Screening of yeast strains capable of hyperproducing amylolytic enzymes

Summary Fifteen strains of yeast, which produced an extracellular amylolytic enzymes, were isolated from nature. One of them produced more than 100 times the enzyme activity in comparison with the 14 strains and the extremely hyperproducing strain of yeast was identified asCandida sp. 347. Paper chromatograms of the amylolytic enzyme demonstrated activity of amyloglucosidase. The optimum pH for activity of the enzyme was 5.5–6.0 and optimum temperature was 60°C.     

Preparation and properties of glucose isomerase of Streptomyces kanamyceticus cells entrapped in calcium alginate

Summary The properties of glucose isomerase in native, heat-treated and immobilized cells of Streptomyces kanamyceticus after heat and mineral treatment have been compared. The optimum pH for glucose isomerase in native cells was shifted from 8.2 to 8.6 by heat treatment and immobilization. There is no change in the optimum temperature (90°C) for activity of the enzyme by the above treatment. Heat-treated cells and immobilized cells show greater pH and thermal stability of the enzyme. The Km values of the enzyme of native cells, heat-treated cells and immobilized heat-mineral-treated cells are 208 mM, 212 mM and 166 mM respectively; Mg⁺⁺ and Co⁺⁺ enhance the activity of isomerase in all cases.     

Studies on the alkalophilicStreptomyces with extracellular xylanolytic activity

Summary An alkalophilicStreptomyces which produced xylanase, isolated from soil, grew in a temperature range of 15–37°C. The pH optimum for growth was 10 and no growth occurred at pH 7. On a simple wheat bran medium the microorganism exhibited maximum enzyme secretion of 12 U/ml at pH 10. The enzyme had a broad pH optimum of 4.8–10 and the optimum temperature of 50°C. It was completely inactivated at 60°C in 2 h. The enzyme hydrolyzed xylan to a mixture of oligomeric products indicating that the main activity was of the endoxylanase type. The culture filtrate had no cellulase activity.     

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.     

Production and properties of fibrinolytic enzyme in solid state cultures of Fusarium pallidoroseum

Summary Ten local fungal isolates were screened for their ability to produce extracellular fibrinolytic enzyme activity in solid state and skaken cultures. Fusarium pallidoroseum was the most active in wheat bran solid cultures. Maximum activity was obtained at 25 °C and 50% moisture content. Addition of 2% casein to the culture increased the activity by 1.7-folds. The 65% ammonium sulphate fraction showed the highest fibrinolytic activity it was further purified by gel filtration on Sephadex G-100 followed by rechromatography of the most active peak on DEAE-cellulose. The pure enzyme was highly active on human fibrin and showed an optimum reaction temperature of 40 °C and pH 7. The enzyme was relatively sensitive to heat treatment at 55 °C and strongly inhibited by EDTA, it restored its activity by adding cobalt ions to the reaction.     

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.     

Production of mold lactase

A process for production of mold lactase was developed. Tests were carried out in pilot and industrial scale with an Aspergillus niger strain selected after screening a number of molds.A computer coupled autoanalyzer system was used for monitoring enzyme formation in the pilot fermentor. Lactase production was investigated using different pH- and temperature-profiles. A. niger lactase has an acid pH optimum, a high temperature optimum and good stability. It does not require any metal ions. It is suitable for immobilization for hydrolysis of lactose in acid whey.Three-fold enhancement of lactase production was obtained by mutagenizing A. niger using NTG as mutagenic agent.The lactases produced by the mutants have the same pH and temperature optima and stability but the growth properties of the mutants were different from those of the original strain.Sufficient specific activity of the enzyme preparation for immobilization was obtained by purifying the enzyme by selective adsorption on Na-Ca-silicate.     

Solid state fermentation for production of higher titres of thermostable alpha-amylase with two peaks for pH optima byBacillus licheniformis M27

Summary Bacillus licheniformis M27 produced 21, 000 units of alpha-amylase/g dry bacterial bran under solid state fermentation in wheat bran medium enriched with 3.3% di-ammonium hydrogen phosphate. The crude enzyme, with temperature optimum at 90°C in 0.5% starch solution, showed pH optima at 6.5–7.0 and 9.5 and over 75% activity over the pH range 6.0–10.5.     

High activity xylanase from thermotolerantStreptomyces T7: Cultural conditions and enzyme properties

Summary A thermotolerantStreptomyces T₇ produced 70–72 U/ml of extracellular xylanase activity when grown at 50°C in submerged culture, in à medium containing 5% wheat bran as a carbon source. Among the various sugars tested, maltose showed the highest activity of 8 U/ml. Pure xylan was less effective as an inducer as compared to wheat bran. Ammonium sulphate at a concentration of 0.7% was found to be optimum for maximum yield of the enzyme. The optimum period and pH for maximum production were 72th and 7.0, respectively. The culture filtrate was devoid of amylase, cellulase and B-xylosidase activity. The xylanase was exceptionally stable and did not show any loss in activity after storage at 50°C at pH 5.0 for 6 days.     

Partial purification and characterization of a NADPH dependent tetrahydroalstonine synthase from Catharanthus roseus cell suspension cultures

A new enzyme was discovered which specifically hydrogenates the iminium form of cathenamine at position 21 to yield the heteroyohimbine alkaloid tetrahydroalstonine. The enzyme was partially purified (35-fold) from Catharanthus roseus cell suspension cultures. It was shown to use exclusively NADPH as reductant, the pH optimum is at 6.6, the temperature optimum at 30°C, the half life of the soluble enzyme preparation is 26 min at 37°C, and the molecular weight is 81 000 ± 3%. Evidence is presented for the occurrence of two distinct and different cathenamine reductases, one reducing the iminium form of this central intermediate to give tetrahydroalstonine, the other one reducing cathenamine to yield ajmalicine. Tetrahydroalstonine synthase was present in cell suspension cultures of C. ovalis, C. roseus, Picralima nitida, Rhazya stricta, and Vinca herbacea. Dedicated to Prof. Dr. Franz Lingens on the occasion of his 60th birthday     

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.     

High activity extracellular glucose (xylose) isomerase from a Chainia species

Summary A sclerotia-forming actinomycete of the genus Chainia secreted high levels of glucose (xylose) isomerase when grown in submerged culture on a wheat bran - yeast extract medium. Maximum activity (4 units/ml) was obtained after 3–4 days when the cell bound activity was 0.19 units/ml. The two enzymes differed significantly in pH optima (extracellular, 9.5; cell-bound, 7.0) and in their adsorption behaviour on CM and DEAE celluloses. Both Mg⁺⁺ and Co⁺⁺ are required by the cell-bound enzyme for its optimum activity while either Mg⁺⁺ or Co⁺⁺ is necessary for the extracellular enzyme.NCL Communication 3320     

The isomaltulose synthesising enzyme ofSerratia plymuthica

Summary An intracellular enzyme was located inSerratia plymuthica which produced isomaltulose from sucrose. The enzyme was purified giving a preparation with a specific activity of 1,285. It has pH and temperature optima of 6.0 and 30°C, respectively. The enzyme was stable retaining 100% activity after 2 weeks at 30°C. It had an isoelectric point at pH 9.0, a Mr of 79,500 and the Km for sucrose was 65.3mM. The enzyme converted 40% (w/v) sucrose to isomaltulose with an efficiency of 87%.     

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)     

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.     

Growth conditions of a pectinolyticAspergillus fumigatus for degumming of natural fibres

Summary Optimum growth conditions forA. fumigatus strain 4 when citric pectin was the sole carbon source were at a temperature of 45°C, pH 4.0 and an incubation time from 36 to 42h. Under these conditions no cellulase activity was found. When orange pulp was the sole carbon source, optimum polygalacturonase activities were found when the fungus was cultured for 36 h at 45°C and a pH 3.0 to 4.5.     

Lactase production in continuous culture by Trichoderma reesei Rut-C30

Summary Trichoderma reesei Rut-C30 was found to produce extracellular lactase when grown on lactose medium. Maximum enzyme levels in continuous culture were observed at dilution rates (D) between 0.02 and 0.027 hr^-1. The enzyme productivity reached 27.3 U/L hr at D = 0.027 hr^-1. Lactase synthesis appears to be inducible and subject to catabolite repression. Optimal growth temperature and pH for enzyme production were 28°C and pH 5. Maximum enzyme activity was observed at 63°C and pH 4.6. The apparent K_m, based on the nitrophenyl-galactopyranoside assay was estimated as 0.4 mM. The enzyme is suitable for lactose hydrolysis in acid whey.     

A thermostable endo-1,4-/B-glucanase from Myceliophthora thermophila

Summary Endo-1,4-/B-glucanase from a thermophilic fungusMyceliophthora thermophila has been isolated and purified to homogeneity. The molecular weight of the enzyme was 52,000 with a pI of 4.7, pH-optimum 5.0–6.0, and specific activity 61 IU/mg (40°, pH 5.0); this activity is 2.4 times higher than that of the enzyme fromTrichoderma reesei. The new endoglucanase is very thermostable; its half-life at 65°C is 170 hours, which is 300 times higher than that of the enzyme fromT. reesei.     

Studies on xylan degrading enzyme from Chainia

Summary The sclerotial actinomycete Chainia (NCL 82-5-1) secreted extracellular xylanase in submerged culture in media containing yeast extract and wheat bran or commercial xylan. A high activity (28 IU/ml) of xylanase was obtained in 72 h on a medium containing 3% xylan. Only a single species of xylanase (i.e. without isoenzymes) was detected by polyacrylamide gel electrophoresis. It had an optimum pH of 5.0 and optimum temperature of 65°C. It was stable at pH 6.0 to heating at 60°C for 10 min. Its pI was 8.0 and the K_m was 0.4%. The results are discussed in relation to xylanase reported from actinomycetes such as Streptomyces xylophagus.     

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.