Antimicrobial and antioxidant activities of Bacillus mojavensis I4 lipopeptides and their potential application against the potato dry rot causative Fusarium solani.

Lipopeptides are diverse metabolites produced by various bacterial and fungal genera. They are known for their antimicrobial and surfactant activities with diverse environmental, pharmaceutical, and also agronomic applications as biocontrol agents. In this study, a PCR was used to confirm the presence of NRPS genes in Bacillus mojavensis I4. This bacterial strain could produce diverse lipopeptides which belong to the fengycin, and surfactin families. The antioxidant activity of I4 biosurfactants was determined through four different in vitro assays. Furthermore, antimicrobial activity assays indicated that I4 lipopeptides exhibited marked inhibitory activity against several bacterial and fungal strains. Further treatment of potato dry rot causative pathogen Fusarium solani with I4 lipopeptides demonstrated a remarkable reduction in the fungal penetration by almost 80% after 15 days of incubation. The findings suggest that I4 lipopeptide is a potential biocontrol agent during potato tuber storage.

Preventive effect of Bacillus mojavensis levan against carbon tetrachloride and cisplatin toxicity: in vivo study.

This study is the first to investigate the hepato- and nephron-preventive effect of levan from Bacillus mojavensis (BM-levan) against toxicity induced by carbon tetrachloride (CCl4) and cisplatin. Thirty-six male albino rats weighing between 230 and 250 g were used for this experiment. The groups received multiples doses of BM-levan and were compared to the untreated group. The in vitro and in vivo biological potentials of BM-levan were evaluated by measuring its antioxidant capacity as well as its hepato- and nephron-protective activities in rat models. The investigations highlighted a significant in vitro antioxidant activity indicated by the radical-scavenging capacity, the reducing power, and the total antioxidant activity measurement. In addition, results demonstrate that BM-levan supplementation during 8 weeks (100 mg/kg body weight) significantly (p < 0.05) decreased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and remarkably (p < 0.05) attenuated the altered lipid profile by decreasing the levels of triglycerides (TG) and total cholesterol (TC), LDL cholesterol (LDL-C) and by enhancing the HDL cholesterol (HDL-C) content, when compared with the CCl4 group. BM-levan also reduced the content of plasma renal biomarkers (urea, creatinine, and uric acid) in the cisplatin-treated group. Moreover, BM-levan inhibited hepatic and renal oxidative stress generated by CCl4 and cisplatin administration, through the enhancement of the antioxidant catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and the diminishment of lipid peroxidation. The harmful effects of CCl4 or cisplatin on hepatic and renal histology were found to be decreased by the addition of BM-levan. Therefore, BM-levan has proved promising for biomedical applications thanks to its in vitro and in vivo antioxidant properties.

Hybrid levan-Ag/AgCl nanoparticles produced by UV-irradiation: properties, antibacterial efficiency and application in bioactive poly(vinyl alcohol) films.

Foodborne diseases caused by resistance of microorganisms to multiple antimicrobial agents have emerged as a major public health concern around the world. The search for potential antimicrobials has resulted in the emergence of metal nanoparticles for protection against these infections. In this study an eco-friendly and green approach was used to biosynthesize hybrid Ag/AgCl nanoparticles (NPs), using levan from Bacillus mojavensis as a stabilizing/reducing agent, with a high efficiency against a broad spectrum of foodborne bacteria as well as biofilm formations. The morphology and physicochemical characteristics of levan-Ag/AgCl NPs were investigated by transmission electron microscopy (TEM), X-ray diffraction (XRD), UV-vis spectroscopy (UV), dynamic light scattering (DLS) and thermogravimetric analysis (TGA). The hybrid levan-Ag/AgCl was evaluated for antibacterial activity against foodborne pathogenic bacteria (Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Listeria monocytogenes, Enterococcus faecalis, Bacillus subtilis and Bacillus thuringiensis). The study demonstrated the strong efficiency of hybrid levan-Ag/AgCl NPs as a potent inhibitor against all tested strains, with much higher activity against Gram-negative than Gram-positive bacteria. Furthermore, bacterial strains were found to be highly sensitive to hybrid levan-Ag/AgCl NPs in comparison to the tested antibiotics. As a possible application of levan-Ag/AgCl NPs as an additive in packaging, PVA films with different amounts of hybrid levan-Ag/AgCl NPs were prepared by casting and their antibacterial, mechanical, and optical properties and ability to expand the shelf life of beef meat were explored. Interestingly, the amount of Ag leached out from films was below the permissible limit. This work demonstrates the strong antibacterial action of hybrid levan-Ag/AgCl NPs and their potential use in bioactive packaging material.

Structural elucidation and interfacial properties of a levan isolated from Bacillus mojavensis.

A strain with high exopolysaccharide (EPS) production was isolated from soil and identified as Bacillus mojavensis based on the 16S rRNA gene sequencing and biochemical properties. The EPS produced simultaneously with the growth phase reached a maximum of 22 g/L after attaining a stationary phase with sucrose used as sole carbon source. B. mojavensis EPS (BM-EPS) was recovered, fractionated by ethanol precipitation and analysed by NMR and methylation analyses. The BM-EPS was found to be composed of (ß2 â†’ 6)-Fruf residues, characteristic of a levan, with an average molecular weight of 2.3 MDa. A homogeneous micro-porous and rough structure matrix was observed by SEM of the freeze-dried powdered sample. A concentration-dependent water-soluble nature was observed, with good water (5.3 g/g) and oil (36 g/g) holding capacities. The levan displayed good emulsification activity with excellent stability against food grade oil, thus favoring it as a promising emulsifying agent to food industries.

Characterization and production optimization of biosurfactants by Bacillus mojavensis I4 with biotechnological potential for microbial enhanced oil recovery.

Response surface methodology was applied to optimize the production of biosurfactants from Bacillus mojavensis I4 using Box-Behnken design with four variables. The optimal variable combination was 3% of glucose as carbon source, 0.6% of glutamic acid as nitrogen source, temperature of 35 °C and 10 g/l of NaCl which yielded to an optimal production of 4.12 g/l. Compositional analysis and FTIR spectrum revealed that the extracted biosurfactants was a lipopeptides. The biosurfactants achieved a critical micelle concentration value of 100 mg/l. Moreover, the extracted biosurfactants were effective at recovering up to 89.2% of motor oil from sand beach and achieved a dispersion rate of 78% of the initial diameter of the oil. These findings suggested the potential use of I4 biosurfactants in the oil industry.

ß-Chitin and chitosan from squid gladius: Biological activities of chitosan and its application as clarifying agent for apple juice.

Chitin is the second most abundant polysaccharide in biomass after cellulose and the term chitosan usually refers to a family of polymers obtained after chitin deacetylation. The aim of this work was the preparation and the characterization of chitin and chitosan from the gladius (pen) of the European squid (Loligo vulgaris). A high level of deproteinization (more than 80%) was recorded using Alcalase® with an enzyme/protein ratio of 10U/mg. The demineralization of the gladius was completely achieved within 8h at room temperature in HCl. 13C NMR, FTIR, and XRD diffractograms of prepared chitin and chitosan were taken and then degree of deacetylation of chitosan was calculated using 13C CP/MAS-NMR Spectroscopic. Further, in vitro antioxidant capacity of chitosan was evaluated on 1,1-diphenyl-2-picrylhydrazyl method (IC50=3.2mgmL-1) and the ß-carotene bleaching assay (IC50=3.3mgmL-1). Antimicrobial activity was also investigated and assays indicated that prepared chitosan exhibited marked inhibitory activity against all microbial strains tested. Additionally, chitosan was tested such as clarifying agent for apple juice and showed powerful clarification capability, without affecting nutritional value. Furthermore, the results suggested that prepared chitosan could be used as alternative additive in pharmaceutical preparations and food industry.

Anionic lipopeptides from Bacillus mojavensis I4 as effective antihypertensive agents: Production, characterization, and identification.

A new isolated Bacillus mojavensis strain I4 was found as producer of biosurfactants by different screening methods, such as parafilm M test, hemolytic activity, oil displacement test, emulsification index, surface tension, and lipase production assay. Enhanced biosurfactants production was obtained using glucose and glutamic acid as carbon and nitrogen sources, respectively. The optimal production of the biosurfactants was obtained by using a C/N ratio of 17, pH of 7.0, and temperature of 37°C. The surface tension was reduced to 29 mN/m and the emulsification index E24 of 62% was achieved after 72 h of culture. The purified biosurfactants showed stability with regard to surface tension reduction and emulsification in a wide range of temperatures (4-120°C), pH (4-10), and salinity (2-12% of NaCl). The thin-layer chromatography showed that the produced biosurfactants were lipopeptides. The biosurfactants were characterized as a group of anionic lipopeptides with zeta potential measurement. Chromatographic characterization using HPLC revealed that I4 lipopeptides contained numerous isoforms and surfactin was the major component. Moreover, the I4 lipopeptides showed interesting angiotensin-converting enzyme-inhibitory activity.

Optimization of polysaccharides extraction from watermelon rinds: Structure, functional and biological activities.

In the present work, optimization of hot water extraction, structural characteristics, functional properties, and biological activities of polysaccharides extracted from watermelon rinds (WMRP) were investigated. The physicochemical characteristics and the monosaccharide composition of these polysaccharides were then determined using chemical composition analysis, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and gas chromatography-flame ionization detection (GC-FID). SEM images showed that extracted polysaccharides had a rough surface with many cavities. GC-FID results proved that galactose was the dominant sugar in the extracted polysaccharides, followed by arabinose, glucose, galacturonic acid, rhamnose, mannose, xylose and traces of glucuronic acid. The findings revealed that WMRP displayed excellent antihypertensive and antioxidant activities. Those polysaccharides had also a protection effect against hydroxyl radical-induced DNA damage. Functional properties of extracted polysaccharides were also evaluated. WMRP showed good interfacial dose-dependent proprieties. Overall, the results suggested that WMRP presents a promising natural source of antioxidants and antihypertensive agents.

Valorisation of smooth hound (Mustelus mustelus) waste biomass through recovery of functional, antioxidative and antihypertensive bioactive peptides.

Concerns over the environmental and waste disposal problems created by the large amounts of by-products generated from fish processing industries are increasing worldwide. The bioconversion of those marine waste by-products through the enzymatic hydrolysis of their protein content offers the possibility for the development of bioactive peptides for use in various biotechnological applications. The present study aimed to investigate and evaluate the biological and functional properties of smooth hound (Mustelus mustelus) protein hydrolysates (SHPHs) obtained by treatment with intestinal and gastric enzyme preparations from M. mustelus viscera and porcine pancreatin. The results revealed that the SHPHs exhibited different degrees of hydrolysis and antioxidant activity. The hydrolysate produced by the intestinal crude extract presented the highest rate of antioxidative activity, showing an IC50 value of 1.47 ± 0.07 mg/mL in 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assays. The alkaline protease extract from the intestine of M. mustelus produced hydrolysate with the highest angiotensin I-converting enzyme (ACE) inhibitory activity (82 ± 1.52% at 2 mg/mL). All the protein hydrolysates showed excellent solubility and interfacial properties that were governed by pH. The major amino acids detected in SHPHs were glutamic acid/glutamine, aspartic acid/asparagine, histidine and arginine, followed by methionine, phenylalanine, serine, valine and leucine. Overall, the results indicated that smooth hound by-products can be used to generate high value-added products, thus offering a valuable source of bioactive peptides for application in wide range of biotechnological and functional food applications.

Purification, identification and structural modelling of DPP-IV inhibiting peptides from barbel protein hydrolysate.

Inhibition of DPP-IV may improve glycemic control in diabetics by preventing the rapid breakdown and there by prolonging the physiological action of incretin hormones. Barbel muscle protein hydrolysate (BMPH) was noted to exhibit DPP-IV inhibitory activity, with an IC50 value of 1.94mg/mL. It was fractionated into five major fractions (FI-FV) by size exclusion chromatography using a Superdex peptide. The FIII fraction was noted to display the highest inhibitory activity, with an IC50 value of 1.23mg/mL, and was, therefore, further fractionated by RP-HPLC. Four major peptide sub-fractions were selected. The results revealed that the SF4 sub-fraction showed the highest DPP-IV inhibitory activity, with an IC50 value of 0.21mg/mL. This sub-fraction was submitted to RP-HPLC, ESI-MS, and ESI-MS/MS analyses. The findings indicated that SF4 consisted of two peptides (IC50=96µg/mL), namely PP1 and PP2, whose structures were identified as Trp-Ser-Gly (330Da) and Phe-Ser-Asp (349Da), respectively. This is the first report of these sequences from barbel proteins. The structural modelling through docking simulations results with DPP-IV showed that the Trp-Ser-Gly peptide bound to DPP-IV with high affinity. Overall, the results suggested that BMPH can be considered as a promising natural source of DPP-IV inhibitory peptides.

Screening and Molecular Identification of New Microbial Strains for Production of Enzymes of Biotechnological Interest

ABSTRACT: This research focused on isolation, identification and characterization of new strains of fungi and bacteria, which were able to produce extracellular xylanase, mannanase, pectinase and α-amylase. Fungi isolates were identified on the basis of analyses of 18S gene sequencing and internal transcribed spacer region. The closest phylogenetic neighbors according to 18S gene sequence and ITS region data for the two isolates M1 and SE were Aspergillus fumigatus and Aspergillus sydowii, respectively. I4 was identified as Bacillus mojavensis on the basis of the 16S rRNA gene sequencing and biochemical properties. The enzyme production was evaluated by cultivating the isolated microorganisms in liquid-state bioprocess using wheat bran as carbon source. Two fungi (M1, and SE) and one bacterium (I4) strains were found to be xylanase producer, and several were proven to be outstanding producers of microbial xylanase. The strains producing xylanase secreted variable amounts of starch-debranching enzymes and produced low level β-mannan-degrading enzyme systems. The bacterium strain was found to be capable of producing pectinolytic enzymes on wheat bran at high level. Some of the strains have good potential for use as sources of important industrial enzymes.

Assessment of pectinase production by Bacillus mojavensis I4 using an economical substrate and its potential application in oil sesame extraction.

Carrot (Daucus carota) peels, local agricultural waste product, is rich in lignocellulolytic material, including pectin which can act as an inducer of pectinase production. Pectinolytic enzymes production by Bacillus mojavensis I4 was studied in liquid state fermentation using carrot peel as a substrate. Medium composition and culture conditions for the pectinase production by I4 were optimized using two statistical methods: Taguchi design was applied to find the key ingredients and conditions for the best yield of enzyme production and The Box-Behnken design was used to optimize the value of the four significant variables: carrot peels powder, NH4Cl, inoculum size and incubation time. The optimal conditions for higher production of pectinase were carrot peels powder 6.5 %, NH4Cl 0.3 %, inoculum level 3 % and cultivation time 32 h. Under these conditions, the pectinase experimental yield (64.8 U/ml) closely matched the yield predicted by the statistical model (63.55 U/ml) with R (2) = 0.963. The best pectinase activity was observed at the temperature of 60 °C and at pH 8.0. The enzyme retained more than 90 % of its activity after 24 h at pH ranging from 6.0 to 10.0. The enzyme preserved more than 85 % of its initial activity after 60 min of pre-incubation at 30-40 °C and more than 67 % at 50 °C. The extracellular juice of I4 was applied in the process of sesame seeds oil extraction. An improvement of 3 % on the oil yield was obtained. The findings demonstrated that the B. mojavensis I4 has a promising potential for future use in a wide range of industrial and biotechnological applications.

Recovery, viscoelastic and functional properties of Barbel skin gelatine: investigation of anti-DPP-IV and anti-prolyl endopeptidase activities of generated gelatine polypeptides.

The characteristics and functional properties of gelatine from freshwater fish skin (Barbus callensis) were investigated. The gelatine extraction efficiency was improved by an acid-swelling process in the presence of barbel crude acid protease extract. Barbel skin gelatine (BSG) contained 92.15% protein, 0.31% lipid and 0.72% ash. The amino acid profile of BSG showed a high percentage of imino acids. The electrophoretic profile showed that BSG is mainly composed of α- and ß-components. BSG showed an excellent solubility and possessed interfacial properties, which were governed by the protein concentration. Biological activities of the hydrolysates obtained after digestion of BSG with several commercial proteases were evaluated. The results suggested that these hydrolysates are a good source of natural inhibitors of dipeptidyl peptidase-IV and prolyl endopeptidase and could potentially be used as dietary ingredients in the management of type 2-diabetes and/or neuropathological disorders.

Production of xylooligosaccharides by immobilized His-tagged recombinant xylanase from Penicillium occitanis on nickel-chelate Eupergit C.

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0-4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.

Alkaline xylanases from Bacillus mojavensis A21: production and generation of xylooligosaccharides.

Medium composition and culture conditions for the xylanases production by Bacillus mojavensis A21 were optimized using two statistical methods: Plackett-Burman design applied to find the key ingredients and conditions for the best yield of enzyme production and Box-Behnken design used to optimize the value of the four significant variables: barley bran, NaCl, agitation, and cultivation time. The optimal conditions for higher production of xylanases were barley bran 18.66 g/l, NaCl 1.04 g/l, speed of agitation 176 rpm and cultivation time 34.08 h. Under these conditions, the xylanase experimental yield (7.45 U/ml) closely matched the yield predicted by the statistical model (7.23 U/ml) with R² = 0.98. The medium optimization resulted in a 6.83-fold increase in xylanase production compared to that of the initial medium. Best xylanase activity was observed at the temperature of 50 °C and at pH 8.0. The enzyme retained more 96% of its activity after 24 h at pH ranges from 7.0 to 90.0. The enzyme preserved more 80% of its initial activity after 60 min of pre-incubation from 30 °C to 60 °C. The main hydrolysis products yielded from corncob extracted xylan were xylobiose and xylotriose, suggesting the good potential of strain A21 in xylooligosaccharides production.

Functional, antioxidant and film-forming properties of tuna-skin gelatin with a brown algae extract.

Characteristics and functional properties of gelatin from skin of Atlantic Bluefin tuna (Thunnus thynnus) were investigated. The gelatin was extracted by an acid-swelling process in the presence of different concentrations of commercial pepsin, followed by subsequent heating. The extraction yield was higher when increasing concentrations of pepsin were used during the swelling process. Emulsion activity index, foam formation ability and foam stability of gelatin increased with the increase of gelatin concentration. Antioxidant properties (ferric-reducing ability and DPPH-radical-scavenging capacity) of gelatin-based edible films containing aqueous or methanolic extracts of brown algae (Cystoseira barbata) were also assessed. For comparative purposes, tuna-skin gelatin edible film with BHA was studied. Antioxidant properties of the films were increased significantly when natural extracts were added. Extracts of brown algae could be useful additives to obtain edible films from tuna-skin gelatin with interesting functional and antioxidant properties.

Chicken feathers: a complex substrate for the co-production of alpha-amylase and proteases by B. licheniformis NH1.

This study is concerned with the co-production of alkaline proteases and thermostable alpha-amylase by some feather-degrading Bacillus strains: B. mojavensis A21, B. licheniformis NH1, B. subtilis A26, B. amyloliquefaciens An6 and B. pumilus A1. All strains produced both enzymes, except B. pumilus A1, which did not exhibit amylolytic activity. The best enzyme co-production was obtained by the NH1 strain when chicken feathers were used as nitrogen and carbon sources in the fermentation medium. The higher co-production of both enzymes by B. licheniformis NH1 strain was achieved in the presence of 7.5 g/l chicken feathers and 1 g/l yeast extract. Strong catabolic repression on protease and alpha-amylase production was observed with glucose. Addition of 0.5% glucose to the feather medium suppressed enzyme production by B. licheniformis NH1. The growth of B. licheniformis NH1 using chicken feathers as nitrogen and carbon sources resulted in its complete degradation after 24 h of incubation at 37 degrees C. However, maximum protease and amylase activities were attained after 30 h and 48 h, respectively. Proteolytic activity profiles of NH1 enzymatic preparation grown on chicken feather or casein-based medium are different. As far as we know, this is the first contribution towards the co-production of alpha-amylase and proteases using keratinous waste. Strain NH1 shows potential use for biotechnological processes involving keratin hydrolysis and industrial alpha-amylase and proteases co-production. Thus, the utilization of chicken feathers may result in a cost-effective process suitable for large-scale production.

Low-cost fermentation medium for alkaline protease production by Bacillus mojavensis A21 using hulled grain of wheat and sardinella peptone.

Media composition and culture conditions for surfactant stable alkaline protease production by Bacillus mojavensis A21 were optimized using two statistical methods. Plackett-Burman design was applied to find the optimal ingredients and conditions to improve yields. Response surface methodology (RSM), including central composite design, was used to determine the optimal concentrations and conditions. The results indicated that several components, including hulled grain of wheat (HGW), sardinella peptone (SP), NaCl, CaCl(2), MgSO(4), K(2)HPO(4), KH(2)PO(4), agitation, culture temperature and initial medium pH, had significant effects on production. The statistical model was constructed via central composite design (CCD) using four selected variables (HGW, NaCl, KH(2)PO(4) and K(2)HPO(4)). Under the proposed optimized conditions, the protease experimental yield (1860.63U/mL) closely matched the yield predicted by the statistical model (1838.60U/mL) with R(2)=0.98. An overall 14.0-fold increase in protease production was achieved using the optimized medium (HGW 30.0g/L, SP 1.0g/L, NaCl 2.0g/L, KH(2)PO(4) 1.0g/L, K(2)HPO(4) 0.3g/L, CaCl(2) 2.0g/L, MgSO(4) 1.0g/L and pH 9.0, compared with the unoptimized basal medium (starch 10.0g/L, yeast extract 2.0g/L, KH(2)PO(4) 0.1g/L, K(2)HPO(4) 0.1g/L, CaCl(2) 0.5g/L and pH 8.0; 137U/mL). A successful and significant improvement (14-fold) in the production of protease by the A21 strain was accomplished using cheap carbon and nitrogen substrates (HGW and SP), which may result in a significant reduction in the cost of medium constituents.

A novel alpha-amylase from Bacillus mojavensis A21: purification and biochemical characterization.

alpha-Amylase from Bacillus mojavensis A21 (BMA.2) was purified to homogeneity by ultrafiltration, Sephadex G-75 gel filtration and Sepharose mono Q anion exchange chromatography, with a 15.3-fold increase in specific activity and 11% recovery. The molecular weight of the BMA.2 enzyme was estimated to be 58 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. The optimum temperature and pH were 80 degrees C and 6.5, respectively. BMA.2 belonged to the EDTA-sensitive alpha-amylase, but its activity was not stimulated by the presence of Ca2+ ions. The major end-products of starch hydrolysis were maltohexaose, maltopentaose and maltotriose. The N-terminal amino acid sequence of the first ten amino acids of the purified alpha-amylase was ASVNGTLMQY. Compared to sequences of other amylases, the ten amino acid sequence contains Val at position 3, while amylases from Bacillus licheniformis NH1 and Bacillus sp. SG-1 have Leu and Thr at position 3, respectively.

Fibrinolytic serine protease isolation from Bacillus amyloliquefaciens An6 grown on Mirabilis jalapa tuber powders.

In this study, Mirabilis jalapa tuber powder (MJTP) was used as a new complex organic substrate for the growth and production of fibrinolytic enzymes by a newly isolated Bacillus amyloliquefaciens An6. Maximum protease activity (1,057 U/ml) with casein as a substrate was obtained when the strain was grown in medium containing (grams per liter) MJTP 30, yeast extract 6, CaCl(2) 1, K(2)HPO(4) 0.1, and K(2)HPO(4) 0.1. The strain was also found to grow and produce extracellular proteases in a medium containing only MJTP, indicating that it can obtain its carbon, nitrogen, and salts requirements directly from MJTP. The B. amyloliquefaciens An6 fibrinase (BAF1) was partially purified, and fibrinolytic activity was assayed in a test tube with an artificial fibrin clot. The molecular weight of the partially purified BAF1 fibrinolytic protease was estimated to be 30 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for the caseinolytic activity were 60 degrees C and 9.0, respectively. The enzyme was highly stable from pH 6.0 to 11.0 and retained 62% of its initial activity after 1 h incubation at 50 degrees C. However, the enzyme was inactivated at higher temperatures. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, suggesting that BAF1 is a serine protease.