Isolation, optimization, and partial purification of amylase from Chrysosporium asperatum by submerged fermentation

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at 30 degrees C and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at 50 degrees C for 20 min, and no inhibitory effect of Ca+2 ions on amylase production was observed.     

Density-dependent changes in hexose transport, glycolytic enzyme levels, and glycolytic rates, in uninfected and murine sarcoma virus-transformed rat kidney cells

In normal rat kidney (NRK) cell cultures, increased cell density results in a decrease in the rates of hexose transport, glucose utilization, and lactate production and an increase in the level of hexokinase activity. A murine sarcoma virus (Kirsten)-transformed cell line (KNRK) showed little or no density-dependent variation in sugar uptake, glucose consumption, or lactate production. On the other hand, hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were elevated in dense transformed cultures as compared to sparse or uninfected cultures. In another virus-transformed cell line (ts339/NRK) exhibiting temperature-dependent morphology, growth pattern, and transport of 2-deoxy- -glucose, the levels of glycolytic enzyme activity were related to cell density but not to the culture temperature. The lack of correlation between glycolytic enzyme activity and lactate production by either uninfected or murine sarcoma virus-transformed cultures supports the suggestion that enhanced growth and/or hexose transport capacity rather than elevated glycolytic enzyme activity are responsible for the increased rate of lactate production by virus-transformed NRK cells.     

Regulation of connective tissue collagenase production: stimulators from adult and fetal epidermal cells

We have examined the ability of primary adult rabbit skin cells to regulate collagenase production in vitro. Dermal cells constitutively produce collagenase in culture, and enzyme production by these cells can be influenced by epithelial cells. Co-culture with skin epidermal cells resulted in more enzyme production by dermal cells, whereas co- culture with corneal epithelial cells yielded less enzyme activity. Connective tissue cells from a different source, cornea, also produced collagenase when co-cultured with skin epidermal cells, although the stromal cells alone made no enzyme. The drug cytochalasin B had very little influence on collagenase production by dermal cells, either alone or in co-culture with epidermal cells, but did significantly potentiate enzyme production by corneal stromal cells responding to epidermal effector molecules. Epidermal-cell-conditioned medium from both fetal and adult rabbit skin was a potent source of stimulators (apparent mol wt 20,500 and 55,000) of connective-tissue-cell collagenase production. Stimulator production by epidermal cultures was cell density dependent. Optimal production of stimulators occurred in adult cultures containing 10(6) epidermal cells/ml of medium, and in fetal cultures containing 10(5) cells/ml. Inhibitors of connective tissue cell enzyme production were not detected in conditioned medium from either adult or fetal epidermal cells.     

Effects of oxygen and nutrients limitation on ajmalicine production and related enzyme activities in high density cultures of Catharanthus roseus

Oxygen and nutrient limitation was investigated in order to identify the origin of a lower specific ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density bioreactor cultures with dissolved oxygen (DO) concentration of 15% and 85% of air saturation, with respect to alkaloid formation and related enzymes activities. Oxygen had an evident effect on ajmalicine production: in the high DO cultures production was more than 5 times higher than in the low DO cultures. The difference in ajmalicine production between high and low DO could not be explained by the enzyme activity profiles. Moreover, the productivity in the high density culture could not restored to the level of a low density culture (at a high DO) by increasing the DO alone. The effect of nutrient limitation was studied with response surface methodology in shake flask cultures. Nutrient limitation could not be demonstrated to be responsible for the productivity loss. Alkaloid and enzyme measurements in the shake flask cultures supported previous findings that the tryptamine pathway may regulate alkaloid production, provided that the terpenoid pathway is sufficiently active. (c) 1994 John Wiley & Sons, Inc.     

Production physiology and properties of a novel fungal fibrinolytic enzyme.

A potent extracellular fibrinolytic enzyme was obtained from cultures of the imperfect fungus Fusarium semitectum under certain growth conditions. Nitrate addition to cultures increased enzyme production. The enzyme showed a versatile proteolytic activity against several protein substrates including casein, gelatin, haemoglobin, bovine serum albumin, and fibrin from both buffalo and human sources. Optimal fibrinolysis occurred at pH values around 7.0. The fibrinolytic activity exhibited marked heat stability in enzyme samples heated at 60 degrees C, and retained more than 40% of its activity in samples heated to 100 degrees C for 10 min. Fibrinolysis proceeded optimally in the temperature range between 50--60 degrees C. Copper ions significantly activated the enzyme. Other biochemical properties are also reported.     

Production of cellulolytic enzymes containing cinnamic acid esterase from Schizophyllum commune

To develop enzyme preparations capable of digesting plant biomass, we examined the production of cinnamic acid esterase as well as cellulolytic and xylanolytic enzymes in cultures of Schizophyllum commune. The cinnamic acid esterase was produced in the cultures containing solid cellulosic substrates, with production being enhanced by delignifying the wood powder. This indicates that these esterases are produced by cellulose, despite their substrates being phenolic compounds. Cellulolytic and xylanolytic enzymes, with the exception of α-arabinofuranosidase, were also produced in cultures containing cellulosic substances. These results show that enzyme preparation can have high activity of cinnamic acid esterase and cellulolytic and xylanolytic enzymes when S. commune is incubated in the presence of cellulose. These enzyme preparations will be useful for digesting plant biomass and for releasing cinnamic acid derivatives from plant cell walls.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Synthesis and excretion of alpha-amylase in vgb+ and vgb- recombinant Escherichia coli: a comparative study

Synthesis and excretion of alpha-amylase is investigated in batch cultures of Escherichia coli JM103[pMK57] (vgb-) and E. coli JM103[pMK79] (vgb+). While total production and excretion of alpha-amylase were promoted in Luria broth (LB) (excretion being as high as 87%), cell-mass-specific production of the enzyme was promoted in M9 in bioreactor cultures and in LB in shake flask cultures. Low aeration and agitation rates and presence of starch were conducive to alpha-amylase synthesis in E. coli JM103[pMK79]. Two-stage bioreactor operating strategies that will improve alpha-amylase production are proposed. The potential of these strategies is demonstrated via two-stage shake flask cultures.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Production and detection of muramidase and acetylglucosaminidase from Agaricus bisporus

The production and regulation of extracellular bacteriolytic enzymes of Agaricus bisporus are being studied to understand better the nutrition of this fungus and to identify factors that regulate the selectivity of mushroom compost as a growth medium. Both muramidase (EC.3.2.1.17) and N -acetyl-β- D -glucosaminidase (β-GlcNAcase, EC.3.2.1.30) have been detected in liquid cultures of A. bisporus , and in cultures fruiting in sterile and non-sterile compost. A turbidometric assay, based on the decrease in optical density of suspended Bacillus subtilis bacterial cell walls, was used to measure muramidase production by A. bisporus . A colorimetric assay was used to measure β-GlcNAcase. Both bacteriolytic enzyme activities were produced on a range of sole carbon sources, including killed freeze-dried B. subtilis cells. Muramidase activity was highest in axenic compost cultures. Bacteriolytic enzyme activity peaked as the first group of fruit bodies was harvested in both sterile and non-sterile compost.     

Production and Heat Stability of Staphylococcal Nuclease

No correlation existed between numbers of organisms and nuclease activity in laboratory-grown cultures of Staphylococcus aureus. Nuclease production was inhibited by anaerobic incubation and stimulated by aeration. Strains of S. aureus varied in the production of nuclease. The optimum pH for enzyme production was 8.3 and employment of a tris(hydroxymethyl)aminomethane buffer system resulted in increased production of the enzyme as compared with a phosphate buffer. The nuclease was extremely heat-stable and had a D value of 16.6 min at 130 C.     

Lysine decarboxylase activity and alkaloid production in Heimia salicifolia cultures

Levels of lysine decarboxylase, thought to exercise control over the biosynthesis of alkaloids derived from the amino acid, have been determined in alkaloid-producing and non-producing cell and organ cultures of Heimia salicifolia. The level of this enzyme has also been measured in cultures grown in the presence and absence of light. In chlorophyllous cell cultures enzyme activity correlates positively with chlorophyll; in shoot cultures the activity also parallels alkaloid production.     

Mixed cultures of Serratia marcescens and Kluyveromyces fragilis for simultaneous protease production and COD removal of whey

AIMS: The aim of this study was to investigate the behaviour of a Serratia marcescens-Kluyveromyces fragilis mixed culture in whey, with the objective of proteases production and organic waste reduction. METHODS AND RESULTS: Discontinuous aerobic fermentations in whey were carried out using individual pure cultures and mixed cultures of S. marcescens and K. fragilis. Cell growth, protease production, lactose and proteins consumption and COD/TOC reduction were monitored. Lactose and protein content of the fermenting medium was almost depleted in the mixed cultures, achieving a reduction in the organic content much higher than in both pure cultures. Interestingly, proteolytic activity in the mixed cultures was similar to that obtained for S. marcescens in pure culture. In addition, protease stability was increased in the mixed cultures. Kinetic models were developed fitting well with the experimental results. CONCLUSIONS: Mixed cultures were found to maintain the achievements of each individual fermentation, yielding a high and stable production of proteases and a significant reduction of COD/TOC. SIGNIFICANCE AND IMPACT OF THE STUDY: Mixed cultures tested in this work have shown a synergistic effect with possible industrial applications. These results lead to a gain in the chain value for enzyme production with an environmentally friendly operation.     

Plasminogen activator in chick embryo muscle cells: induction of enzyme by RSV, PMA and retinoic acid.

To explore the generality of the effects of sarcoma viruses, tumor-promoting phorbol esters and retinoic acid, we have studied plasminogen activator production in differentiating chick myogenic cultures. Although slightly higher than in chick fibroblast cultures, the level of spontaneously synthesized enzyme is low; it reaches a peak shortly after maximum cell fusion has been completed and then declines. Rous sarcoma virus (RSV) transformation of differentiating myotubes was accomplished by infecting myoblasts with a temperature-sensitive mutant, maintaining cultures at the nonpermissive temperature until completion of fusion and shifting to permissive temperatures at selected times thereafter. RSV transformation, phorbol myristate acetate (PMA) and retinoic acid all induced high levels of plasminogen activator production by differentiating myotubes in the absence of DNA synthesis. In comparison with fibroblasts, virus-induced enzyme synthesis by myogenic cultures proceeded more slowly but ultimately reached comparably high levels. Whereas cAMP strongly repressed RSV- and PMA-induced plasminogen activator production by chick fibroblasts, it weakly stimulated enzyme synthesis by myotubes. This suggests that enzyme induction by RSV and PMA is not mediated primarily through effects on cAMP metabolism.     

Kinetics and regulation of beta-lactamase formation in methylotrophic bacterium

The regulation and kinetics of formation of an inducible, cell-bound oxacillin-hydrolyzing beta-lactamase (M-OXA) by a methanol-grown Pseudomonas strain were investigated in batch, chemostat, and two-stage continuous cultures. The extent to which enzyme production occurs declines at increased growth rates, and the rate of M-OXA beta-lactamase production follows a kinetic pattern that is partially growth independent and partially inversely growth linked. Growth and enzyme formation are regulated differently by medium constituents. The initial steps of M-OXA beta-lactamase synthesis takes place during the exponential growth phase, and active enzyme is produced by induced nonproliferating cells, probably through a turnover mechanism.     

Isolation and partial properties of cellulose-decomposing strain of Cytophaga sp. LX-7 from soil

A new facultatively anaerobic, Gram-negative bacterium, Cytophaga sp. LX-7, degrading crystalline cellulose completely, was isolated from soil by dilution plating on cellodextrin agarose plates. This strain could excrete extracellularly all three types of cellulase and cellulosic substrates were the strongest inducer of endocellulase with CMC-liquefying activity production. No reducing sugar was found in cultures of cellulose during incubation. An enzyme which degrades crystalline cellulose was detected in cultures of cellulose by measuring the formation of soluble carbohydrate but was not detected by determining the reducing sugar released. This strain also synthesized cell-bound cellobiose oxidizing enzyme which was previously noted only in fungi. Both cellulose and soluble sugars could promote the production of cellobiose oxidizing enzyme.     

Effect of medium supplements, illumination and superoxide dismutase on the production of coccoid forms of Campylobacter jejuni ATCC 29428

Factors influencing the production of coccoid forms in cultures and suspensions of a strain of the enteric pathogen Campylobacter jejuni during storage in air were investigated. Addition of blood or a supplement containing ferrous sulphate, sodium metabisulphite and sodium pyruvate minimized conversion of rods to coccoid forms in cultures. Exposure of cultures to light during storage in air increased the rate of production of coccoid forms. Ultraviolet radiation was shown to effect the viability of cells in suspensions but the increase in production of coccoid forms was low after irradiation. The presence of hydrogen peroxide and its dissociation products in bacterial suspensions increased conversion to coccoid forms. Addition of active superoxide dismutase, a superoxide anion scavenging enzyme, minimized production of coccoid forms in suspensions stored in air. Coccoid forms contained a lower level of superoxide dismutase than rods. It is deduced that a decreased level of the enzyme in cells is linked with production of coccoid forms.     

Effect of medium supplements, illumination and superoxide dismutase on the production of coccoid forms of Campylobacter jejuni ATCC 29428

Factors influencing the production of coccoid forms in cultures and suspensions of a strain of the enteric pathogen Campylobacter jejuni during storage in air were investigated. Addition of blood or a supplement containing ferrous sulphate, sodium metabisulphite and sodium pyruvate minimized conversion of rods to coccoid forms in cultures. Exposure of cultures to light during storage in air increased the rate of production of coccoid forms. Ultraviolet radiation was shown to effect the viability of cells in suspensions but the increase in production of coccoid forms was low after irradiation. The presence of hydrogen peroxide and its dissociation products in bacterial suspensions increased conversion to coccoid forms. Addition of active superoxide dismutase, a superoxide anion scavenging enzyme, minimized production of coccoid forms in suspensions stored in air. Coccoid forms contained a lower level of superoxide dismutase than rods. It is deduced that a decreased level of the enzyme in cells is linked with production of coccoid forms.     

Metabolic roles of peptone and yeast extract for the culture of a recombinant strain ofEscherichia coli

Summary The influence of complex compounds on the growth of a recombinant strain ofEscherichia coli containing the gene encoding glyceraldehyde 3-phosphate dehydrogenase, as well as the production of this enzyme have been studied. Batchwise cultures led to an accumulation of acetate, which was not utilized in a yeast extract-free medium. After glucose exhaustion, growth stopped and enzyme activity decreased. Whereas yeast extract allowed acetate assimilation and growth, peptone stabilized the enzymatic activity. The addition of both compounds resulted in optimal performances for enzyme production.