Biosynthesis of hydrolytic enzymes during cocultivation of macro- and micromycetes

Effects of co-cultivation of higher Basidiomycetes and Phycomycetes on the biosynthesis of cellulases, amylases, and proteases was studied. Four optimal pairs of fungal cultures were selected. Of these, three pairs belonged to higher fungi, and one pair was constituted by fungi of distinct ecological groups, a macromycete and a micromycete. The activities of amylase and protease were 1.5 to 2 times higher, and the activity of cellulase was lower during the growth of higher fungi associations. The mixed association of the macromycete Schizophyllum commune and the micromycete Mucor sp. was the most active producer of hydrolytic enzymes. During the growth of this mixed association, a fourfold and 1.5-fold increases were observed in the activity of endoglucanase and protease, respectively, paralleled by stimulation of amylase formation.     

Exocellular enzyme activity of dermatophytes and other fungi isolated from ruminants in Southern Iraq

Sixteen fungal species were isolated from 182 specimens collected from four ruminants (buffalo, camel, cattle and sheep) in Southern Iraq. Fungi represented by five species of dermatophytes and eleven species of other fungi were screened for the activity of four enzymes; keratinase, proteinase, lipase and amylase. Keratinase was found to be produced by all of the dermatophytes and non-dermatophytes, except for Paecillomyces variottii and Scytalidium lignicola. However, high keratinase activity was expressed by the dermatophytic species particularly by Trichophyton mentagrophytes var. erinacei and Microsporum gypseum. Three dermatophytes viz. M. gypseum, T. verrucosum and T. mentagrophytes var. nodulare were capable of producing protease, lipase and amylase. Although, T. mentagrophytes var. erinacei showed high protease activity, it did not produce lipase and amylase. On the contrary most of the non-dermatophytic species revealed protease and lipase activities higher than the dermatophytes. The Curvularia spp. isolates showed the highest protease and amylase activity, while Aspergillus parasiticus revealed the highest activity of lipase and amylase. No correlation was observed between enzyme activity and the growth rate of the examined fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of extracellular proteinases—protein C activators of blood plasma—by the micromycete Aspergillus ochraceus during submerged and solid-state fermentation

The conditions for the submerged and solid-state fermentation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C of human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state fermentation, a protein C-activating proteinase with a pI of 6.0–6.3 was identified.     

Effect of iso-osmotic levels of salts and PEG-6000 on enzymes in germinating pea seeds

Effects of iso-osmotic levels of salts (NaCl, CaCl₂, Na₂SO₄) and PEG-6000 on the activity of hydrolytic and nitrogen assimilatory enzymes in pea embryo axis and coty ledon were studied. The activity of nitrate reductase and nitrite reductase in embryo axis and cotyledon and the activity of protease and α-amylase in cotyledon decreased with decreasing medium osmotic potential as compared to control at all the stages of seedling growth. The activity of protease and amylase increases with increasing levels of stress in embryo axis. Sodium chloride induced, stress had more deleterious effects on the activity of nitrate reductase, nitrite reductase and αamylase followed by other salts and PEG-6000. On the other hand, CaCl₂ induced salt stress was more depressive for protease activity. The maximum increase in the activity of protease and amylase was observed in embryo axis at higher concentration of salts and PEG-6000.     

The relation of extracellular amylase,mycelium, and time,in some thermophilic and mesophilic humicola species

All four fungi studied attained approximately the same dry weight of mycelium in starch-yeast extract medium. Only about one-fourth the amount of mycelia was produced in yeast extract alone (starch omitted). However, the initial growth rate ofH. grisea var.thermoidea was greater than the other three fungi. Extracellular amylase was produced by all four fungi, butH. lanuginosa produced 8 to 12 times as much as the other three. Maximum extracellular amylase was found before autolysis with these three fungi, but after autolysis withH. lanuginosa. Extracellular amylase was detected in YE medium (lacking starch), but in very low amounts (approximately one-eighth the amount observed as when starch was present). Increasing the amount of starch in the medium increased extracellular amylase. However, when the starch concentration was kept constant, increasing the concentration of yeast extract had no effect on extracellular amylase.Contribution No. 59 from the Botany Section, The Department of Biology. Portion of a thesis presented by the senior author in partial fulfillment for the M.S. degree.     

Extracellular amylase activities ofRhizomucor pusillus andHumicola lanuginosa at initial stages of growth

Among thermophilic fungi,Rhizomucor Pusillus andHumicola lanuginosa have been reported to be among the most prolific producers of amylase, an apparently heat stable enzyme vital to the incorporation of carbon from macromolecular sources such as starch. Yet the highest levels of extracellular amylase in starch-yeast cultures of these fungi were measured after most of the growth had occurred; pre-growth levels appeared to be very small. Since these low levels are the significant ones for growth, a procedure was devised to measure them: 1.162×10^–2 units (mg maltose/ml/min) were measured after two days of growth ofR. pusillus and 6.230×10^–3 units measured after four days of the slower-growingH. lanuginosa. Re-assays of these after dialysis to remove most of the reducing sugars gave 1.689 × 10^–2 units and 1.234 × 10^–2 units, respectively, with all correlation coefficients 0.96 or better.     

Antagonistic and plant growth promoting activities of endophytic and soil actinomycetes

The objective of this study was the isolation and screening of actinomycete isolates for antagonistic potential and plant growth promoting activities. A total of 321 isolates were recovered from different plants, their rhizospheric soils and non-rhizospheric soils of Punjab and Himachal Pradesh regions. Out of these, 62 were endophytic, 156 were rhizospheric and 103 were non-rhizospheric isolates. In primary screening (dual culture assay), 83 isolates antagonised one or more test phytopathogenic fungi. From these active isolates, 20 were found to be antagonistic in well diffusion assay (secondary screening) and most of them demonstrated broad spectrum inhibitory activity against five to six test fungi. Studies on plant growth promoting activities revealed that 12 showed abilities to produce indole acetic acid, 10 produced siderophores and 12 showed ammonia production. Phosphate solubilisation was observed in five isolates and four fixed atmospheric N₂. In addition, production of hydrolytic enzymes such as chitinase, amylase, cellulase and protease was demonstrated by five, twenty, eleven and eleven isolates, respectively. The results of this study indicate that these isolates may be used as biocontrol and plant growth promoting agents. Morphological and chemotaxonomic studies revealed that all the active isolates belonged to the genus Streptomyces     

Detection of extracellular protease activity in different species and genera of ectomycorrhizal fungi

In northern forest ecosystems, most soil nitrogen (N) is in organic form and forest trees are largely dependent on ectomycorrhizal (ECM) fungi and their degradative abilities for N uptake. The ability of ECM fungi to acquire N from organic substrates should, therefore, be a widespread trait given its ecological importance. However, little is known about the degradative abilities of most ECM fungi as they remain untested due to problems of isolation or extremely slow growth in pure culture. In this paper, we present data on extracellular protease activity of 32 species of ECM fungi, most of which have not previously been cultured. Milk powder plates and zymograms were compared for detecting protease activity in these intractable species. In total, 29/32 of the species produced extracellular protease activity, but detection was method dependent. Growth on milk powder plates detected protease activity in 28 of 32 species, while zymograms only detected proteases in Amanita muscaria, Russula chloroides, Lactarius deterrimus and Lactarius quieticolor. The study supports the hypothesis that protease excretion is a widespread physiological trait in ECM fungi and that this ability is of considerable significance for nitrogen uptake in forest ecosystems.     

Correlation between the fatty acid composition and the activity of extracellular enzymes from Bacillus caldolyticus

Summary Bacillus caldolyticus, grown at 70°C, produces a highly active extracellular amylase and protease. Both enzymes are formed either within the membrane, or at its inner surface. The activity of both extracellular enzymes was found to decline drastically when brain-heart infusion was omitted from the medium. A simultaneous increase of both enzymes inside the cell was observed. The shifting in extra- and intra-cellular activity was caused by changes in membrane composition due to the increase of anteiso-odd and n-even, and the decrease of iso-odd fatty acids. Membrane composition and enzymic activity could be influenced by the addition of either leucine or iso-leucine as precursors for the synthesis of branched-chain fatty acids: In presence of leucine the anteiso-odd and n-even fatty acids returned to their normal level, while the iso-odd fatty acids increased. Simultaneously the extracellular protease activity increased, and the intracellular activity declined. Growth in amylose-medium supplied with leucine lead to a decrease of both the intra- and extracellular amylase, and changes in the fatty acid composition of the membrane which could not be restored by transfer of the organism to complete media. Addition of iso-leucine first lead to a sharp decrease of extracellular protease and a drastic increase of intracellular protease activity, accompanied by an increase of anteiso-odd and n-even fatty acids, and a decrease of iso-odd compounds. After the second growth in presence of iso-leucine the intra- and extra-cellular protease activity was reversed, and thus showed a return to the starting situation. The reversal is accompanied by the preferential incorporation of fatty acids with a higher melting point into the membrane. Extracellular amylase activity was found to increase after the first growth with iso-leucine, and to decline sharply after the second culture with iso-leucine, together with a very high intracellular amylase activity at that point. Extra- and intra-cellular amylase activity both declined upon growth in complete medium, while the fatty acid distribution remained different from the initial composition.     

A modified protoplast-regeneration protocol facilitating the detection of cloned exoenzyme genes in Bacillus subtilis

Incorporation of starch or casein into protoplast-regeneration medium facilitated shotgun cloning of α-amylase and neutral protease genes from an unidentified Bacillus sp. in Bacillus subtilis by polyethylene glycol-induced protoplast transformation. This modification and the use of the plasmid vector pPL603b enabled us to simultaneously select for promoter-bearing recombinant plasmids that expressed amylase or protease activity. The inserts were found to be 4 and 4.6 kb, respectively. Although protease activity directed by the cloned gene was only 2- to 4-fold higher than for the donor strain, that of α-amylase was 28-fold higher.     

The Abundance and Structure of the Root-Associated Microbial Complexes of Two Greenhouse Rose Cultivars

The study of the root-associated microbial complexes of affected and healthy rose plants of two cultivars (Grand gala and Royal velvet) grown in a greenhouse showed that the biomass of eukaryotic microorganisms in the rhizoplane and rhizosphere of healthy rose plants and in the surrounding soil was considerably lower than in the same loci of affected plants. In contrast, the biomass of root-associated prokaryotic microorganisms was higher in the case of healthy than in the case of affected rose plants. The root-associated bacterial complexes of both affected and healthy rose plants were dominated by the genera Arthrobacter, Rhodococcus, and Myxobacterium and did not contain phytopathogenic bacteria. The root-associated fungal complex of healthy roses was dominated by fungi of the genus Trichoderma, whereas that of the affected rose plants was dominated by the species Aureobasidium microstictum. The affected cane cuttings and cankers occurring on affected canes were found to contain Coniothyrium fuckelii (the causal fungus of rose stem canker) and sclerotia of Botrytis cinerea (the causal fungus of gray rot). The micromycete complex of healthy rose plants was not so diverse as was the micromycete complex of affected rose plants.     

Postprandial changes in pH and enzyme activity from the stomach and intestines of Tilapia rendalli (Boulenger, 1897), Oreochromis mossambicus (Peters, 1852) and Clarias gariepinus (Burchell, 1822)

The ability to utilise dietary components differs among fish species. Digestive enzymes may be used to determine the efficiency of the digestive process. In this study, the activities of the digestive enzymes in Tilapia rendalli, Oreochromis mossambicus and Clarias gariepinus were explored. Protease, amylase, lipase and cellulase activities were measured in different parts of the digestive tract of the three fish species. The pH dynamics along the digestive tract were monitored. In all fish species, the presence of food led to a reduction in stomach pH, whereby pH values of 1.54, 1.58 and 2.01 were recorded 12 h after feeding in O. mossambicus, T. rendalli and C. gariepinus, respectively. Protease and amylase activities were significantly higher (P < 0.05, anova ) in the tilapias than in C. gariepinus. The tilapias may be pre‐adapted to produce more protease and amylase to digest plant material, which is more difficult to digest than animal matter. In all species amylase activity was significantly higher in the proximal intestine than in the other parts of the digestive tract (P < 0.05, anova ). The highest protease activity was recorded in the distal intestines. This is because of the alkaline pH recorded in the proximal and distal intestines, which favours amylase and protease activity, respectively. Lipase activities were significantly higher (P > 0.05) in C. gariepinus than in both tilapias. Marginal cellulase activities were recorded in all species. It is inferred here that phylogeny and not diet may be the main factor influencing enzyme activities, as all fish were fed a similar diet.     

福寿螺和田螺消化酶活性比较

为比较福寿螺(Pomacea canaliculata(Lamarck,1828))和当地中国圆田螺(Cipangopaludina chinensis(Gray,1832))消化能力的差异,探索福寿螺成功入侵的机制,以田螺为对照,测定了1—4龄的福寿螺和田螺的胃和肝脏的消化酶——纤维素酶(羧甲基纤维素法)、淀粉酶(3,5-二硝基水杨酸法)和脂肪酶(滴定法)的活性。结果表明:1)相同年龄的福寿螺胃和肝脏中的消化酶活性明显高于田螺。其中,纤维素酶活性分别高出1.00—2.11倍、1.66—2.84倍;淀粉酶活性分别高出1.53—3.47倍、1.47—1.80倍;脂肪酶活性分别高出2.07—4.73倍、6.13—9.93倍。2)在生长发育过程中,福寿螺胃和肝脏中的消化酶活性变化幅度(51.2%—131.2%)明显高于田螺(23.3%—47.1%)。3)福寿螺的各种消化酶之间存在协同作用。如福寿螺的淀粉酶活性与脂肪酶活性呈极显著正相关(胃中r=0.736**、肝脏中r=0.867**)。此外,胃中的淀粉酶活性还与纤维素酶活性呈显著正相关关系(r=0.696*)。相应地,田螺胃中的淀粉酶和脂肪酶之间也存在显著的正相关关系(r=0.706*),而肝脏中的纤维素酶与脂肪酶活性呈显著负相关(r=-0.593*)。4)福寿螺对纤维素类和淀粉类物质都有较强的消化能力,且能较好地消化脂肪类物质,而田螺能消化纤维素类和淀粉类物质,对脂肪的消化能力却很弱。福寿螺的纤维素酶和淀粉酶活性分别是田螺的2.42和1.88倍,脂肪酶活性达到了5.66倍。可见,福寿螺具有较高的消化酶活性,且各消化酶之间存在正协同性。这可能是导致福寿螺食量大、食性杂,使其能快速生长和成功入侵的重要原因之一。     

The influence of fungal food quality on the growth and fecundity of Folsomia candida (Collembola: Isotomidae)

Summary The Basidiomycete fungi Coriolus versicolor and Hypholoma fasciculare were grown in liquid media containing 2, 20, 200 and 2,000 ppm nitrogen (as asparagine) and fed to cultures of Folsomia candida. Collembola feeding on both species of fungi exhibited trends of increased moulting and egg laying rates up to 200 ppm N and an inhibition of growth and fecundity at 2,000 ppm N. The differences in moulting rates between individual treatments were small for both species of fungi and not all the pair wise comparisons of treatments were significantly different. Egg laying rates of collembola fed C. versicolor showed a highly significant response to all levels of N in the growth medium and egg production at 200 ppm N was over three times higher than at 2 ppm N. Collembola fed H. fasciculare showed a less marked fecundity response to the different nitrogen levels and egg production at 200 ppm was approximately 1.5 times higher than at 2 ppm N. Both moulting and egg laying rates were significantly affected by the species of fungus presented as food to the collembola. The patterns of growth and reproduction of starved control groups of F. candida as well as those fed the test fungi demonstrate the adaptability of this species to changes in the quality and quantity of available food.     

Characteristics and properties of a caldo-active bacterium producing extracellular enzymes and two related strains

Summary The caldo-active strain YT-P was found to produce a variety of extracellular enzymes, including an amylase and a protease, which were further examined. With azo-casein as a substrate, optimum conditions with respect to enzyme and substrate concentration were determined for the protease. The optimum temperature was found to be 70°C, with a sharp decline to both lower and higher temperatures. The enzyme was found to be extremely heat-stabile, with unaltered activity after 8 hours at 80°C.Optimum conditions for the amylase were also examined. This enzyme was shown to be less heat-stabile, though the temperature optimum was again at 70°C. The activity or stability was not influenced by absence or presence of Ca-ions. The main activity of the amylase was found in the 20–40% ammonium sulfate fraction, which also contained the bulk of the proteolytic enzyme.This strain growth optimally on a variety of carbon sources at 72°C. Typical submicroscopical features are the double-layered cell wall, and a cytoplasmic membrane with a varying number of small dots and dot-free patches.Furthermore the nutritional requirements and submicroscopical features of two other strains, YT-G and YT-F, are described and compared to strain YT-P.Based on the fatty acid composition of the three spore forming caldo-active strains we suggest that they belong to the genus Bacillus, and propose the names B. caldolyticus for strain YT-P, B. caldovelox for strain YT-F, and B. caldotenax for strain YT-G.     

Extracellular enzyme production by haploids,heterocaryons and diploids of Aspergillus nidulans

Summary Extracellular amylase, lipase and protease produced by haploids, diploids and heterocaryons of Aspergillus nidulans were analysed. Three morphologically normal strains and 8 morphologic mutants as well as various genetic combinations of the 11 strains were examined in solid culture medium containing specific substrates. The enzyme production of each strain was determined by measuring the halo around the colony. It was observed that the colonies showing less growth also showed more alterations in enzyme production. The compact strains (BVIII and B6) and the slow-growing heterocaryons (pp+M32 and pp+M35) showed the highest enzymatic index for the three enzymes simultaneously. If colony growth is not considered, then for amylase and protease the highest values were reached by some diploid and heterocaryons and for lipase by one morphological strain. The results showed that morphological mutants and some combinations could be used for higher production of amylase, lipase and protease.     

Characterization of Aeromonas hydrophila strains and their evaluation for biodegradation of night soil

Among 67 psychrotrophic bacterial isolates of Leh, India screened for production of hydrolytic enzymes at 10 °C, four belonging to Aeromonas hydrophila were characterized and evaluated for biodegradation of night soil. All strains produced metalloproteases on a variety of carbon and nitrogen sources. Strains LA1 and LA15 also produced α-amylase and PC5 both α- & β-amylase. No amylase was produced by PN7, however it produced lipase. Casein and glucose induced maximum enzyme activity (protease and amylase) in LA15 and PC5, respectively. In LA1, maximum induction of protease was observed with casein and of amylase with maltose. Corn oil/tributyrin served as the best inducers for protease and lipase production by PN7. A. hydrophila strains were found to be psychrotrophic with optimum growth and enzyme activity at 20 and 37 °C, respectively. Maximum biodegradation of night soil was observed by strain LA1 at 5–20 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Separation and partial purification of extracellular amylase and protease from Bacillus caldolyticus

Summary The extracellular amylase and protease from Bacillus caldolyticus can be concentrated by ammonium sulfate precipitation after growth on either solid or in liquid media containing starch, glucose, and brain-heart infusion. Using the Diaflo ultrafiltration system with membranes of various permeability, the enzymes could be separated from each other by extensive flushing with buffer. Best results were obtained with the 50–70% ammonium sulfate fraction as starting material, yielding 72% of the total amylase activity in the low molecular weight fraction (UM-10 fraction: 10000–30000), while 54 and 25% respectively of the protease were retained in the two high molecular weight fractions (50000–100000, and more than 100000). Similar results were obtained with the 20–50% ammonium sulfate fraction, while the fraction of 0–20% saturation contained a low molecular weight protease. The native amylase seems to consist of a number of sub-units, which after extensive flushing accumulate in the fraction with an approximate molecular weight between 10000 and 30000. The enzyme could also be precipitated from cell-free liquid media with ammonium sulfate, followed by separation and purification on ultra-filtration cells. According to the specific activity of the UM-10 fractions a 400-fold purification was obtained compared to the amylase activity of the cell-free medium.Direct concentration and separation from liquid media, omitting ammonium sulfate treatment, was also found to be possible, although prolonged flushing with buffer was necessary to obtain satisfactory separation.During purification from the ammonium sulfate fractions, amylase activity was found to decrease but could be restored by Ca-ions. At 70°C, a final concentration of 0.5 mM CaCl₂, was sufficient for full restoration, while three times that amount was necessary at 80°C. Determination of the K _m-values for Ca at different temperatures resulted in an asymptotically increasing curve at temperatures beyond 75°C. Addition of Ca had a pronounced effect on the stability of the amylase at 80°C but not at 90°C. Protease activity and stability was not affected by Ca-ions.     

Production of extracellular enzymes in the entomopathogenic fungus Verticillium lecanii

This study investigates the mechanisms as well as strategies for purification and characterization of potential enzymes involved in pathogenesis of entomopathogenic fungi. The test strain of Verticillium lecanii that was screened, during the present investigation, proved to be an efficient producer of protein and polysaccharide degrading enzymes (amylase, protease, and lipase), hence indicating versatility in biochemical mechanisms. Halo zones produced colony growth of V. lecanii on agar confirmed activity of protease, amylase and lipase enzyme by the V. lecanii isolate. Enzymatic Index (EI) observed were: Protease – 2.195, Amylase- 2.196, Lipase- 2.147. Spectrophotometric analysis of enzymatic activity of V.lecanii at five different pH – 3, 5, 7, 9, 11 revealed that highest proteolytic activity of the V. lecanii isolate was reported at pH 7 and 9 whereas proteolytic activity was minimum at acidic pH 3. Maximum amylolytic activity of V. lecanii on the 7^th day of inoculation was at pH 3 i.e. in an acidic environment in contrast to neutral pH 7. Maximum lipolytic activity of V. lecanii was found at pH 7. Since enzyme production in entomopathogenic fungi is specific and forms an important criterion for successful development as well as improvement of mycoinsecticides, hence a significant conclusion from the present analysis is the degree of variation in secretion of enzymes in test strain of Verticillium lecanii.     

Fermentative production of extracellular amylase from novel amylase producer,Tuber maculatum mycelium,and its characterization

Abstract

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22?±?2?°C) under static conditions which resulted in 1.41?U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50?°C. The enzyme showed good thermostability at 50?°C by retaining 98% of the maximal activity after 100?min of incubation. The amylase activity was marginally enhanced in presence of Cu²⁺ and Na⁺ and slightly reduced by K⁺, Ca²⁺, Fe²⁺, Mg²⁺, Co²⁺, Zn²⁺, and Mn²⁺ ions at 1?mM concentration.