Purification and characterization of the protease enzymes of Streptomyces thermovulgaris grown in rapemeal-derived media

When grown in a particulate-free, protein-rich medium derived from rapemeal (termed medium B), Streptomyces thermovulgaris produced multiple protease enzymes. The main protease activity was attributed to two types of serine protease, denoted as SV1 and SV2. A metallo protease component (SV3) and an azocaseinase component (SV4) were also present. Protease SV1 had a molecular weight of 30 kDa and a pI of 5.8. Protease SV2 was characterized by a high thermostability in the presence of calcium ions and had a pI of 8.4. This enzyme had a molecular weight of 60 kDa, but we suggest that this is the dimeric form, with 30 kDa being the monomer unit. The method chosen for initial downstream processing influenced both the yield and type of protease purified. When cell-free supernatant fluid was concentrated using ultrafiltration, rather than acetone precipitation, a higher percentage and a greater range of proteases were recovered. The medium used for the growth of Strep. thermovulgaris also appeared to affect the type of protease produced. A more diverse range of proteases were produced on rapemeal-derived medium when compared to yeast extract medium.     

Characterization of growth and product formation by a thermophilic streptomycete grown in a particulate rapemeal-derived liquid medium

Streptomyces thermoviolaceus was grown in a glutamate salts medium and the applicability of DNA and a number of intracellular dehydrogenases as indicators for growth in a particulate repemeal salts medium were assessed. Only NADH dehydrogenase proved unsuitable but analysis of the rates of increase of activities of this enzyme during batch culture suggested that energy metabolism was an important factor in defining the onset of secondary metabolism and as a causal element of the biphasic pattern of growth exhibited by this streptomycete. Growth, substrate utilization and production of extracellular products could readily be analysed in the particulate rapemeal salts medium. Free sugars and carbohydrates were utilized predominantly in early exponential phase but thereafter the rapemeal proteins were metabolized as observed by rises in protease activities and levels of ammoniacal nitrogen. Growth in rapemeal-derived media produced titres of the antibiotic granaticin which were at least as high (usually higher) as most of the growth substrates tested suggesting that rapemeal may have considerable potential for the exploitation of natural products. Particulate rapemeal medium also resulted in high yields of commercially important extracellular enzymes.     

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.     

Directed biosynthesis of proteolytic enzymes in Actinomyces thermovulgaris]

The biosynthesis of proteolytic enzymes in the thermophilic culture of Actinomyces thermovulgaris, strain T-54, was directed by changing the composition of the medium and the temperature of cultivation. A temperature of 40 degrees C is optimal for the growth and production of neutral and alkaline proteases. The maximum activity of acid proteases was found during the growth on a complex medium at 30 degrees C. An increase of temperature to 50 degrees C during the cultivation of the microorganism on a chemically defined medium resulted in its secondary growth and a sharp rise in the activity of alkaline and neutral proteases.     

Nutritional regulation of protease production by the feather-degrading bacterium Chryseobacterium sp. kr6

The effects of nutritional conditions on growth and protease production by the feather-degrading Chryseobacterium sp. kr6 were investigated. Higher growth was observed on feather-containing or tryptone (TR) medium when compared to casein (CA) or glucose-nitrogen (GN) base medium. Protease production occurred during growth on feather-containing and TR media, whereas no protease activity was detected on CA or GN medium, indicating that protease production is not constitutive, depending on the presence of specific complex nitrogen sources. Supplementation of whole feathers (WF) medium with glucose (WFG) or NH(4)Cl (WFN) did not result in major differences in growth and protease production, whereas soluble protein was lower in supplemented media. Glucose consumption and growth were higher on WFG than on GN medium, suggesting that the absence of a specific complex nitrogen source limited bacterial growth. On WF medium, this strain grew closely attached to the feather structures, initially on the barbules and subsequently on the feather rachis. It was observed, through zymogram analysis, that strain kr6 produced diverse proteolytic enzymes in response to different growth substrates. These results were confirmed by the differential behaviors of crude proteases towards protease inhibitors.     

Repeated batch production of alkaline protease by solid-state fermentation using urethane foam as carriers

By immobilizing fungi on a urethane foam carrier (UFC), a novel solid-state fermentation system was developed in order to produce repeatedly industrial useful enzymes. In this study, alkaline protease was produced by growing Aspergillus oryzae 460 (ATCC 20386) in a flask scale. Repeated batch production of alkaline protease was carried out by exchanging a part of the culture broth with fresh medium every 12 hr. The effects of feeding medium composition, and feeding volume were examined. Alkaline protease production stopped in the early phase at high values of soluble starch feeding rate and culture broth dilution rate. The enzyme production continued longer when 10 to 30 g/l polypepton was added to the feeding medium. Using soluble starch solution as feeding medium, a maximum activity of 520,000 U/l-bulk volume alkaline protease was obtained at culture time of 168 hr where the culture conditions of soluble starch concentration and feeding volume were 100 g/l and 0.025 l/l-bulk volume/dose, respectively. Production of the enzyme continued for 300 hr and total aklaline protease activity reached 870,000 U/l-bulk volume by adding 30 g/l polypepton to the fresh medium.     

Identification and primary characterization of specific proteases in the digestive juice of Archachatina ventricosa

The profile of sedimentation on a 4-20% (w/v) linear sucrose gradient of the digestive juice of the mollusk Archachatina ventricosa revealed the presence of at least four specific proteases. A first peak, corresponding to a sedimentation coefficient of 3.9 S, contained two endoproteases that could be assayed, one with Leu-pNA and the other with Met-pNA. Their activity was maximal at pH 8.0 and increased in the presence of Ca(2+) ions. Both enzymes were inhibited by the chelating agent 1,10-phenanthroline but their thermal inactivation kinetics were different. A second protease peak was observed at 6.8 S and corresponded to a metallo-endoprotease that hydrolyzed with a maximal activity at pH 8.0 only the amide bonds of peptide substrates having a threonine residue at the P1' position. A last protease peak identified at 9.0 S contained a protease that preferentially acted on tripeptides, such as Val-Pro-Leu (diprotin B) and Thr-Val-Leu, releasing the C-terminal residue. Unlike the proteases identified in the two other peaks, its activity was maximal at acid pH (5.0) and was inhibited by the serine protease inhibitors. Together these results show the potential of A. ventricosa as a source of specific proteases.     

Enhancement of long-chain acyl-CoA hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators

The present study has confirmed previous findings of long-chain acyl-CoA hydrolase activities in the mitochondrial and microsomal fractions of the normal rat liver. In addition, experimental evidence is presented in support of a peroxisomal localization of long-chain acyl-CoA hydrolase activity. (a) Analytical differential centrifugation of homogenates from normal rat liver revealed that this activity (using palmitoyl-CoA as the substrate) was also present in a population of particles with an average sedimentation coefficient of 6740 S, characteristic of peroxisomal marker enzymes. (b) The subcellular distribution of the hydrolase activity was greatly affected by administration of the peroxisomal proliferators clofibrate and tiadenol. The specific activity was enhanced in the mitochondrial fraction and in a population of particles with an average sedimentation coefficient of 4400 S, characteristic of peroxisomal marker enzymes. Three populations of particles containing lysosomal marker enzymes were found by analytical differential centrifugation, both in normal and clofibrate-treated rats. Our data do not support the proposal that palmitoyl-CoA hydrolase and acid phosphatase belong to the same subcellular particles. In livers from rats treated with peroxisomal proliferators, the specific activity of palmitoyl-CoA hydrolase was also enhanced in the particle-free supernatant. Evidence is presented that this activity at least in part, is related to the peroxisomal proliferation.     

一株产纤溶酶菌株的分离鉴定及其纤溶组分分析

【目的】筛选性能良好的产纤溶酶菌株,对菌株进行多项分类鉴定,分析其纤溶酶系的组成特征及纤溶能力。【方法】通过酪蛋白培养基初筛,琼脂-纤维蛋白双层平板复筛,从海泥、土壤等环境中筛选纤维蛋白降解菌,以尿激酶为标准测定纤溶酶活性。通过形态学、生理生化特征研究,结合16S rDNA基因序列分析菌株种类及系统分类地位。通过SDS-PAGE和纤维蛋白酶谱法分析胞外纤溶酶系的组成特征。【结果】筛选到一株能降解纤维蛋白的细菌CNY16,鉴定其为沙福芽孢杆菌(Bacillus safensis)。该酶为胞外酶,SDS-PAGE和纤维蛋白酶谱结果表明该纤溶酶系有至少两种分子量大小不同的纤溶酶,分别约33 kD和23 kD。能有效溶解血块中纤维蛋白,并且对红细胞无降解作用。【结论】细菌CNY16是一株新的纤溶酶产生菌,纤溶酶活性及稳定性较好,具有潜在开发价值。为获取新型纤溶酶提供了一种新的菌源。     

Production of proteinases in the process of developing cultures of Streptomyces thermovulgaris T-54

The secretion of some proteolytic enzymes by Streptomyces thermovulgaris T 54 was studied using artificial chromogenic substrates of proteinases. Maximum accumulation of the enzymes hydrolysing Z-Glu-pNA and Z-Ala-Ala-Leu-pNA occurred during autolysis of the culture. Two peaks of activity were observed, when DNP-Gly-Gly-Ile-Arg was used as a substrate, one of them being correspondent to prevalence of dense colonies in the culture and the other to the prevalence of friable networks of hyphae.     

Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores

A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.     

Regulation of Neutral Protease Productivity in Bacillus subtilis: Transformation of High Protease Productivity

A transformable strain of Bacillus subtilis 6160, a derivative of B. subtilis 168, produces three kinds of casein hydrolytic enzymes (alkaline protease, neutral protease, and esterase) in a culture medium. B. natto IAM 1212 produces 15 to 20 times as much total proteolytic activity as does B. subtilis. Extracellular proteases produced by the two strains were separated into each enzyme fraction by diethylaminoethyl-Sephadex A-50 column chromatography. The difference in the total protease activities of extracellular proteases between the two strains was due to the amount of neutral protease. The ratios of neutral protease activity to alkaline protease activity (N/A) were 1.1 in B. subtilis 6160 and 13.0 in B. natto IAM 1212. Enzymological and immunological properties of alkaline protease and neutral protease obtained from the two strains were quite similar or identical, respectively. Specific activities measured by an immunological analysis of the two neutral proteases against casein were also equal. A genetic character of high protease productivity in B. natto IAM 1212 was transferred to B. subtilis 6160 by the deoxyribonucleic acid-mediated transformation. Among 73 transformants that acquired high protease productivity, 69 produced a higher amount of neutral protease and the ratios of N/A were changed to 15 to 60. Three other strains were transformed in the productivity of neutral protease and alpha-amylase simultaneously, and one showed considerable change in the production of alkaline protease and neutral protease. The specific activities (casein hydrolytic activities/enzyme molecules) of neutral proteases from the representative four transformants were equal to those of the two parental strains. These results suggested the presence of a specific gene(s) that participated in the productivity of neutral protease in B. subtilis.     

Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp

A novel keratin-degrading bacterium Stenotrophomonas sp. strain D-1, isolated from deer fur, produced two types of extracellular proteins: proteolytic and disulfide bond-reducing. The results on the biochemical properties suggest that this protease belongs to the serine protease, and the disulfide bond-reducing protein could be the disulfide reductase type. None of these enzymes showed keratinolytic activity independently. However, after mixing of the two enzymes, the keratinolytic activity was increased tremendously (more than 50-fold) over that of the protease only. This keratinolytic activity was more than 2-fold higher than that of the combination with proteinase K (also known for its high keratinolytic activity). Since the two enzymes discovered in this study acted cooperatively and resulted in higher keratinolytic activity, a new mechanism of keratin degradation has been revealed. To our knowledge, this is the first report on the cooperative action of two enzymes resulting in the effective degradation of keratin.     

Screening and isolation of an organic solvent-tolerant bacterium for high-yield production of organic solvent-stable protease

Forty-three strains were screened from crude oil-contaminated samples by toluene and cyclohexane enrichment in medium. Ten of these strains demonstrated high protease activity on skim-milk agar. Among them, the PT121 isolate, identified as Pseudomonas aeruginosa, was selected based on its extracellular protease stability in the presence of hydrophilic organic solvents. The crude protease also retained most of its activity up to at least 14 days in the presence of various organic solvents at 50% concentration, and the protease activity in production medium was 10,876U/ml after 72h incubation. This protease showed high activity as a catalyst for aspartame precursor Cbz-Asp-Phe-NH2 synthesis in the presence of 50% dimethylsulfoxide (DMSO).     

Wall-associated processing of extracellular enzymes of Staphylococcus simulans biovar staphylolyticus

Staphylococcus simulans biovar staphylolyticus produces a staphylolytic glycylglycine endopeptidase (lysostaphin) and a micrococcolytic endo-β-N-acetylglucosaminidase (hexosaminidase) as proenzymes that are proteolytically processed through multiple intermediates to their mature forms by an extracellular sulfhydryl protease. Analysis of protease production by immunoblots using antiserum prepared against purified protease and by renaturing activity gels using gelatin as the substrate has revealed that the lysostaphin-processing protease also is produced as a proenzyme, which appears to be autocatalytically processed. Very little proprotease could be detected in supernatants from cultures of S. simulans biovar staphylolyticus, which suggested that the protein was being processed before it was released to the culture medium. Analysis of wall-associated proteins revealed that processing of proprotease occurred primarily in the cell wall. Furthermore, processing of prolysostaphin and prohexosaminidase also occurred in the cell wall matrix.     

Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through Plackett-Burman and response surface methodological approaches

The proteolytic enzymes are the most important group of commercially produced enzymes. The production of alkaline protease was optimized using a newly isolated Bacillus sp. RKY3. The fermentation variables were selected in accordance with the Plackett-Burman design and were further optimized via response surface methodological approach. Four significant variables (corn starch, yeast extract, corn steep liquor, and inoculum size) were selected for the optimization studies. The statistical model was constructed via central composite design (CCD) using three screened variables (corn starch, corn steep liquor, and inoculum size). An overall 2.3-fold increase in protease production was achieved in the optimized medium as compared with the unoptimized basal medium. Enzyme activity increased significantly with optimized medium (939 u ml(-1)) when compared with unoptimized medium (417 u ml(-1)).     

Overproduction of extracellular protease activity by Streptomyces C5-A13 in fed-batch fermentation

Summary Streptomyces C5-A13, a non-sporulating, pleiotropic mutant of the anthracycline-producing strain, Streptomyces C5, overproduces extracellular proteolytic activity against the substrate azocasein. This extracellular protease activity was produced primarily during the stationary phase. This appears to be an effect related to growth rate rather than to glucose repression, because only very high concentrations of glucose appear to inhibit protease synthesis. Production of extracellular protease activity was stimulated by the presence of carbonate anions in the medium. The optimal concentration of soluble carbonate was 60–80 mM and the stimulation by carbonate was shown not to be due to a pH effect. Approximately 3200–3500 units of extracellular azocaseinase activity were produced per millititre of culture broth using partially optimized fed-batch fermentation processes. This value represents about ninefold greater activity than produced under shake flask conditions.     

An organic solvent-, detergent-, and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45 degrees C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over abroad range of temperatures (45-70 degrees C) and pH (8-10) range with an optimum activity at pH 10 and 65 degrees C. It was comparatively stable in the presence ofa relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45 degrees C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.