Namita 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.

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.

Biotechnological applications and prospective market of microbial keratinases

Keratinases are well-recognized enzymes with the unique ability to attack highly cross-linked, recalcitrant structural proteins such as keratin. Their potential in environmental clean-up of huge amount of feather waste has been well established since long. Today, they have gained importance in various other biotechnological and pharmaceutical applications. However, commercial availability of keratinases is still limited. Hence, to attract entrepreneurs, investors and enzyme industries it is utmost important to explicitly present the market potential of keratinases through detailed account of its application sectors. Here, the application areas have been divided into three parts: the first one is dealing with the area of exclusive applications, the second emphasizes protease dominated sectors where keratinases would prove better substitutes, and the third deals with upcoming newer areas which still await practical documentation. An account of benefits of keratinase usage, existing market size, and available commercial sources and products has also been presented.

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).

Green Enzymatic Synthesis of L-Ascorbyl Fatty Acid Ester: An Antioxidant

Abstract In green chemistry, biocatalysis using microwaves is a very attractive tool for various regioselective syntheses. L-Ascorbyl fatty acid esters were synthesized by an immobilized lipase from Bulkholderia multivorans using microwaves, with a dynamically enhanced rate of reaction and appreciable yield of around 80%. Microwave radiation had no effect on enzymic inactivation, however, synergism between microwaves and biocatalyst was observed.

Selective disruption of disulphide bonds lowered activation energy and improved catalytic efficiency in TALipB from Trichosporon asahii MSR54: MD simulations revealed flexible lid and extended substrate binding area in the mutant

TALipB (33 kDa) is a solvent stable, enantioselective lipase from Trichosporon asahii MSR54. It is cysteine-rich and shows activation in presence of thiol reducing agents. DIANNA server predicted three disulphide bridges C53-C195 (S1), C89-C228 (S2) and C164-C254 (S3) in the enzyme. Selective disruption of disulphide bonds by cysteine to alanine mutations at Cys53 and Cys89 of S1 and S2 bonds resulted in enzyme activation. Mutant mTALipB (S1+S2) showed increase in specific activity by ∼4-fold (834 mM/mg) and improved Vmax of 6.27 μmol/ml/min at 40 °Con pNP caprate. Temperature optima of mTALipB shifted from 50 to 40 °C and activation energy decreased by 0.7 kcal mol(-1). However, the mutant was less thermostable with a t1/2 of 18 min at 60 °C as compared to t1/2 of 38 min for the native enzyme. Mutant also displayed an improved activity on all pNP esters and higher enantiomeric excess (61%) during esterification of (±) 1-phenylethanol. Far-UV CD analysis showed significant changes in secondary structure after S-S bridge disruption with 7.16% decrease in α-helices and 1.31% increase in β-sheets. In silico analysis predicted two lids (α5 and α9) in TALipB. Molecular dynamic simulations at 40 °C and 50 °C revealed that in the mTALipB, both the lids opened at 40 °C with clockwise and anticlockwise rotations in Lid1 and Lid2, respectively. In the native protein, however, the lid was only partially open even at 50 °C. Concomitant to lid flexibility, there was an extension of accessible catalytic triad surface area resulting in improved catalytic efficiency of the mutant enzyme.