Lipase-catalyzed ammonolysis of trimethylsilylmethyl acetate in organic solvent*

Lipase could catalyze the ammonolysis of trimethylsilylmethyl acetate in organic solvents and Novozym 435 was the best biocatalyst for the reaction. The influences of some factors on the reaction were investigated. Cyclohexane, n-hexane and heptane were found to be suitable reaction media and ammonium carbamate was the best ammonium source. The optimal initial water activity, temperature and pH value were 0.55–0.75, 35°C and 6.5 respectively, under which a substrate conversion of 97.6% could be achieved after reaction for 140 h.     

白葡萄酒活性干酵母对不同氮源利用的研究

选用5种不同的白葡萄酒活性干酵母,以硫酸铵、氯化铵、硝酸铵、尿素、酵母粉等5种物质为氮源,观察其生长量并称量菌体重,以此分析其对氮源利用情况及不同氮源对酵母生长的影响。研究表明:供试菌系在不同氮源中均能生长。不同氮源对酵母的生长速度和生长量有不同影响;不同酵母菌种对不同氮源的利用也有差异。在以酵母粉为氮源的培养基中生长最好。在实验提供的氮源中,酵母粉为供试菌最优氮源,其次是硫酸铵,氯化铵与硫酸铵基本相当,而硝酸铵最差。8#菌种对各种氮源的利用能力相对较强。17#菌种对各种氮源的利用能力最弱。     

The nutrition of colletotrichum gloeosporioides penz

Summary Colletotrichum gloeosporioides could grow and sporulate on a wide range of pH (viz., from 3.0 to 8.5). The best growth was obtained at pH 6.0. Mannitol proved to be the best carbon source. Good growth and sporulation were also observed on maltose, glucose, galactose and sucrose. Nitrates supported better growth than ammonium compounds. Glutamic acid was found to be the best amino acid. Nitrites inhibited the growth completely at acid pH values but they supported growth at alkaline pH. Mannitol-glutamic acid was most suitable carbon-nitrogen combination for growth. Magnesium sulphate was the only sulphur source which was good both for growth and sporulation. The organism could not grow on media lacking carbon, nitrogen or sulphur.     

Production of L-serine by Sarcina albida.

Conditions for the production of microbial L-serine hydroxymethyltransferase and for the conversion of glycine to L-serine were studied. A number of microorganisms were screened for their abilities to form and accululate L-serine from glycine, and Sarcina albida was selected as the best organism. Enzyme activity in this organism as high as 0.12 U/ml could be produced in shaken cultures at 30 degrees C in a medium containing glucose, ammonium sulfate, glycine, yeast extract, and inorganic salts. L-Serine was produced most efficiently by shaking cells at 30 degrees C in a reaction mixture containing 20% glycine, 5 X 10(-3) M formaldehyde, and 3 X 10(-4) M pyridoxal phosphate in yields of 22 mg of broth in 5 days. L-Serine was easily isolated in 84% yields by ion-exchange resin.     

Production of L-serine by Sarcina albida.

Conditions for the production of microbial L-serine hydroxymethyltransferase and for the conversion of glycine to L-serine were studied. A number of microorganisms were screened for their abilities to form and accululate L-serine from glycine, and Sarcina albida was selected as the best organism. Enzyme activity in this organism as high as 0.12 U/ml could be produced in shaken cultures at 30 degrees C in a medium containing glucose, ammonium sulfate, glycine, yeast extract, and inorganic salts. L-Serine was produced most efficiently by shaking cells at 30 degrees C in a reaction mixture containing 20% glycine, 5 X 10(-3) M formaldehyde, and 3 X 10(-4) M pyridoxal phosphate in yields of 22 mg of broth in 5 days. L-Serine was easily isolated in 84% yields by ion-exchange resin.     

The use of positively charged leaving-groups in the synthesis of α-D-linked glucosides. Synthesis of methyl 2,3,4-tri-O-benzyl-6-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside

Quaternary ammonium and triphenylphosphonium salts of 2,3,4-tri-O-benzyl-6-O-(N-phenylcarbamoyl)-D-glucopyranosyl bromide were readily prepared by reaction with tertiary amines and triphenylphosphine under anhydrous conditions. Methanolysis of these salts was studied to determine the conditions of solvent and temperature that would produce the highest yields of α-D-glucosides. The quaternary ammonium salts gave the highest yields with solvents of low dielectric constant and room temperature. The phosphonium salts gave moderate yields with diethyl ether at 50°. The synthesis of methyl 2,3,4-tri-O-benzyl-6-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside by treatment of the quaternary ammonium salt of 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl bromide with methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside was studied as a model for the synthesis of oligosaccharides. The anomeric composition of the disaccharide product could be easily determined from the optical rotation since the specific rotations of both the final product and of the gentiobioside analog are known. Under the best conditions, the yield of disaccharide was low (50%) and the reactions were not completely stereoselective.     

Enzymatic Production of l-Citrulline by Pseudomonas putida

To develop an efficient method for the production of l-citrulline, optimum conditions for the conversion of l-arginine to l-citrulline by microbial l-arginine deiminase and for production of the enzyme were studied. A number of micro-organisms were screened to test their ability to form and accumulate l-citrulline from l-arginine. Pseudomonas putida was selected as the best organism. With this organism, enzyme activity as high as 9.20 units per ml could be produced by a shaking culture at 30 C in a medium containing glucose, ammonium phosphate, l-arginine hydrochloride, yeast extract, peptone, and inorganic salts. Appropriate addition of a surface active agent to the reaction mixture was found to shorten the time required for the conversion. A large amount of l-arginine hydrochloride was converted stoichiometrically to l-citrulline in 62 hr at 37 C. Accumulated l-citrulline was readily isolated in pure form by ordinary procedures with ion-exchange resins. Yields of isolated l-citrulline of over 90.5% from l-arginine hydrochloride were easily attainable.     

马铃薯蛋白酶A抑制剂的纯化与性质

采用硫酸铵沉淀、Sephadex G-50和CM柱层析,从马铃薯汁中纯化蛋白酶A（protease A,PrA）抑制剂,并对其部分性质进行研究。蛋白酶A抑制剂为单亚基,相对分子质量为16．71kDa。热稳定性良好,抑制最佳pH范围为5—5．5,最佳反应时间为1．5h,对PrA抑制类型为竞争和非竞争性混合抑制作用。     

Thermolysin and alpha-chymotrypsin mediated synthesis of tripeptides containing proline

The tripeptides Z-Pro-Leu-Gly-OEt, Z-Pro-Leu-Gly-NH2 and Z-Pro-Leu-Gly-OBzl were synthesized by thermolysin and alpha-chymotrypsin catalysis. The optimum conditions for the couplings between Z-Pro-OH and H-Leu-OEt or H-Leu-Gly-OEt catalysed by thermolysin were determined by a systematic study involving analysis of pH effect, ammonium sulfate concentration, reaction time, enzyme concentration, and relative proportion of the carboxyl and amine components. The best yield obtained for Z-Pro-Leu-OEt was 77% and for Z-Pro-Leu-Gly-OEt, 100%. Z-Pro-Leu-OEt was coupled to H-Gly-OEt, H-Gly-NH2 and H-Gly-OBzl. The best conditions to obtain Z-Pro-Leu-Gly-OEt and Z-Pro-Leu-Gly-NH2 were determined by the study of some factors that affect the reaction yield, such as organic solvent presence, substrate ratio and aqueous and organic solvent ratio. The yield obtained under optimum synthesis conditions was 55% for Z-Pro-Leu-Gly-OEt and 61% for Z-Pro-Leu-Gly-NH2. Z-Pro-Leu-Gly-OBzl was synthesized with 42% yield.     

戊糖乳杆菌发酵培养基氮源条件的优化

对戊糖乳杆菌发酵培养基的氮源条件进行了优化。通过单因素实验及响应面分析优化利用木糖高产乳酸的戊糖乳杆菌发酵培养基的不同氮源组合。优化得到的牛肉膏与柠檬酸氢二铵复合的最佳组成为牛肉膏17.72 g/L,柠檬酸氢二铵1.91 g/L,得到乳酸实际最大产量42.37 g/L。添加玉米浆与酵母粉和无机氮源复合的最佳组成为玉米浆46.54 g/L,酵母粉21.95 g/L,柠檬酸氢二铵9.95 g/L,可得到乳酸最大产量41.06 g/L。通过响应面优化减少了有机氮源的种类。牛肉膏与柠檬酸氢二铵的复合得到了更高的乳酸产量,且减少了有机氮源用量,节约了成本。玉米浆与酵母粉的复合解决了单一玉米浆造成的木糖利用速率过低的问题,同样得到较高浓度的乳酸。     

Mammalian folyl polyglutamate synthetase: partial purification and properties of the mouse liver enzyme

Folyl polyglutamate synthetase has been partially purified from mouse liver, and the general features of this enzyme have been characterized. The purification procedure utilized fractionation with ammonium sulfate, gel filtration, and affinity chromatography on ATP-agarose and resulted in a 350-fold increase in specific activity with 8-20% recovery of enzyme activity. Enzyme could be stabilized by glycerol or by ATP, but stability was not appreciably enhanced by folate. The enzymatic reaction was completely dependent on folate, ATP, and Mg2+ while partial reaction rates were observed in the absence of KCl or beta-mercaptoethanol. Highest reaction rates were observed at pH 8.2-9.5 at 37 degrees C. Chromatography of purified enzyme on calibrated gel filtration columns suggested a molecular weight of 65 000. Mouse liver folyl polyglutamate synthetase coupled [3H]glutamic acid to all of the naturally occurring folates studied. Analysis of the reaction products by high-performance liquid chromatography demonstrated that several folyl oligoglutamates were formed at low substrate concentrations but that only folyl diglutamate was formed at substrate concentrations approaching saturation. Dihydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate, and 5-formyltetrahydrofolate were the best substrates. Folic acid and 5-methyltetrahydrofolate were also substrates for this reaction, but much higher concentrations of these compounds were required to saturate the enzyme. These data suggest that all of the tetrahydrofolyl compounds (except 5-methyltetrahydrofolate) are the monoglutamyl substrates for polyglutamation in vivo and that 5-methyltetrahydrofolate is not likely to be a direct precursor for folate polyglutamates in mouse liver.     

Effects of temperature and fertilizer on activity and community structure of soil ammonia oxidizers

We investigated the effect of temperature on the activity of soil ammonia oxidizers caused by changes in the availability of ammonium and in the microbial community structure. Both short (5 days) and long (6.5, 16 and 20 weeks) incubation of an agricultural soil resulted in a decrease in ammonium concentration that was more pronounced at temperatures between 10 and 25 degrees C than at either 4 degrees C or 30-37 degrees C. Consistently, potential nitrification was higher between 10 and 25 degrees C than at either 4 degrees C or 37 degrees C. However, as long as ammonium was not limiting, release rates of N2O increased monotonously between 4 and 37 degrees C after short-term temperature adaptation, with nitrification accounting for about 35-50% of the N2O production between 4 and 25 degrees C. In order to see whether temperature may also affect the community structure of ammonia oxidizers, we studied moist soil during long incubation at low and high concentrations of commercial fertilizer. The soil was also incubated in buffered (pH 7) slurry amended with urea. Communities of ammonia oxidizers were assayed by denaturant gradient gel electrophoresis (DGGE) of the amoA gene coding for the alpha subunit of ammonia monooxygenase. We found that a polymerase chain reaction (PCR) system using a non-degenerated reverse primer (amoAR1) gave the best results. Community shifts occurred in all soil treatments after 16 weeks of incubation. The community shifts were obviously influenced by the different fertilizer treatments, indicating that ammonium was a selective factor for different ammonia oxidizer populations. Temperature was also a selective factor, in particular as community shifts were also observed in the soil slurries, in which ammonium concentrations and pH were better controlled. Cloning and sequencing of selected DGGE bands indicated that amoA sequences belonging to Nitrosospira cluster 1 were dominant at low temperatures (4-10 degrees C), but were absent after long incubation at low fertilizer treatment. Sequences of Nitrosospira cluster 9 could only be detected at low ammonium concentrations, whereas those of Nitrosospira cluster 3 were found at most ammonium concentrations and temperatures, although individual clones of this cluster exhibited trends with temperature. Obviously, ammonia oxidizers are able to adapt to soil conditions by changes in the community structure if sufficient time (several weeks) is available.     

Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports

Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Partition coefficients (K) of cephalexin in very different aqueous two-phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl ester and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin.     

Synthesis and application of amphoteric starch graft polymer

An amphoteric starch-graft-polyacrylamide (S-g-PAM) was prepared by inverse emulsion polymerization, subsequent hydrolysis reaction and Mannich reaction. The copolymerization was carried out using ammonium persulfate and urea as redox initiator. The reaction conditions and application as flocculant were investigated. Experiments showed that in hydrolysis reaction, a stable emulsion of anionic S-g-PAM with high hydrolysis degree could be obtained in a shorter time when sodium carbonate and sodium hydroxide were used together as hydrolyzing agents. In Mannich reaction, after pre-formation of an aldehyde–amine adduct was added to the anionic emulsion product, the amination degree of amphoteric S-g-PAM could reach 43.6% and the highest solution viscosity was obtained. The application test showed that the results of treatment of several kinds of industrial waste water by amphoteric S-g-PAM were better than those treated with cationic polyacrylamide (PAM), hydrolytic PAM and amphoteric PAM.     

A Glycine-Hcl-Formalin Fixative for Improved Staining of Neuroglia

The pH of fixatives as well as the components of the fixative mixture exert a displacing action on the isoelectric point of tissue proteins, which influences the intensity of staining. Studies of these effects showed that ammonium nitrate, sulfate or chloride could be substituted successfully for ammonium bromide in Cajal's formalin-ammonium bromide fixative. However, the best results from staining with Rio Hortega's silver carbonate and with Cajal's gold-sublimate methods were obtained after fixation in a mixture consisting of: glycine, 1.05 gm; 12V HCl, 14.8 ml; concentrated formalin, 15 ml and distilled water to make 100 ml.     

Purification and Properties of a Bacterial Enzyme Catalyzing Purine Nucleoside Transribosylation

An enzyme catalyzing the ribosyl group transfer from inosine to adenine was purified from Aerobacter cloacae No. 172–1 by means of ammonium sulfate fractionation and DEAE-cellulose and hydroxylapatite column chromatographies. A. cloacae was found to have much activities of this enzyme in its cell free extract. The enzyme activity was increased about 90-fold. By using the purified enzyme, some properties of the reaction were investigated. The enzyme was considerably stable and essentially required inorganic orthophosphate for the reaction. It was suggested that the enzyme might be a purine nucleoside phosphorylase and that the reaction might be a coupled reaction with ribose-1-phosphate as an intermediate. The enzyme could not transfer the ribosyl group between purine and pyrimidine bases.     

Substrate uptake,loss, and reserve in ammonia-oxidizing bacteria (AOB) under different substrate availabilities

The controversial arguments on the true substrate in nitritation kinetics might be due to the cells' dual substrate-transport system. Our experiments revealed that, under ammonia-rich environments, it diffused into the membrane (ammonia was the direct substrate); but, under oligotrophic, ammonium ion was actively transported (ammonium was the direct substrate). Facilitating this change-over, the bacterial composition in the sludge was altered, although the predominant was Nitrosomonas eutropha in most of the six chemostats. Also, the substrate affinity constant (K_s) fell resulting in partial compensation for the reduced availability of substrate. When the environmental ammonia concentration was lower than the cytoplasmic one, a backward diffusion appeared to take place, which probably had the cells accelerate its energy-consuming ammonium transport. The % ammonium oxidizing bacteria (AOB) to the total number of bacteria in the sludge remarkably decreased when cells were grown under oligotrophic environments. This could be evidence of the cellular energy dissipation caused by ammonia loss and recovery. Intracellular total ammonium nitrogen (TAN) accumulations were observed, which gradually increased from a basal value of ∼1 M (for AOB grown under copious environments) to much higher values (grown under oligotrophic environment). It did not affect the reaction kinetics but potentially served as a reserve against famine.     

15-Hydroxyprostaglandin dehydrogenase from human placenta. 1. Isolation and characterization]

15-Hydroxyprostaglandin dehydrogenase was isolated from human term placenta up to a final purification of 380-fold. A spec. act. of 2000 mU/mg of protein was reached. The preparation was not homogeneous as judged by analytical disc electrophoresis. The enzyme could be stored in the presence of 50% glycerol and 10mM 2-mercaptoethanol without any loss of activity for at least one year. A distinct single protein band stained after discontinuous polyacrylamide gel electrophoresis was shown by enzymatic activity staining to correspond to 15-hydroxyprostaglandin dehydrogenase activity. Thus no evidence for the exitstence of isoenzymes was obtained. The protein in the final preparation steps showed neither alcohol dehydrogenase, NAD reductase, nor NADH oxidase activity, nor enzymatic conversion of prostaglandin or 15-oxoprostaglandin in the absence of NAD and NADH. No spontaneous reactions between NAD and prostaglandin or NADH and 15-oxoprostaglandin were detectable in the absence of the enzyme. Ethanol and glycerol slightly inhibited the reaction. Various buffers (Tris/HC1, potassium phosphate, HEPES, and triethanolamine) and salts (ammonium chloride, ammonium sulfate, potassium chloride, and sodium chloride) had different effects on the reaction rate. The pH profile of the reaction shows a plateau between pH 7.0 and 7.8 and a steep maximum at pH 9.5. A linear Arrhenius plot was obtained for the temperature dependence of the reaction from 20 to 37 degrees C. The molar activation enthalpy of the reaction was calculated to be 13.1 kcal/mole. The molecular weight of 15-hydroxyprostaglandin dehydrogenase was estimated to be 32000 -/+ 3000 by gel filtration on Sephadex G-150 in the presence of 10mM mercaptoethanol.     

Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources

Poly(hydroxybutyric acid) (PHB) and other biodegradable polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of cane molasses and corn steep liquor, two of the cheapest substrates available in Egypt, may help to reduce the cost of producing such biopolyesters. In this work, the effect of different carbon sources was studied. Maximum production of PHB was obtained with cane molasses and glucose as sole carbon sources (40.8, 39.9 per mg cell dry matter, respectively). The best growth was obtained with 3% molasses, while maximum yield of PHB (46.2% per mg cell dry matter) was obtained with 2% molasses. Corn steep liquor was the best nitrogen source for PHB synthesis (32.7 mg per cell dry matter), on the other hand, best growth was observed when ammonium chloride, ammonium sulphate, ammonium oxalate or ammonium phosphate were used as nitrogen sources.     

Some Properties of Carbamoyl Phosphate Synthase Partially Purified from the Larvae of Blowfly,Aldrichina grahami

Glutamine and ornithine were found to stabilize effectively carbamoyl phosphate synthase (CPSase) partially purified from the larvae of Aldrichina grahami reared aseptically.

Glutamine, ATP. and Mg ion were required for the enzyme reaction. A high concentration of ammonia could replace the requirement of glutamine; N-acetylglutamate could not enhance the reaction. The apparent Km for ammonium ion, however, was much higher than that for glutamine. The concentration of ATP required for half maximal velocity was 1.0×10^?2 m.

Various kinds of nucleotides of pyrimidines and purines inhibited the enzyme reaction. The reaction product in the assay system radioautographically coincided with citrulline.