Microbial reclamation of squid pen for the production of a novel extracellular serine protease by Lactobacillus paracasei subsp paracasei TKU012

A protease-producing bacterium, strain TKU012, was isolated from infant vomited milk and identified as Lactobacillus paracasei subsp paracasei. Strain TKU012 produced protease when it was grown in a medium containing squid pen powder of marine waste. An extracellular protease was purified from culture supernatant by DEAE-Sepharose and Sephacryl S-100 chromatography. A protease, purified 77-fold to homogeneity in an overall yield of 11%, has a molecular weight of about 49 kDa estimated by SDS-PAGE. The protease was maximally active at pH 10 and 60 degrees C and showed substrate specificity to casein and gelatin. The protease retains 21% and 91% activity in the presence of Tween 20 (2% w/v) and SDS (2mM), respectively. The enzyme activity was reduced in the presence of PMSF and showed 23% sequence coverage rate with metalloprotease of Serratia marcescens. This is the first report of extracellular proteases purified from lactobacilli.     

A serine protease from a detergent-soluble extract of Leishmania (Leishmania) amazonensis

Proteases mediate important crucial functions in parasitic diseases, and their characterization contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of promastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS-PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS-PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was at pH 7.0 and 28 degrees C, using a-N-o-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 degrees C for 12 min and preserved only 20% of its activity after pre-treatment at 37 degrees C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gelatin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species.     

Protease expression in the supernatant of Chinese Hamster Ovary cells grown in serum-free culture

The protease activity secreted by the Chinese Hamster Ovary (CHO-K1) cell line grown in serum-free medium was examined by substrate gel electrophoresis (zymography). The cell line expressed extracellular proteases that were active on gelatin zymograms but not on casein zymograms. The main protease band visible by gelatin zymography was approx. 92 kDa. Incubation of the conditioned medium with aminophenylmercuric acetate (APMA) resulted in the appearance of gelatinase activity at 82 kDa. Incubation of the conditioned media with EDTA significantly decreased the gelatinolytic activity of both the 92 kDa and 82 kDa forms, indicating the gelatinase responsible was a metalloprotease. Immunoblotting of the conditioned medium showed the gelatinase to be the pro- form of matrix metalloprotease-9 (pro-MMP-9), also known as gelatinase B.     

Proteolytic activity of Oidiodendron kalrai.

The physiochemical characteristics of the intracellular proteolytic enzymes of Oidiodendron kalari, a neuropathogenic fungus, were studied. The organism in the yeast phase was grown in a semisynthetic medium containing 1% tryptone, at 37 degrees C for 48 hr, on a gyrotory shaker. The crude extract was prepared by breaking the cells in a French pressure cell and the proteolytci activity was tested against biological substrates. The cell-free extract hydrolyzed casein, hemoglobin, lactalbumin, gelatin, elastin, collagen and purified rabbit renal basement membrane to various degrees. Optimal proteolytic activity was observed at pH 6 and at 32 degrees C. Calcium and EDTA did not affect the enzymatic activity; however, activity was partially inhibited by sulfhydryl-blocking agents and by heat-inactivated horse, calf, and human serum. The extract was totally inactivated by exposure to a temperature of 70 degrees C for 60 min. Storage at -76 degrees C or -15 degrees C for 6 months or at 4 degrees C for 4 weeks did not affect protease activity.     

Changes in particulate-bound protease activity during cold acclimation in Tetrahymena pyriformis

The protease activity, as assayed at pH 8.0 with azocasein as substrate, of a ciliate protozoan Tetrahymena pyriformis NT-1, was found to alter by growing the cells at various constant temperatures or at shifted temperatures. The intracellular protease activity, when cells were grown at either constant 39 degrees C or 15 degrees C, was decreased throughout the growth phase with significant secretion into the medium. On the other hand, when the culture temperature was transferred from 39 degrees C to 15 degrees C, the protease activity in cells was greatly increased up to about 28-fold at 8 h after the shift. There was, however, no secretion into the medium during the cold acclimation after the shift, where no cell division occurred. The elevated protease activity was quickly decreased to the control level when the culture was warmed to 39 degrees C after 8-h chilling, and recovery of normal cell division was seen. The marked increase in the protease activity caused by the shift to 15 degrees C was completely blocked by the addition of either cycloheximide or actinomycin D. The thermally induced enhancement of protease activity was found to occur with no different preference between three protease fractions.     

Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2

A collagenase in the culture supernatant of B. subtilis FS-2, isolated from traditional fish sauce, was purified. The enzyme had a molecular mass of about 125 kDa. It degraded gelatin with maximum activity at pH 9 and a temperature of 50 degrees C. The purified enzyme was stable over a wide range of pH (5-10) and lost only 15% and 35% activity after incubation at 60 degrees C and 65 degrees C for 30 min, respectively. Slightly inhibited by EDTA, soybean tripsin inhibitor, iodoacetamide, and iodoacetic acid, the enzyme was severely inhibited by 2-beta-mercaptoethanol and DFP. The protease from B. subtilis FS-2 culture digested acid casein into fragments with hydrophilic and hydrophobic amino acids as C-terminals, in particular Asn, Gly, Val, and Ile.     

Characterization of a thermosensitive sporulation mutant of Bacillus subtilis affected in the structural gene of an intracellular protease.

A thermosensitive sporulation mutant (ts-15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42 degrees C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts-15R1 and ts-15R2) and were also transmitted together by transformation into the wild type. Immunological studies have shown that when ts-15 is grown at 42 degrees C it synthesizes a 'cryptic' protein with apparently the same antigenic properties as the wild type or as ts-15 mutant grown at the permissive temperature (30 degrees C). The intracellular proteases from the wild type and from ts-15 grown at 30 degrees C and 42 degrees C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts-15 grown at 30 degrees C was 64000--65000 in the absence of sodium dodecylsulfate and 31000--32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts-15 grown at 42 degrees C showed the same molecular weight of 32000--34000 in the presence or in the absence of sodium dodecylsulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts-15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form.     

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.     

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.     

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.     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Regulation of extracellular protease formation by Serratia marcescens.

Heavy cell suspensions of Serratia marcescens, when grown in gelatin-containing media, produce extracellular proteases which increase in specific activity in a linear fashion for 3 to 4h. During partial purification, a single peak of proteolytic activity was demonstrated by Sephadex G-100 chromatography. However, electrophoresis using 5% polyacrylamide gels discloses three proteolytically active bands. Evidence in favor of gelatin acting as an inducer of the 'proteolytic system' was provided by two observations. First, proteolytic activity is only present in media containing gelatin. Secondly, the addition of 10(-4) M rifampicin to cells growing in gelatin-containing medium plus an additional carbon source inhibits protease activity totally, but has no effect on growth. When glycerol is added to a growing cell suspension in gelatin-containing medium, growth increases, but protease specific activity decreases. This 'glycerol effect' is not due to an accumulation of active or inactive enzyme in association with the cell, nor to a decrease in the total number of proteases synthesized. Rather, glycerol, as other utilizable carbohydrates, exerts a repression which can be eliminated by 5 mM dibutyryl cyclic AMP.     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

Production of an extracellular ribonuclease by Pseudomonas maltophilia.

As part of a screening program for pseudomonad enzymes having an industrial interest, we selected ribonuclease (RNase) producing strains. Of the 150 pseudomonads screened, 6 were found to produce an extracellular RNase activity when grown on solid medium. In broth culture, the RNase activity from these six species remained bound to the cells unless gelatin was added to the medium. Gelatin was essential for the release of RNase in the broth culture, but the pH of the medium, addition of potential inducers such as nucleic acids, or addition of cations did not affect this release. However, gelatin did not appear to induce the synthesis of the enzyme. Strain B-88, identified as Pseudomonas maltophilia, was selected for further study of the enzyme. The extracellular RNase isolated from B-88 broth cultures could be separated in two fractions on the basis of the molecular weight by the ultrafiltration technique. The low molecular weight fraction reacts optimally at temperatures between 55 and 60 degrees C and optimal pH values varying from 7.4 to 9.5. At neutral or alkaline pH, the enzyme was stable at temperatures below 37 degrees C but was inactivated at 55 degrees C. The RNase was inhibited by mercury and cobalt and stimulated by magnesium.     

N-Acetyl-d-Glucosamine-Mediated Regulation of Extracellular Protease in the Entomopathogenic Fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana GK2016 grown in a liquid medium incorporating gelatin as the sole carbon and nitrogen source produced an extracellular serine protease (molecular weight, 35,000; pI ca. 10). Without gelatin, B. bassiana could utilize N-acetyl-d-glucosamine (GlcNAc; 2-acetamido-2-deoxy-d-glucose) as the sole source of carbon and nitrogen, and GlcNAc availability increased the storage carbohydrate content in mycelia. Synthesis of protease was repressed in gelatin medium containing GlcNAc at levels of >1.07 mumol mg of fungal dry weight. At levels below this, protease synthesis was initiated; subsequently, free amino nitrogen appeared in the medium and diauxic growth was observed. Slow feeding with GlcNAc (35.34 mug ml h) did not repress protease synthesis nor did GlcNAc accumulate in the medium above 0.5 mg ml. Increasing the rate of release of GlcNAc (83.51 mug ml h) resulted in the accumulation of GlcNAc in the medium to 2.0 mg ml, a 45% increase in growth and a decrease in protease synthesis by about 81%. Free amino acids generated from the hydrolysis of gelatin did not repress protease synthesis. These data are interpreted in terms of known interaction of B. bassiana with insect cuticular components. We suggest that the action of extracellular chitinases synthesized by B. bassiana on insect cuticle, and pursuant release of GlcNAc, may have important consequences on the regulation of other extracellular catabolic enzymes such as the protease.     

Production of C5a antagonist by synovial and peritoneal tissue fibroblasts

Fibroblasts grown from synovial and peritoneal tissues release into the medium an inhibitor of neutrophil chemotaxis. The inhibitor resembles the antagonist previously described in synovial and peritoneal fluids. It is a heat stable (56 degrees C) protein of MW approximately 40 kDa that counteracts the chemotactic activity of zymosan-activated serum or purified C5a but not the peptide chemoattractant F-met-leu-phe. No chemotactic inhibitor was detected in media from skin fibroblast cultures or in formal human sera. It is suggested that the inhibitor is produced locally by synovial and peritoneal fibroblasts and that it might play a role in the regulation of inflammation at sites lined with mesothelium.     

Glucocorticoid regulation of chloroquine nonsensitive insulin degradation in cultured fetal rat hepatocytes

The influence of cortisol and other culture conditions on insulin degradation by the chloroquine-sensitive pathway and the chloroquine-nonsensitive pathway (CNP) was investigated in fetal rat hepatocytes during 3 days of culture. The proportions of the chloroquine nonsensitive release of 125I-insulin degradation products into the conditioned medium/h increased from the 1st to the 3rd day of culture, i.e. from 19 to 50% by cells grown in the presence of cortisol and from 17 to 82% by those grown in the absence of cortisol. Replacement of the conditioned medium with the respective fresh medium dramatically enhanced cellular insulin degradation by CNP, i.e. from 22 to 58%, and 19 to 85% in cells grown for 2 days in the presence and absence of cortisol, respectively. Thus, the conditioned medium contained some factor(s) that inhibited CNP. Therefore, we used the inhibited insulin and alpha-casein degradation by papain in vitro as an assay to investigate the nature of the putative anti-(insulin) protease. Cycloheximide completely prevented the appearance of anti-papain activity in the medium. Conditioned medium obtained from cells grown in the presence of cortisol contained about 2-fold more anti-papain activity than the medium that was obtained in the absence of the steroid. The release of anti-papain activity also declined with time from 1 to 3 days of culture and showed an inverse relationship with the magnitude of cellular insulin degradation by CNP. The inhibition of papain-mediated insulin degradation by the anti-(insulin) protease was noncompetitive. The anti-(insulin) protease was nondialyzable (up to the 10-kDa exclusion limit) and inactivated by heat treatment at 50 degrees C for 30 min. These results suggest that fetal hepatocytes synthesize and secrete a glucocorticoid-regulated heat-labile low molecular mass (less than 25 kDa) anti-(insulin) protease, which may contribute to the suppression of insulin degradation caused by the enzymes involved in CNP.     

Fish protein hydrolysis by a psychrotrophic marine bacterium isolated from the gut of hake (Merluccius hubbsi)

The aims of this study were to identify a psychrotrophic bacterium, strain CR41, producing a cold adapted protease during growth at low temperatures and to evaluate the ability of the cells to hydrolyze hake fish protein. The strain was isolated from the intestinal tract of hake collected from the San Jorge Gulf (Patagonia, Argentina) and it was identified as Pseudoalteromonas. Growth and fish protein hydrolysis were determined using an aerated simple mineral medium plus 10% fish protein concentrate. Proteolytic activity was measured at 7 and 22 degrees C during culture in the concentrate. Protease production started in the exponential growth phase and reached a maximum during stationary phase. Protease activity at 7 degrees C was lower than at 22 degrees C. After 8 h of incubation, the percentage of hydrolyzed protein was 84% at 7 degrees C and 95% at 22 degrees C. Electrophoresis detection showed that degradation of muscle hake proteins was complete at both temperatures, and in gelatin zymograms extracellular activity showed two proteolytic bands with apparent molecular masses of approximately 31.6 and 62 kDa.     

Properties of adenosine monophosphate deaminase of Candida albicans.

Adenosine monophosphate deaminase (AMPD; EC 3.5.4.6) catalyses the hydrolysis of adenosine monophosphate (AMP) to commensurate amounts of inosine monophosphate (IMP) and ammonia. The production of AMP deaminase in Candida albicans was measured in Lee's medium grown cultures. The highest AMPD activity was observed at 24 h of growth. The enzyme had an optimum pH and temperature at 6-7 and 28 degrees C, respectively. This enzyme was inhibited under iron-limited growth conditions as well as by protease inhibitors. The AMPD of C. albicans showed a moderate increase in activity when cultures were grown in the presence of the divalent cations Mg2+, Ca2+, and Zn2+. Moreover, ADP, ATP, adenine, adenosine, deoxyribose and hypoxanthine increased the enzyme activity. Cultures grown in trypticase soy broth exhibited maximum AMPD activity compared with those grown in Sabouraud dextrose broth or Lee's medium.     

Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities

Bacillus proteolyticus CFR3001 isolated from fish processing wastes (both fresh water and marine) produced an alkaline protease. The optimum conditions for cell growth and protease production were 37 degrees C, 96 h, agitation speed of 100 rpm and medium pH 9. The partially purified protease obtained from had specific activity of 22.05 at 37 degrees C was active between 40 degrees C and 50 degrees C and lost >20% of its activity around 60 degrees C. Its molecular weight was approximately 29 kDa and it inhibited the growth of several pathogenic organisms such as Escherichia coli, Listeria monocytogenes, Bacillus cereus and Yersinia enterocolytica. The scanning electron microscopy (SEM) studies revealed that the protease produced by B. proteolyticus CFR3001 lysed the cells of these pathogenic bacteria.