CMCase production by Spicellum roseum in liquid and solid culture

Summary CMCase was produced by 7 strains of Spicellum roseum in both liquid and wheat bran solid substrate cultures. No growth occurred above 35°C. Maximum enzyme production occurred at 30°C, whereas best enzyme activity occurred at pH 5.0 and 50°C. In liquid cultures of S. roseum, NRRL strains 13103, 13104, and 13106 produced activities of ca. 1.1, 1.5, and 1.5 mg glucose per hr/ml culture supernate at 1 week and 2.9, 1.5, and 2.1, respectively at 3 weeks compared to Trichoderma reesei NRRL 11236 (MCG77), which produced activities of 2.8 and 1.3 at 1 and 3 weeks.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Comparative study of amylolytic enzymes production byAspergillus niger in liquid and solid-state cultivation

Summary Amylolytic enzymes produced by a strain ofAspergillus niger cultivated on cassava starch in liquid or solid culture were found to be mainly glucoamylases. For the same initial amount of substrate, the glucoamylase activity increased even after 60 h of culture on solid medium whereas it decreased in liquid culture. Some characteristics of the amylases produced in both culture conditions were compared. The pH optima for enzymes produced in solid and liquid cultures were 4.5 and 5.0 respectively. Glucoamylase synthetized in solid cultures was significantly more thermostable than that from liquid culture and was maximally active at 70°C compared to 50°C for the enzyme from liquid cultures. The Km values expressed as mg soluble starch/100 ml were 0.1% for crude enzyme from solid culture and 0.057% for crude enzyme from liquid culture.     

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.     

Xylanase production in solid-state fermentation: a study of its properties

Summary Xylanase production by Aspergillus niger van Tieghem was studied in solid-state cultivation. The screening of substrates was carried out in column incubators aerated with humidified air at 30°C. Results of physiological studies showed that the best yield of xylanase was 2500 U/g dry matter on a mixture of straw+bran 1:1 at 70% of moisture content.In order to compare some properties of the xylanase produced in both liquid and solid cultures, A. niger was also grown on xylan in submerged cultures. The enzymes produced in solid and liquid cultures have an optimum pH of about 3.8 and 4.5, respectively. Xylanase synthetized in solid fermentation is a little more thermostable than that from liquid culture and is maximally active at 50° C, compared to 45° C for enzyme from liquid culture.     

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     

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH₄)₂SO₄ and soybean flour as nitrogen sources and KH₂PO₄ at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.     

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%.     

The properties of immobilized whole cell ofHumicola spp. with rifamycin oxidase activity

Summary The whole cell ofHumicola spp. ATCC 20620 with rifamycin oxidase activity was immobilized by copolymerization with acrylamide. The whole cell was defatted by treatment with acetone to reduce the diffusional resistance through the cell membrane. The recovery of enzyme activity after the immobilization step was about 50%. The acetone-defatted cell showed the maximum activity at pH 7.5 for both free and the immobilized forms. No appreciable activity loss could be detected when stored at 4 °C and pH 7.8 for one month, while the half life at 40 °C and pH 8 was decreased to about 8 days. The apparent Km values of rifamycin oxidase for the free and immobilized acetonedefatted cells were 0.3mM and 0.6mM, respectively. The enzyme demonstrated substrate inhibition, but the degree of substrate inhibition was different between two forms of the enzyme preparation. A complete substrate inhibition was observed for the immobilized cell, whereas the enzyme activity was partially inhibited at high substrate concentration in the acetone-defatted cells.     

Hydrolysis of animall fats by lipase at temperatures below their melting points

Summary We have studied the hydrolysis of high melting animal fats by the lipase fromCandida rugosa at temperatures between 20°C and 37°C without the addition of surfactants or organic solvents. To establish the practical applications of this process we investigated the optimal conditions of the reaction at high substrate concentrations (50% fat w/v) to achieve 95% hydrolysis (or better) in 24 hours. Experiments were conducted in solid emulsions without constant stirring (500 ml total reaction volume). Under all conditions tested, edible pork lard was a better substrate than inedible beef tallow yielding up to 96% hydrolysis with as low as 0.3 g lipase/Kg fat or 98% hydrolysis with 0.5 g lipase/Kg fat. The optimum temperature for the hydrolysis of edible pork lard was around 30°C. Inedible beef tallow and pork lard did not exhibit a clear optimum temperature. Inedible lard gave results intermediate between those of edible lard and inedible beef tallow.     

Extracellular protease activity in antibiotic-producingStreptomyces thermoviolaceus

Production of secondary metabolites was investigated in the thermophilic streptomyceteStreptomyces thermoviolaceus grown at 45°C in a fermenter. Extracellular protein was secreted into the culture medium at the same time as an antibiotic granaticin; both were synthesized during the second slower phase of biphasic growth, which is most apparent at 45°C for this organism. Protease, assayed as azocaseinase, was identified as one component of, and marker for, the excreted protein. The effects of different growth temperatures revealed that the synthesis of extracellular protein, like that of the antibiotic, was maximal in cultures grown between 37° and 45°C, whereas protease activity was greatest in 50°C grown cultures. A method was devised, based on acetone precipitation, for concentrating the protease activity from culture supernatants. Characterization of the concentrated activity using inhibitors suggested the presence of a serine and a metallo-type protease. A peptide substrate specific for the metallo-protease showed that it had a pH optimum for activity of 6.5–7.0. Approximately 50% of the activity was lost after 80 min of incubation at 70°C. Although calcium (5 mM) promoted increased thermotolerance such that around 65% of the activity remained after 100 min at 70°C, it seems that manganese and/or zinc may be more important for enzyme activity.     

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.     

Induction of freezing tolerance in alfalfa cell suspension cultures

The effects of ABA, 2,4-D, kinetin and cold exposure on the cold hardiness of Medicago sativa L. cell suspensions were investigated. Cultures treated with 5×10^–5 M ABA at 2°C for 4 weeks in the absence of kinetin showed a 50% survival after freezing to –12.5°C, whereas cultures grown at 25°C under normal conditions tolerated freezing to only –3°C. The optimum ABA treatment of 5×10^–5 M for 4 weeks was effective only in combination with cold exposure. Of six cell lines tested, all showed different degrees of induced cold hardiness. The results suggest that ABA alone cannot induce freezing tolerance on alfalfa cell suspension cultures and that the deletion of kinetin and combination of low temperature and ABA is critical for the induction of cold hardiness in alfalfa cell suspension cultures.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ 50% killing temperature     

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.     

Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m³ fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4–30°C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70–75°C. The enzyme was almost thermostable (91–92%) at pH 6.5 and 9.0 after 41 h preincubation at 55°C and lost only 20–33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0. Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55°C and pH 6.5. Correspondence to: J. Gomes     

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.     

Properties of laccase purified from nitrogen limited culture of white-rot fungus Coriolus hirsutus

Laccase produced by nitrogen-limited culture of Coriolus hirsutus was purified to electrophoretic homogeneity (133-fold) with an overall yield of 40%. The molecular mass of the enzyme was determined as 82 kDa by SDS-PAGE and 80 kDa using gel filtration. It had a pI of 3.50. With ferulic acid and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as the substrate, the enzyme had optimal activity at pH 4.0 and 2.5, respectively. The enzyme was stable in the range pH 5.5 to 7.0 at 30 °C for 1 h. The enzyme was optimally active at 70 °C and it lost all activity within 15 min at 80 °C. The apparent Km value of enzyme toward ABTS was 67 °M and had highest affinity toward sinapinic acid. The enzyme was totally inhibited by 0.01 mM cysteine.     

Chemical modification of xylanase from Chainia sp. (NCL 82.5.1)

Summary The high molecular weight xylanase from Chainia (NCL 82. 5. 1) is extracellular, cellulase-free and stable at alkaline pH (pH 8.0) at 50°C. The enzyme showed inhibition by N-bromosuccinimide(NBS) and by cysteine-specific reagents p-hydroxy mercuric-benzoate(PHMB) and N-ethyl maleimide(NEM) implying that tryptophan and cysteine are present at or near the active site of the enzyme. The enzyme was reversibly inhibited by low concentrations (0.5 M) of guanidine hydrochloride (Gdn.HCl) indicative of the presence of a carboxylate group in the active site of the enzyme. Kinetics of inactivation of enzyme by Gdn.HCl revealed that the essential carboxylate residues are present at the substrate-binding region of the enzyme.     

Production of dextranase byLipomyces starkeyi

Summary The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.     

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.     

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.