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Rani Gupta

Publications and source records attributed to Rani Gupta.

17 recordsLinked to original sources
PubMed2006

Microbial keratinases and their prospective applications: an overview.

Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide "keratin" recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.

Bacteria↗
PubMed2005

A simple activity staining protocol for lipases and esterases.

A simple activity staining protocol for rapid detection and differentiation of lipases and esterases was developed based on pH drop due to fatty acids released following lipolysis. Though the detection of lipolysis as a function of drop in pH is not new, the present method has been made more sensitive by the judicious selection of the initial pH of the chromogenic substrate, which has been set near the end point of the dye so that even a slight drop in pH results in immediate color change. In the present case, the dye phenol red was taken, which has the end point at pH 7.3-7.4 where the color is pink. A slight drop due to fatty acid release results in yellow coloration. The assay has high reproducibility and can detect as low as 0.5 p-NPP enzyme units within 15 min. In addition, this method can be used for various lipidic substrates such as oils and tributyrin, making it suitable for both lipases and esterases.

Burkholderia↗
PubMed2005

Single-step purification of lipase from Burkholderia multivorans using polypropylene matrix.

Lipase from Burkholderia multivorans was purified with high yields directly from fermentation broth by a single-step purification protocol involving adsorption and desorption. The crude enzyme (lyophilized powder) from B. multivorans was loaded on Accurel (Membrana, Germany), a polypropylene matrix, using butanol as the solvent in a buffer at pH 9.0 and ambient temperature for a period of 12 h. The enzyme adsorbed onto the matrix with high specific activity (33 units mg(-1) protein). This was followed by desorption of the enzyme from the matrix using Triton X-100 as the eluent. The enzyme was finally recovered by precipitation with acetone (50%, v/v). Thus, an overall enzyme yield of 66% with a 3.0-fold purification was obtained. The purity of the enzyme was ascertained by SDS-PAGE. The phenomenon of adsorption and desorption on Accurel was studied for three more lipases, viz. Mucor meihei lipase (Sigma-Aldrich Co.), Lipolase (Novo Nordisk, Denmark) and Pseudomonas aeruginosa lipase (laboratory isolate).

1-Butanol↗
PubMed2005

Concurrent sorption of Zn(II), Cu(II) and Co(II) by Oscillatoria angustissima as a function of pH in binary and ternary metal solutions.

This paper reports biosorption of Zn(II), Cu(II) and Co(II) onto O. angustissima biomass from single, binary and ternary metal solutions, as a function of pH and metal concentrations via Central Composite Design generated by statistical software package Design Expert 6.0. The experimental design revealed that metal interactions could be best studied at lower pH range i.e. 4.0-5.0, which facilitates adequate availability of all the metal ions. The sorption capacities for single metal decreased in the order Zn(II)>Co(II)>Cu(II). In absence of any interfering metals, at pH 4.0 and an initial metal concentration of 0.5 mM in the solution, the adsorption capacities were 0.33 mmol/g Zn(II), 0.26 mmol/g Co(II) and 0.12 mmol/g Cu(II). In a binary system, copper inhibited both Zn(II) and Co(II) sorption but the extent of inhibition of former was greater than the latter; sorption values being 0.14 mmol/g Zn(II) and 0.27 mmol/g Co(II) at initial Zn(II) and Co(II) concentration of 1.5 mM each, pH 4.0 and 1mM Cu(II) as the interfering metal. Zn(II) and Co(II) were equally antagonistic to each others sorption; Zn(II) and Co(II) sorption being 0.23 and 0.24 mmol/g, respectively, at initial metal concentration of 1.5 mM each, pH 4.0 and 1mM interfering metal concentration. In contrast, Cu(II) sorption remained almost unaffected at lower concentrations of the competing metals. Thus, in binary system inhibition dominance observed was Cu(II)>Zn(II), Cu(II)>Co(II) and Zn(II) approximately Co(II), due to this the biosorbent exhibited net preference/affinity for Cu(II) sorption over Zn(II) or Co(II). Hence, the affinity series showed a trend of Cu(II)>Co(II)>Zn(II). In a ternary system, increasing Co(II) concentration exhibited protection against the inhibitory effect of Cu(II) on Zn(II) sorption. On the other hand, the inhibitory effect of Zn(II) and Cu(II) on Co(II) sorption was additive. The model equation for metal interactions was found to be valid within the design space.

Algorithms↗
PubMed2005

Keratinolytic potential of Bacillus licheniformis RG1: structural and biochemical mechanism of feather degradation.

Keratinolytic Bacillus licheniformis RG1 was used to study the mechanism of keratinolysis. Scanning electron microscopy studies revealed that bacterial cells grew closely adhered to the barbules of feathers, completely degrading them within 24 h. Biochemical studies indicated that the Bacillus strain produced an extracellular protease, which had keratinolytic potential. The extracellular keratinolytic activity (425 U) was synergistically enhanced by the addition of intracellular disulfide reductases (1712 U). However, these enzymes alone (keratinase and disulfide reductase), without live bacterial cells, failed to degrade the feather. Complete feather degradation was obtained only when living bacterial cells were present, emphasizing that bacterial adhesion plays a key role during the degradation process. The bacterial cells probably provide a continuous supply of reductant to break disulfide bridges. In addition, sulfite detected in the extracellular broth during feather degradation indicated that sulfitolysis may also play a role in feather degradation by the bacterium.

Animals↗
PubMed2004

Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RG1 using statistical methods involving response surface methodology.

A 3.5-fold increase in keratinase production by Bacillus licheniformis RG1 was achieved by using statistical methods involving Plackett-Burman design and response surface methodology. Eight variables were screened using Plackett-Burman design. Of these, glucose, peptone and glutathione were found to affect the response signal positively, whereas CaCl(2) had a negative effect. Further interaction of these factors, along with phosphate and incubation time, was studied using response surface methodology. An optimum keratinase production of 1295 units/mg dry weight was obtained with the following medium composition: 1% glucose, 1% peptone, 1% phosphate, 0.05% glutathione, 0.5% feather and 2% inoculum under shaking at 250 rev./min with an incubation period of 72 h at 37 degrees C. Keratinase production was found to be a function of biomass and maximum production occurred during the stationary phase.

Algorithms↗
PubMed2004

A glycerol-inducible thermostable lipase from Bacillus sp.: medium optimization by a Plackett-Burman design and by response surface methodology.

The production of a neutral lipase from a Bacillus sp. was improved tremendously (193-fold) following media optimization involving both the "one-at-a-time" and the statistical designing approaches. The present lipase was poorly induced by oils, instead its production was induced in the presence of sugars and sugar alcohols, mainly galactose, lactose, glycerol, and mannitol. A high inoculum density of 15% v/v (A550 = 0.8) led to maximum lipase production. Interestingly, the enzyme induction was growth independent, a property very different from most of the lipases investigated to date. The optimal composition of the growth medium to achieve maximum lipase production was determined to be as follows: NH4Cl, 35 g x L(-1); glycerol, 10 mL x L(-1); K2HPO4, 3 g x L(-1); KH2PO4, 1 g x L(-1); MgSO4.7H2O, 0.1 g x L(-1); glucose, 2 g x L(-1); MgCl2, 0.6 mmol x L(-1), with 15% inoculum density and an incubation period of 24 h. About 62 U x mL(-1) of enzyme production was achieved in the optimized medium.

Ammonium Chloride↗
PubMed2003

Lipase assays for conventional and molecular screening: an overview.

Lipases are versatile biocatalysts that can perform innumerable different reactions. Their enantio-, chemo- and stereo-selective nature makes them an important tool in the area of organic synthesis. Unlike other hydrolases that work in aqueous phase, lipases are unique as they act at the oil/water interface. Besides being lipolytic, lipases also possess esterolytic activity and thus have a wide substrate range. Hence, the lipase assay protocols hold a significant position in the field of lipase research. Lipase activity can be estimated using a wide range of assay protocols that differ in terms of their basic principle, substrate selectivity, sensitivity and applicability. As the value of these enzymes continues to grow and new markets are exploited, development of new or improved enzymes will be a key element in the emerging realm of biotechnology. Hence, development of faster and simpler protocols incorporating newer and more specific substrates is the need of the hour. In this endeavour, methods that could be adopted for molecular screening occupy an important position. Here, an overview of the lipase assay protocols is presented with emphasis on the assays that can be adopted for the molecular screening of these biocatalysts.

Enzyme Activation↗
PubMed2003

Lipase mediated upgradation of dietary fats and oils.

In the present scenario, fats and oil modification is one of the prime areas in food processing industry that demands novel economic and green technologies. In this respect, tailored vegetable oils with nutritionally important structured triacylglycerols and altered physicochemical properties have a big potential in the future market. In this context, it is well established that lipases especially microbial lipases, which are regiospecific and fatty acid specific, are of immense importance and hence could be exploited for retailoring of vegetable oils. Further, of the bulk available, cheap oils could also be upgraded to synthesize nutritionally important structured triacylglycerols like cocoa butter substitutes, low calorie triacylglycerols, PUFA-enriched and oleic acid enriched oils. It is also possible to change the physical properties of natural oils to convert them into margarines and hard butter with higher melting points or into special low calorie spreads with short or medium chain fatty acids. Today, by and large, fat and oil modifications are carried out chemically following the method of directed inter-esterification. The process is energy intensive and non-specific. Lipase mediated modifications are likely to occupy a prominent place in oil industry for tailoring structured lipids since enzymatic modifications are specific and can be carried out at moderate reaction conditions. However, as a commercial venture, lipases are yet to be fully exploited. Once the technologies are established, the demand of lipases in oil industry is expected to increase tremendously in the near future for specific modifications of fats and oils to meet the changing consumers' dietary requirements.

Animals↗
PubMed2003

Microbial biomass: an economical alternative for removal of heavy metals from waste water.

Today indiscriminate and uncontrolled discharge of metal contaminated industrial effluents into the environment has become an issue of major concern. Heavy metals, being non-biodegradable and persistent, beyond a permissible concentration form unspecific compounds inside the cells thereby causing cellular toxicity. The only alternative to remove them from the wastewater is by immobilizing them. The conventional methods adopted earlier for this purpose included chemical precipitation, oxidation, reduction, filtration, electrochemical treatment, evaporation, adsorption and ion-exchange resins. These methods require high energy inputs especially when it refers to dilute solutions. Here microbial biomass offers an economical option for removing heavy metals by the phenomenon of biosorption. Non-living or dead biomass sequester metal(s) on their cell surface due to certain reactive groups available like carboxyl, amine, imidazole, phosphate, sulphydryl, sulfate and hydroxyl. The process can be made economical by procuring spent biomass from industry or naturally available bulk biomass. A batch or a continuous process of removal of heavy metals directly from effluents can be developed in a fixed bed reactor using the immobilized biomass. Further biosorption potential of the biomass can be improved by various physical and chemical treatments. The availability of variety of microbial biomass and their metal binding potential makes it a economical and sustainable option for developing effluent treatment process for removal and recovery of heavy metals.

Absorption↗
PubMed2003

Serum nitrate levels as an index of endothelial function in pre-eclamsia and normal pregnancy.

The study was conducted in St. John's Medical College Hospital and Department of Physiology, with the aim of studying the serum nitrate levels in pre-eclampsia and normal pregnancy. The total number of subjects studied in various groups were 159, control (n = 55), first trimester (n = 13), second trimester normal (n = 42), second trimester pre-eclampsia/PET (n = 5), third trimester normal (n = 32), third trimester pre-eclampsia/PET (n = 12). The serum nitrate was measured by one step enzymatic assay using Nitrate reductase from Aspergillus species. The nitrate levels in the third trimester pre-eclamptic group was found to be significant lower (P = 0.02), as compared to normal subjects, however the renal functions were normal in all the subjects.

Adult↗
PubMed2002

Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology.

The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K(m),V(max), K(cat), activation energy (E(a)) and temperature coefficient (q(10)) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michaelis-Menten constant (K(m)) for casein decreased with corresponding increase in V(max), as reaction temperature was raised from 45-60 degrees C. The protease exhibited K(m) of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60 degrees C, respectively, whereas V(max) values at these temperatures were 74.07, 99.01, 116.28, and 120.48 microg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45 degrees C until 60-65 degrees C followed by thermal inactivation. Likewise, the energy of activation (E(a)) was more between 45-55 degrees C (9747 cal/mol) compared to E(a) between 50-60 degrees C (4162 cal/mol).

Bacillus↗
PubMed2002

Microwave-assisted rapid characterization of lipase selectivities.

A rapid screening procedure for characterization of lipase selectivities using microwaves was developed. The rate of reaction of various commercial lipases (porcine pancreas, Mucor miehei, Candida rugosa, Pseudomonas cepacia) as well as lipases from laboratory isolates-Bacillus stearothermophilus and Burkholderia cepacia RGP-10 for triolein hydrolysis was 7- to 12-fold higher in a microwave oven as compared to that by pH stat. The esterification of sucrose/methanol and ascorbic acid with different fatty acids was also achieved within 30 s in a microwave using porcine pancreas, B. stearothermophilus SB-1 and B. cepacia RGP-10 lipases. The relative rates and selectivity of the lipases both for hydrolytic and synthesis reactions remains unaltered. However, the rate of reaction was dynamically enhanced when exposed to microwaves. Microwave-assisted enzyme catalysis can become an attractive procedure for rapid characterization of large number of enzyme samples and substrates, which otherwise is a cumbersome and time-consuming exercise.

Animals↗
PubMed2002

Optimization of alkaline protease production from Bacillus sp. by response surface methodology.

High yields (1939 U/ml) of an alkaline protease were obtained in batch fermentation of a Bacillus sp. using a response surface methodology. The interaction of four variables, viz., starch, peptone, incubation time, and inoculum density, suggested inoculum density to be an insignificant variable. However, incubation time had a profound effect on protease yields at all the concentrations of carbon and nitrogen used. The response surface raised and flattened with increase in time of incubation, and maximum protease production up to 1939 U/ml was obtained after 96 h of incubation. The model equation obtained was validated experimentally at maximum starch (15 mg/ml) and peptone (7.5 mg/ml) concentration with increased incubation time up to 144 h in the presence of minimum inoculum density (1%). An overall 2.6-fold increase in protease production was obtained as compared with mean observed response (750 U/ml) at zero level of all variables.

Analysis of Variance↗
PubMed2002

Statistical media optimization and production of ITS alpha-amylase from Aspergillus oryzae in a bioreactor.

The production of an intermediate temperature-stable (ITS) alpha-amylase from Aspergillus oryzae was studied by using a central composite design with three independent variables, viz., starch, yeast extract, and K(2)HPO(4). The model equation provided a suitable model for the response surface for alpha-amylase production, and, from the optimal concentrations of the medium components, a model was predicted, which was then used for enzyme production in a 150-L bioreactor. In the bioreactor studies, the enzyme yields (161 U/ml) were similar to that of the shake flask (133 U/ml); however, the time required for maximum alpha-amylase production in the bioreactor was reduced to 48 h compared with 120 h in shake flask cultures. An increased level of phosphate in the medium and low inoculum size were necessary to control the excessive foaming in the bioreactor; however, control of the pO(2) level and agitation was not mandatory for enzyme production. The peak enzyme production coincided with the increase in pH of the fermentation broth and was maximal when the pH of the system was above 7.5. Thus, in the present study, pH acted as an indicator of the initiation or end of the enzyme synthesis or of the fermentation cycle.

Aspergillus oryzae↗
PubMed1996

Polyamines as modulators of microcycle conidiation in Aspergillus flavus.

Since polyamines (PAs) play a potential role in the regulation of growth and developmental processes in a wide variety of organisms, we have examined the influence of the PAs putrescine (Put) and spermidine (Spd) and the PA biosynthetic inhibitors alpha-difluoromethylornithine (DFMO), alpha-difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and cyclohexylamine (CHA), singly and in combinations on microcycle conidiation (MC) in Aspergillus flavus. The exogenous application of the diamine Put (concentrations ranging from 0.1 to 5 mM) caused a sharp decline of MC in a dose-dependent fashion, but induced vegetative growth. However, the triamine Spd (0.1-5 mM) had a minimal effect on MC and induced a shift from MC to normal condition. PA inhibitors, especially DFMO, MGBG and CHA, produced greater inhibition of MC and complete inhibition of MC was observed at 5 mM of these inhibitors. DFMA even at 5 mM had only a weak inhibitory effect on MC. DFMO also inhibited conidial germination and germ tube growth. MGBG and CHA, while having an inhibitory effect on MC, induced vegetative growth. The inhibitory effect of PA inhibitors was partially reversed by exogenous Put or Spd, with Spd being more effective than Put. The analysis of free PA levels during various phases of MC revealed that undifferentiated spores contained a high Put/Spd ratio and there was a dramatic decrease in Put/Spd ratio before and during microcycle conidiophore maturity. The change in spermine titres could not be detected. These observations imply that Put is essential for vegetative growth, while Spd is involved in MC, and that a low Put/Spd ratio seems to be important for spore differentiation to MC.

Aspergillus flavus↗