Efficient immobilization of epoxide hydrolase onto silica gel and use in the enantioselective hydrolysis of racemic para-nitrostyrene oxide

Epoxide hydrolase from Aspergillus niger (E.C. 3.3.2.3) was immobilized by covalent linking to epoxide-activated silica gel under mild conditions. A very easy procedure allowed to prepare an immobilized biocatalyst with more than 90% retention of the initial enzymatic activity. Immobilized and free enzyme showed very similar behaviour with respect to the effect of pH on activity and stability. One benefit of immobilizing epoxide hydrolase from A. niger on silica gel was the enhanced enzyme stability in the presence of 20% DMSO. The kinetic resolution of racemic para-nitrostyrene oxide was investigated by using this new immobilized biocatalyst. The enantioselectivity of the enzyme was not altered by the immobilization reaction: both unreacted epoxide and formed diol were obtained with very high ee (99 and 92%, respectively). In addition, the biocatalyst could be easily separated from the reaction mixture and re-used for over nine cycles without any noticeable loss of enzymatic activity or change in the enantioselectivity extent. The activity of immobilized AnEH was retained for several months.     

Microbiological transformations 50: selection of epoxide hydrolases for enzymatic resolution of 2-, 3- or 4-pyridyloxirane

A study aimed to select efficient epoxide hydrolases (EHs) allowing to achieve the enzymatic resolution of 2-, 3- and 4-pyridyloxirane (1–3) has been achieved, using 2-pyridyloxirane 1 as test substrate. Five thus selected EH-sources that showed interesting enantioselectivity were looked at in more detail for the conversion of 1–3.     

A novel enantioselective epoxide hydrolase from Agromyces mediolanus ZJB120203: Cloning,characterization and application

A new strain Agromyces mediolanus ZJB120203, capable of enantioselective epoxide hydrolase (EH) activity was isolated employing a newly established colorimetric screening and chiral GC analysis method. The partial nucleotide sequence of an epoxide hydrolase (AmEH) gene from A. mediolanus ZJB120203 was obtained by PCR using degenerate primers designed based on the conserved domains of EHs. Subsequently, an open reading frame containing 1167 bp and encoding 388 amino acids polypeptide were identified. Expression of AmEH was carried out in Escherichia coli and purification was performed by Nickel-affinity chromatography. The purified AmEH had a molecular weight of 43 kDa and showed its optimum pH and temperature at 8.0 and 35 °C, respectively. Moreover, this AmEH showed broad substrates specificity toward epoxides. In this study, it is demonstrated that the AmEH could unusually catalyze the hydrolysis of (R)-ECH to produce enantiopure (S)-ECH. Enantiopure (S)-ECH could be obtained with enantiomeric excess (ee) of >99% and yield of 21.5% from 64 mM (R,S)-ECH. It is indicated that AmEH from A. mediolanus is an attractive biocatalyst for the efficient preparation of optically active ECH.     

Optimization of fermentation conditions for gluconic acid production using Aspergillus niger immobilized on cellulose microfibrils

The optimisation of gluconic acid fermentation using immobilized Aspergillus niger on a highly porous cellulose support is described. Experimental results showing the effects of variations in oxygen partial pressure, glucose concentration and biomass concentration have been obtained with a continuous recirculation reactor. Levels of dissolved oxygen and glucose concentrations during fermentation significantly affect the production and fermentation time. The optimum biomass requirement on a porous cellulose support has been estimated to be 0.234 mg cm⁻² for efficient bioconversion. Increasing the quantum of biomass beyond this value resulted in an overgrown biofilm, which affected productivity adversely. Morphological characteristics of immobilized A. niger have also been investigated.     

黑曲霉F044脂肪酶的分离纯化及酶学性质研究

黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAESepharoseFastFlow阴离子交换层析和SephadexG-75凝胶过滤层析得到电泳纯的脂肪酶,纯化倍数为73·71倍,活性回收率为34%。对纯化脂肪酶性质研究表明:该脂肪酶分子量约为35~40kD,水解橄榄油的最适温度和最适pH分别为45℃和7·0,在60℃以下和pH2·0~9·0之间有很好的稳定性。该脂肪酶的水解活性对Ca2 表现明显的依赖性,而Mn2 、Fe2 和Zn2 对脂肪酶则有显著的抑制作用。在最适条件下水解pNPP的Km和Vmax分别为7·37mmol/L和25·91μmol/(min·mg)。其N-端的15个氨基酸序列为Ser(Glu/His)-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。     

Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44 h, glycerol (20 g L⁻¹) and corn steep liquor (CSL) (30 g L⁻¹) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2 g L⁻¹ h⁻¹) and continuous feeding of CSL (1 g L⁻¹ h⁻¹) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5±0.8 g L⁻¹; maximum EH production was 351.2±13.1 U L⁻¹ with a specific activity of 14.3±0.5 U g⁻¹. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.     

Influence of pH on the expression of a recombinant epoxide hydrolase in Aspergillus niger

The filamentous fungus Aspergillus niger was investigated in relation to its ability to produce a soluble epoxide hydrolase (EH) (E.C. 3.3.2.3) belonging to the microsomal EH family. This EH is a highly useful biocatalyst for kinetic resolution of racemic epoxides to give enantiopure building blocks. The production of EH on an industrial scale is still a major challenge and is linked to various optimization processes. In this work, production of protein and organic acids as a function of pH and cultivation time was investigated. The production of EH was highest (1000 U/L for p-nitrostyrene oxide) under acidic fermentation conditions (pH value of about 3). The metabolic flux toward production of organic acids and thereby acidification of the environment increased with an increasing pH value. At pH 7, nearly 50% of total carbon of the substrate was incorporated into organic acids, mainly gluconic and oxalic acid. Finally, the addition of protease inhibitors, antioxidants and cryoprotectants was investigated in relation to the stability of the EH during the downstream process. The determination of the pH dependence during fermentation and understanding of the parameters influencing the stability of the enzyme has allowed us to optimize intracellular expression. The EH has been easily isolated from the biomass with high activity (1.67 U/mg lyophilisate) in a robust process.     

Physiological aspects of free and immobilized Aspergillus niger cultures producing citric acid under various glucose concentrations

Similarities and differences between cultures of free and immobilized Aspergillus niger were identified under various glucose concentrations. Growth and citric acid production rates were compared, and the macro-morphology and fine structure of the mycelia examined to determine which parameters were significant in the production of citric acid. With free cultures the diameter of pellets was inversely related to glucose concentration, while biomass levels were lower for immobilized cultures than the equivalent free cultures. Rates of citric acid production were higher with immobilized mycelium, especially at higher glucose levels. The morphology that characterized high citric acid productivity was that of swollen hyphal tips which were seen at concentrations over 100 kg/m³ glucose in both free and immobilized mycelium. Although there is a characteristic morphology associated with high productivity it does not account for the difference observed between free and immobilized mycelia. The increased glucose uptake and productivity was not due to an increased surface area either, since the immobilized system was slightly lower in total surface area than the equivalent free cultures. The major difference was in the mean diffusion path in the two systems.     

Immobilization of the Solanum tuberosum epoxide hydrolase and its application in an enantioconvergent process

Five supports have been evaluated for the immobilization of the epoxide hydrolase from Solanum tuberosum (StEH) by adsorption. The highest immobilization yield (90-99%) and the maximum EH (epoxide hydrolase) activity (0.6 U g^-1 wet support) were obtained by ionic adsorption onto DEAE-cellulose. Although the activity recovered upon immobilization of StEH onto DEAE-cellulose was low, a notable stabilization factor of 6.9 at 65°C was obtained. In addition, the immobilized StEH showed a higher temperature for maximal activity (57°C) and the optimal pH (5.0) was shifted one unit towards the acidic region as compared to the free enzyme. Immobilized StEH was successfully reused in six consecutive hydrolytic kinetic resolutions of rac-pCSO without noticeable loss in activity. Finally, the sequential use of immobilized StEH with the immobilized EH from Aspergillus niger (AnEH) in a repeated batch reactor, operated for five cycles, enabled the enantioconvergent preparation of the corresponding (R)-diol, which was thus obtained with an ee of 89% and an overall yield of 100%.     

Immobilization of the epoxide hydrolase from Aspergillus niger

Three methods for the immobilization of the epoxide hydrolase from the fungus Aspergillus niger were tested. The highest immobilization yield (90%) and retention of activity (65%) were obtained by adsorption onto DEAE-cellulose compared to adsorption onto hydrophobic porous polypropylene and covalent linkage using Eupergit resin. The enzymatic properties of the immobilized enzyme were similar to those of the free enzyme with respect to the effect of temperature and pH on both activity and stability as well as the effect of solvent (DMF) on activity. The kinetic parameters were affected leading to lower K _M(app) and higher Vm _(app).     

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l⁻¹ with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg l ⁻¹ with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.     

Mild hydrolysis of nitriles by the immobilized nitrilase from Aspergillus niger K10

The cell free extract from the nitrile-hydrolyzing strain Aspergillus niger K10 (0.25 mg of protein) was adsorped onto a 1 mL HiTrap Butyl Sepharose column. The benzonitrile-hydrolyzing activity of the immobilized enzyme (about 1.6 U/mg of protein) was stable at pH 8 and 35 °C within the examined period (4 h). The enzyme load on the above column was increased 18 times in order to achieve high nitrile conversion. This enzyme preparation was used for the conversion of 3-cyanopyridine and 4-cyanopyridine under the above conditions. The initial substrate conversion was nearly quantitative. The activity was fairly stable; the conversion of 3-cyanopyridine decreased to 70% after 15 h, while the conversion of 4-cyanopyridine was 60% of the initial value after 39 h. The former substrate was converted into nicotinic acid and nicotinamide (molar ratio approximately 16:1) and the latter one into isonicotinic acid and isonicotinamide (molar ratio approximately 3:1).     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

Biocatalytic resolution of nitro-substituted phenoxypropylene oxides with Trichosporon loubierii epoxide hydrolase and prediction of their enantiopurity variation with reaction time

Biocatalytic resolution of 3-(2′-nitrophenoxy)propylene oxide (1a), 3-(3′-nitrophenoxy)propylene oxide (1b) and 3-(4′-nitrophenoxy)propylene oxide (1c) were exploited by using lyophilized cells of yeast Trichosporon loubierii ECU1040 with epoxide hydrolase (EH) activity, which preferentially hydrolyzes (S)-enantiomers of the epoxides (1a–c), yielding (S)-diols and (R)-epoxides. The activity increased as the nitro group in the phenyl ring was shifted from 4′-position (1c) to 2′-position (1a). When the substrate concentration of 1a was increased from 10 to 80 mM, the E-value increased at first, until reaching a peak at 40 mM, and then decreased at higher concentrations (>40 mM). The optically active epoxide (R)-1a was prepared at gram-scale (97% ee, 41% yield). Furthermore, a simple method was developed to predict the enantiomeric excess of substrate (ee_s) at any time of the whole reaction course based on the ee_s value determined at a certain reaction time at a relatively lower substrate concentration. This will be helpful for terminating the reaction at a proper time to get both higher optical purity and higher yield of the remaining epoxides.     

Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation

Mutants of Aspergillus niger NCIM 1207, isolated by subjecting conidia to UV-irradiation, were tested for the production of lipase (glycerol ester hydrolase EC 3.1.1.3). Mutants UV-10 and ANCR-1 showed seven fold and five fold enhanced productivity of enzyme, respectively, over the wild strain in shake flask culture when grown in SOB medium containing 1% olive oil. Maximum lipase activity (41 IU/ml) was obtained in the culture broth when UV-10 was grown in medium supplemented with 0.5% Triton X-100. A higher concentration of oil in the medium did not help lipase production in the case of mutant UV-10. Similarly no increase in enzyme levels was observed when mutant UV-10 was grown in medium supplemented with glucose. However, the addition of glucose in the medium resulted in increased levels of lipase production by wild strain, Aspergillus niger NCIM 1207.     

黑曲霉脂肪酶：基因克隆、表达及体外复性

根据黑曲霉F044脂肪酶N-端氨基酸序列,运用生物信息学方法,找到与黑曲霉脂肪酶基因同源的候选基因A84689。根据该基因序列,设计引物直接PCR扩增得到黑曲霉脂肪酶全长基因anl。anl全长1044bp,含3个内含子,编码297个氨基酸(含信号肽27个氨基酸),与其它脂肪酶基因没有明显同源性。将编码成熟脂肪酶的anl连接到pET28a载体上得到重组表达质粒,转化大肠杆菌BL21(De3),诱导表达并纯化出目的蛋白。通过大量稀释和DEAESepharoseFastFlow层析相结合的方法,变性后的纯化蛋白在体外实现再折叠复性。     

Enantioselective resolution of racemic styrene oxide at high concentration using recombinant Pichia pastoris expressing epoxide hydrolase of Rhodotorula glutinis in the presence of surfactant and glycerol

The reaction medium was optimized to accomplish epoxide hydrolase-catalyzed, batch enantioselective hydrolysis of racemic styrene oxide at high initial substrate concentrations. The recombinant Pichia pastoris containing the epoxide hydrolase gene of Rhodotorula glutinis was used as the biocatalyst. Enantiopure (S)-styrene oxide with 98% ee was obtained with 41% yield (maximum yield = 50%) from 1.8 M racemic styrene oxide at pH 8.0, 4 degrees C in the presence of 40% (v/v) Tween 20 and 5% (v/v) glycerol.     

Effect of water activity on invertase production in solid state fermentation by improved diploid strains of Aspergillus niger

Invertase production under solid state fermentation (SSF) was determined using two overproducing mutants (Aw96-3 and Aw96-4) isolated previously from the wild type strain Aspergillus niger C28B25, as well as one diploid (DAR1) and two autodiploid strains (AD96-3 and AD96-4) constructed by parasexual crossings among these mutants. Using polyurethane foam (PUF) as an inert carrier, two initial water activity (Aw) values were evaluated (0.99 and 0.96). At Aw=0.99, maximal activity was reached by diploid AD96-4 (48.91 IU/ml) representing 30- and 13-fold increases with respect to maximal values achieved by the wild type and the haploid parental mutant (Aw96-4), respectively. Similar levels were achieved by this strain at Aw=0.96. However, diploid DAR1 only produced high levels of invertase at Aw=0.96 (43.90 IU/ml), whereas strain AD96-3 reached its highest production (31.10 IU/ml) at Aw=0.99. Both productivity and yields were also analysed for every strain at each Aw value.     

Microbial transformations of flavanone and 6-hydroxyflavanone by Aspergillus niger strains

Flavanone (1) and 6-hydroxyflavanone (2) were subjected to transformation by means of Aspergillus niger strains (one wild and three UV mutants). For both substrates the biotransformation resulted in reduction of the carbonyl group (products 5 and 7) and dehydrogenation at C-2 and C-3 (3 and 8). Additionally, for flavanone (1) reduction of C-4 together with hydroxylation at C-7 (6) and dehydrogenation at C-2, C-3 along with hydroxylation at C-3 (4) were observed.     

Enantioselective epoxide hydrolase activity of a newly isolated microorganism, Sphingomonas echinoides EH-983, from seawater

A marine microorganism, Sphingomonas echinoides EH-983, which possesses epoxide hydrolase (EH) activity was isolated from seawater and characterized. The EH of S. echinoides EH-983 preferentially metabolized (R)-enantiomer when the racemic styrene oxides were supplied as substrates. The optimal pH and temperature for the enantioselective hydrolysis by whole-cells ofS. echinoides EH-983 were 7.0 and 20 °C, respectively. When kinetic resolution was conducted with a racemic mixture of styrene oxides at an initial concentration of 40 mM, enantiopure (S)-styrene oxide was obtained in 180 min with a yield of 21.3%. To our best knowledge, S. echinoides EH-983 is the first marine microorganism that is reported to have EH activity.