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)     

Purification and properties of an endopolygalacturonase produced byRhizopus stolonifer

Summary A polygalacturonase from culture filtrates of a strain ofRhizopus stolonifer was purified about 80 fold by ethanol precipitation, followed by ion exchange chromatography (CM-Sepharose 6B) and gel filtration (Sephadex G-100). The purified preparation was homogeneous when examined by PAGE. The enzyme is an endopolygalacturonase with an optimum catalytic activity at pH 5.0 and 45°C, and a molecular weight of 57,000±500 daltons. The activity was stimulated by Fe⁺⁺⁺, Mg⁺⁺, Co⁺⁺, and inhibited by Mn⁺⁺ and Zn⁺⁺. The enzyme was stable in the pH range of 3.0 to 5.0. The purified enzyme was specific for nonmethoxylate polygalacturonic acid, with K_m and V_max values respectively of 0.19 mg/ml and 1.3 mol/g/min. In addition, this enzymatic preparation degraded pectic substances in organge peel.     

Extracellular invertase from Aspergillus athecius: Isolation and immobilization

Summary A fungal strain isolated from soil and identified asAspergillus athecius, when grown on moistened wheat bran produced large amounts of extracellular invertase. Most of the invertase from the moldy bran was easily extracted by low ionic strength buffer (0.005 M, pH 5.7). The crude invertase immobilized on DEAE cellulose showed not only increased activity (45%) but also greater thermal and storage stability than the free enzyme. The free and the bound enzymes showed a temperature optimum of 50–55°C and a pH optimum of 5.7 and 4.8 respectively. The K_m app. of the bound enzyme was lower than that of the free enzyme.     

Solid state fermentation of straw withNeurospora crassa for CMCase and β-glucosidase production

Summary CMCase and -glucosidase were produced by the mutantNeurospora crassa 40b cultivated on untreated wheat straw in a solid state fermentation. Best enzyme activities were observed when the growth medium was composed of wheat straw mixed with certain mineral solutions at a ratio 1:2 (w/v). A partially purified enzyme preparation showed optimum enzyme activities of CMCase and -glucosidase at pH 4.0 and 5.0 and temperature 50 and 60°C respectively. The apparent Km values for the same enzymes were 16.8 g/l and 1.03x10^–4 M respectively. At optimum growth and enzyme assay conditions yields as high as 586.2 U CMCase and 58.4 U -glucosidase per gram of straw were obtained.     

Production and localization of cellulases and β-glucosidase from the thermophilic fungusThielavia terrestris

Summary The enzyme production and localization ofThielavia terrestris strains C464 and NRRL 8126 were compared to determine their optimum temperature and pH for cellulase activity. High levels of intracellular -glucosidase activity were detected in the former strain. The intracellular -glucosidase of both strains were more thermostable than the extracellular enzyme; the half life ofT.terrestris (C464) endoglucanase activity at 60°C was greater than 96 hrs.     

Isolation of aClostridium thermocellum gene encoding a thermostable β-1, 3-glucanase (laminarinase)

Summary AClostridium thermocellum gene directing the synthesis of a thermostable -glucanase was localized on a 1.9-kb DNA fragment by subcloning intoEscherichia coli plasmid vectors. The enzyme was highly efficient in degrading glucans with alternating -1, 3- and -1,4-linkages such as lichenan and barley glucan. It was also active towards the -1, 3-glucan laminarin, but lacked activity on cellulosic substrates and -glucans. The enzyme was therefore classified as -1, 3-glucanase (laminarinase) and the corresponding gene was designatedlicA. With barley -glucan as substrate the enzyme had a pH optimum around pH 6.5 and a temperature optimum at 65°C. It was stable for several hours at 60°C in the absence of substrate.     

Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at –15°C) was induced when 50 M ABA or 3.2 M mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at –15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.Abbreviations ABA abscisic acid - MFD mefluidide - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ killing temperature for 50% of the embryos     

Characteristics of yeast β-galactosidase immobilized on calcium alginate gels

Summary Saccharomyces anamensis having -galactosidase activity, has been immobilized in calcium alginate gel matrix that retained 78.6% enzyme activity to that of native cells. Optimum pH(7.0) was negligibly affected by immobilization. K_m values for immobilized and native cells were 119 mM and 102 mM respectively. Protective agents like dithioerythritol, bovine serum albumin, enhance the enzyme activity when added prior to immobilization. Immobilized cells can be stored in refrigeration(4°C) for 42 days without a significant loss of enzyme activity.     

Cellobiose hydrolysis using glutaraldehyde-treated mycelia of Aspergillus terreus Thom

Summary The use of glutaraldehyde-treated mycelial pellets of Aspergillus terreus Thorn as a reusable form of -glucosidase was studied. The -glucosidase activity of these pellets has a pH optimum of 4.8 and as compared to untreated mycelia show decreased leakage of enzyme, higher Vmax, greater half-life at 65°C and increased reusability.     

Glucose isomerase activity of streptomyces kanamyceticus

Summary Streptomyces kanamyceticus produces a significant level of intracellular glucose isomerase when grown in submerged culture. The optimum temperature for enzyme activity is 90°C, but the optimum pH is changed by the kinds of buffer solution used. The activity is higher at pH 7.0–9.5. Treatment of cells with cetyl trimethyl ammonium bromide extracts almost the same amount of the enzyme as ultrasonic treatment. The selection of the method of treatment for enzyme extraction depends, however, on the nature of cell growth in synthetic or complex medium.     

Characterization of fructose 6 phosphate phosphoketolases purified from Bifidobacterium species

An extracellular chitin deacetylase activity has been purified to homogeneity from autolyzed cultures of Aspergillus nidulans. This enzyme is an acidic glycoprotein with a pI of 2.75 and a 28% (wt/wt) carbohydrate content. The apparent M _r of the enzyme estimated by SDS/PAGE and Superose 12 (f.p.l.c.) was around 27,000. The enzyme had an optimum pH at 7.0 and was stable in the pH range 4.0–7.5. Its optimum temperature of reaction was 50°C, and it was stable from 30° to 100°C after 1 h of preincubation. The enzyme hydrolyzed glycol chitin and oligomers of N-acetylgucosamine and to a lesser extent chitin, colloidal chitin, carboxymethylchitin, and an -1 3,1 6-N-acetylgalactosamine-galactan among other substances with amido groups, but the enzyme did not hydrolyze peptide bonds. The role of this enzyme could be deacetylation of chitin oligosaccharides during autolysis, after action of endochitinase on cell walls.     

Characterization of thermostable α-glucosidase from Clostridium thermohydrosulfuricum 39E

Summary Clostridium thermohydrosulfuricum 39E produced a cell-bound -glucosidase. It was partially purified 140-fold by solubilizing with Triton X-100, ammonium sulfate treatment, DEAE-Sepharose CL-6B, octyl-Sepharose and acarbose-Sepharose affinity chromatography. The optimum temperature for the action of the enzyme was at 75°C. It had a half-life of 35 min at 75°C, 110 min at 70°C and 46 h at 60°C. The enzyme was stable at pH 5.0–6.0 and had an optimum pH at 5.0–5.5. It hydrolyzed the -1,4-linkages in maltose, maltotriose, maltotetraose and maltohexaose, the rate decreasing in order of higher-sized oligosaccharides. The enzyme preparation also hydrolyzed the -1,6 linkages in isomaltose and isomaltotriose. It rapidly hydrolyzed p-nitrophenyl -d-glucoside (pNPG). The K _m values for maltose, isomaltose, panose, maltotriose, and pNPG were 1.85, 2.95, 1.72, 0.58, and 0.31 mm, respectively, at pH 5.5 and 60°C. The enzyme produced glucose from all these substrates. The enzyme preparation did not require any metal ion for activity. The -glucosidase activity was inhibited by acarbose. Offprint requests to: B. C. Saha     

Production kinetics and stability properties of ϖ(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase is a key enzyme that channels primary metabolites to a tripeptide common to cephalosporin and cephamycin biosynthesis inStreptomyces clavuligerus. Time-course studies indicated that theS. clavuligerus ACV-synthetase was stable during the cephamycin C fermentation: the enzyme was produced early in the growth phase and its activity remained high up to 96 h of growth. The detection of crude ACV-synthetase activity in older cultures was best achieved with an assay medium supplemented with 5 mM phosphoenolpyruvate, at lower ATP concentrations. During storage at 4°C, a progressive decrease in the stability of crude ACV-synthetase was observed with increasing culture age. Although a proteinolytic activity with a pH optimum at 8.2 was detected in crude cell-free extracts, no significant variation was observed in its activity with increasing culture age to account for the instability of ACV-synthetase in vitro. Addition of proteinase inhibitors did not improve the stability of the enzyme. However, a stabilization cocktail containing dithiothreitol. MgCl₂, the three substrate amino acids, and glycerol increased the stability of the enzyme isolated from cultures grown for 30–40 h, which was shortly after the appearance of antibiotics in the culture fluid. This stabilized enzyme retained half of its initial activity after 6 days at 4°C.     

A thermostable β-glucosidase isolated from a bacterial species of the genus Thermus

Summary An extremely thermophilic aerobic bacterium which produced -glucosidase was isolated from soil collected at the Yudanaka hot spring in Japan. It was identified as belonging to the genus Thermus. Production of -glucosidase by this bacterium was stimulated by the addition of cellobiose or laminaribiose to the medium. The optimum pH and temperature of the enzyme were 4.5–6.5 and 85° C respectively. The enzyme was stable in the pH range of 4.5–7.0 at 70° C for 2 h and the half-life at 75° C was 5 days. The K _m value of the enzyme for p-nitrophenyl--d-glucopyranoside, determined at 70° C in 0.1 M sodium phosphate buffer (pH 6.5), was 0.28 mM while the K _m was 2.0 mM for cellobiose. The enzyme effectively hydrolysed cellobiose at 70° C and the conversion yields of cellobiose to glucose were 95%, 93% and 90% at substrate concentrations of 5%, 10% and 15%, respectively.     

Immobilization of glucoamylase on naphthyl cotton cloth

Summary Aspergillus niger glucoamylase was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme was cross-linked with glutaraldehyde. The immobilized glucoamylase exhibited greater pH dependence though the optimal pH did not change. The immobilized glucoamylase in a packed bed column completely hydrolysed 5% soluble starch at a specific velocity of about 4. Used naphthyl cloth could be regenerated by heating in 2 N NaOH at 100°C for 1 hour.     

Production of active human interferon-β inE. coli I. Preferential production by lower culture temperature

Summary Unusually low culture temperature, such as 20°C, was shown to be preferable for the synthesis of active human interferon- (IFN-) inE. coli harboring a recombinant plasmid. TheE. coli cells cultured at 20°C gave 8.6-fold higher IFN- activity than those cultured at 37°C. However, almost the equal amounts of IFN- protein were accumulated in both cells cultured at 20°C and at temperature higher than 20°C, suggesting that IFN- might exist as an active form in the cells cultured at 20°C, while as a rather denatured form in the cells cultured at higher temperature.     

Purification and properties of heat stable α-amylase from Bacillus brevis

Summary An extracellular -amylase has been isolated from a continuous culture of a thermophilic strain of Bacillus brevis. This enzyme was purified eightfold and obtained in electrophoretically homogenous form. The enzyme had a molecular weight of about 58000, a pH optimum from 5.0 to 9.0 and a temperature optimum at 80°C. The half-life of the purified enzyme in the presence of 5 mM CaCl₂ at 90° C and pH 8.0 was 20 min. The K _m value for soluble starch was calculated to be 0.8 mg/ml.     

Extrinsic factors potassium chloride and glycerol induce thermostability in recombinant anthranilate synthase from Archaeoglobus fulgidus

Thermostable anthranilate synthase from the marine sulfate-reducing hyperthermophile Archaeoglobus fulgidus has been expressed in Escherichia coli, purified, and characterized. The functional enzyme is an ₂₂ heterotetrameric complex of molecular mass 150±15 kDa. It is composed of two TrpE (50 kDa) and two TrpG (18 kDa) subunits. The extrinsic factors glycerol (25%) and potassium chloride (2 M) stabilized the recombinant enzyme against thermal inactivation. In the presence of these extrinsic factors, the enzyme was highly thermostable, exhibiting a half-life of thermal inactivation of about 1 h at 85°C. The kinetic constants for the enzyme under these conditions were: K_m (chorismate) 84 M, K_m (glutamine) 7.0 mM, k_cat 0.25 s^–1, and pH optimum 8.0. The enzyme was competitively, though non-cooperatively, inhibited by tryptophan.     

Purification and characterization of an extracellular polygalacturonase from the thermophilic fungus,Thermomyces lanuginosus

A polygalacturonase was purified from the thermophilic fungus, Thermomyces lanuginosus to apparent homogeneity by ultrafiltration, acetone precipitation and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60 °C. The apparent K_M with potassium pectate was 0.67 mg/ml and the V_max was 7.2 × 10⁵ mol/min/mg protein. The apparent molecular weight of the enzyme was 59 kDa and it contained approximately 10% carbohydrate. The enzyme was completely stable at room temperature (32 ± 3 °C) and retained about 50% activity at 50 °C for 6 h. The zymogram of the purified enzyme revealed two activity bands, one of which was a major one. Polyclonal antibodies raised against the enzyme did not show any immunological relatedness with other mesophilic polygalacturonases.     

Production and properties ofβ-glucosidase byNeurospora sitophila

Growth at 25°C and pH 5.50 favour the production of-glucosidase. De-fatted oilseed flour and Tween 80 enhanced the production of-glucosidase, Lactose, gentibiose, gentibiose-acetate, laminarabiose and xylobiose induced-glucosidase activity. Precipitation of the culture filtrate with (NH₄)₂SO₄ resulted in 26-fold purification with 67% recovery. The optimum pH and temperature for activity were 5.0 to 5.4 and 55°C respectively. The enzyme was stable at 40°C with half-life at 12 h at 50°C. TheK _m andV _max for the hydrolysis ofp-nitrophenyl--d-glucoside at 40°C H 5.0 are 0.28mm and 0.60 U/mg protein, respectively.