Production of pectolytic and cellulolytic enzymes byfusarium oxysporum andF. moniliforme under different cultivation conditions

Six-day incubation was most suitable for production of pectolytic and cellulolytic enzymes byFusarium on different culture media. Czapek’s medium favoured maximum production of polygalacturonase (PG) and cellulase (C_x), peptone dextrose gave highest yields of pectin methyl galacturonase (PMG) withF. oxysporum. Cole’s medium was found to be poor for the enzyme production by both organisms. A positive correlation was observed between the growth rate of the pathogenic forms and their enzyme production. InF. oxysporum the PG secretion was maximum at pH 4.5 and inF. moniliforme at pH 5.0. PMG production optimum was at pH 5.5. No PG and PMG were produced above pH 7. InF. oxysporum the C_x activity was highest at pH 5.5 and inF. moniliforme at pH 4.5. Maximum PG and PMG activities were recorded at 35 °C in both pathogens. The C_x activity of both organisms was maximum at 45 °C but some carboxymethyl cellulose hydrolysis was found even at 60 °C.     

Lipase activity of two seed-borne fungi of sesamum (Sesamum indicum Linn.)

Macrophomina phaseolina andPhoma nebulosa secreted lipase which varied with the medium.M. phaseolina produced more lipase thanP. nebulosa. Sesamum seed meal caused stimulation of lipase production. The lipase secreted by both organisms showed maximum activity at pH 5.O. The optimum temperature for activity of lipase secreted byM. phaseolina was found to be 40 °C, while for that ofP. nebulosa it was 30 °C.     

Production of saccharifying enzyme using the wastewater of a Schochu distillery

A saccharifying enzyme was produced by Aspergillus awamori var. kawachi using wastewater generated by a Shochu distillery. The production of the saccharifying enzyme was of the non-growth associated type, and 80 U of activity per ml of broth was obtained in about 6 d of flask cultivation. Since the Shochu distillery wastewater contained high concentrations of volatile fatty acids that were converted to their free forms and severely inhibited cell growth at low pH, the optimum initial pH ranged from 4.5 to 6.0. It is suggested that cell autolysis facilitated the release of the saccharifying enzyme, but a released protease digested the saccharifying enzyme with a subsequent decrease in activity. The saccharifying enzyme was easy to purify, and the purified enzyme was homogeneous when analyzed by disc electrophoresis. The molecular weight was estimated to be 54,000 Da by SDS-PAGE, and the isoelectric point was found to be pH 3.6 by isoelectric focusing. The optimum temperature and pH for the reaction ranged from 50 to 55°C and 4.5 to 5.5, respectively. The saccharifying enzyme could not digest raw starch. The hydrolyzate of soluble starch hydrolyzed by the saccharifying enzyme was composed of two to four oligosaccharides. From these results and the amino acid sequence in the N-terminal, the enzyme produced was concluded to be α-amylase.     

Preparation of Triglyceride Hydroperoxides

Corticium rolfsii AHU 9627, which we isolated from a tomato stem, is one of the most promising producers of a raw starch saccharifying enzyme. The effects of the cultural conditions and medium components on the enzyme production were investigated. The enzyme production was improved by increasing both the concentrations of carbon sources and organic nutrients in the medium. Under the optimum cultural conditions, the enzyme activity of the culture supernatant against raw starch reached a maximum after 8-days incubation at 27°C and the activity reached 80 units per ml (when determined at 40°C and pH 4.0). The optimal pH and temperature for the enzyme reaction were 4.0 and 65°C, respectively. The saccharifying reaction was scarcely inhibited even with a high substrate concentration, and raw starch was rapidly hydrolyzed into glucose.     

Purification and characterization of a novel solvent-tolerant lipase from Fusarium heterosporum

A microorganism producing a solvent-tolerant lipase was identified as Fusarium (F.) heterosporum. The lipase was purified from the culture filtrate to homogeneity as judged by disc-PAGE and SDS-PAGE. The purification included SP-Sephadex chromatography, gel filtration and isoelectric focusing, and the recovery yield was 38%. The lipase was a monomeric protein with a molecular weight of 31 kDa estimated by SDS-PAGE, and a pI of 7.0. The optimum pH at 40°C and optimum temperature at pH 5.6 were 5.5–6.0 and 45–50°C, respectively, when olive oil was used as the substrate. The lipase was stable over a pH range of 4–10 at 30°C for 4 h, and up to 40°C at pH 5.6 for 30 min. Furthermore, the enzyme was not inactivated even after incubation at 30°C in 50% solvent such as dimethylsulfoxide (DMSO), hexane, benzene and ether for 20 h. The activity did not decrease in a reaction with stirring in a mixture containing 50% DMSO or dimethylformamide. The lipase preferably reacted on middle-chain fatty acid triglycerides (6≤C≤12), and cleaved only 1,3-ester bonds of triolein. The enzyme had an N-terminal sequence of Ala-Val-Thr-Val-Thr-Thr-Gln-Asp-Leu-Ser, which has not previously been found in any other protein. We compared the properties of lipases from F. heterosporum and another strain F. oxysporum.     

Enhancing the cyclodextrin production by synchronous utilization of isoamylase and α-CGTase

Cyclodextrins (CD) are cyclic α-1,4-glucans composed of glucose units, and they have multiple applications in food, pharmaceuticals, cosmetics, agriculture, chemicals, etc. CD are usually produced by cyclodextrin glycosyltransferase (CGTase) from starch. In the present study, a simultaneous conversion approach was developed to improve the yield of CD from starch by conjunction use of isoamylase with α-CGTase. The isoamylase of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The biochemical characterization of the enzyme showed that the optimum temperature and pH of the recombinant enzyme was 50 °C and 5.5, respectively, and it maintained 60 %, 85 % and 78 % relative activity at 30 °C, 40 °C and 60 °C, respectively. When the recombinant isoamylase and α-CGTase were used simultaneously to convert potato starch (15 %, w/v) into CD, the optimum conditions were found to be: 10 U of α-CGTase and 48 U of isoamylase per gram of substrate, with reaction temperature of 30 °C and pH 5.6. On the optimum condition, the total yield of CD reached 84.6 % (w/w) after 24 h, which was 31.2 % higher than transformation with α-CGTase alone. This is the first report of synchronous bioconversion of CD by both α-CGTase and isoamylase, and represents the highest efficiency of CD production reported so far.     

Growth and toxigenicity of Fusaria of the sporotrichiella section as related to environmental factors and culture substrates

Isolates ofFusarium poae, F. sporotrichioides, F. sporotrichioides var.chlamydosporum andF. sporotrichioides var.tricinctum made their best growth on PDA substrates at 24 °C, but good growth was also made at 18 °C and 30 °C. At 35 °C growth made by theF. sporotrichioides var.chlamydosporum was quite good, and superior to that of the other fungi. Moderate growth was made by all fungi at 12 °C and byF. sporotrichioides var.tricinctum also at 6 °C, while growth of the other fungi at that temperature was slight. At low temperatures toxic isolates of all butF. sporotrichioides grew better than non-toxic isolates, and growth of all isolates usually was better in light than in darkness up to temperatures of 18 °C. F. poae andF. sporotrichioides produced highest toxicity on rabbit skins when grown at 5–8 °C,F. sporotrichioides var.tricinctum at 15–20 °C. Darkness always favoured toxin development at all temperatures. In a comparison of 3 liquid substrates, overall toxin production was stronger on a starch substrate than on Czapek's or carbohydrate-peptone substrates. Among grain substrates, barley gave highest overall toxicity, which was again favoured by darkness.F. poae isolates were most toxic when derived from soil,F. sporotrichioides isolates when derived from barley. Further tests with 8 liquid substrates confirmed thatF. poae andF. sporotrichioides produce stronger toxicity at 8 °C than at 25 °C, and substrates favoured toxin production at pH 5.6 more than at pH 3.8 or 7.2. At pH 5.6 the isolates induced marked changes in the pH level of the substrate on which they grew. No relation was found to exist between the vigour of growth made by any of these fungi under various environmental conditions and the severity of the toxiç reaction their extracts produced on rabbit skins.     

Acid-stable α-Amylase of Black Aspergilli

When black Aspergilli were cultivated in appropriate condition, culture filtrate showed dextrinizing activity even after acid treatment such as pH 2.5, at 37°C for 30 minutes. It suggested the existence of acid-stable dextrinizing amylase. To isolate this enzyme paper el-ectrophoretic procedure was carried out and the spot which showed acid-stable dextrinizing activity was obtained in addition to α-amylase and glucoamylase spots. This new amylase was purified by fractional precipitation with ammonium sulfate, rivanol and acetone, and was obtained in a crystalline form.     

Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047

An endosymbiont Halobacterium salinarum MMD047, which could produce high yields of amylase, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% sucrose. The enzyme was found to be produced constitutively even in the absence of starch. The optimum temperature and pH for the enzyme production was 40°C and 8.0, respectively. The enzyme exhibited maximum activity in pH range of 6∼10 with an optimum pH of 9.0. The enzyme was stable at 40°C and the enzyme activity decreased dramatically above 50°C. Based on the present findings, the enzyme was characterized as relatively heat sensitive and alkalophilic amylase which can be developed for extensive industrial applications.     

Purification an α-galactosidase from Coriolus versicolor with acid-resistant and good degradation ability on raffinose family oligosaccharides

An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg^?1. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4–50 °C and at a pH range of 2–5. Among the metal ions tested, Cu²⁺, Cd²⁺ and Hg²⁺ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag⁺ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.     

Characterization of the amylolytic system ofCandida strains

Some physical and chemical properties ofα-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) produced in semisolid fermentation byCandida fennica FTPT-1829,C. famata FTPT-1539 andC. fennica FTPT-8903 were determined. The optimum temperature values were 42, 44, 48 °C and 60, 50, 50 °C forα-amylase and glucoamylase excreted byC. fennica 8903,C. fennica 1829 andC. famata 1539, respectively. The optimum pH values for all strains were 5.0 and 6.0 forα-amylase and glucoamylase, respectively. The degradation of pullulan by all the yeast species indicates debranching activity. This research was carried out with financial support fromPrPq/UFMG. The second author received grants fromRHAE/CNPq.     

Ethanol production from raw starch by simultaneous fermentation using Schizosaccharomyces pombe and a raw starch saccharifying enzyme from Corticium rolfsii

Alcoholic fermentation from raw corn starch using Schizosaccharomyces pombe AHU 3179 and a raw starch saccharifying enzyme (RSSE) from Corticium rolfsii AHU 9627 was investigated. The optimum ethanol production was achieved at pH 3.5, 27°C and under the yeast cell concentration of 2.7 × 10⁹ cells/ml. Addition of RSSE 5 units (as glucoamylase)/g raw corn starch was found sufficient. Under these optimum conditions, 18.5% (v/v, at 15°C) ethanol was obtained from 30% raw corn starch (30.8% as glucose) after incubation for 48 h.     

Production of ethanol from sugars in wood hydrolysate byFusarium oxysporum

Wood hydrolysate used for ethanol production by two strains ofFusarium oxysporum contained 2.3% (w/v) reducing sugars (xylose and glucose). Ethanol production at the optimum reducing sugar concentration of 54.8 g/l medium, at pH 5.5, and 30°C was 12.3 g/l and 11.7 g/l byF. oxysporum D-140 and NCIM-1072, respectively in shake flasks during 96 h fermentation. The maximum production of ethanol under optimum cultural conditions, and in the presence of yeast extract plus minerals, was 13.2 g/l medium byF. oxysporum D-140 over 108 h fermentation.

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Résumé L'hydrolysat de bois utilisé pour la production d'éthanol par deux souches deFusarium oxysporum contenait 2.3% (poids/vol.) de sucres réducteurs (xylose et glucose). La production d'éthanol, à la concentration optimum en sucres réducleurs de 54.8 g par litre de milieu à pH 5.5 et à 30°C était de 12.3 g/l et 11.7 g/l respectivement chezF. oxysporum D-140 et NCIM-1072, en flacons agités pendant 96 h de fermentation. La production maximum d'éthanol, dans les conditions optimum de culture, et en prosence d'extrait de levure et de minéraux a mit de 13.2 g par litre de milieu chezF. oxysporum D-140 en 108 h de lermentation.

     

Highly stable laccase from repeated-batch culture of Funalia trogii ATCC 200800

The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55–90°C, which was maximal at 80–95°C. It was highly stable within the temperature intervals 20–50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0–9.0. NaN₃ and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.     

Secretion of alpha-amylase by Rumex virus tumors in vitro; properties and assay

The existence of an extracellular α-amylase, which is secreted by virus tumors from the roots of Rumex acetosa grown in vitro, has been demonstrated and the properties of the enzyme have been studied. As far as the authors are aware, this is the first enzyme demonstrated to be secreted from intact cells of higher plants.An assay for the quantitative determination of this enzyme is presented. This assay is based on the spectrophotometric determination of the decrease in color of the starch-iodine complex due to the activity of the amylase on the starch substrate.Among the factors discussed which affect the density of the starch-iodine complex are iodine concentration, temperature, and ionic strength.The optimum pH for activity of this enzyme is 4.6, both in acetate and citrate-phosphate buffers.At pH 4.6 in 0.02 M acetate buffer, the Q₁₀ for the hydrolysis of starch by the enzyme is 1.6 in the range of 20–40 °C. The activation energy in this range calculated by the Arrhenius equation is 8000 cal./ mole.This α-amylase is protected by calcium and is sensitive to low pH values as are the cereal α-amylases.The energy of activation for the heat denaturation of the enzyme is 43,000 cal./mole, as calculated by the Arrhenius equation.     

Existence of acid and alkaline phosphohydrolase activity in the phytoflagellate Ochromonas danica

Cell homogenates of light-grown Ochromonas danica contained distinct non-specific non-phosphate-repressible acid and alkaline phosphohydrolase activities. Acid phosphohydrolase activity had a broad pH range of 2.0–5.0 and the optimum for alkaline phosphohydrolase activity was pH 8.6 Acid phosphohydrolase (pH 3.6) activity had an optimum temperature of 55°C; the alkaline enzyme activity had an optimum temperature of 37–40°C.     

Purification and properties of mycodextranase from Bacillus circulans NHB-1

A Gram-positive bacterium, Bacillus circulans, isolated from soil was found to produce an enzyme hydrolyzing nigeran (mycodextran, alternating α-1,3- and α-1,4-linked glucan). The molecular weight of the purified enzyme was 120,000 and its isoelectric point was 8.30. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 7.0 and up to 50°C. The K_m (mg/ml) for nigeran was 1.37. The enzyme specifically hydrolyzed the nigeran into nigerose and nigeran tetrasaccharide by an endo-type action, indicating that it is a mycodextranase (EC 3.2.1.61) cleaving only the α-1,4-glucosidic linkages in nigeran. The N-terminal amino acid sequence of the purified enzyme of B. circulans (APTVYEAESAAKTGGV) was different from that of the mycodexstranase purified from Streptomyces sp. J-13-3 (XDPGDPTDPDPSGVGATLPF).     

Purification and enzymatic properties of α-galactosidase from Penicillium janthinellum

α-Galactosidase (E.C.3.2.1.22) from Penicillium janthinellum was purified by precipitation and fractionation with ammonium sulphate, cold acetone or ethanol, calcium phosphate gel, and column chromatographies on Sephadex G-100 and G-200. The enzyme was purified about 110.39-fold when Sephadex G-100 was used. α-Galactosidase exhibited the optimum pH and temperature at 4.5 and 60°C, respectively. The optimum enzyme stability was obtained at pH 3.5 for 24 h (at room temperature). The enzyme was found to be thermostable below 65°C up to 40 minutes and was gradually inactivated by increasing the temperature above this degree. The MICHAELIS constant was 0.55 mM for p-nitrophenyl-α-D-galactoside. The α-galactosidase activity was strongly inhibited by Hg⁺⁺ and slightly activated by Mn⁺⁺. The results show the possibility of producing a thermostable enzyme from a low-priced agricultural product, for instance, lupine.     

Kinetic of the production of polygalacturonase and pectin lyase by two closely relatedFormae speciales ofFusarium oxysporum

The kinetic of thein vitro production of polygalacturonase and pectin lyase of two closely related fungi,Fusarium oxysporum f.sp.lycopersici andF. oxysporum f.sp.radicis-lycopersici, was examined under various culture conditions such as the source of carbon, the pH, and the age of cultures. Over a 5-day period, the production of these enzymes by various isolates of the sameforma specialis (f. sp.) ofF. oxysporum was not significantly different (P ≥ 0.05). However, the amount of the enzymes produced differed markedly between both f. sp. The different carbon sources added to the culture media, such as citrus pectin, apple pectin, tomato cell wall fragments, andd-galacturonic acid, proved to be higher pectinase inducible substrates than sucrose and glucose. For both fungi, polygalacturonase and pectin lyase activities were optimal at pH 5.0 and 8.0, respectively. Furthermore, pectin lyase production had a partial Ca²⁺ requirement in contrast to polygalacturonase production which was limited by Ca²⁺. In most experiments performed, the production of polygalacturonase appeared superior withF. oxysporum f.sp.radicislycopersici than withF. oxysporum f.sp.lycopersici. On the other hand, pectin lyase production ofF. oxysporum f.sp.lycopersici was approximately 10-fold greater than that byF. oxysporum f.sp.radicis-lycopersici in media supplemented withd-galacturonic acid.     

Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Ağri,Turkey

A novel amylolytic enzyme producing thermophilic bacterial strain KP1 from the Diyadin hot spring water in A?ri, Turkey, was isolated in the present study. Phylogenetic analysis based on the partial 16S rRNA gene, biochemical and physiological tests revealed that the strain KP1 belongs to the genus Anoxybacillus. The pH and temperature optima for the α-amylase production by Anoxybacillus sp. KP1 were 8.0 and 50°C, respectively, where the maximum growth was obtained at the 28^th hour of incubation and the highest α-amylase activity was obtained at the 40^th hour of incubation (8979.6 U/mL). The optimum pH and temperature for the enzyme activity were 8.0 and 60°C, respectively. The maximum α-amylase production was secreted in the presence of 2% (w/v) soluble starch (10837.7 U/mL). Among the various organic and inorganic nitrogen sources tested, while keeping the beef extract concentration constant, casamino acid (14310.6 U/mL), urea (14126 U/mL), and tryptone (13217.2 U/mL) at a concentration of 2% gave the maximum α-amylase production. The enzyme activity was enhanced in the presence of 1.5 mM Mn²⁺ (123%), whereas it was strongly inhibited 1.5 mM by Hg²⁺. Inhibition by 89% was obtained also with sodium dodecyl sulphate (1%). The enzyme was found to be relatively stable at a range of pH and temperature.