Green extraction of puromycin-based antibiotics from Streptomyces albofaciens (MS38) for sustainable biopharmaceutical applications.

Background: Microbial secondary metabolites have shown promise as a source of novel antimicrobial agents. In this study, we aimed to isolate, characterize, and evaluate the antimicrobial activity of compound from a novel Streptomyces albofaciens strain MS38. The objective was to identify a potential bioactive compound with broad-spectrum antimicrobial properties. Methods: The isolated strain MS38 on starch casein agar was characterized using morphological, physiological, and molecular identification techniques. The compound was obtained from the fermented broth through extraction with n-butanol and further purification using silica gel column chromatography and high-performance liquid chromatography (HPLC). Structural elucidation was conducted using Ultraviolet (UV), Infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS) techniques. The antimicrobial activity was evaluated using the agar well diffusion method and the microplate Alamar blue assay (MABA). Results: The isolated strain MS38 was identified as novel S. albofaciens based on morphological characteristics and confirmed by 16S sequences analysis and MALDI-TOF MS. The compound obtained from the fermented broth exhibited substantial antimicrobial activity against a variety of pathogenic bacteria and fungi. Structural analysis revealed a complex chemical structure with characteristic functional groups indicative of potential antimicrobial properties. The compound demonstrated strong activity against both Gram-positive (Staphylococcus Spp.) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, as well as fungi, including Candida albicans and Trichophyton rubrum. Conclusion: This study successfully isolated and characterized a bioactive compound from a novel S. albofaciens MS38. The compound exhibited significant antimicrobial activity against a range of pathogenic microorganisms. These findings underscore the importance of exploring microbial biodiversity for the discovery of novel antimicrobial agents. This study contributes to the growing knowledge of microbial secondary metabolites with potential therapeutic value.

Isolation, Purification, and Characterization of Heparinase from Streptomyces variabilis MTCC 12266.

Arterial/venous thrombosis is the major cardiovascular disorder accountable for substantial mortality; and the current demand for antithrombotic agents is extensive. Heparinases depolymerize unfractionated heparin (UFH) for the production of low molecular-weight heparins (LMWHs; used as anticoagulants against thrombosis). A microbial strain of Streptomyces sp. showing antithrombotic activity was isolated from the soil sample collected from north India. The strain was characterized by using 16S rRNA homology technique and identified as Streptomyces variabilis MTCC 12266 capable of producing heparinase enzyme. This is the very first communication reporting Streptomyces genus as the producer of heparinase. It was observed that the production of intracellular heparinase was [63.8 U/mg protein (specific activity)] 1.58 folds higher compared to extracellular heparinase [40.28 U/mg protein]. DEAE-Sephadex A-50 column followed by Sepharose-6B column purification of the crude protein resulted 19.18 folds purified heparinase. SDS-PAGE analysis of heparinase resulted an estimated molecular-weight of 42 kDa. It was also found that intracellular heparinase has the ability to depolymerize heparin to generate LMWHs. Further studies related to the mechanistic action, structural details, and genomics involved in heparinase production from Streptomyces variabilis are warranted for large scale production/purification optimization of heparinase for antithrombotic applications.

Isolation and purification of antibacterial compound from Streptomyces levis collected from soil sample of north India.

During the screening programme for microbial cultures producing antimicrobial agents, an active microbial strain of Streptomyces was isolated from the agricultural soil of Narnaul, Haryana India. Physiological, biochemical characteristics and 16S ribosomal RNA sequence homology studies revealed that it was similar to Streptomyces levis (sequence similarity 100%). The microbial strain was submitted to Genomebio Technologies Pvt. Ltd., Pune, Maharashtra, India under Accession No. EU124569. The isolated strain was found to produce extracellular active compound showing strong antimicrobial activity against Klebsiella pneumoniae MTCC 109, Pseudomonas aeruginosa MTCC 741 and Staphylococcus aureus MTCC 96. The antibacterial compound was successfully isolated and purified. Structure elucidation of antibacterial metabolite with EI-MS/ HRMS showed molecular ion peak at m/z 686 [M+H]+. Whereas, elemental analysis of the said compound showed C = 61.31, H = 8.61, N = 2.04 and O = 28.02, and indicated a molecular formula of C35H59NO12. The presence of 'chromone' nucleus in the compound's chemical structure was confirmed by using 1HNMR studies. The present study reports the purification of potential antibacterial compound from Streptomyces levis isolated from the unexplored soil of north India and warrants for further characterization of this potential compound for optimum utilization for antimicrobial purposes.

Response Surface Methodology-Genetic Algorithm Based Medium Optimization, Purification, and Characterization of Cholesterol Oxidase from Streptomyces rimosus.

The applicability of the statistical tools coupled with artificial intelligence techniques was tested to optimize the critical medium components for the production of extracellular cholesterol oxidase (COD; an enzyme of commercial interest) from Streptomyces rimosus MTCC 10792. The initial medium component screening was performed using Placket-Burman design with yeast extract, dextrose, starch and ammonium carbonate as significant factors. Response surface methodology (RSM) was attempted to develop a statistical model with a significant coefficient of determination (R2 = 0.89847), followed by model optimization using Genetic Algorithm (GA). RSM-GA based optimization approach predicted that the combination of yeast extract, dextrose, starch and ammonium carbonate at concentrations 0.99, 0.8, 0.1, and 0.05 g/100 ml respectively, has resulted in 3.6 folds increase in COD production (5.41 U/ml) in comparison with the un-optimized medium (1.5 U/ml). COD was purified 10.34 folds having specific activity of 12.37 U/mg with molecular mass of 54 kDa. The enzyme was stable at pH 7.0 and 40 °C temperature. The apparent Michaelis constant (Km) and Vmax values of COD were 0.043 mM and 2.21 µmol/min/mg, respectively. This is the first communication reporting RSM-GA based medium optimization, purification and characterization of COD by S. rimosus isolated from the forest soil of eastern India.

Coumarin-benzimidazole hybrids as a potent antimicrobial agent: synthesis and biological elevation.

Molecular hybridization approach is an emerging tool in drug discovery for designing new pharmacophores with biological activity. A novel, new series of coumarin-benzimidazole hybrids were designed, synthesized and evaluated for their broad spectrum antimicrobial activity. Among all the synthesized molecules, compound (E)-3-(2-1H-benzo[d]imidazol-1-yl)-1-((4-chlorobenzyl)oxy)imino)ethyl)-2H-chromen-2-one showed the most promising broad spectrum antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Proteus vulgaris. In addition, it has showed no cytotoxicity and hemolysis at 10 times the MIC concentration. SAR studies indicate that position of the chlorine atom in the hybrid critically determines the antibacterial activity.

Isolation, Screening, and Identification of Novel Isolates of Actinomycetes from India for Antimicrobial Applications.

The search for novel bioactive compounds from the natural environment has rapidly been gaining momentum with the increase in multi-drug resistant (MDR) pathogens. In the present study, the antimicrobial potential of novel actinomycetes has been evaluated by initial screening of six soil samples. Primary and secondary screening was performed against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans, Candida tropicalis, Trichophyton rubrum, and other MDR bacterial and fungal test strains, thirteen active isolates were selected for further study. Microbial strains were identified on the basis of growth conditions and other biochemical characters. Five most active microbial strains were identified using 16S rRNA sequence homology and designated as Streptomyces xanthophaeus MTCC 11938, Streptomyces variabilis MTCC 12266, Streptomyces xanthochromogenes MTCC 11937, Streptomyces levis EU 124569, and Streptomyces sp. NCIM 5500. Four antibacterial and three antifungal compounds isolated from the above five isolates were purified and partially characterized using UV absorption and IR spectra. Two antibacterial metabolites, belong to chromone and peptide antibiotic, respectively. The antifungal compounds were found to be of non-polyene nature. In conclusion, we study the isolation of novel bacterial strains of actinomycetes for producing novel compounds having antibacterial and antifungal activities from the unexplored agro-ecological niches of India. Also, this study paves the way for further characterization of these isolates of Streptomyces sp. for their optimum utilization for antimicrobial purposes.

Purification and characterization of chitinase from Streptomyces violascens NRRL B2700.

Chitinase is one of the important enzymes as it is directly linked to Chitin that has wide applications in industrial, medical and commercial fields for its biocompatibility and biodegradability. Here, we report extracellular chitinase production by Streptomyces violascens NRRL B2700 under submerged fermentation condition. Chitinase production started after 10 h of incubation and reached to maximum level at 72 h of cultivation. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that maltose, xylose, fructose, lactose, soybean meal and ammonium nitrate served as good carbon and nitrogen sources to enhance chitinase yield by 1.6 to 6 fold. Medium supplemented with 1% colloidal chitin produced high chitinase concentration (0.1714 U/mg). The enzyme chitinase was purified from the culture broth by 75% ammonium sulphate precipitation, DEAE-cellulose ion-exchange and sephadex G-100 gel filtration. The molecular mass of the purified chitinase was 65 kDa as estimated by SDS-PAGE. The apparent Michaelis constant (K(m)) and the maximum rate (V(max)) of the enzyme for colloidal chitin were 1.556 mg/mL and 2.680 µM/min/mg, respectively suggested high affinity towards-chitin. Possibly, it is the first report on production of chitinase from S. violascens NRRL B2700. The findings were encouraging, especially for cost effective production, and further warrants media and purification optimization studies for enhanced yield.

Strategies for Fermentation Medium Optimization: An In-Depth Review.

Optimization of production medium is required to maximize the metabolite yield. This can be achieved by using a wide range of techniques from classical "one-factor-at-a-time" to modern statistical and mathematical techniques, viz. artificial neural network (ANN), genetic algorithm (GA) etc. Every technique comes with its own advantages and disadvantages, and despite drawbacks some techniques are applied to obtain best results. Use of various optimization techniques in combination also provides the desirable results. In this article an attempt has been made to review the currently used media optimization techniques applied during fermentation process of metabolite production. Comparative analysis of the merits and demerits of various conventional as well as modern optimization techniques have been done and logical selection basis for the designing of fermentation medium has been given in the present review. Overall, this review will provide the rationale for the selection of suitable optimization technique for media designing employed during the fermentation process of metabolite production.

Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO4, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500.

Isolation, characterization and antifungal docking studies of wortmannin isolated from Penicillium radicum.

During the search for a potent antifungal drug, a cell-permeable metabolite was isolated from a soil isolate taxonomically identified as Penicillium radicum. The strain was found to be a potent antifungal agent. Production conditions of the active compound were optimized and the active compound was isolated, purified, characterized and identified as a phosphoinositide 3-kinase (PI3K) inhibitor, commonly known as wortmannin (Wtmn). This is very first time we are reporting the production of Wtmn from P. radicum. In addition to its previously discovered anticancer properties, the broad spectrum antifungal property of Wtmn was re-confirmed using various fungal strains. Virtual screening was performed through molecular docking studies against potential antifungal targets, and it was found that Wtmn was predicted to impede the actions of these targets more efficiently than known antifungal compounds such as voriconazole and nikkomycin i.e. 1) mevalonate-5-diphosphate decarboxylase (1FI4), responsible for sterol/isoprenoid biosynthesis; 2) exocyst complex component SEC3 (3A58) where Rho- and phosphoinositide-dependent localization is present and 3) Kre2p/Mnt1p a Golgi alpha1,2-mannosyltransferase (1S4N) involved in the biosynthesis of yeast cell wall glycoproteins). We conclude that Wtmn produced from P. radicum is a promising lead compound which could be potentially used as an efficient antifungal drug in the near future after appropriate structural modifications to reduce toxicity and improve stability.

A modified Lumry-Eyring analysis for the determination of the predominant mechanism underlying the diminution of protein aggregation by glycerol.

Aggregation of aspartate-ß-semialdehyde dehydrogenase (ASD) was analyzed by applying modified Lumry-Eyring with nucleated polymerization (LENP) model. Intrinsic nucleation time scales were determined. In absence of glycerol, ASD undergoes concentration and time-dependent polymerization into low-molecular weight soluble aggregates and thereafter condensation into insoluble aggregates. In the presence of increasing solvent glycerol concentration, the aggregation becomes more and more nucleation dominated, with slower polymerization to low-molecular weights soluble aggregates, without any condensation into insoluble aggregates. Effective nucleus size as well as the number of monomers in each irreversible growth event were sensitive to the changes in solvent glycerol concentration. Glycerol-directed diminution of aggregation appears to be largely due to the inhibition of rearrangement (decreased nucleation rearrangement rate coefficient, K r,x ) because of compaction induced due to preferential hydration, thus, preventing the soluble aggregates from locking into irreversible soluble nuclei. Appreciably decreased K r,x (as compared to nucleation dissociation constant, K d,x ), appears to be responsible for increased nucleus size at higher solvent glycerol concentration. This study explains how modified LENP model can be applied to determine the predominant mechanism responsible for the diminution of aggregation by polyhydric alcohols (glycerol).

Production, purification and characterization of cholesterol oxidase from a newly isolated Streptomyces sp.

Cholesterol oxidase production (COD) by a new isolate characterized as Streptomyces sp. was studied in different production media and fermentation conditions. Individual supplementation of 1 % maltose, lactose, sucrose, peptone, soybean meal and yeast extract enhanced COD production by 80-110 % in comparison to the basal production medium (2.4 U/ml). Supplementation of 0.05 % cholesterol (inducer) enhanced COD production by 150 %. COD was purified 14.3-fold and its molecular weight was found to be 62 kDa. Vmax (21.93 µM/min mg) and substrate affinity Km (101.3 µM) suggested high affinity of the COD for cholesterol. In presence of Ba(2+) and Hg(2+) the enzyme activity was inhibited while Cu(2+) enhanced the activity nearly threefold. Relative activity of the enzyme was found maximum in triton X-100 whereas sodium dodecyl sulfate inactivated the enzyme. The enzyme activity was also inhibited by the thiol-reducing reagents like Dithiothreitol and ß-mercaptoethanol. The COD showed moderate stability towards all organic solvents except acetone, benzene and chloroform. The activity increased in presence of isopropanol and ethanol. The enzyme was most active at pH 7 and 37 °C temperature. This organism is not reported to produce COD.

[Purification and chemical characterization of antimicrobial compounds from a new soil isolate Streptomyces rimosus MTCC 10792].

A new isolate of Streptomyces sp. from soil of state Chhattisgarh (India) having broad spectrum antibacterial and antifungal activity was obtained. The active strain was identified as Streptomyces rimosus subsp. rimosus with accession number MTCC 10792 based on physiological, biochemical characteristics and 16S rRNA sequence homology studies. Antimicrobial compound produced by S. rimosus was tested against the drug resistance pathogens by the Bauer and Kirby method. The crude active metabolite was extracted using solvent n-butanol and purified by silica column chromatography and HPLC method. The physicochemical characteristics of the one purified compound viz. color, melting point, solubility, elemental analysis; ESIMS, IR,UV, 1HNMR, 13CNMR and chemical reactions have been investigated. Purified antimicrobial compound produced by S. rimosus MTCC 10792 at concentration 25 µg/ml showed antitubercular activity against Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis H37Ra as well as broad activity against all tested bacterial and fungal pathogens.

Enhanced antibiotic production by Streptomyces sindenensis using artificial neural networks coupled with genetic algorithm and Nelder-Mead downhill simplex.

Antibiotic production with Streptomyces sindenensis MTCC 8122 was optimized under submerged fermentation conditions by artificial neural network (ANN) coupled with genetic algorithm (GA) and Nelder-Mead downhill simplex (NMDS). Feed forward back-propagation ANN was trained to establish the mathematical relationship among the medium components and length of incubation period for achieving maximum antibiotic yield. The optimization strategy involved growing the culture with varying concentrations of various medium components for different incubation periods. Under non-optimized condition, antibiotic production was found to be 95 microgram/ml, which nearly doubled (176 microgram/ml) with the ANN-GA optimization. ANN-NMDS optimization was found to be more efficacious, and maximum antibiotic production (197 microgram/ml) was obtained by cultivating the cells with (g/l) fructose 2.7602, MgSO4 1.2369, (NH4)2PO4 0.2742, DL-threonine 3.069%, and soyabean meal 1.952%, for 9.8531 days of incubation, which was roughly 12% higher than the yield obtained by ANN coupled with GA under the same conditions.

Microbial heparin/heparan sulphate lyases: potential and applications.

Heparin/heparan sulphate glycosaminoglycans (HSGAGs) are composed of linear chains of 20-100 disaccharide units of N-acetylated D: -glucosamine α (1-4) linked to glucuronic acid. HSGAGs are widely distributed on the cell surface and extracellular cell matrix of virtually every mammalian cell type and play critical role in regulating numerous functions of blood vessel wall, blood coagulation, inflammation response and cell differentiation. These glycosaminoglycans present in this extracellular environment very significantly influence the blood coagulation system and cardiovascular functions. Recent studies have investigated the mechanism by which cancer causes thrombosis and emphasizes the importance of the coagulation system in angiogenesis and tumour metastasis. Heparan sulphate/heparin lyases or heparinases are a class of enzymes that are capable of specifically cleaving the (1-4) glycosidic linkages in heparin and heparan sulphate to generate biologically active oligosaccharides with substantially significant and distinct clinical, pharmaceutical and prophylactic/therapeutic applications. Bioavailability and pharmacokinetic behaviour and characteristics of these oligosaccharides vary significantly depending on the origin/nature of the substrate (heparin or heparan sulphate-like glycosaminoglycans), the source of enzyme and method of preparation. Various microorganisms are reported/patented to produce these enzymes with different properties. Heparinases are commercially used for the depolymerization of unfractionated heparin to produce low molecular weight heparins (LMWHs), an effective anticoagulant. Individual LMWHs are chemically different and unique and thus cannot be interchanged therapeutically. Heparinases and LMWHs are reported to control angiogenesis and metastasis also. This review catalogues the degradation of HSGAGs by microbial heparin/heparan sulphate lyases and their potential either specific to the enzymes or with the dual role for generation of oligosaccharides for a new generation of compounds, as shown by various laboratory or clinical studies.

Purification and characterization of the enzyme cholesterol oxidase from a new isolate of Streptomyces sp.

An extracellular cholesterol oxidase (cho) enzyme was isolated from the Streptomyces parvus, a new source and purified 18-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 20 U/mg with a 55 kDa molecular mass. The enzyme was stable at pH 7.2 and 50 °C. The enzyme activity was inhibited in the presence of Pb(2+), Ag(2+), Hg(2+), and Zn(2+) and enhanced in the presence of Mn(2+). The enzyme activity was inhibited by the thiol-reducing reagents (DTT, ß-mercaptoethanol), suggesting that disulfide linkage is essential for the enzyme activity. The enzyme activity was found to be maximum in the presence of Triton X-100 and X-114 detergents whereas sodium dodecyl sulfate fully inactivated the enzyme. The enzyme showed moderate stability towards all organic solvents except acetone, benzene, chloroform and the activity increased in the presence of isopropanol and ethanol. The K(m) value for the oxidation of cholesterol by this enzyme was 0.02 mM.

Maximizing the native concentration and shelf life of protein: a multiobjective optimization to reduce aggregation.

A multiobjective optimization was performed to maximize native protein concentration and shelf life of ASD, using artificial neural network (ANN) and genetic algorithm (GA). Optimum pH, storage temperature, concentration of protein, and protein stabilizers (Glycerol, NaCl) were determined satisfying the twin objective: maximum relative area of the dimer peak (native state) after 48 h of storage, and maximum shelf life. The relative area of the dimer peak, obtained from size exclusion chromatography performed as per the central composite design (CCD), and shelf life (obtained as turbidity change) served as training targets for the ANN. The ANN was used to establish mathematical relationship between the inputs and targets (from CCD). GA was then used to optimize the above determinants of aggregation, maximizing the twin objectives of the network. An almost fourfold increase in shelf life (~196 h) was observed at the GA-predicted optimum (protein concentration: 6.49 mg/ml, storage temperature: 20.8 °C, Glycerol: 10.02%, NaCl: 51.65 mM and pH: 8.2). Since no aggregation was observed at the optimum till 48 h, all the protein was found at the dimer position with maximum relative area (64.49). Predictions of the finally adapted network also reveal that storage temperature and solvent glycerol concentration plays key role in deciding the degree of ASD aggregation. This multiobjective optimization strategy was also successfully applied in minimizing the batch culture period and determining optimum combination of medium components required for most economical production of actinomycin D.

Purification and characterization of a novel heparin degrading enzyme from Aspergillus flavus (MTCC-8654).

A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/microg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 degrees C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co2+, Mn2+ ions, and reducing agents (beta-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K (m) and V (max) as 2.2 x 10(-5 )M and 30.8 mM min(-1), respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.

Studies on the production of actinomycin-D by Streptomyces griseoruber--a novel source.

AIMS: To isolate and characterize bioactive metabolites produced by a micro-organism isolated from a soil sample associated with the roots of a medicinal plant, Azadirachta indica. METHODS AND RESULTS: Morphological, cultural, physiological and 16S rRNA homology studies revealed that the organism showed 99% similarity with Streptomyces griseoruber NBRC 12873. One bioactive metabolite (Py2) isolated from the fermented broth was characterized as actinomycin-D (act-D). It showed high activity against various gram-positive and gram-negative bacterial cultures, Mycobacterium tuberculosis H37Rv and human neoplastic cells in vitro using standard protocols. CONCLUSIONS: The isolated strain S. griseoruber produced act-D predominantly (210 mg l(-1), c. 88% of the crude) under nonoptimized growth conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptomyces griseoruber may be exploited as a potential source for the commercial production of act-D, as this strain is not reported to produce act-D. Further investigations on the strain for commercial application will be of immense pharmaceutical importance.

Response surface methodology for optimization of medium components in submerged culture of Aspergillus flavus for enhanced heparinase production.

AIMS: Aim of the study was to develop a medium for optimal heparinase production with a strain of Aspergillus flavus (MTCC-8654) by using a multidimensional statistical approach. METHODS AND RESULTS: Statistical optimization of intracellular heparinase production by A. flavus, a new isolate, was investigated. Plackett-Burman design was used to evaluate the affect of medium constituents on heparinase yield. The experimental results showed that the production of heparinase was dependent upon heparin, the inducer; chitin, structurally similar to heparin and NH(4)NO(3,) the nitrogen source. A central composite design was applied to derive a statistical model for optimizing the composition of the fermentation medium for the production of heparinase enzyme. The optimum fermentation medium consisted of (g l(-1)) Mannitol, 8.0; NH(4)NO(3), 2.5; K(2)HPO(4), 2.5; Na(2)HPO(4), 2.5; MgSO(4).7H(2)O, 0.5; Chitin, 17.1; Heparin, 0.6; trace salt solution (NaMoO(4).2H(2)O, CoCl(2).6H(2)O, CuSO(4).5H(2)O, FeSO(4).7H(2)O, CaCl(2)), 10(-4) mol l(-1). CONCLUSIONS: A 2.37-fold increase in heparinase production was achieved in economic and effective manner by the application of statistical designs in medium optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: Heparinase production was doubled by statistical optimization in a cost-effective manner. This heparinase can find application in pharmaceutical industry and for the generation of low-molecular-weight heparins, active as antithrombotic and antitumour agents.