Studies on the growth of a thermotolerant yeast strain,Kluyveromyces marxianus IMB3, on sucrose containing media

A thermotolerant alcohol-producing yeast strain, Kluyveromyces marxianus IMB3 was shown to grow on sucrose (10% [w/v]) containing media at 45 °C. Under such conditions the organism reached stationary phase within 20 hours and yielded ethanol concentrations in the region of 33g/L. During growth on sucrose containing media the organism was found to produce a cell- associated activity capable of hydrolysing sucrose. This activity was shown to have a Km of 5.0mM when sucrose was used as the substrate. In addition the enzyme was shown to have a pH optimum of 5.0 and a temperature optimum of 50–55 °C and under those conditions the enzyme was shown to be relatively thermostable.     

Partial characterization of β-glucosidase activity produced by Kluyveromyces marxianus IMB3 during growth on cellobiose-containing media at 45°C

Summary We report- the partial characterization of a -glucosidase produced during growth of the thermotolerant yeast, K. marxianus IMB3 on lactose-containing media at 45°C. The enzyme had Km values of 1.1mM and 14.8mM for the substrates p-nitrophenyl--D-glucoside and cellobiose, respectively. The enzyme had a pH optimum of 5.5 and was optimally active at 50°C. It was stable up to 125 hours at 25°C and 35°, with half-lives of 45 hours and 2 hours at 45°C and 50°C, respectively. The enzyme was inhibited to varying degrees in the presence of metal ions and was completely inactivated by Hg²⁺. Ethanol concentrations [1–10% (v/v)] had little effect on activity. Glucose (20mM) caused inhibition when p-nitrophenyl--D-glucoside was used as substrate, whereas lactose at similar concentrations had no effect.     

Membrane enzyme reactor with simultaneous separation using electrophoresis

A membrane enzyme reactor with simultaneous separation was investigated. Enzymes, urease and aspartase, were immobilized by a porous polytetrafluoroethylene membrane. Electrical field was applied in the medium while the reaction was carried out. Products with electrical charge could be separated through the membrane from the reaction medium as they were formed. Reaction behavior was analyzed by a simple model considering both pore-migration and reaction in the skelton of the membrane. According to the analysis the inherent reaction rate of the immobilized enzymes decreases significantly. This is probably caused by the structural variation of enzymes. For the case of urease, the change of pH inside the membrane may also cause the decrease of the reaction rate. The model analysis showed that the enzyme content in the membrane and the residence time of the substrate in the membrane governed overall extent of reaction.List of Symbols e g (dm³)^–1 enzyme concentration in the membrane - L cm membrane thickness - K _m mM Michaelis constant - Rate mmol · min^–1 · g^–1 rate of product formation per unit weight of enzyme - S mM substrate concentration - S _in mM inlet substrate concentration - S _out mM outlet substrate concentration - u cm · min^–1 migration rate - V V voltage between the electrodes - V _m mmol · min^–1 · g^–1 maximum reaction rate - X conversion - z cm distance from the surface inside the membrane - void fraction of the porous membrane - tortuosity of the membrane - min space time     

Phytate dephosphorylation by free and immobilized cells ofSaccharomyces cerevisiae

Summary Saccharomyces cerevisiae in the form of baker's yeast, cells cultivated on a yeast extract-peptone-glucose medium, as well as cells immobilized in 18% (w/v) polyacrylamide gel showed the ability to hydrolyze 1.727 mM sodium phytate solution at 45°C, pH 4.6, in a stirred tank reactor. Seventy percent yield of dephosphorylation was observed after 2 h using a baker's yeast concentration of 5.8 g dry matter per 100 ml. Hydrolytic activity at 1.8–2.0 M Pi min^–1 was observed between 1st and 3rd h of the reaction in cells cultured 24 or 48 h. No inhibition by the substrate was found at sodium phytate concentrations of 0.587–1.727 mM. After 1.5 h of hydrolysis a single, well distinguished peak ofmyo-inositol-triphosphate was the main product found. By means of immobilization the stability of the biocatalyst was enhanced 3.3-fold and reached its half-life at 64 ninety-minute runs.     

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha)

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-dl-arginine-p-nitroanilide (dl-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors — soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 °C and was stable from pH 4.0 to pH 10.0 when incubated at 20 °C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of dl-BAPNA by the chinook salmon enzyme was 60 °C, however, the enzyme was unstable at temperatures above 40 °C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS–PAGE.     

Ornithine transcarbamylase fromLactobacillus buchneri NCDO110

Ornithine transcarbamylase (OTCase) (E.C.2.1.3.3) was partially purified fromLactobacillus buchneri NCDO₁₁₀. It was stabilized by the presence of glycerol. The optimal pH for enzyme activity is 8.5. The positive cooperativeness was observed among ornithine molecules at pH values different from the optimum. The Mr of the enzyme was calculated to be 162,000 by gel filtration on Ultrogel ACA-34. Maximum activity occurred at 35°C. G^* of the reaction was calculated from Arrhenius plot. The values were 9100 cal mol^–1 below 35°C, and 4300 cal mol^–1 above 35°C. The Km value for carbamylphosphate was 7.1×10^–4 M, and the Km for ornithine was 1.6×10^–3 M, under the conditions described here. Dead-end inhibition analysis was performed with norvaline, which is a structural analogue of ornithine. Norvaline acted as a noncompetitive inhibitor when carbamylphosphate was the variable substrate, and as a competitive inhibitor when ornithine was the variable substrate. The results are consistent with a ping-pong mechanism.     

Serine proteinase from Bacillus brevis: lytic action on intact yeast cells

Summary A serine proteinase which showed lytic acitivity against either intact cell or cell wall preparations of Candida utilis has been isolated from Bacillus brevis culture filtrate by affinity chromatography on bacitracin-silochrome and phenylboronale-Sepharose. Both its proteolytic and lytic activities were completely abolished by inhibitors of serine proteinases, including phenylmethylsul-phonylfluoride, the inhibitor from Actinomyces janthinus, and duck ovomucoid. The optimum pH range for the enzyme is 7.5–9.0, the optimum temperature 40°–50°C, its pI value 8.6 and motecular weight 28000. The amino acid composition of this proteinase is similar to that of serine proteinase from B. amyloliquefaciens (subtilisin BPN), its N-terminal amino acid sequence being identical to that of BPN through 21 residues. The enzyme cleaves chromogenous substrates for subtilisins but shows no activity on a substrate for trypsin. By means of both turbidimetry and electron microscopy the enzyme studied was shown to cause yeast cell lysis.     

Cell-bound cholinesterase inTrichoderma harzianum

Cell-bound cholinesterase enzyme activity is reported for the first time in the mycelium ofTrichoderma harzianum. This enzyme hydrolyzes both the acetylcholine and the butyryl thiocholine esters. TheK _m andV _max for choline ester are 0.69 mM and 1.0 nmol acid released min^–1 g^–1 protein. However, the thiocholine ester has aK _m value of 2.2 mM andV _max value of 3.33 nmol product formed min.^–1 g^–1 protein. The enzyme is inhibited by eserine, a true classical cholinesterase inhibitor.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Regulation in the chemolithotrophthiobacillus neapolitanus: Fructose-1,6-diphosphatase

Summary Partially purified fructose diphosphatase from the obligate chemolithotroph,Thiobacillus neapolitanus has been characterized, and some of its regulatory properties described. The enzyme had a high effinity for its substrate, but was inhibited by substrate at concentrations above 1 mM. The enzyme had an absolute requirement for a divalent cation. In the absence of EDTA there was a single pH optimum in the alkaline range between 8.5 and 9.5; in the presence of EDTA there was considerable was activity at both neutral and alkaline pH. This diphosphatase was inhibited by AMP at 10^–4 M or greater-, the lower the pH, the greater the AMP inhibition. Treatment of the enzyme with 5×10^–5 Mpara hydroxy mercuribenzoate allowed retention of full catalytic activity while abolishing considerable AMP inhibition. Exposure of the enzyme to several concentrations of urea had no effect on the AMP inhibition. Homocystine (0.06 mM) and coenzyme A (0.1 mM) had no effect. At 1 mM, PEP caused 60% inhibition, 2, 3-diphosphoglyceric acid produced 26% inhibition, and pyruvate had no effect.     

Human plasma uridine diphosphate galactose-glycoprotein galactosyltransfertase. Purification, properties and kinetics of the enzyme-catalysed reaction.

The soluble galactosyltransferase of human plasma catalysed the transfer of galactose from UDP-galactose to high- and low-molecular-weight derivatives of N-acetylglucosamine, forming a beta-1-4 linkage. The enzyme was purified by using (NH4)2SO4 precipitation and affinity chromatography on an alpha-lactalbumin-Sepharose column. The galactosyltransferase was maximally bound to this column in the presence of N-acetylglucosamine, and the enzyme was eluted by omitting the amino sugar from the developing buffer. The molecular weight of the enzyme was estimated to be 85000 by gel filtration. The assay conditions for optimum enzymic activity was 30 degrees C and pH7.5. Mn2+ ion was found to be an absolute requirement for transferase activity. The Km for Mn2+ was 0.4 mM and that for the substrate, UDP-galactose, was 0.024 mM. The Km for the acceptors was 0.21 mM for alpha1-acid glycoprotein and 3.9 mM for N-acetylglucosamine. In the presence of alpha-lactalbumin, glucose became a good acceptor for the enzyme and had a Km value of 2.9 mM. Results of the kinetic study indicated that the free enzyme reacts with Mn2+ under conditions of thermodynamic equilibrium, and the other substrates are added sequentially.     

Immobilized 4-aminophenyl 1-thio-β- -galactofuranoside as a matrix for affinity purification of an exo-β- -galactofuranosidase

An alternative and fast method for the purification of an exo-β- -galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β- -galactofuranoside affinity chromatography system and specific elution with 10 mM -galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM -galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β- -galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and K_m and V_max values of 0.311 mM and 17 μmol h⁻¹ μg⁻¹ respectively, when 4-nitrophenyl β- -galactofuranoside was employed as the substrate.     

A new acid carboxypeptidase,O-1, fromAspergillus oryzae

A new acid carboxypeptidase was purified fromAspergillus oryzae grown on solid bran culture medium. The purified enzyme was found to be homogeneous by disc gel electrophoresis at pH 9.4 and isoelectric focusing. The enzyme was termedA. oryzae acid carboxypeptidase O-1 with isoelectric point 4.08. The substrate specificity of the new enzyme was investigated with proangiotensin, angiotensin, and bradykinin. Even when the proline was present at the penultimate position of the peptide, the enzyme rapidly hydrolyzed the carboxyterminal Pro-X (X=amino acid) peptide bond. TheK _m andk _cat values for angiotension (–Pro⁷–Phe⁸) at pH 3.7 and 30°C were 0.2 mM and 1.7 sec^–1, respectively.     

Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast

Preparation and purification of substrate amounts of radioactive as well as non-radioactive dolichyl diphosphate N-acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I-III). The test conditions are described in detail. All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35-fold and 70-fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+. The Km values for UDP-N-acetylglucosamine for enzyme I and II were 15 and 10 muM, respectively, and for GDP-mannose (enzyme III) 7 muM. The apparent Km values for the lipophilic acceptor was 180 muM for enzyme I (dolichyl phosphate), 40 muM for enzyme II (dolichyl diphosphate N-acetylglucosamine) and 17 muM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol X h-1 X mg protein-1. All reactions were inhibited by nucleoside diphosphates.     

Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml^–1 of digitonin and 64 µg ml^–1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml^–1 of protease, whereas activity strongly decreases in the presence of 64 µg ml^–1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.     

Enzymes of asparagine synthesis in maize roots

An asparagine synthetase which is active with either glutamine or NH ₄ ⁺ has been found in maize (Zea mays L.) roots. Unlike the enzyme obtained from legume cotyledons, the maize-root enzyme is only slightly more efficient with glutamine (Km, 1.0 mM) than with NH ₄ ⁺ (Km, 2.0–3.0 mM). The activity of this enzyme is higher in the mature root than in the root-tip region, i.e. root cells develop a capacity to make asparagine from glutamine or NH ₄ ⁺ as they mature. -Cyanoalanine synthetase is also present in maize roots. The apparent Km for cysteine is 2.6 mM and for cyanide is 0.57 mM. The enzyme is more active in the root tip than in mature root tissue. Thus, if asparagine were made in the root tip, the cyanide pathway could represent the mechanism of synthesis. It is our contention, however, that this potential is not realized under normal conditions because ¹⁴C-experiments performed previously have indicated a limited availability of both CN and cysteine in the maize root.     

Production and properties of ∝-mannosidase from aspergillus sp.

Summary Aspergillus sp NCIM 508 produced 22 U/L of extracellular -mannosidase activity in a medium containing 8 % brewer's yeast cells. The optimum period and pH range for maximum production of the enzyme were 7 days and 4.0–6.0, respectively. The optimum pH and temperature for enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable for 24 h at 28°C, in the pH range 6.0–7.0. The enzyme retained 100 and 65 % of its original activity after heating for 15 min at 45 and 55°C, respectively. The Km and V_max for p-nitrophenyl--D- mannoside (PNPM) were 71M and 7.5 × 10^–2 moles/min/mg, respectively. The enzyme was strongly inhibited by 1 mM Hg⁺⁺ and Cu⁺⁺ and partially by Co.⁺⁺ (NCL Communication No.; 5780)     

Microbiological degradation of the herbicide dicamba

Summary Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba byP. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100–2000 mg dicamba L^–1 (0.5–9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl^– per mol of dicamba degraded. The cultures had an optimum pH 6.5–7.0 for dicamba degradation. Growth studies at 10°C, 20°C, and 30°C yielded activation energy values in the range of 19–36 kcal mol^–1 and an average Q₁₀ value of 4.0. Compared with the pure cultureP. paucimobilis, the consortium was more active at the lower temperature.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Purification, carbohydrate composition and kinetic properties of the constitutive yeast acid phosphatase

Constitutive acid phosphatase was purified from yeast cells grown in a medium supplied with 100 mM phosphate. Specific activity of the pure enzyme was 63.5 mumol/min X mg. The enzyme contains 42.5% of protein, 55% of mannose and 2.5% of N-acetylglucosamine. The carbohydrate chains are N-glycosidically linked to the protein. The pure enzyme shows non-linearity in the Lineweaver-Burk plot, thus indicating the presence of two enzyme forms with Km values of about 0.65 mM and 8.5 mM. The pH optimum of the enzyme is at pH 3.3. The enzyme is much more sensitive to heat denaturation than the repressible acid phosphatase.