Gene cloning, expression, and characterization of the Bacillus amyloliquefaciens PS35 lipase

Abstract

Lipases are enzymes of immense industrial relevance, and, therefore, are being intensely investigated. In an attempt to characterize lipases at molecular level from novel sources, a lipase gene from Bacillus amyloliquefaciens PS35 was cloned, heterologously expressed in Escherichia coli DH5α cells and sequenced. It showed up to 98% homology with other lipase sequences in the NCBI database. The recombinant enzyme was then purified from E. coli culture, resulting in a 19.41-fold purification with 9.7% yield. It displayed a preference for long-chain para-nitrophenyl esters, a characteristic that is typical of true lipases. Its optimum pH and temperature were determined to be 8.0 and 40 °C, respectively. The half-lives were 2.0, 1.0 and 0.5 h at 50 °C, 60 °C and 70 °C, respectively. The metal ions K+ and Fe3+ enhanced the enzyme activity. The enzyme displayed substantial residual activity in the presence of various tested chemical modifiers, and interestingly, the organic solvents, such as n-hexane and toluene, also favored the enzyme activity. Thus, this study involves characterization of B. amyloliquefaciens lipase at molecular level. The key outcomes are novelty of the bacterial source and purification of the enzyme with desirable properties for industrial applications.

Overexpression of Lipase Gene from <i>Alcaligenes</i> sp. JG3 and its Activity toward Hydrolysis Reaction

“…Both of the unit activity and specific activity for crude extract and second washed protein was lower than purified Lip.JG3 due to the presence of many other proteins that might interfere with the hydrolysis reaction although the amount of the protein itself was higher. On another report, a similar pattern of hydrolysis activity from purified lipase had higher activity than the crude extract from Bacillus amyloliquefaciens PS35 [23].…”

All‐around management of a fungal isolate obtained from cheese spoilage as an environmental source: Direct approach from an undergrad student to a biotechnological characterization

“…) the best results were obtained with sonication with 211 U/L. Centering the analysis on this method, diverse reports indicate that the use of sonication is an efficient method for cell lysis for organisms with resistant cell membranes and has been used by several authors, with some mentions that physical methods are the most common at industrial level . The discussion about sonication presented by Klimek et al .…”

“…8) the best results were obtained with sonication with 211 U/L. Centering the analysis on this method, diverse reports indicate that the use of sonication is an efficient method for cell lysis for organisms with resistant cell membranes and has been used by several authors, with some mentions that physical methods are the most common at industrial level [29,34]. The discussion about sonication presented by Klimek et al (2011) [35] refers that the use of it depends on factors such as the percentage of chitin in the cell walls which can lead to the variation in effectiveness from the same lysis method between organisms; however, said methods do not leave residues and by not having a direct interaction with the enzyme, like some detergents may have for the inherent presence of charges that could affect the integrity or enzymatic yield of the enzyme of interest, comprise in our case a preferable technique.…”

Search for new cultured lipophilic bacteria in industrial fat-containing wastes

“…Representatives of Bacillus are among the most common producers of bacterial lipases [3]. The lipolytic enzymes of B. subtilis and B. amyloliquefaciens are of significant biotechnological interest and, therefore, many of them have been identified, cloned, and characterized to date [34][35][36][37][38][39]. In addition, B. subtilis and B. amyloliquefaciens are known for their activity as plant growth promoting rhizobacteria (PGPR) and are widely used to stimulate growth and counteract numerous plant pathogens [40][41][42].…”