Sapna Bradoo

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.

Parametric optimization and biochemical regulation of extracellular tannase from Aspergillus japonicus

Abstract Aspergillus japonicus produces maximum extracellular tannase activity after 24 h incubation. The enzyme is produced constitutively on simple and complex sugar substrates but activity is doubled in the presence of tannic acid as sole carbon source. Parametric optimization of the enzyme yielded 1·13-fold increase in enzyme production (33·06 U/ml) at 30°C and pH 6·6 with 0·2% glucose and 2% tannic acid in Czapek-Doxs minimal medium. The enzyme shows strong end-product repression with gallic acid.

A hyper-thermostable, alkaline lipase from Pseudomonas sp. with the property of thermal activation

A hyper-thermostable, alkaline lipase from a newly-isolated, mesophilic Pseudomonas sp. was optimal at pH 11 and at 90 °C. It had a half-life of more than 13 h at 90 °C. It was activated by 30% when heated at 90 °C for 2 h. The enzyme had a greater affinity for mustard oil (Km=40 mg ml−1) than for olive oil (Km=140 mg ml−1).

Partitioning and resolution of mixture of two lipases from Bacillus stearothermophilus SB-1 in aqueous two-phase system

The partitioning and resolution of two lipases (acidic and neutral) was achieved in an aqueous two-phase system from crude culture supernatant of Bacillus stearothermophilus SB-1. The effects of changes in PEG molecular weight, varying PEG/phosphate system, pH and sodium chloride concentration were examined on the partition coefficient, K in PEG/phosphate system. The best system was PEG 6000 (70% w/v)/phosphate (40% w/v) and NaCl (3% w/v) at pH 7.0 where the total lipase partitioned to the top PEG phase. The two lipases were then selectively eluted by addition of fresh salt phase in a pH dependent manner with acidic lipase moving to the bottom phase at pH 4.0 with 5.27-fold purification while neutral lipase shifted to the bottom phase at pH 6.0 with 15.25-fold purity. The two lipases were easily recovered from the salt phase by immobilisation on accurel. Thus the mixture of two lipases was resolved and purified into acidic and neutral within 2 h with complete recycling of both the phase components. It has also been found that addition of PEG 6000 leads to an increase in the enzyme activity of the lipase mixture.

Two acidothermotolerant lipases from new variants of Bacillus spp.

Two acidothermotolerant lipases from new isolates of Bacillus stearothermophilus SB-1 and Bacillus licheniformis SB-3 are reported. In addition, a thermotolerant, neutral lipase from Bacillus atrophaeus SB-2 that hydrolyses castor oil is also reported. The lipase from B. stearothermophilus SB-1 retained 70% activity and that from B. licheniformis SB-3 retained 50% activity at pH 3.0 at 50 °C. In addition, at 100 °C B. stearothermophilus SB-1 lipase had a half life of 25 min at pH 3.0 and 15 min at pH 6.0. Lipase activity was markedly stimulated by glycerol in case of B. stearothermophilus SB-1 and by diethylether in cases of B. atrophaeus SB-2 and B. licheniformis SB-3. The lipases varied in their substrate specificity towards triacylglycerols. The rate of hydrolysis of neem oil with B. stearothermophilus SB-1 and B. atrophaeus SB-2 lipases was, respectively, nearly 4-fold and 2-fold more than with olive oil.

Microwave Assisted Synthesis and Antibacterial Activity of New Quinolone Derivatives

Summary. A series of novel 6-fluoro-7-(5-alkyl-1,3,4-thiadiazol/oxadiazol-2-ylsulfanyl)-4-quinolone-3-carboxylic acids were synthesized using microwave irradiation. The compounds were tested for their in vitro antibacterial activity. All compounds containing the 1,3,4-thiadiazole/oxadiazole moiety at position 7 showed promising antibacterial activity.Zusammenfassung. Einige neue 6-Fluoro-7-(5-alkyl-1,3,4-thiadiazol/oxadiazol-2-sulfanyl)-4-chinolon-3-carbonsäuren wurden unter Bestrahlung mit Mikrowellen synthetisiert. Die Verbindungen wurden hinsichtlich ihrer antibakteriellen Aktivität in vitro untersucht. Sämtliche Verbindungen mit 7-ständigen 1,3,4-Thiadiazol/Oxadiazol-Einheiten zeigten vielversprechende Aktivität.

Rapid purification of extracellular tannase using polyethylene glycol–tannic acid complex

This paper reports on a rapid procedure for partial purification of extracellular tannase using a combination of tannic acid and PEG‐6000. A pH‐dependent protein precipitation was obtained within 30 min. Monitoring the primary precipitation curve of the culture filtrate at varying pH, a second step was designed that yielded an approximately eight‐fold increase in the enzyme purification. This procedure is of importance as no temperature and pH monitoring is required and a very fast precipitation is obtained without any alteration in the enzymatic properties of the precipitant.