Lipase Engineering

Lipases enantioselectivity can be improved physically, chemically, or genetically. Physically, lipases can be modified by cross-linking, crystallization, and immobilization that involves attaching the lipase onto an insoluble solid support. Immobilization enhances the stability of lipase and facilitates lipase recovery. Details of lipase immobilization methods are discussed in Chapter 3. 

Supercritical Fluids Technology in Lipase Catalyzed Processes 

Directed evolution is a powerful tool used to improve lipase properties that does not depend on a comprehensive understanding of the relationship between enzyme structure and function. It rather depends on simple, yet powerfnl, random mutation and selection. The targeted genes are exposed to iterative cycles of random mutagenesis, expressed in an appropriate host and subsequently screened (Johannes and Zhao, 2006). Bacillus lipase was engineered by directed evolntion, where a lip gene was cloned and expressed in E. coli. The mutagenesis was executed by error-prone polymerase chain reaction (PCR). The mutation enhanced the specific activity of the lipase by twofold (Khurana et al., 2011). 

Adlercreutz, P. 1994. Enzyme-Catalysed Lipid Modification. Biotechnology Genetic Engineering Reviews 12 231-254. 

Al-Zuhair, S., F. W. Ling, and L. S. Jun. 2007. Proposed Kinetic Mechanism of the Production of Biodiesel from Palm Oil Using Lipase. Process Biochemistry 42 (6) 951-960. 

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Lipase Engineering Database is available as http //www.led.uni-stuttgardt.de/. 

Despite the various uses for lipases, the availability of lipases with specific characteristics, their stability, and operational properties limits their uses in many applications, especially in the biosynthesis of molecules in organic media. Researchers have managed to overcome some of these shortcomings through reaction medium engineering and lipase engineering physically, chemically, or genetically. 

In 1989, two enzymes based on genetic engineering techniques were introduced, ie, a cloned alkaline protease (IBIS) and a protein engineered Subtihsin Novo (Genencor, California). Lipase and ceUulase types of detergent enzymes have also begun to appear. 

Apart from proteases, cutinases and lipases were demonstrated to hydrolyse PA. Hydrolysis of PA with a lipase (not further specified) was confirmed by using FTIR analysis and dye-binding assays [32]. Several reports have assessed the potential of cutinases for PA functionalisation, and a fungal cutinase from F. solani was successfully genetically engineered towards higher activity on a PA oligomer and PA [23-25]. 

Patel, M.T., Nagarajan, R. and Kilara, A. (1996) Lipase-catalyzed biochemical reactions in novel media—a review. Chemical Engineering Communications, 153, 365 04. 

The lipid components of food are known to be critical in the development of much of a food s flavor. Modifications to lipid modifying enzymes such as lipases have led to new products useful in the rapid preparation of other food components Chapter 13,14), Better utilization of lipid constituents in food products can be gained from a better understanding of the thermodynamic and physicochemical characteristics of emulsions. Significant advancement in emulsion chemistry and food engineering have recently appeared in the literature and are an important portion of this volume Chapter 19),... 

Genetic engineering. The X-ray structures are known for many hydrolases, allowing for modeling of the substrate in the active site as well as structurally based, random or rational protein mutation to magnify or invert enantioselectivity. An example of the latter is provided by the rational design of a mutant of Candida antarctica lipase (CALB), which, instead of the wild-type R-selectivity, displayed... 

We collaborated with Professor Palligamai Vasudevan of the Chemical Engineering Department of the University of New Hampshire on a study of immobilization of lipases on CoFoam. Immobilization was performed at the Hydrophilix facility in Portland, ME. Approximately 2 g lipase (from porcine pancreas and Mucor miehei) were stirred into 500 ml deionized water. The enzyme solution was emulsified with an equal volume of a methylene diisocyanate (MDI)-based hydrophilic polyurethane... 

Numerous structural and mechanistic studies have been made with them.620 629 633-635 The gene for a lipase from Candida rugosa has been synthesized using codons that maximize its expression in Saccharomyces cerevisiae and which allow for further genetic engineering of the lipase.636... 

A protein-engineered variant (Lipolase Ultra) has since been developed with enhanced performance at low temperatures. At low temperatures, fatty stains become more troublesome to remove. Lipases therefore have an even more valuable role in cool washes. Although it is more expensive to produce, the manufacturer believes it to render more value, especially in low-temperature washes. 

R. J. Kazlauskas, Elucidating structure-mechanism relationships in lipases prospects for predicting and engineering catalytic properties, Trends Biotechnol. 1994, 12, 464-72. 

Kondo, M., K. Rezaei, F. Temelli and M. Goto, On-Line Extraction-Reaction of Canola Oil with Ethanol by Immobilized Lipase in Supercritical Carbon Dioxide, Industrial Engineering Chemistry Research, 41, 5770-5774 (2002). 

Flow cytometry is well suited for the analysis of enzyme activity and kinetics at the single cell level (Watson and Dive, 1994). Flow cytometric assays for numerous enzymes including esterases, proteases, peroxidases, lipases, and oxidoreductases3 are available and are widely used in research and clinical practice. To date, flow cytometry has not been widely exploited as a screening tool for enzyme engineering purposes, but this is rapidly changing. 

Gordillo, M. A., Montesinos, J. L., Casas, C., Valero, F., Lafuente, J., and Sola, C. 1998a. Improving lipase production from Candida rugosa by a biochemical engineering approach. Chem. Phys. Lipids, 93,131-142. 

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