Fungus fruit body lytic enzyme from a myxomycete,Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).     

Production of lipase fromAspergillus tamarii and its compatibility with commercial detergents

The production of extracellular lipase byAspergillus tamarii isolated from seeds ofNigella sativa and its compatibility with commercial detergents were studied. Optimal conditions for production were: a cultivation period of 6d, cultivation temperature 30°C, pH 7.0 (0.1 mol/L phosphate buffer) and 0.15% olive oil. The crude enzyme showed a high level of pH stability but was not thermostable. The enzyme retained more than 90% of its activity in the presence of some commercial detergents.     

Bacillus licheniformis protease-catalyzed peptide synthesis via the kinetically controlled approach using the carbamoylmethyl ester as an acyl donor in anhydrous acetonitrile

Summary The couplings ofN-protected amino acid esters with amino acid amides proved to be carried out in anhydrous acetonitrile in the presence ofBacillus licheniformis protease (subtilisin Carlsberg) immobilized on Celite. The maximal peptide yields were obtained with the immobilized enzyme prepared through lyophilization from a pH 10.7 buffer solution. A series of dipeptide syntheses and several segment condensations were achieved generally in high yields by the combined use of the immobilized enzyme prepared from this pH and the carbamoylmethyl ester as the acyl donor.     

Adenyl cyclase in cell-free extracts ofEscherichia coli

The adenyl cyclase enzyme system was detected in the cells ofEscherichia coli disrupted by sonic treatment. This enzyme activity is located mainly in the cell fraction sedimenting at 2,000×g, i.e. in the cytoplasmic membrane fraction. A prolonged sonication treatment of the cell suspension was followed by the disappearance of activity in the membrane preparation. The pH optimum of the adenyl cyclase inEscherichia coli was on the alkaline side, around pH 9.     

Localisation and distribution of alcohol-cytochrome 553 reductase in acetic acid bacteria

Alcohol-cytochrome 553 reductase was detected in several strains of the mesoxydans and oxydans group ofAcetobacter. A similar enzyme, but with a higher optimum pH, was detected inAcetobacter aceti (liquefaciens) and in two strains ofGluconobacter.Intermittent ultrasonic disruption ofAcetobacter aceti cells, strainsrancens T-5 andmobilis 6428, showed that the alcohol-cytochrome 553 reductase was mainly localized on the cell hull. The NADP-linked aldehyde dehydrogenase appeared to be present as a cytoplasmic component.The oxidation rate of ethanol and acetaldehyde by intact resting cells which have been grown with either glucose or ethanol as a carbon source under either neutral or acidic conditions was nearly identical. The ethanol oxidizing enzyme system thus behaved as constitutive enzymes, as would be expected if they were bound to the cell hull.The results support the hypothesis that the alcohol-cytochrome 553 reductase is one of the important components of the enzyme system responsible for the physiological production of acetic acid from ethanol by acetic acid bacteria.     

Extracellular proteases ofHendersonula toruloidea Scytalidium hyalinum andScytalidium japonicum

Proteolytic activity was demonstrated in all the six clinical isolates ofHendersonula toruloidea, five ofScytalidium hyalinum and one ofS. japonicum, screened, by using bovine serum albumin as the substrate. Assay of crude enzyme filtrates showed that enzyme activity increased with the age of the organism reaching a peak after twelve days of growth. The optimum temperature and pH for maximal enzyme activity were 37 °C and 5.6 respectively. The maximum dry weight of mycelia recorded wereH. toruloidea, 135 mg;S. hyalinum, 100 mg; andS. japonicum 103 mg/50 ml broth after 22 days of growth. Proteases may play a role in the pathogensis of infection caused by these fungi by hydrolyzing keratinized tissues such as stratum corneum and nails of humans.     

Isolation and characterization of β-galactosidase fromLactobacillus crispatus

β-Galactosidase was isolated from the cell-free extracts ofLactobacillus crispatus strain ATCC 33820 and the effects of temperature, pH, sugars and monovalent and divalent cations on the activity of the enzyme were examined.L. crispatus produced the maximum amount of enzyme when grown in MRS medium containing galactose (as carbon source) at 37°C and pH 6.5 for 2 d, addition of glucose repressing enzyme production. Addition of lactose to the growth medium containing galactose inhibited the enzyme synthesis. The enzyme was active between 20 and 60°C and in the pH range of 4–9. However, the optimum enzyme activity was at 45°C and pH 6.5. The enzyme was stable up to 45°C when incubated at various temperatures for 15 min at pH 6.5. When the enzyme was exposed to various pH values at 45°C for 1 h, it retained the original activity over the pH range of 6.0–7.0. Presence of divalent cations, such as Fe²⁺ and Mn²⁺, in the reaction mixture increased enzyme activity, whereas Zn²⁺ was inhibitory. TheK _m was 1.16 mmol/L for 2-nitrophenyl-β-d-galactopyranose and 14.2 mmol/L for lactose.     

Characteristics of trehalase inCandida tropicalis

Summary The trehalase content of different yeasts varies widely. A strain ofCandida tropicalis was found to be the best source of this enzyme among the yeasts tested. The trehalase activity in this yeast could be increased 8.5 times by growing it on trehalose rather than glucose. Thus trehalase is an adaptive enzyme inC. tropicalis. It was found that the amount of trehalase which could be solubilized increased with increasing pH during autolysis of the cells, none being released from the cell debris at pH 4.5 and most at pH 6.3. Some evidence was obtained to show that the solubilization was caused by an enzyme. The stability of trehalase under various conditions was studied. A partial purification was achieved by precipitation with 40% ethanol at a temperature of −18°C. The maximum temperature of the enzyme was 48°C., and the optimum pH ranged from 4.1 to 5.3     

Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus<Emphasis Type="Italic"> Sporotrichum thermophile</Emphasis>

A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0±1.5 kDa, with a mass of 33±1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55–60 °C. The purified esterase was stable at the pH range 5.0–7.0. The enzyme retained 70% of activity after 7 h at 50 °C and lost 50% of its activity after 45 min at 55 °C and after 12 min at 60 °C. Determination of k _cat/K _m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5-O-trans-feruloyl--l-arabinofuranoside (NPh-5-Fe-Araf) 2-fold more efficiently than NPh-2-Fe-Araf. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.     

Methionine-repressible homoserine dehydrogenase of serratia marcescens: Purification and properties

Summary Serratia marcescens Sa-3 possesses two homoserine dehydrogenases and neither has any aspartokinase activity unlike the case ofEs-cherichia coli enzymes. The two enzymes have been separated. One of them is active with either NAD^– or NADP⁺ and has been purified about 180-fold to homogeneity. This enzyme is completely repressed by the presence of 1mm methionine or homoserine in the growth medium, but its activity is unaffected by any amino acid of the aspartate family either singly or together. In many of its properties (such as pH optimum, K_m for substrate and cofactors), it resembles its counterpart inE. coli K₁₂. Potassium ions stabilize the enzyme but are not essential for activity. Its molecular weight is around 155,000 as determined by gel filtration and approximately 76,000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme has two subunits (polypeptide chains) in the molecule: 8m urea has no effect on enzyme activity. This enzyme represents approximately 30% of the total homoserine dehydrogenase activity ofS. marcescens unlike inSalmonella typhimurium andE. coli K₁₂ where it is a minor or a negligible component.     

Partial purification of an enzyme hydrolyzing indole-3-acetamide from rice cells

The activity of indole-3-acetamide (IAM) hydrolase from rice cells was enriched ca. 628-fold by gel filtration and anion exchange column chromatography. The molecular masses of the IAM hydrolase estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis were approximately 50.5 kD and 50.0 kD, respectively. The enzyme exhibited maximum activity at pH 6.0–6.5. The enzyme was stable against heat treatments between 4 and 50°C and works optimally at 52°C. The activity remained constant at 4°C for at least 143 days. The purified enzyme fraction hydrolyzed indoleacetic acid ethyl ester (Et-IAA) in addition to IAM and its homologue, 1-naphthalene-acetamide, but not indole-3-acetonitrile. Km values of the enzyme were 0.96 mM and 0.55 mM for IAM and Et-IAA, respectively. Although the molecular mass of the enzyme was very similar to that of IAM hydrolase of Agrobacterium tumefaciens involved in tumor formation, the biochemical properties of the enzyme including its high Km value were considerably different from those of the A. tumefaciens enzyme. Based on these enzyme properties, we will discuss whether the amidohydrolase is involved in auxin biosynthesis in rice cells.     

Purification and characterization of an extracellular chitobiase fromTrichoderma harzianum

Chitobiase (EC 3.2.1.29), from the culture filtrate ofTrichoderma harzianum, was purified in sequential steps by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The physical and biochemical properties of the enzyme have been determined. The native enzyme has a molecular weight of 118 kDa when determined by gel filtration, and 64 kDa by SDS-PAGE. The enzyme catalyzed the hydrolysis of N,N-diacetylchitobiose andp-nitrophenyl--N-acetyl glucosamine with apparent Km of 575 µM and 235 µM, respectively. The pH optimum for the enzyme was pH 5.5, and maximum activity was obtained at 50°C. Glucosamine and N-acetylglyucosamine strongly inhibited the enzyme.     

Characterization ofZymomonas mobilis alkaline phosphatase activity inEscherichia coli

Zymomonas mobilis phoA gene encoding alkaline phosphatase was expressed inEscherichia coli CC118 carrying the recombinant plasmid pZAP1. The pH optimum for this enzyme was 9.0 and showed a peak activity at 42°C. This enzyme required Zn²⁺ for its catalytic activity; however, Mg²⁺ or Ca²⁺ significantly affected the activity. This enzyme was found to be ethanolabile, and ethanol inhibition was reversed by addition of Zn²⁺. Kinetics ofZ. mobilis alkaline phosphatase production inE. coli CC118 (pZAP1) showed that the enzyme activity was growth associated and localized in the cellular fraction, and the maximum activity was found in the stationary phase.     

Characterization of cytochrome oxidase purified from rat liver

The purpose of this study was to characterize the physical properties of cytochromec oxidase from rat liver. The enzyme was extracted from isolated mitochondria with nonionic detergents and further purified by ion-exchange chromatography on DEAE Bio-Gel A. The purified enzyme contained 9.64 nmol heme a/mg protein and one iron atom plus one copper atom for each heme a. The specific activity of the final preparation was 146 µmol of ferrocytochromec oxidized/min · mg protein, measured at pH 5.7. The spectral properties of the enzyme were characteristic of purified cytochrome oxidase and indicated that the preparation was free of cytochromesb, c, andc ₁. In analytical ultracentrifugation studies, the enzyme sedimented as a single component with anS ^20,w of5.35S. The Stokes radius of the enzyme was determined by gel filtration chromatography and was equal to 75 Å. The molecular weight of the oxidase calculated from its sedimentation coefficient and Stokes' radius was 180,000, indicating that the active enzyme contained two heme a groups. The purified cytochrome oxidase was also subjected to dodecyl sulfate-polyacrylamide gel electrophoresis in order to determine its components. The enzyme was resolved into five polypeptides with the molecular weights of I, 27,100; II, 15,000; III, 11,900; IV 9800; and V, 9000.     

Alpha-tocopheryl acetate hydrolase in chicken liver. Characterisation and properties

An enzyme catalysing the hydrolysis ofa-tocopheryl acetate was characterised in chicken liver. The enzyme was localised in the microsomes, had an optimum pH 8.6 and aK _m value of 0.5 mM. The enzyme did not hydrolyse retinyl acetate, cholesteryl acetate and ethyl acetate, thus indicating a high degree of specificity.a-Tocopheryl acetate hydrolase required bile salts as a specific co-factor. The results suggested a role for this enzyme in the absorption of vitamin E.     

Partial purification and characterization of a mannuronan-specific alginate lyase fromPseudomonas aeruginosa

Production of a thick exopolysaccharide coat (alginate) by mucoid strains ofPseudomonas aeruginosa has been shown to contribute to the pathogenicity and persistence of these bacteria in the lungs of patients with cystic fibrosis. Previous studies have shown that some mucoidP. aeruginosa strains produce an enzyme(s) capable of degrading this alginate coat. In this study, an alginate lyase from mucoidP. aeruginosa strain WcM#2 was isolated and characterized. Lyase production was enhanced by the addition of 0.2–0.3m NaCl to the growth media. The lyase was eluted from an alginate-Sepharose affinity column with 0.5m NaCl, which can serve as a simple one-step purification protocol for obtaining semi-pure functional alginate lyase. Fractionation of the enzyme preparation on a Sephadex G-75 sizing column showed that the enzyme has an apparent molecular weight of 40,000, whereas sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested a molecular weight of approximately 43,000. The affinity-purified enzyme had a pH optimum of 9.0, its activity was enhanced in the presence of 0.3m NaCl, and it showed substrate specificity for polymannuronic acid blocks. These results demonstrate the presence of a mannuronan-specific alginate lyase inP. aeruginosa that differs in several respects from previous reports ofP. aeruginosa alginate lyases.     

Production and characterization of the milk-clotting protease of<Emphasis Type="Italic"> Myxococcus xanthus</Emphasis> strain 422

The cheese industry is seeking novel sources of enzymes for cheese production. Microbial rennets have several advantages over animal rennets. (1) They are easy to generate and purify and do not rely on the availability of animal material. (2) The production of microbial clotting enzymes may be improved by biotechnological techniques. In this work, the biochemical characterization of a novel milk-clotting extracellular enzyme from Myxococcus xanthus strain 422 and a preliminary evaluation of its cheese-producing ability are reported. Strain 422 was selected from four M. xanthus strains as the best producer of extracellular milk-clotting activity, based on both its enzyme yield and specific milk-clotting activity, which also afforded lower titration values than enzymes from the three other M. xanthus strains. The active milk-clotting enzyme from M. xanthus strain 422 is a true milk-clotting enzyme with a molecular mass of 40 kDa and a pI of 5.0. Highest milk-clotting activity was at pH 6 and 37 °C. The enzyme was completely inactivated by heating for 12 min at 65 °C. The crude enzyme preparation was resolved by anion-exchange chromatography into two active fractions that were tested in cheese production assays of compositional (dry matter, fat content, fat content/dry-matter ratio, and moisture-non-fat content) and physicochemical properties (firmness, tensile strength, pH and Aw) of the milk curds obtained. Purified protein fraction II exhibited a significantly higher milk-clotting ability than either protein fraction I or a total protein extract, underlining the potential usefulness of M. xanthus strain 422 as a source of rennet for cheese production.     

Regulation of acid phosphatases in anAspergillus niger pacC disruption strain

AnAspergillus niger strain has been constructed in which the pH-dependent regulatory gene,pacC, was disrupted. ThepacC gene ofA. niger, like that ofA. nidulans, is involved in the regulation of acid phosphatase expression. Disruptants were identified by a reduction in acid phosphatase staining of colonies. Southern analysis demonstrated integration of the disruption plasmid at thepacC locus and Northern analysis showed that the disruption strain produced a truncatedpacC mRNA of 2.2 kb (as compared to 2.8 kb in the wild type). The strain carrying thepacC disruption was used to assign thepacC gene to linkage group IV; this was confirmed by CHEF electrophoresis and Southern analysis. This strain further allowed us to determine which extracellular enzyme and transport systems are under the control ofpacC inA. niger. Expression of theA. niger pacC wild-type gene and the truncatedpacC gene showed that, in contrast to the auto-regulated wild-type expression, which was elevated only at alkaline pH, the truncatedpacC gene was deregulated, as high-level expression occurred regardless of the pH of the culture medium. Analysis of the phosphatase spectrum by isoelectric focussing and enzyme activity staining both in the wild-type and thepacC disruptant showed that at least three acid phosphatases are regulated by thepacC. For the single alkaline phosphatase no pH regulation was observed.     

Cell growth and α-amylase production characteristics ofBacillus subtilis

Growth, differential rate of α-amylase synthesis and production characteristics ofBacillus subtilis DP 1 (isolate from starch materials) in comparison with 10Bacillus strains were examined in batch fermentation. The effect of the carbon and nitrogen source was evaluated with regard to cell growth and enzyme production. The pH optimum of enzyme activity was 6.5 and temperature optimum 60°C.     

Production of a raw starch saccharifying amylase byBacillus alvei grown on different agricultural substrates

Maximum activity of the amylase ofBacillus alvei was attained after growth of the organism on sorghum starch. Rice, corn, yam, cassava and potato starch gave high enzyme activities as did soluble starch. Glucose, maltose and glycerol were less effective. Optimum conditions for both growth and enzyme production were pH 6.8 at 40°C.