Isolation,partial purification,biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis,Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37?°C for 48?h with a constant agitation of 120?rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1?M ice cold CaCl₂ and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60?s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production.     

Screening,selection and development of Bacillus subtilis apr-IBL04 for hyper production of macromolecule alkaline protease

Bacillus subtilis microbe is commonly found in soil and produces proteases on nitrogen and carbon-containing sources and increases the fertility rate by degrading nitrogenous organic materials. The present study was aimed to develop hyper producing mutant strain of B. subtilis for the production of proteases, to improve the process variables by the response surface methodology (RSM) under central composite design (CCD) and the production of protease by the particular mutant strain in a liquid state fermentation media. The mutation of the strain was carried out using ethidium bromide. Pure B. subtilis strain was collected and screened for hyper-production of protease. The production of protease by mutant B. subtilis strain was optimized by varying temperature, inoculum size, pH and incubation time under liquid state fermentation. The CCD model were found to be reliable with r² of 0.999. The maximum enzyme activity of B. subtilis IBL-04 mutant with 3 mL/100 mL inoculum size, 72 h fermentation time, pH 8, and 45 °C temperature was developed with enzyme activity 631.09 U/mL, indicates 1–7-fold increase in enzyme activity than the parent strain having 82.32 U/mL activity. These characteristics render its potential use in industries for pharmaceutical and dairy formulation.     

Enhanced production of fibrinolytic enzyme by a new Xanthomonas oryzae IND3 using low-cost culture medium by response surface methodology

Cardiovascular diseases (CVDs) cause high mortality throughout the world. Existing fibrinolytic agents are highly expensive and have many side effects. Microbial fibrinolytic enzymes are very much considered as novel therapeutic candidate for the treatment of CVDs. Reports on fibrinolytic enzyme from Xanthomonas sp. is lacking. This study reports fibrinolytic enzymes from Xanthomonas oryzae IND3 as it shows hyperactivity on fibrin-agarose plates. This organism utilized various agro-industrial wastes for enzymes production. Among all, cow dung enhanced more enzyme production, hence it was used as the low-cost substrate for statistical optimization of fibrinolytic protease in Solid state fermentation. Response surface methodology was employed to optimize the factors and enhanced yield by 4-fold. The interactions among the variables, viz, sucrose, yeast extract, and pH of the medium were investigated using Central Composite Design (CCD). The predicted fibrinolytic enzyme activity was 2340 U/g, and the observed fibrinolytic enzyme activity was 2294?±?12.8?U/g. The fibrinolytic enzyme degraded blood clot in vitro completely. This study is the first report on statistical optimization of fibrinolytic enzyme production in SSF from Xanthomonas sp. The crude extract has immense activity on proteinaceous wastes. The production of fibrinolytic protease using the low-cost substrate could reduce the production cost of enzyme.     

Isolation and Molecular Characterisation of Alkaline Protease Producing Bacillus thuringiensis

Proteases are of particular interest because of their action on insoluble keratin substrates and generally on a broad range of protein substrates. Proteases are one of the most important groups of industrial enzymes used in detergent, protein, brewing, meat, photographic, leather, dairy, pharmaceutical and food industry. In the present study, the organism isolated from the protein rich soil sample was identified by biochemical and molecular characterisation as Bacillus thuringiensis and further optimum conditions for alkaline protease synthesis were determined. The growth conditions for B. thuringiensis was optimised by inoculating into yeast extract casein medium at different pH and incubating at different temperatures. The maximum protease production occurred at pH 8 and at 37 °C. B. thuringiensis showed proteolytic activity at various culture conditions. Optimum conditions for the protease activity were found to be 47 °C and pH 8. In the later stage, the blood removing action of crude and partially purified protease was found to be effective within 25 min in the presence of commercial detergents indicating the possible use of this enzyme in detergent industry. Enzyme also showed good activity against hair substrate keratin and can be used for dehairing.     

Optimization of protease production by endophytic fungus,Alternaria alternata,isolated from an Australian native plant

Endophytes are recognised as potential sources of novel secondary metabolites, including enzymes and drugs, with applications in medicine, agriculture and industry. There is a growing need for new enzymes, including proteases, for use in industry that can function under a variety of conditions. In this study, three fungal endophytes (Alternaria alternata, Phoma herbarum and an unclassified fungus), were isolated from the Australian native plant, Eremophilia longifolia, and assessed for production of proteases. The lyophilised growth media obtained after fungal fermentation were analysed for protease production using enzyme activity assays. Protease production was optimised by assessing the effects of temperature, pH, carbon source and nitrogen source on activity. A. alternata showed the greatest protease activity in a wide range of pH (3–9). The broadest activity between 9 and 50 °C was observed at pH 7, suggesting a neutral protease. Overall, the optimum conditions were 37 °C and pH 7 with a maximum specific activity value of 69.86 BAEE units/mg. The characteristics demonstrated by this fungal endophyte showed that it is a potential source of an enzyme with particular application in the dairy industry. However, further studies of the tolerance to higher temperatures and pH will indicate whether the enzyme is suitable to such applications.     

Inoculation effect of thermophilic microorganisms on protease production through solid-state fermentation under non-sterile conditions at lab and bench scale (SSF)

The production of protease enzyme was evaluated through the solid state fermentation (SSF) of soy fibre, a waste product that acted as a sole substrate for the fermentation, at a laboratory and bench scale using a 500-mL (batch size 115 g) and 10-L (batch size 2300 g) bioreactors. The objective was to assess the effect of the inoculation of the thermophilic bacteria Thermus sp. on the production of the enzyme when working at laboratory and bench scale under non-sterile conditions, since scaling-up and the need of sterilization are the main challenges of SSF, preventing its industrial development. Results revealed that the inoculation led to a substantial increase in the protease obtained on both scales when compared to non-inoculated fermentation. The maximum protease activities increased as a result of the inoculation from 500 to 800 and from 350 to 670 U/g dry matter of soy fibre in the lab and bench scale bioreactors, respectively. Finally, a very good correlation was found between the protease activities obtained and the fermentation most relevant parameters: oxygen uptake rate (R ² = 0.81) and temperature (R ² = 0.82). In this work, we have demonstrated that inoculation is effective even under non-sterile conditions at the kg scale and that this strain is able to compete with autochthonous microbiota and increase the protease production to levels higher than those previously reported in literature.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.     

Effect of pH on the metabolic flux of Klebsiella oxytoca producing 2,3-butanediol in continuous cultures at different dilution rates

The efficiency of the bioconversion process and the achievable end-product concentration decides the economic feasibility of microbial 2,3-butanediol (2,3-BDO) production. In 2,3-BDO production, optimization of culture condition is required for cell growth and metabolism. Also, the pH is an important factor that influences microbial performance. For different microorganisms and substrates, it has been shown that the distribution of the metabolites in 2,3-BDO fermentation is greatly affected by pH, and the optimum pH for 2,3-BDO production seems dependently linked to the particular strain and the substrate employed. Quantification analysis of intracellular metabolites and metabolic flux analysis (MFA) were used to investigate the effect of pH on the Klebsiella oxytoca producing 2,3-BDO and other organic acids. The main objectives of MFA are the estimation of intracellular metabolic fluxes and the identification of rate-limiting step and the key enzymes. This study was conducted under continuous aerobic conditions at different dilution rates (0.1, 0.2, and 0.3 h^?1) and different pH values (pH 5.5 and 7.0) for the steady-state experimental data. In order to obtain the flux distribution, the extracellular specific rates were calculated from the experimental data using the metabolic network model of K. oxytoca. Intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.     

The Involvement of hybrid cluster protein 4, HCP4, in Anaerobic Metabolism in Chlamydomonas reinhardtii

The unicellular green algae Chlamydomonas reinhardtii has long been studied for its unique fermentation pathways and has been evaluated as a candidate organism for biofuel production. Fermentation in C. reinhardtii is facilitated by a network of three predominant pathways producing four major byproducts: formate, ethanol, acetate and hydrogen. Previous microarray studies identified many genes as being highly up-regulated during anaerobiosis. For example, hybrid cluster protein 4 (HCP4) was found to be one of the most highly up-regulated genes under anoxic conditions. Hybrid cluster proteins have long been studied for their unique spectroscopic properties, yet their biological functions remain largely unclear. To probe its role during anaerobiosis, HCP4 was silenced using artificial microRNAs (ami-hcp4) followed by extensive phenotypic analyses of cells grown under anoxic conditions. Both the expression of key fermentative enzymes and their respective metabolites were significantly altered in ami-hcp4, with nitrogen uptake from the media also being significantly different than wild-type cells. The results strongly suggest a role for HCP4 in regulating key fermentative and nitrogen utilization pathways.     

Tourmaline ceramic balls stimulate growth and metabolism of three fermentation microorganisms

Effects of tourmaline ceramic balls on growth and metabolism of Saccharomyces cerevisiae, Lactobacillus acidophilus and Aspergillus oryzae were studied. Treatments with 3, 6, 9 or 12 g of tourmaline ceramic balls in a 50 ml culture showed significant stimulation of the growth of the three microorganisms. In optimal treatments with 12 g of tourmaline balls, the growth of S. cerevisiae, L. acidophilus, and A. oryzae was increased by 34, 32 and 10%, respectively. After 72 h fermentation of S. cerevisiae, total carbohydrate content in the culture medium was decreased by 65% and ethanol production was increased by 150%. Total carbohydrate content was decreased by 80% and the pH value was decreased by 0.3, as a result of organic acid production in the medium of L. acidophilus after 72 h fermentation. In the case of A. oryzae, enzyme activities of protease and amylase were increased by 90 and 31%, respectively, after 96 h fermentation. Results indicated that tourmaline stimulates initiation of growth in the early lag stage and increases production of metabolites at a later stage of fermentation. The strong stimulatory effect of tourmaline on growth, utilization of substrates and production of metabolites in the three microorganisms suggests a potential application in the fermentation industry.     

On farm production of arbuscular mycorrhizal fungi inoculum using lignocellulosic agrowastes

The present study evaluated the efficiency of lignocellulosic agrowastes produced in Brazil as substrates for production of on farm AMF inoculum and tested different diluents and inoculation techniques. In a first experiment, Sorghum bicolor seedlings were colonized with Rhizophagus clarus or Claroideoglomus etunicatus and transplanted to 20 L bags containing sugarcane bagasse (SC), king palm leaf sheets (KP), or barley hulls (BH) mixed (1:1:1 or 2:1:1, v/v/v) with sand and rice shell. SC and KP were conducive for production of spores and infectious propagules. A number of infectious propagules obtained were greater than with BH and ranged from 233–350 propagules cm^?3 for both isolates in SC and KP at the1:1:1 mix dilution. Number of spores of both fungi was affected mainly by the SC agrowaste, and spore densities were significantly higher compared to KP and BH. In a second experiment, SC was mixed with soil or sand and inoculation consisted of transplanting colonized seedlings or adding soil inoculum. Number of propagules tended to differ for each fungus according to the inoculation technique or diluent. It is concluded from the data that SC and KP are suitable agrowastes to be incorporated in substrates for producing AMF inoculum using the on farm method.     

Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820?×?10³ U/L and extracellular protease activity of 172?×?10³ U/L were obtained at the 16th?hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.     

Purification and characterization of a new metal protease which hydrolyzes the cyclic decapeptide,gramicidin S

We screened a strain which can produce a new protease. The strain, Lactobacillus sp. no. 1, was isolated from a natural environment as an organism which could utilize gramicidin S as a sole nitrogen source. This strain was proved to produce much protease because it formed a large halo on a plate containing casein, and the protease was purified using ion exchange column chromatography. The amino-terminal amino acid sequence of the hydrolyzed products by the cleavage of gramicidin S was determined by a protein sequencer, and sizes of those products were analyzed by a mass spectrometer. The protease could cleave two peptide bonds between l-Orn-l-Leu in gramicidin S. These cleavage sites were different from other reported cleavage sites of gramicidin S by protease. The molecular weight of the protease was 42,000 by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the enzyme activity were 5.5 and 45°C, respectively. The enzyme activity was inhibited by EDTA, but not by phenylmethylsulfonyl fluoride (PMSF). Because the reported protease that can hydrolyze gramicidin S was a serine protease and the cleavage site was different from that of this protease from Lactobacillus sp. no. 1, we concluded that this enzyme was a new type of metal protease which can cleave both linear and cyclic peptide substrates with a unique substrate specificity.     

Nutritional factors affecting organic solvent-tolerant alkaline protease production by a newBacillus cereus strain 146

An organic solvent-tolerant bacterium designated as 146 capable of producing an organic solvent-stable alkaline protease was isolated from contaminated soil of a wood factory. The strain was a Gram-positive, spore-forming, nitrate-positive, rod-shaped organism capable of hydrolysing gelatine, starch, skim milk and identified asBacillus cereus. Activity of the protease was drastically increased in the presence of 1–decanol, isooctane, n-dodecane and n-tetradecane, but reduced in the presence of ethyl acetate, benzene, toluene, 1-heptanol, ethylbenzene and hexane. The bacterium was shown to require lactose as a carbon source and peptone as a nitrogen source. The optimum fermentation condition for the production of alkaline protease was in the presence of beef and yeast extract. Optimum pH was determined to be at 10.0 at incubation temperature of 37 °C for 48 h. Results from the studies suggest that 146 is a new strain of Bacillus cereus capable of producing organic solvent-tolerant alkaline protease with potential use in industries.     

Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S⁰). Growth rates were highest on media containing peptides and S⁰, with or without maltose. Growth did not occur on the peptide medium without S⁰. S⁰ had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S⁰ with or without maltose were the same, suggesting that S⁰ is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S⁰ in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S⁰-reducing enzyme in this organism and the mechanism of the S⁰ dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.     

Superoxide dismutase, protease and lipase expression in clinical isolates of Staphylococcus aureus: a tool for antimicrobial drug discovery

The rising incidents of invasive infections due to multidrug resistant Staphylococcus aureus necessitate the exploration of newer targets for development of antibiotics. Pathogenicity of S. aureus is attributed to a wide range of virulence factors. The aim of this study was to screen the production of three virulence factors viz. extracellular protease, extracellular lipase and superoxide dismutase in human pathogenic strains of S. aureus for development of a test panel which could aid in screening of natural products of plant and microbial origin. 27 clinical isolates were compared for their enzyme expression profiles of which eight were finally selected. Sau G5 was the only protease producing organism selected in the test panel, while Sau G3 and Sau G9 were best SOD producers and Sau G16, Sau G18, Sau G22, Sau A5 and Sau A2 exhibited highest expression among different groups of clinical staphylococci.     

Cloning and functional expression of the mitochondrial alternative oxidase gene (aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L. lactis as a host strain. Expression of aox1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. lactis showed that the alternative respiratory pathway operates and improves significantly the microorganism's response to oxidizing stress conditions as it enhances biomass production, suppresses lactate formation, and leads to accumulation of large amounts of nisin.     

Identification of a New Marine Bacterial Strain SD8 and Optimization of Its Culture Conditions for Producing Alkaline Protease

While much attention has been given to marine microorganisms for production of enzymes, which in general are relatively more stable and active compared to those from plants and animals, studies on alkaline protease production from marine microorganisms have been very limited. In the present study, the alkaline protease producing marine bacterial strain SD8 isolated from sea muds in the Geziwo Qinhuangdao sea area of China was characterized and its optimal culture conditions were investigated. Strain SD8 was initially classified to belong to genus Pseudomonas by morphological, physiological and biochemical characterizations, and then through 16S rDNA sequence it was identified to be likely Pseudomonas hibiscicola. In addition, the culture mediums, carbon sources and culture conditions of strain SD8 were optimized for maximum production of alkaline protease. Optimum enzyme production (236U/mL when cultured bacteria being at 0.75 mg dry weight/mL fermentation broth) was obtained when the isolate at a 3% inoculum size was grown in LB medium at 20 mL medium/100mL Erlenmeyer flask for 48h culture at 30°C with an initial of pH 7.5. This was the first report of strain Pseudomonas hibiscicola secreting alkaline protease, and the data for its optimal cultural conditions for alkaline protease production has laid a foundation for future exploration for the potential use of SD8 strain for alkaline protease production.     

Investigations on alkaline protease production with B. subtilis PE-11 immobilized in calcium alginate gel beads

Bacillus subtilis PE-11 cells were immobilized in calcium alginate and used for the production of alkaline protease. The influence of alginate concentration, different cations, concentration of cation, curing time, bead diameter and nutrient strength on alkaline protease production and stability of biocatalyst were investigated. Repeated batch fermentations of immobilized cells in shake flasks were carried out with the optimized parameters such as; 3% alginate, 0.25 M calcium chloride with 1 h curing time, 3.24 mm bead diameter and 0.75% glucose and 0.75% peptone as nutrients. The results indicated that, a good level of enzyme was maintained for a period of about 9 days. The immobilized cells of B. subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation.