Biotransformations with Rhizopus arrhizus and Geotrichum candidum for the preparation of (S)-atenolol and (S)-propranolol

(+/-)-Atenolol/(+/-)-propranolol and their acetates were incubated with the fungus Rhizopus arrhizus and Geotrichum candidum separately for different time intervals to afford (S)-atenolol/(S)-propranolol in good optical yield. The time and pH for this biotransformation was optimised. The present biodegradations using Rhizopus arrhizus and Geotrichum candidum provides a simple and useful method to obtain (S)-atenolol and (S)-propranolol which are active enantiomers of the beta-adrenergic blockers.     

Water activity and substrate concentration effects on lipase activity

Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.     

Environmental factors affecting the degradation of Dyfonate by soil fungi.

The ability of selected fungi to degrade the soil insecticide Dyfonate (O-ethyl S-phenyl ethylphosphonodithioate) into water-soluble, noninsecticidal metabolites was found to be dependent on the supply of nutrients, incubation time, temperature, pH, as well as other factors. With yeast extract as the carbon source (5 g/liter) and ammonium nitrate (1 g/liter) as the nitrogen source, both Rhizopus arrhizus and Penicillium notatum degraded the insecticide to a larger extent than with any other combination of nutrients used. With glucose as the carbon source, concentrations of ammonium nitrate above 5 g/liter inhibited the degradation of Dyfonate by R. arrhizus. Time-course studies on the metabolism of the insecticide indicated that Dyfonate was first absorbed by the fungal mycelium, where it was metabolized followed by the release of water-soluble, noninsecticidal, breakdown products into the culture media. The degradation appeared to involve the breakdown of Dyfonate into ethyl acetate soluble metabolites, such as ethylethoxyphosphonothioic acid, ethylethoxyphosphonic acid, methyl phenyl sulfoxide, and methyl phenyl sulfone. These compounds were then further degraded into water-soluble products. The optimum conditions for the degradation of the insecticide by R. arrhizus were observed at pH 6.0 to 7.0 and at 15-25 degrees C. Aged fungal mycelia were as active as mycelia in the logarithmic growth phase.     

非水溶剂中Rhizopus arrhizus脂肪酶催化合成三种酯的最佳条件(英文)

以少根根霉 (Rhizopusarrhizus)脂肪酶为催化剂 ,有机溶剂为反应介质 ,合成了 3种短链脂肪酸酯 .研究了反应温度、溶剂、底物浓度、底物摩尔比、吸水剂用量等因素对酯化反应的影响 .确定了3种酯的最佳合成条件 :(1)己酸乙酯 :反应温度为 4 0℃ ,环己烷为溶剂 ,0 2 5mol L底物浓度 ,酸醇摩尔比为 1∶1 2 ;(2 )乙酸异丙酯 :5 0℃ ,环己烷为溶剂 ,0 15mol L底物浓度 ,摩尔比为 1∶1;(3)乙酸异戊酯 :5 0℃ ,异辛烷为溶剂 ,0 2 0mol L底物浓度 ,摩尔比为 1∶1.三种酯合成时均需 0 12 5g ml的0 5nm分子筛为吸水剂 ,在 8h后 ,合成酯转化率达到 97%～ 99% .     

EVALUATION OF BIOTECHNOLOGICAL POTENTIALS OF SOME INDUSTRIAL FUNGI IN ECONOMICAL LIPID ACCUMULATION AND BIOFUEL PRODUCTION AS A FIELD OF USE

Considering the vast number of scientific reports on various potential uses of fungi, there was an attempt to select the best lipid producer of some fungi at optimized conditions (Aspergillus versicolor, Rhizopus oryzae, Rhizopus arrhizus, Tramates versicolor). The aim was to offer new fields of use to the industries already culturing and using such materials. Aspergillus versicolor mycelia were found to be accumulating the highest amount of lipids. Experiments to improve lipid accumulation and transesterification properties were performed in molasses medium; the first steps were testing the effects of different pH values and different nitrogen sources on lipid accumulation. Various concentrations of KNO₃ (0.5, 1.0, 1.5 gL^?1) and molasses (6%, 8%, 10%) were tried in order to find the optimum carbon and nitrogen requirements. Maximum lipid content was 22.8% in the samples containing 6% molasses solution and 1.0 gL^?1 KNO₃ at pH 4 after 10 days of incubation. The highest fatty acid ethyl ester yield of these samples was 77% (5.0 ethanol:oil, 0.4 sulfuric acid:oil at 30°C for 6 hr). Since the crude lipids were rich in C16 and C18 fatty acids, this was considered as suitable feedstock for biodiesel production.     

Microbial hydroxylation of natural drimenic lactones.

Incubation of confertifolin and isodrimenin with Mucor plumbeus, Aspergillus niger or Rhizopus arrhizus gave in good yields the corresponding 3 beta-hydroxy derivatives. From isodrimenin, the known natural 7 alpha-hydroxy derivative (futronolide) was also obtained and its structure was definitely established by X-ray crystallographic study of its acetate derivative.     

Enzymatic synthesis of lysophosphatidic acid and phosphatidic acid

Immobilised 1,3-specific lipase from Rhizopus arrhizus was used as catalyst for the esterification of -glycero-3-phosphate and fatty acid or fatty acid vinyl ester in a solvent-free system. With lauric acid vinyl ester as acyl donor, a_w<0.53 favored the synthesis of lysophosphatidic acid (1-acyl-rac-glycero-3-phosphate, LPA1) and the spontaneous acyl migration of the fatty acid on the molecule. Subsequent acylation by the enzyme resulted in high phosphatidic acid (1,2-diacyl-rac-glycero-3-phosphate, PA) formation and high total conversions (>95%). With oleic acid, maximum conversions of 55% were obtained at low water activities. Temperatures below melting point of the product favored precipitation and resulted in high final conversion and high product ratio [LPA/(PA+LPA)]. Thus, LPA was the only product with lauric acid vinyl ester as acyl donor at 25°C. Increased substrate ratio ( -glycero-3-phosphate/fatty acid) from 0.05 to 1 resulted in a higher ratio of LPA to PA formed, but a lower total conversion of -glycero-3-phosphate. Increased amounts of enzyme preparation did not result in higher esterification rates, probably due to high mass-transfer limitations.     

Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).     

应用反向线点杂交技术鉴定临床常见曲霉属和毛霉目真菌

目的应用反向线点杂交技术(reverse line blot hybridization,RLB)快速鉴定临床常见的曲霉属和毛霉目真菌。方法收集我院真菌和真菌病研究中心保存的5种曲霉菌(烟曲霉、黄曲霉、黑曲霉、土曲霉、构巢曲霉)和7种毛霉目真菌(冻土毛霉菌、总状毛霉菌、卷枝毛霉菌、少根根霉、小孢根霉、微小根毛霉、伞状犁头霉),共计98株菌株。利用真菌通用引物ITS1和ITS4对菌株进行PCR扩增,用12个真菌种特异性探针与扩增后产物进行反向线点杂交。将RLB结果与真菌传统形态学鉴定结果、ITS区DNA测序结果进行比较。结果 RLB可以正确鉴定98株实验菌株,与形态学方法和ITS区测序方法鉴定结果100%一致,种特异性探针之间未见交叉杂交,显示出该方法的高度敏感性和特异性。8株阴性对照菌株(白念珠菌、茄病镰刀菌、尖端赛多孢、马尔尼菲青霉、疣状瓶霉、棒曲霉、日本曲霉以及雅致小克银汉霉),使用RLB方法无法鉴定。通过烟曲霉基因组DNA浓度10倍倍比稀释法验证RLB的敏感性为1.8×10-3 ng/μL。结论 RLB技术为实验室早期快速诊断、鉴定临床常见的曲霉属和毛霉目真菌提供参考。     

Nutrition of tempe moulds

The nutritional requirements in vitro of Amylomyces rouxii NRRL 3160, Rhizopus arrhizus NRRL 1469, R. arrhizus NRRL 1526, R. oligosporus NRRL 2710, R. oligosporus NRRL 5905, R. oligosporus NRRL A-10457, R. oryzae IMI 215407, R. oryzae NRRL 3563 and R. stolonifera NRRL A-2293 were investigated. All strains grew in glucose-ammonium-salts medium without addition of vitamins. None utilized, as sole carbon and energy source, raffinose or stachyose, the main flatulence-associated oligosaccharides in soybean. All the strains, except Rhizopus oryzae IMI 215407, utilized one or more long chain fatty acids as sole sources of carbon and energy but only the R. arrhizus strains, R. oryzae NRRL 3563 and R. stolonifera , were able to use glycerol as sole carbon and energy source. Phytic acid was used neither as a source of carbon nor of phosphate by any strain.     

Carboxymethyl-substituted bifunctional chelators: preparation of aryl isothiocyanate derivatives of 3-(carboxymethyl)-3-azapentanedioic acid, 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic++ + acid, and 1,4,7,10-tetraazacyclododecane-N,N',N",N'-tetraacetic acid for use as protein labels

3-(Carboxymethyl)-3-azapentanedioic acid (NTA), 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic acid (EGTA), and 1,4,7,10-tetraazacyclododecane-N,N',N",N'-tetraacetic acid (DOTA) structures having a 4-nitrophenyl substituent attached via an alkyl spacer to the methylene carbon atom of one carboxymethyl arm of the chelator were obtained by alkylation of 4-nitrophenylalanine with bromoacetic acid (NTA), by reductive alkylation of 1,8-diamino-3,6-dioxaoctane with (4-nitrophenyl)-pyruvic acid followed by alkylation with bromoacetic acid (EGTA), and by alkylation of the trimethyl ester of 1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid with the methyl ester of alpha-bromo-4-(4-nitrophenyl)pentanoic acid and subsequent saponification (DOTA). The nitrophenyl-substituted chelators were converted to the corresponding amines by hydrogenation then reacted with thiophosgene to give the protein-reactive aryl isothiocyanate derivatives.     

富马酸生产菌少根根霉的诱变筛选

以实验室原少根根霉为出发菌种,通过紫外线和LiCl诱变处理,发现当紫外线照射时间为3min,并在质量分数4％LiCl的平板中培养,可诱变出富马酸高产菌。利用溴甲酚绿加塑料小管的平板进行初筛,其生成的富马酸通过塑料小管底部渗透到指示培养基中,产生变色圈,根据变色圈大小可初步判断诱变株的产酸能力,大大缩短了筛选时间。在葡萄糖质量浓度为80g/L时,诱变后的3#菌种在发酵72h后能产生55．02g/L的富马酸,比原菌种的富马酸产量提高了2．49倍。     

Lipases have similar water activity profiles in different reactions

The shape of the profiles of enzyme activity versus water activity for four different lipases were independent of the reaction used to determine the activity. The profile for each lipase (Rhizopus arrhizus, Pseudomonas sp., Candida rugosa and Lipozyme) in esterification, hydrolysis and transesterifications profiles were the same. In transesterification the yield was unaffected by the water activity but the hydrolysis yield increased with increasing aW .     

少根根霉原变种发酵生产薯蓣皂苷元

从穿龙薯蓣Dioscorea nipponica中分离得到的一少根根霉原变种Rhizopus arrhizus var. arrhizus菌株,能实现薯蓣皂苷的生物转化。用该菌株发酵穿龙薯蓣D. nipponica生产薯蓣皂苷元,采用高效液相色谱法测定薯蓣皂苷元的含量,其总得率可达3.00%。运用该菌株发酵制备薯蓣皂苷元,操作简单,环保,且得率高。     

少根根霉(Rhizopus arrhizus)脂肪酶基因在毕赤酵母中的分泌表达

利用PCR技术从少根根霉中扩增出脂肪酶基因(包括前导序列和成熟肽),并将其连接到酵母分泌表达载体pPIC9K中,转化毕赤酵母GS115。利用抗生素G418从重组阳性克隆中筛选得到高拷贝的转化子。在5 L的发酵罐中,当碳源耗尽后开始流加甲醇诱导脂肪酶的表达,经过96 h培养后发酵液上清液中重组脂肪酶(rRAL)的表达量约为90 mg/L。rRAL经过超滤,SP-Sepharose离子交换层析和Butyl-Sepharose疏水层析纯化。纯化后的蛋白在SDS-PAGE上为单一条带,表观分子量为32 kDa,比酶活为1 543 U/mg。N-端序列分析表明rRAL是经过加工后的产物。同时没有发现全长的Rhizopus arrhizus脂肪酶(RAL)被分泌表达。     

Mechanism of aluminum interference on uranium biosorption by Rhizopus arrhizus

Uranium competitive uptake experiments by Rhizopus arrhizus were carried out at three different solution pH levels and in the presence of different concentrations of competing aluminum ions in order to examine the competing ion effect. The coion effect became more pronounced as the coion concentration in solution and pH level increased. A preliminary examination of the effect of aluminum on the rate of uranium uptake was also completed. Results showed that the presence of aluminum does not interfere with the kinetics of uranium uptake by R. arrhizus. Electron microscopic and energy dispersive X-ray analyses were also performed on samples of the biomass. The combination of spectral data and the information from the equilibrium studies and the kinetic studies suggested that aluminum interferes with the uranium biosorptive uptake capacity of R. arrhizus by the precipitation of a metastable amorphous hydroxy polymeric precipitate through a mechanism we refer to as steric competition. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 16-27, 1997.     

Evidence for selective hydrolysis of aliphatic copolyesters induced by lipase catalysis

High molar mass random poly(butylene succinate-co-butylene sebacate), P(BS-co-BSe), and poly(butylene succinate-co-butylene adipate), P(BS-co-BA), with different composition, were synthesized and subjected to enzymatic hydrolysis by Lipase from Mucor miehei or from Rhizopus arrhizus. The enzymatic hydrolysis of P(BS-co-BSe)s and P(BS-co-BA)s films produced a mixture of water-soluble monomers and co-oligomers that were separated and identified by on-line high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Optimization of the HPLC analysis allowed the separation of isobar co-oligomers, differing only for the co-monomers sequence. Oligomers with the same monomer composition and molar mass but different sequence were identified by HPLC/ESI-MS-MS on-line analysis. The results obtained show a preferential hydrolytic cleavage induced by the lipases used. In particular, these enzymes prefer cleaving sebacic ester bonds in P(BS-co-BSe) copolymers, whereas succinic ester bonds appear to be hydrolyzed faster than adipic ester bonds in P(BS-co-BA) copolyesters. 1H NMR analysis further substantiates these findings. The primary products generated by lipase hydrolysis of polyester films underwent further degradation at longer reaction times. The HPLC/ESI-MS analysis of these mixtures at various times provided the first evidence that lipase catalysis is active also in water solution, a hydrophobic effect induced by the aliphatic units of these polyesters.     

Blends of aliphatic polyesters. VI. Lipase-catalyzed hydrolysis and visualized phase structure of biodegradable blends from poly(epsilon-caprolactone) and poly(L-lactide)

Phase-separated biodegradable polymer blends were prepared from poly(epsilon-caprolactone) (PCL) and poly(L-lactide) (PLLA), and Rhizopus arrhizus lipase-catalyzed hydrolysis and phase structure of the blend films were investigated. Gravimetry revealed that the lipase-catalyzed hydrolysis of PCL in PCL- and PLLA-rich phases is disturbed by the presence of PLLA. Polarimetry confirmed the occurrence of a predominant hydrolysis of PCL and subsequent removal of the hydrolyzed water-soluble PCL oligomers in the blend films. Gravimetry and gel permeation chromatography of the non-blended PLLA film indicated that R. arrhizus lipase has no catalytic effect on the hydrolysis of PLLA. The phase structure of the blend films could be visualized by selective enzymatic removal of one component and subsequent scanning electron microscopic observation.     

Chiral synthesis of alpha-phenylpyridylmethanols with Rhizopus arrhizus

The bioreduction of 2-benzoylpyridine (1a) with Rhizopus arrhizus afforded (S)-(+)-alpha-phenyl-2-pyridylmethanol (2a) in 82% enantiomeric excess (e.e.) while the asymmetric hydrolysis of its racemic acetate resulted in the antipode (R)-(-)-2a with 24% optical purity.     

Comparison of biosorption properties of different kinds of fungi for the removal of Gryfalan Black RL metal-complex dye

Three kinds of filamentous fungi (Rhizopus arrhizus, Trametes versicolor, Aspergillus niger) were tested for their ability to adsorb Gryfalan Black RL metal-complex dye as a function of pH, temperature and dye concentration. R. arrhizus and T. versicolor exhibited the maximum dye uptake at pH 2.0 and at 25 degrees C while A. niger performed the highest dye biosorption at pH 1.0 and at 35 degrees C. Sorption capacity of each biosorbent increased with increasing initial dye concentration. Among the three fungi, R. arrhizus was the most effective biosorbent showing a maximum dye uptake of 666.7 mg g(-1). The Langmuir model described the equilibrium data of each dye-fungus system accurately in the concentration and temperature ranges studied. Kinetic analysis indicated that both adsorption kinetics and internal diffusion played an important role on controlling the overall adsorption rate for each fungus. Thermodynamic analysis verified that A. niger biosorption was endothermic while the others were exothermic.