Rani Gupta

Bacterial lipases: an overview of production, purification and biochemical properties

Abstract Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures. Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia. Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source. Bacterial lipases are mostly extracellular and are produced by submerged fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common. Lipases are serine hydrolases and have high stability in organic solvents. Besides these, some lipases exhibit chemo-, regio- and enantioselectivity. The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.

Microbial α-amylases: a biotechnological perspective

Abstract Amylases are one of the most important and oldest industrial enzymes. These comprise hydrolases, which hydrolyse starch molecules to fine diverse products as dextrins, and progressively smaller polymers composed of glucose units. Large arrays of amylases are involved in the complete breakdown of starch. However, α-amylases which are the most in demand hydrolyse α-1,4 glycosidic bond in the interior of the molecule. α-Amylase holds the maximum market share of enzyme sales with its major application in the starch industry as well as its well-known usage in bakery. With the advent of new frontiers in biotechnology, the spectrum of α-amylase application has also expanded to medicinal and analytical chemistry as well as in automatic dishwashing detergents, textile desizing and the pulp and paper industry. Amylases are of ubiquitous occurrence, produced by plants, animals and microorganisms. However, microbial sources are the most preferred one for large scale production. Today a large number of microbial α-amylases are marketed with applications in different industrial sectors. This review focuses on the microbial amylases and their application with a biotechnological perspective.

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.

Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor

Alkaline protease production in Bacillus mojavensis was improved up to 4.2-fold in a 14 l bioreactor during validation of a predicted statistical model. The final enzyme yield in the bioreactor was 2389 U ml−1 obtained within 10–12 h compared to 558 U ml−1 after 24 h in shake flask cultures. Analysis of variance (ANOVA) of face-centered central composite design showed a high coefficient of determination (R2) value of 0.9473, thus ensuring a satisfactory adjustment of the quadratic model with the experimental data. The coordinates of three factors (casamino acids, inoculum age and agitation) were positive, whereas negative coordinates were obtained for other two factors (glucose and incubation time). Protease production was subjected to catabolite repression by glucose (>2 mg ml−1). The response surface curves predicted increased levels of casamino acids (12 mg ml−1) and midlevel of glucose (2 mg ml−1) as best C/N combination for optimal enzyme production (1133 U ml−1) within 18 h at high agitation rates (>250 rpm) and low inoculum density (A550 nm≅0.250). Protease production was drastically reduced at low agitation rates. The present study provides useful information about the regulation of protease synthesis through manipulation of various physicochemical factors.

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.

A rapid plate assay for screening L-asparaginase producing micro-organisms

A pH and dye‐based fast procedure for screening l‐asparaginase‐producing micro‐organisms is reported. The procedure is suitable for bacterial and fungal screening. The results are obtained within 24 and 48 h for bacteria and fungi, respectively. The results correlate with quantitative estimations in culture broths.

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.

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H2O2, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.

Zn2+ biosorption by Oscillatoria anguistissima

Abstract Oscillatoria anguistissima showed a very high capacity for Zn 2+ biosorption (641 mg g −1 dry biomass at a residual concentration of 129·2 ppm) from solution and was comparable to the commmercial ion-exchange resin IRA-400C. Zn 2+ biosorption was rapid, pH dependent and temperature independent phenomenon. Zn 2+ adsorption followed both Langmuir and Freundlich models. The specific uptake (mg g −1 dry biomass) of metal decreased with increase in biomass concentration. Pretreatment of biomass did not significantly affect the biosorption capacity of O. anguistissima. The biosorption of zinc by O . anguistissima was an ion-exchange phenomenon as a large concentration of magnesium ions were released during zinc adsorption. The zinc bound to the biomass could be effectively stripped using EDTA (10 mM) and the biomass was effectively used for multiple sorption–desorption cycles with in-between charging of the biomass with tap water washings. The native biomass could also efficiently remove zinc from effluents obtained from Indian mining industries.

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 CaCl2 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 °C. Keratinase production was found to be a function of biomass and maximum production occurred during the stationary phase.