional and structural knowledge of prokaryotic XODs remain limited (compared with their well-known eukaryotic counterparts), thereby hindering their biochemical analysis and industrial application. Using genetic and biochemical analyses, we identified and characterised recombinant XOD (CcXOD_AB) from Cellulosimicrobium cellulans ATCC21606. Bioinformatics analysis suggests that CcXOD_AB shares low amino acid sequence identities with other XODs. The purified enzyme exhibits the maximum activity at 55 °C and pH 8.0. In addition, CcXOD_AB exhibits moderate thermostability and retains 80.65 % of the original activity after 30 min of incubation at 60 °C. Ca^{2 +} has a slight inhibitory effect, whereas Co^{2 +} and Mn^{2 +} have a strong inhibitory effect on XOD_AB activity. In particular, low Ba²⁺ and Mg^{2 +} concentrations have no effect, whereas high Mg^{2 +} (≥10 mM) and Ba²⁺ (≥2 mM) concentrations show an inhibitory effect on enzyme activity. The K_m and V_max values for xanthine are 131.29 ± 11.09 μmol•L⁻¹ and 15.23 ± 0.65 μmol•L^-1 min⁻¹, respectively. Results indicate that CcXOD_AB is a novel enzyme with potential industrial application.     

Release of short chain fatty acids from cream lipids by commercial lipases and esterases

Lipases and esterases are frequently used in dairy production processes to enhance the buttery flavour of the end product. Short chain fatty acids, and especially butanoic acid, play a key role in this and different enzymes with specificity towards short chain fatty acids are commercially available as potent flavouring tools. We have compared six lipases/esterases associated with buttery flavour production. Although specificity to short chain fatty acids was ascribed to each enzyme, clear differences in free fatty acid profiles were found when these enzymes were applied on cream. Candida cylindraceae lipase was the most useful enzyme for buttery flavour production in cream with the highest yield of free fatty acids (57 g oleic acid 100 g⁻¹ fat), no release of long chain fatty acids and specificity towards butanoic acid.     

MELDB: a database for microbial esterases and lipases

MELDB is a comprehensive protein database of microbial esterases and lipases which are hydrolytic enzymes important in the modern industry. Proteins in MELDB are clustered into groups according to their sequence similarities based on a local pairwise alignment algorithm and a graph clustering algorithm (TribeMCL). This differs from traditional approaches that use global pairwise alignment and joining methods. Our procedure was able to reduce the noise caused by dubious alignment in the distantly related or unrelated regions in the sequences. In the database, 883 esterase and lipase sequences derived from microbial sources are deposited and conserved parts of each protein are identified. HMM profiles of each cluster were generated to classify unknown sequences. Contents of the database can be keyword-searched and query sequences can be aligned to sequence profiles and sequences themselves.     

Asymmetric transformation of enol acetates with esterases from Marchantia polymorpha

Two esterases catalyzing the asymmetric hydrolysis of enol acetates to give optically active -alkylated ketones were isolated from cultured cells of Marchantia polymorpha by a three-step procedure: hydrophobic chromatography, anion exchange chromatography and gel-filtration chromatography. These esterases had opposite stereoselectivities on protonation of the enol intermediate in the hydrolysis and one of them obviously reversed the stereoselectivity when the chain length and the bulkiness of substituents at the β-position to the acetoxyl group were increased. The internal amino acid sequences of peptide fragments obtained by the proteolysis of the esterases with lysyl endopeptidase had no similarity to those of other hydrolytic enzymes.