Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326

Bacillus cereus MCM B-326, isolated from buffalo hide, produced an extracellular protease. Maximum protease production occurred (126.87+/-1.32 U ml(-1)) in starch soybean meal medium of pH 9.0, at 30 degrees C, under shake culture condition, with 2.8 x 10(8) cells ml(-1) as initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25-65 degrees C and pH 6-12, with maximum activity at 55 degrees C and pH 9.0. The enzyme required Ca(2+) ions for its production but not for activity and/or stability. The partially purified enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be effectively used to remove hair from buffalo hide indicating its potential in leather processing industry.     

Purification and characterization of myofibril-bound serine protease from lizard fish (Saurida undosquamis) muscle

Myofibril-bound serine protease (MBSP) from lizard fish (SAURIDA UNDOSQUAMIS: Synodontidae) skeletal muscle was purified to homogeneity with higher purification (1260-fold) and higher recovery (7%) than our previous report in lizard fish (Saurida wanieso). The new purification method combines a heat-treatment for dissociation from washed myofibrils, acid-treatment at pH 5.0 before and after lyophilization, and alcohol-treatment, followed by two column chromatographies. The molecular mass of the enzyme was estimated to be 50 kDa under non-reducing conditions and 28 kDa under reducing conditions by SDS-PAGE. The N-terminal amino acid sequence of the MBSP was determined to be 22 residues (IVGGYEXEAYSKPYQVSINLGY) and the sequence showed high homology to carp and other fish trypsins (64-77%), but did not show high homology to carp MBSP (41%). The enzyme activity was inhibited by serine protease inhibitors such as Pefabloc SC, leupeptin, TLCK and native protein inhibitors (soybean trypsin inhibitor, alpha(1)-antitrypsin and aprotinin). The purified enzyme specifically hydrolyzed at the carboxyl side of the arginine residue of synthetic 4-methyl-coumaryl-7-amide substrate. When purified MBSP was stored at -35 degrees C in the presence of 50% ethylene glycol (V/V), the enzyme activity was entirely preserved over 6 months and stable against freezing and thawing. Activities for both casein and the synthetic substrate were most active at pH 9.0, and the enzyme was most active approximately 55 degrees C with casein and between 35 and 45 degrees C for synthetic substrate. When myofibrils were incubated with purified MBSP, myosin heavy chain was mostly degraded approximately 55 degrees C, but the degradation of actin was very slow.     

A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713

An alkaline protease was isolated from culture filtrate of B. subtilis NCIM 2713 by ammonium sulphate precipitation and was purified by gel filtration. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 8.0 and temperature 70 degrees C. The purified protease had molecular weight 20 kDa, Isoelectric point 5.2 and km 2.5 mg ml(-1). The enzyme was stable over the pH range 6.5-9.0 at 37 degrees C for 3 hr. During chromatographic separation this protease was found to be susceptible to autolytic degradation in the absence of Ca2+. Ca2+ was not only required for the enzyme activity but also for the stability of the enzyme above 50 degrees C. About 62% activity was retained after 60 min at pH 8.0 and 55 degrees C. DFP and PMSF completely inhibited the activity of this enzyme, while in the presence of EDTA only 33% activity remained. However, it was not affected either by sulfhydryl reagent, or by divalent metal cations, except SDS and Hg2+. The results indicated that this is a serine protease.     

Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80 degrees C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80 degrees C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80 degrees C with a half-life of 4 h at 90 degrees C and 1.5 h at 100 degrees C.     

Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production.

A novel protease, hydrolyzing azocasein, was identified, purified, and characterized from the culture supernatant of the fish pathogen Yersinia ruckeri. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity was detected in peptone but not in Casamino Acid medium. Its synthesis appeared to be under catabolite repression and ammonium control. The protease was purified in a simple two-step procedure involving ammonium sulfate precipitation and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein indicated an estimated molecular mass of 47 kDa. The protease had characteristics of a cold-adapted protein, i.e., it was more active in the range of 25 to 42 degrees C and had an optimum activity at 37 degrees C. The activation energy for the hydrolysis of azocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42 degrees C. The enzyme had an optimum pH of around 8. Characterization of the protease showed that it required certain cations such as Mg(2+) or Ca(2+) for maximal activity and was inhibited by EDTA, 1,10-phenanthroline, and EGTA but not by phenylmethylsulfonyl fluoride. Two N-methyl-N-nitro-N-nitrosoguanidine mutants were isolated and analyzed; one did not show caseinolytic activity and lacked the 47-kDa protein, while the other was hyperproteolytic and produced increased amounts of the 47-kDa protein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal anti-47-kDa-protease serum, and zymogram analyses showed that protease activity was present in 8 of 14 strains tested and that two Y. ruckeri groups could be established based on the presence or absence of the 47-kDa protease.     

Isolation and some properties of a serine protease from the fruits of Cudrania cochinchinensis (Lour.) Kudo et Masam

An endopeptidase (Cudrania protease) with a molecular mass of 76 kDa has been purified from the fruits of Cudrania cochinchinensis (Lour.) Kudo et Masam. The enzyme was stable between pH 6 and 10 at 30 degrees C for 60 min. The enzyme activity was inhibited by diisopropyl fluorophosphate, chymostatin, and aprotinin, but not by EDTA or pepstatin. These results indicated that the enzyme was a serine protease.     

An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Production, purification, and characterization of a novel thermostable serine protease from soil isolate, Streptomyces tendae

An isolate of Streptomyces tendae produced a extracellular protease which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 21 kDa. Optimum activity was at 70 degrees C and pH 6. It was stable at 55 degrees C for 30 min and between pH 4 and 9. It was resistant to neutral detergents and organic solvents such as Triton X-100, Tween 80, methanol, ethanol, acetone, and 2-propanol at 5% (v/v). The enzyme was completely inhibited by 5 mM PMSF, indicating it to be a serine protease. N-terminal amino acid sequence did not show any homology with other known proteolytic enzymes. The protease may therefore be a novel neutral serine protease, which is stable at high temperature and over a broad range of pH.     

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40 degrees C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu2, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.     

Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

Some properties of the extracellular protease produced by the psychotrophic bacterium Pseudomonas fluorescens strain AR-11.

The major extracellular protease from Pseudomonas fluorescens strain AR-11 has been partially purified by a factor of 300 by a combination of DEAE-cellulose ion-exchange chromatography and gel filtration. The enzyme had a molecular weight of 38 400 and exhibited optimum activity with isoelectrically precipitated casein substrate at pH 6.5 with Km - 0.13 mM. The protease was strongly inhibited by a number of heavy metal ions at the 10 mM level and also inhibited by thiol agents, while 10 mM EDTA led to slight activation. Optimum activity was retained, amounting to 33% of the maximum activity at 4 degrees C and 72% at 20 degrees C. Heat inactivation studies in which the isolated protease was heated at high temperature before subsequent incubation at 35 degrees C with substrate showed that for 50% inactivation 25 s heating at 130 degrees C or 17 s at 140 degrees C of 8.5 s at 150 degrees C was requried. The combination of high stability to heat treatments and retention of considerable activity at low incubation temperatures indicates that such a protease might have considerable significance in the processing and subsequent storage of food and other products.     

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.     

Purification and characterization of a thermostable alkaline protease from alkalophilic Thermoactinomyces sp. HS682.

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatographies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25,000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0. Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu2+ and Hg2+. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37 degrees C for 60 min. The optimum pH was pH 11.5-13.0 at 37 degrees C and the optimum temperature was 70 degrees C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl2, it showed maximum proteolytic activity at 80 degrees C and stability from pH 4-12.5 at 60 degrees C and below 75 degrees C at pH 11.5. The stabilization by Ca2+ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of microbial serine protease, although alanine of the NH2-terminal amino acid was deleted.     

Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).     

Purification and characterization of a small size protease from Bacillus sp. APR-4

A thermostable extracellular protease of Bacillus sp. APR-4 was purified by size-exclusion and ion-exchange chromatographic methods and its properties were studied. The purified enzyme had a specific activity of 21,000 U/mg of protein and gave single band on SDS/PAGE with a molecular mass of 16.9 KDa. This protease had an optimal pH of 9 and exhibited its highest activity at 60 degrees C. The enzyme activity was inhibited by EDTA, suggesting the presence of metal residue at the active site. Ca2+ (5 mM) had stabilising effect on the activity of protease, but Cu2+ (5 mM) had inhibitory effect. The enzyme exhibited highest specificity towards casein (1%) and had a Km of 26.3 mg/ml and a Vmax of 47.6 U/mg with casein as a substrate. The stability of this enzyme was evaluated in the presence of some organic solvents and the enzyme was stable in methanol, petroleum ether and ethanol. Detergents (Wheel, Farishta) had stimulatory effect on the activity of this enzyme.     

Isolation and purification of an extracellular protease from a new strain of Bacillus subtilis, viz.NCIM 2711

A new extracellular protease having a prospective application in the food industry was isolated from Bacillus sUbtilis NCIM 2711 by (NH4)2SO4 precipitation from the cell broth. It was purified using DEAE-Cellulose and CM-Sephadex C-50 ion-exchange chromatography. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 7.0 and temperature 55 degrees C with Km 1.06 mg/ml. The enzyme was stable over a pH range 6.5-8.0 at 30 degrees C for 1 hr in presence of CaCl2 x 2H2O. At 55 degrees C, the enzyme retained 60% activity up to 15 min in presence of CaCl2 x 2H2O. EDTA and o-phenanthroline (OP) completely inhibited the enzyme activity while DFP, PMSF and iodoacetamide were ineffective. The enzyme was completely inhibited by Hg2+ and partially by Cd2+, Cu2+, Ni2+, Pb2+ and Fe2+. The OP inhibited enzyme could be reactivated by Zn2+ and Co2+ up to 75% and 69% respectively. It is a neutral metalloprotease showing a single band of 43 kDa on SDS-PAGE.     

Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101

A novel haloalkaliphilic, thermostable serine protease was purified from the extreme halophilic archaeon, Halogeometricum borinquense strain TSS101. The protease was isolated from a stationary phase culture, purified 116-fold with 18% yield and characterized biochemically. The molecular mass of the purified enzyme was estimated to be 86 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10.0 in 20% NaCl. The enzyme had high activity over the pH range from 6.0 to 10.0. Enzymatic activity was strongly inhibited by 1 mM phenyl methylsulfonyl fluoride, but activity was increased 59% by 0.1% cetyltrimethylammonium bromide. The enzyme exhibited relatively high thermal stability, retaining 80% of its activity after 1 h at 90 degrees C. Thermostability increased in the presence of Ca2+. The stability of the enzyme was maintained in 10% sucrose and in the absence of NaCl.     

Laundry detergent compatibility of the alkaline protease from Bacillus cereus

The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C.     

Isolation of a cadmium-releasing bacterium and characterization of its novel protease

Microorganisms were screened for their ability to release cadmium from scallop hepatopancreas, which is the main residue after removing of the edible parts of scallop. The isolated strain, 23-0-11, identified as Arthrobacter nicotinovorans, secreted a protease which released cadmium from scallop hepatopancreas into the liquid medium. The molecular mass of the enzyme was estimated to be 27 kDa. The sequence of the 15 N-terminal amino acids of the protease showed no close similarity with any other protein. Compared with a commercial enzyme, the purified protease had greater ability to release cadmium. The enzyme activity was greatest at 50 degrees C and pH 7.0, and was enhanced in the presence of Ca(2+), Mg(2+) and Mn(2+), while being strongly inhibited by Co(2+). The inhibition profile by the serine protease inhibitor, phenylmethylsulphonyl fluoride (PMSF), confirmed that the protease belonged to the serine protease family.     

Optimal protease production condition for Prevotella ruminicola 23 and characterization of its extracellular crude protease

In this study, Prevotella ruminicola 23 (ATCC 19189), a ruminal proteolytic bacterium, was used as protease producer to examine the optimal condition for protease production. The best carbon and nitrogen sources for the maximum growth were glucose with peptone. Both sucrose and glucose could stimulate high protease production. Casein and peptone are better nitrogen sources for protease production than other choice in this study. The best enzyme production condition was 18-20 h incubation which was at late log phase in the broth of 5% glucose or sucrose as carbon source with 0.1% ammonium chloride and 0.2% peptone as nitrogen sources. Most of the protease activity was secreted into broth (65%) and on cell surface (18%). The optimal temperature and pH for protease reaction were 40 degrees C and pH 6.8, respectively. After incubation for 6h, the crude extract maintained 50% of original protease activity at 30 and 50 degrees C, and protease activity was stable between pH 6 and 8. The protease inhibitor test showed that serine, aspartic acid and metallo-protease inhibitors could cause inhibition of proteolysis. Protein feedstuff degradation experiments suggested that protease in crude extract had higher degradation ability on fish meal, whey, and feather meal (2.39, 2.60 and 1.76 micromol aminoacid/mg enzyme/h) in comparison to soybean meal and blood meal (1.11 and 1.09 micromol aminoacid/mg enzyme/h). The protease in the crude extract should have application potential in term of improving utilization of fish meal and feather meal for monogastric animals.