Continuous Asymmetric Reduction Of 4-Oxoisophorone By Thermophilic Bacteria Using A Hollow Fiber Reactor

Continuous asymmetric reduction of 4-oxoisophorone by the thermophilic bacterium Thermomonospora curvata JTS321 was examined using three reactor systems: packed bed, fluidized bed and hollow fiber. T. curvata was immobilized in polyacrylamide-hydrazide gels when used in the packed and fluidized bed reactors. Of the three reactor systems, the highest productivity (964 mg.1^-1.h^-1) was observed in the fluidized bed reactor. However, many cells grew outside of the gel matrix, causing product contamination. The productivity of the hollow fiber reactor was 504 mg.1^-1.h^-1; the problem of cell contamination of the product was avoided, as the molecular cut-off of the hollow fibers (400 000) was of an appropriate size to prevent cell leakage to the product stream. We therefore consider that the hollow fiber reactor is most suitable for continuous microbial conversions.     

Continuous Asymmetric Reduction Of 4-Oxoisophorone By Thermophilic Bacteria Using A Hollow Fiber Reactor

Continuous asymmetric reduction of 4-oxoisophorone by the thermophilic bacterium Thermomonospora curvata JTS321 was examined using three reactor systems: packed bed, fluidized bed and hollow fiber. T. curvata was immobilized in polyacrylamide-hydrazide gels when used in the packed and fluidized bed reactors. Of the three reactor systems, the highest productivity (964 mg.1^-1.h^-1) was observed in the fluidized bed reactor. However, many cells grew outside of the gel matrix, causing product contamination. The productivity of the hollow fiber reactor was 504 mg.1^-1.h^-1; the problem of cell contamination of the product was avoided, as the molecular cut-off of the hollow fibers (400 000) was of an appropriate size to prevent cell leakage to the product stream. We therefore consider that the hollow fiber reactor is most suitable for continuous microbial conversions.     

Continuous stereospecific Δ-3-keto-steroid reduction by PAAH bead-entrapped Clostridium paraputrificum cells

The development of a continuous anaerobic process for stereospecific Δ⁴-3-keto-steroid reduction by immobilized Clostridium paraputrificum cells cells is described. Following a study on conditions for cell growth and sporulation, spores of C. paraputrificum were aseptically immobilized in PAAH beads. Conditions for cell growth and induction in the immobilized state were determined, as well as the medium composition required to maintain a stabilized immobilized cell population. The effect of the concentration of ethylene glycol added as selected cosolvent on reaction kinetics, substrate solubility, specific activity, and cell growth, was investigated. A 10% (v/v) cosolvent input provided maximal activity along with enhanced solubility of the steroidal substrate. It was shown that cell growth was enhanced in the presence of the added cosolvent in addition to its effect on substrate solubility and enzymic activity. The immobilized cells readily performed Δ⁴, as well as 3-keto steroid reduction of several steroids, including ADD, AD, 16-dehydroprogesterone, progesterone, and hydrocortisone. It was shown that repeated batch-wise reduction cycle—in the presence of the cosolvent—resulted in rapid loss of activity, while the continuous uninterrupted process permitted the attaining of full bioconversion level, maintained stable for at least the period of 5 days of continuous operation tested.     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.     

Comparative study of production of dextransucrase and dextran by cells of Leuconostoc mesenteroides immobilized on Celite and in calcium alginate beads

In fed-batch fermentation, cells of L. mesenteroides immobilized on three types of Celite were used to produce dextransucrase (DS) followed by production of dextran. A layer of calcium alginate on the porous Celite R630 particles improved their mechanical stability, increased the amount of soluble DS produced and decreased the cell leakage from the highly porous support. Enzyme production with the immobilized cell cultures was significantly affected by both pore and particle size. Immobilized cultures using Celite R648 (average particle radius of 200 mum and pore size of 0.14 mum) produced the highest total enzymatic activity, followed by Celite R633, alginate-coated Celite R630, Celite R630, and then calcium alginate beads. Culture of free cells produced about 18% more total enzymatic activity than immobilized cells in calcium alginate beads, but about 64% less than immobilized cells on Celite R630. It is expected that larger amounts of enzymatic activity than measured are immobilized inside the alginate-coated Celite R630 and calcium alginate beads due to the mass transfer limitation conferred by the dextran product formed therein. The dextran yield from conversion of sucrose to dextran and fructose with all such enzyme-enriched, immobilized-cell cultures was higher than that obtained from free-cell culture under similar conditions.     

Immobilization of Saccharomyces cerevisiae for Ethanol Fermentation on γ-Alumina Particles using a Spray-Dryer

Saccharomyces cerevisiae was immobilized on γ-alumina particles with binder polymer by a spray-drying process. Batch fermentations of sucrose to ethanol were performed using the immobilized cells. The optimum pH was 4, and this helped to minimize microbial contamination and facilitate electrostatic attraction between the γ-alumina particles and the yeast cells. Addition of yeast extract resulted in high ethanol conversion. The reasons for differences between cellulose acetate phthalate (CAP) and styrene-maleic acid co-polymer (SMC) as binders on ethanol conversion were not apparent. The release of binder with the SMC from the γ-alumina was less than that from the CAP. Presoaking of γ-alumina particles in resin for binder solution before the immobilization using a spray-dryer was effective in achieving high ethanol conversion.     

Chemoenzymatic production of 1,5-dimethyl-2-piperidone

A chemoenzymatic process for the preparation of 1,5-dimethyl-2-piperidone (1,5-DMPD) from 2-methylglutaronitrile (MGN) has been demonstrated. MGN was first hydrolyzed to 4-cyanopentanoic acid (4-CPA) ammonium salt using the nitrilase activity of immobilized Acidovorax facilis 72W cells. The hydrolysis reaction produced 4-CPA ammonium salt with greater than 98% regioselectivity at 100% conversion, and at concentrations of 170–210 g 4-CPA/l. Catalyst productivities of at least 1000 g 4-CPA/g dry cell weight (dcw) of immobilized cells were achieved by recycling the immobilized-cell catalyst in consecutive stirred-batch reactions. After recovery of the immobilized cell catalyst for reuse, the 4-CPA ammonium salt in the aqueous product mixture was directly converted to 1,5-DMPD by low-pressure catalytic hydrogenation in the presence of added methylamine.     

Enantioselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester

The key chiral intermediate 3,5-dihydroxy-6-(benzyloxy) hexanoic acid, ethyl ester 2a, was made by the stereoselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester 1. Among various microbial cultures evaluated, cell suspensions of Acinetobacter calcoaceticus SC 13876 reduced 1 to 2a. The reaction yield of 85% and optical purity of 97% was obtained using glycerol-grown cells. The substrate was used at 2 g l⁻¹ and cells were used at 20% (w/v, wet cells) concentrations. The optimum pH for the reduction of 1 to 2a was 5.5 and the optimum temperature was 32°C. Cell extracts of A. calcoaceticus SC 13876 in the presence of NAD⁺, glucose, and glucose dehydrogenase reduced 1 to the corresponding monohydroxy compounds 3 and 4 [3-hydroxy-5-oxo-6-(benzyloxy) hexanoic acid ethyl ester 3, and 5-hydroxy-3-oxo-6-(benzyloxy) hexanoic acid ethyl ester 4]. Both 3 and 4 were further reduced to 2a by cell extracts. Reaction yield of 92% and optical purity of 99% were obtained when the reaction was carried out in a 1-l batch using cell extracts. The substrate was used at 10 g l⁻¹. Product 2a was isolated from the reaction mixture in 72% overall yield. The GC and HPLC area % purity of the isolated product was 99% and the optical purity was 99.5%. The reductase which converted 1 to 2a was purified about 200-fold from cell extracts of A. calcoaceticus SC 13876. The purified enzyme gave a single protein band on SDS-PAGE corresponding to 35,000 daltons.     

Enzymatic resolution of (R, S)-Naproxen in water-saturated ionic liquid

A water-saturated ionic liquid has been exploited for resolution of (R, S)-Naproxen by lipase-catalyzed hydrolysis to enhance the conversion and facilitate product recovery. From the enantioselectivity and activity of lipase, water-saturated [bmim]PF₆ (1-butyl-3-methylimidazolium hexafluorophosphate) was selected as the best reaction medium. To prevent the dissolution of lipase in the ionic liquid, a weakly polar, amorphous multiporous silica YWG-C₆H₅ was used as a support for immobilization. The production of (S)-Naproxen was initially performed in a batch reactor containing 20 mL of substrate solution. After 72 h reaction, 98.2% enantiomeric excess of the (S)-Naproxen was obtained with 28.3% hydrolysis conversion. The unconventional solvent properties of ionic liquids have been exploited in reaction medium recycling, product recovery and water recruiting schemes. In a repetitive batch reaction system, the immobilized lipase could be repeatedly used for 5 times with only a slight reduction in reaction conversion.     

Biodegradation of phenol by Trichosporon cutaneum cells covalently bound to polyamide granules

Polyamide granules with high specific area were used for covalent immobilization of Trichosporon cutaneum R57. In order to increase the concentration of active (amino) groups necessary for cell immobilization, the polyamide (PA) sorbent was chemically modified. The optimal conditions for covalent immobilization of the cells were determined. Phenol degradation was studied with chemically immobilized cells. For comparison, parallel experiments were carried out with physically immobilized and free cells. Both covalently-bound and free cells fully degraded phenol at concentrations up to 1·0 g/litre. The optimal pH of phenol degradation by covalently bound cells was 6·0. The number of cycles of effective phenol degradation by immobilized cells was studied. The results obtained for covalently bound Trichosporon cutaneum R57 cells on PA granules clearly show the possibility for their application for the purification of waste water containing phenol.     

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines

Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell‐loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell‐loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

In vitro capsaicin production by immobilized cells and placental tissues of Capsicum annuum L. grown in liquid medium

Capsaicin, from green pepper fruits is used in formulated foods and in pharmaceuticals. Cell cultures of Capsicum annuum L. were obtained from seedlings on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. In vitro-grown cells and placental tissues from fruits were immobilized in calcium alginate. Immobilized cells and placental tissues produced capsaicin which leached out into the medium. Immobilized placental tissue exhibited greater potentiality for capsaicin synthesis than immobilized cells. Production reached a level of 1345 μg capsaicin g⁻¹ of immobilized placenta on the 14th day of culture. Production of capsaicin, on replenished nutrient medium in immobilized placenta was 2400 μg on the 30th day. Ferulic acid fed to immobilized placenta at 2.5 mM level increased capsaicin production by 2-fold by the 5th day of the culture period. Of the elicitors used, curdlan was effective on capsaicin production in immobilized cells. Extracts of Aspergillius niger and Rhizopus oligosporus stimulated capsaicin production in immobilized placental tissues.     

木荷花挥发性成分的GC-MS分析

采用无水乙醚超声萃取得到新鲜木荷(Schima superba)花浸膏提取物,顶空固相微萃取富集挥发性成分,气相色谱-质谱联用仪分析,归一化法计算各组分的相对含量.鉴定出挥发性化合物中的51个成分,约占相对总含量的99%;挥发性成分中含氧化合物的含量超过93%,其中主要的化合物及其相对含量为酮代异佛尔酮(26.33%)、氧化芳樟醇(19.53%)、环氧芳樟醇(8.80%)、3,7-二甲基-2,6-辛二烯-1-醇(8.23%)、白藜芦素(7.89%)、4-羟基3,5,5-三甲基-2-环己烯-1-酮(6.54%)、2,6,6-三甲基-1,4-环己二酮(4.06%)、苯乙醇(2.17%)、2-甲基-2-壬烯-1-醇(2.04%)等.     

Production of L-phenylacetyl carbinol by immobilized yeast cells: I. Batch fermentation

Immobilization of Saccharomyces cerevisiae ATCC 834 within alginate beads enhances microbiological conversion of benzaldehyde to L-phenylacetyl carbinol (L-PAC), a precursor employed for synthesis of L-ephedrine. Yields of 90% L-PAC on benzaldehyde (initially 0.6% in medium) were obtained with immobilized cells, in contrast to about 10% with free cells which tend to form pellets in the presence of benzaldehyde. The predominant favorable action of immobilization appears to be a reduction in the toxic or inhibitory effects of benzaldehyde. With an initial benzaldehyde concentration of about 0.6% in the medium the optimum cell mass concentration was observed to be about 28 g cell mass (immobilized) per liter of medium.     

Silicone-immobilized cells ofGeotrichum sp. G38 used in biotransformation

The conditions for the reduction of dibenzoyl by silicone-immobilizedGeotrichum sp. G38 were examined, and optimal concentrations of stannous octanoate, ethyl silicate, water and entrapped biomass for the reaction were established. The optimum pH and temperature of the reaction medium were 7.0, and 35‡C (free cells) or 40‡C (immobilized cells), respectively. The immobilized cells showed higher activity than free cells under optimum conditions. The extent of conversion remained greater than 90% even after immobilized cells had been recycled 28 times. When silicone-immobilizedGeotrichum sp. G38 was used in the reduction of ethyl benzoylacetate, the (R)-enantiomer was obtained in an 81% enantiomeric excess compared with 49% enantiomeric excess using free cells.     

Continuous two step bioconversion of 4-oxoiso-phorone (OIP) to 4-hydroxy-2,2,6-trimethylcyclo-hexanone (4-HTMCH) by thermophilic bacteria

Summary The continuous two step conversion of 4-oxoisophorone(OIP) to 4-hydroxy-2,2,6-trimethylcyclohexanone(4-HTMCH) via dihydrooxoisophorone(DOIP) was carried out using two types of thermophilic growing cells, Thermomonospora curvata and Bacillus stearothermophilus, in sequential connected continuous stirred tank reactor(CSTR) and hollow fiber reactor system. For more than 80 hours operation, 46% of OIP was converted to 4-HTMCH with productivity of 179 mg/h/l.     

Comparative study of d-xylose conversion to ethanol by immobilized growing or non-growing cells of the yeast Pachysolen tannophilus

Summary The direct conversion of d-xylose to ethanol was investigated using immobilized growing and non-growing cells of the yeast Pachysolen tannophilus. Both preparations produced ethanol from d-xylose, however the d-xylose conversion to ethanol was much better with immobilized growing cells. Ethanol concentration up to 22.9 g/l and ethanol yield of 0.351 g/g of d-xylose were obtained in batch fermentation by immobilized growing cells whereas only 17.0 g/l and 0.308 g/g of d-xylose were obtained by immobilized non-growing cells. With continuous systems, immobilized growing cells were necessary for the long-term operation, since a steady state ethanol concentration of 17.7 g/l was maintained for only one week by immobilized non-growing cell reactor. With simultaneous control of aeration rate and concentrations of nitrogen sources in feed medium, immobilized growing cells of P. tannophilus showed excellent performance. At a residence time of 25 h, the immobilized cell reactor produced 26.9 g/l of ethanol from 65 g/l of d-xylose in feed medium.     

Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

Production of cellulase by freely suspended and immobilized cells of Trichoderma reesei

Trichoderma reesei (QM 9123) was immobilized within the open porous network of reticulated polyurethane foam matrices, and the growth pattern, glucose consumption and cellulase production were compared with those of freely suspended cells. It was found that the method of immobilization was simple and had no detrimental effect on cell activity. Various production media, to be used after the cultivation of T. reesei were tried. It was found that a nitrogen source-free production medium gave the highest enzyme titers of 1.5 × 10³ FPA U l⁻¹. Similar results were obtained with both freely suspended and immobilized cells.     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。