Production of neutral and alkaline extracellular proteases by the thermophilic fungus, Scytalidium thermophilum, grown on microcrystalline cellulose

Extracellular proteases produced by Scytalidium thermophilum, grown on microcrystalline cellulose, were most active at pH 6.5–8 and 37–45 °C when incubated for 60 min. Highest protease activity was at day 3 where endoglucanase activity was low. Protease activity measurements with and without the protease inhibitors, p-chloromercuribenzoate, PMSF, antipain, E-64, EDTA and pepstatin A, suggest production of thiol-containing serine protease and serine proteases. Endoglucanase and Avicel-adsorbable endoglucanase activity in culture medium was not significantly affected by protease inhibitors.     

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.     

Properties of extremely thermostable proteases from anaerobic hyperthermophilic bacteria

Summary Hyperthermostable proteases were characterized from five archaeobacterial species (Thermococcus celer, T. stetteri, Thermococcus strain AN 1, T. litoralis, Staphylothermus marinus) and the hyperthermophilic eubacterium Thermobacteroides proteolyticus. These proteases, which were found to be of the serine type, exhibited a preference for phenylalanine in the carboxylic side of the peptide. The enzymes from Thermococcus stetteri and T. litoralis hydrolysed most substrates (peptides) tested. All proteases were extremely thermostable and demonstrated optimal activities between 80 and 95°C. The pH optimum was either neutral (T. celer, Thermococcus strain AN 1) or alkaline. The protease of Thermobacteroides proteolyticus was optimally active at pH 9.5. Zymogram staining showed the presence of multiple protease bands for all strains investigated.Offprint requests to: G. Antranikian     

Purification and properties of proteases produced byalternaria alternata (Fr.) Keissl,regulation of production by fructose

A strain ofAlternaria alternata (Fr.) Keissl, when grown on wheat bran Czapek Dox medium was found to secrete one neutral and two alkaline proteases. The purified enzymes were found to be endo peptidases, the alkaline proteases being serine proteases and neutral proteases being cysteine proteases. Fructose when added to the culture medium was found to give rise to a new neutral protease at the expense of the neutral protease produced in the absence of fructose and was also found to enhance the production of alkaline proteases. It also appears that fructose modifies the alkaline proteases with respect to some characteristics such asV _max, Ea etc. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis indicated a significantly altered protein profile in fructose supplemented medium.     

Purification and properties of an alkaline protease of <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

An extracellular alkaline serine protease has been purified from a strain of Aspergillus clavatus, to apparent homogeneity, by ammonium sulfate precipitation and chromatography on Sephadex G-75. Its molar mass, estimated by SDS-PAGE, was 35 kDa. Maximum protease activity was observed at pH 9.5 and 40°C. The enzyme was active between pH 6.0 and 11.0 and was found to be unstable up to 50°C. Calcium at 5 mM increased its thermal stability. The protease was strongly inhibited by PMSF and chymostatin as well as by SDS, Tween 80 and carbonate ion. Substrate specificity was observed with N-p-Tos-Gly-Pro-Arg-p-nitroanilide and N-Suc-Ala-Ala-Ala-p-nitroanilide being active substates. Parts of the amino acid sequence were up to 81% homologous with those of several fungal alkaline serine proteases.     

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7–8 and at temperature close to 35°C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40–45°C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.     

Hieronymain I,a new cysteine peptidase isolated from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae)

A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) by acetone fractionation followed by cation exchange chromatography (FPLC) on CM-Sepharose FF. Homogeneity of the enzyme was confirmed by mass spectroscopy (MALDI-TOF), isoelectric focusing, and SDS-PAGE. Hieronymain is a basic peptidase (pI > 9.3) and its molecular mass was 24,066 Da. Maximum proteolytic activity on casein (>90% of maximum activity) was achieved at pH 8.5–9.5. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group. The N-terminal sequence of hieronymain (ALPESIDWRAKGAVTEVKRQDG) was compared with 25 plant cysteine proteases that showed more than 50% of identity.     

Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Summary While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.     

Purification and some properties of an extracellular acid protease from <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">clavatus</Emphasis>

Acid proteases represent an important group of enzymes, widely used in food, beverage and pharmaceutical industries. For most of these applications the enzymatic preparation must be at least partially purified and free of substances that could change the characteristics of the product or the process. Fungal proteases have replaced other sources because they are easily obtained mainly from Mucor, Rhizopus, Penicillium and Aspergillus species. A strain of Aspergillus clavatus was selected by producing high level of acid protease activity. An extracellular aspartatic protease from this strain was purified 37.2 times with 37% recovery using (NH₄)₂SO₄ fractionation and ion-exchange chromatography. The enzyme was found to be monomeric having a molecular mass of 30.4 kDa. The purified enzyme is an acid protease with optimum pH of 5.5 and temperature for optimum activity of 50 °C. Its high pH stability was verified in the range of 3.5–6.5. The acid protease was strongly inhibited by Hg⁺² and partially inhibited by Cu⁺², Zn⁺² and Mn⁺². The enzyme was sensitive to denaturing agent SDS and activated by thiol-containing reducing agent dithiotreitol (DTT). The protease activity was not influenced by iodoacetic acid, E-64 and PMSF, while it was lightly actived by EDTA and totally inhibited by pepstatin, with a K_i of 7.8 μM, indicating that is an aspartic protease. A. clavatus acid protease presents interesting characteristics for biotechnological process, such as cheese and flavor manufacture and dietary supplements, in which activity and stability in acid pH are required.     

Beta-galactosidase-neuraminidase deficiency: restoration of beta-galactosidase activity by protease inhibitors

Beta-Galactosidase was partially restored by protease inhibitors, leupeptin, chymostatin and E-64 in cultured fibroblasts from three patients with beta-galactosidase-neuraminidase deficiency. Pepstatin did not activate this enzyme. Neuraminidase was not affected by any of these compounds in the culture medium. It was concluded that the activating effect was produced by a specific inhibition of thiol proteases.     

Isolation and Identification of a Microorganism which Produces Non Streptomyces Pepsin Inhibitor and N-Diazoacetyl-dl-norleucine Methylester Sensitive Acid Proteases

Non pepsin inhibitor (S–PI) and diazoacetyl-dl-norleucine methylester (DAN) sensitive acid proteases producing microorganism was isolated from farm soil of Osaka Prefecture.

The isolated strain was identified as Scytalidium lignicolum M–133. When it was aerobically grown on a medium consisting of glucose 5%, meat extract 1.5%, yeast extract 0.1%, KH₂PO₄ 0.2%, MgSO₄·7H₂O 0.05% at pH 3.5 and 25°C, the strain produced two acid proteases, A and B, in the culture broth.

The acid proteases A and B were not at all inactivated by S–PI and DAN. These acid proteases were expected to be a new type of acid protease from the viewpoint of the active site.     

Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus.

Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70 degrees C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.     

Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus

Streptomyces strain K_1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH₂-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.     

Production of neutral and alkaline proteases in batch and chemostat cultures by bacillus mesentericus 76

The kinetics of the bacterial extracellular protease synthesis (neutral and alkaline protease of Bacillus mesentericusstrain 76, R-form) in batch and chemostat cultures under conditions of glucose limitation were investigated. When the medium was supplemented with casein the production of the proteases was significantly higher. Optimal dilution rates for obtaining of two proteases are fixed. The synthesis of both alkaline and neutral proteases is controlled by catabolite repression and induction.     

Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp.

A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S0-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.     

Production in sea-water of thermostable alkaline proteases by a halotolerant strain of Bacillus licheniformis

A halotolerant strain of Bacillus licheniformis, previously isolated from marine sediments, produced high protease activity during the early stationary phase of growth. The use of sea-water in the fermentation medium enhanced the production of this activity of 150%. After a partial purification, three different proteolytic enzymes could be detected, which were alkaline serine proteases, exhibiting optimal activity at pH 9.0 and at 70°C. The proteases were activated by NaCl, with a two, three-fold increase in activity and were stable in presence of 0.7% NaBO3, 0.5% NaClO and 3% H2 O2. © Rapid Science Ltd. 1998     

Adding value to a recalcitrant and problematic waste: the use of dog hair for fungal keratinolytic protease production

Abstract

The demand for keratinolytic proteases has increased in recent years, in view of their applications in the feed, detergent, fertilizer, leather and textile industries. Recently, studies have focussed on exploration of new and inexpensive carbon sources for their production. Among keratin wastes, dog hair presents no utility and is an environmental concern. In this study, we evaluate the feasibility of using white and melanized dog hair (WDH and MDH, respectively) as alternative substrates for protease production by a Fusarium oxysporum strain. The effects of dog hair concentration, cultivation period, and medium pH on alkaline protease production were investigated using a central composite rotary design (CCRD) and response surface methodology (RSM). The optimization process increased protease activity 14.85- and 7.19-fold, using WDH and MDH, respectively. The alkaline proteases produced from WDH and MDH showed distinct biochemical characteristics. To our knowledge, this is the first report on biotechnological use of this problematic, waste residue. Our results open new avenues for conversion of dog hair into other valuable products, especially feed or fertilizer.     

Identification and partial characterization of two cysteine proteases from castor bean leaves (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.) activated by wounding and methyl jasmonate stress

Jasmonates are signaling molecules that play key roles in wound response and regulate the biosynthesis of many defensive proteins, including proteases. In this study, we investigate the effects of wounding and methyl jasmonate (MJ) application on the protein expression pattern of Ricinus communis L. leaves and on proteolytic activity. Gelatin zymography demonstrated that both MJ and mechanical wounding induce alterations in the proteolytic pattern of castor bean leaves (R. communis L.). Expression of two cysteine proteases (38 and 29 kDa) was induced by the treatments employed; however, MJ induced a higher protease level than mechanical wounding during the stress period (24, 48, and 72 h). The increase in protease activity mirrors the decline in soluble protein content and rubisco degradation that may indicate initiation of senescence in castor plants. The 29 kDa protease has an acidic optimal pH; whereas the 38 kDa protease has a neutral optimum activity. Both proteases were almost completely inhibited by E-64 and cystatin. The significant induction of these proteins by MJ suggests a possible role of cysteine proteases in leaf senescence as well as their involvement in regulating both the wound response and MJ in castor bean plants.     

Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae

 In order to direct the persistent expression of recombinant human serum albumin (HSA) from the GAL10 promoter in the yeast Saccharomyces cerevisiae, we carried out periodic feeding of galactose during shake-flask cultures. Unexpectedly, the recombinant protein secreted was observed to undergo rapid degradation, which was apparently accelerated by carbon-source feeding. The extracellular degradation of HSA occurred even in the strain deficient in the major vacuolar proteases PrA and PrB, and in the strain lacking the acidic protease Yap3p (involved in the generation of HSA-truncated fragments). Interestingly, the degradation correlated closely with the acidification of extracellular pH and thus was significantly overcome either by buffering the culture medium above pH 5.0 or by adding amino acid-rich supplements to the culture medium, which could prevent the acidification of medium pH during cultivation. Addition of arginine or ammonium salt also substantially minimized the degradation of HSA, even without buffering. The extracellular degradation activity was not detected in the cell-free culture supernatant but was found to be associated with intact cells. The results of the present study strongly suggest that the HSA secreted in S. cerevisiae is highly susceptible to the pH-dependent proteolysis mediated by cell-bound protease(s) whose activity and expression are greatly affected by the composition of the medium. Received: 23 August 1999 / Received revision: 8 November 1999 / Accepted: 12 December 1999     

A novel organic solvent tolerant protease from a newly isolated Geomicrobium sp. EMB2 (MTCC 10310): production optimization by response surface methodology

Thirty-eight haloalkaliphilic bacterial strains were isolated from Sambhar Salt Lake, India and screened for their ability to secrete haloalkaliphilic proteases. Among them, a moderately halophilic, mesophilic and alkaliphilic potent strain Geomicrobium sp. EMB2 produced an extracellular protease, which was remarkably stable in organic solvents, salt, surfactants, detergents and alkaline pH. Statistically based experimental designs were applied to study the interactions and optimization of medium constituents for efficient protease production by Geomicrobium sp. EMB2. An overall 20-fold increase in protease production was achieved in the optimized medium (721 U/ml) as compared with the unoptimized medium (37 U/ml). The high production level coupled with novel properties makes it a prospective industrial enzyme. The Geomicrobium sp. EMB2 isolate is deposited in Microbial Type Culture Collection, Chandigarh, India with accession number MTCC 10310.