Combination of site-directed mutagenesis and yeast surface display enhances Rhizomucor miehei lipase esterification activity in organic solvent
ABSTRACT To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.
SourceAvailable from: Saisubramanian Nagarajan[Show abstract] [Hide abstract]
ABSTRACT: Fat-splitting enzymes (lipases), due to their natural, industrial, and medical relevance, attract enough attention as fats do in our lives. Starting from the paper that we write, cheese and oil that we consume, detergent that we use to remove oil stains, biodiesel that we use as transportation fuel, to the enantiopure drugs that we use in therapeutics, all these applications are facilitated directly or indirectly by lipases. Due to their uniqueness, versatility, and dexterity, decades of research work have been carried out on microbial lipases. The hunt for novel lipases and strategies to improve them continues unabated as evidenced by new families of microbial lipases that are still being discovered mostly by metagenomic approaches. A separate database for true lipases termed LIPABASE has been created recently which provides taxonomic, structural, biochemical information about true lipases from various species. The present review attempts to summarize new approaches that are employed in various aspects of microbial lipase research, viz., screening, isolation, production, purification, improvement by protein engineering, and surface display. Finally, novel applications facilitated by microbial lipases are also presented.Applied biochemistry and biotechnology 09/2012; DOI:10.1007/s12010-012-9849-7 · 1.69 Impact Factor
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ABSTRACT: Lipases have received great attention as industrial biocatalysts in areas like oils and fats processing, detergents, baking, cheese making, surface cleaning, or fine chemistry [1,2]. They can catalyse reactions of insoluble substrates at the lipid-water interface, preserving their catalytic activity in organic solvents . This makes of lipases powerful tools for catalysing not only hydrolysis, but also various reverse reactions such as esterification, transesterification, aminolysis, or thiotransesterifications in anhydrous organic solvents [4,5]. Moreover, lipases catalyse reactions with high specificity, regio and enantioselectivity, becoming the most used enzymes in synthetic organic chemistry . Therefore, they display important advantages over classical catalysts, as they can catalyse reactions with reduced side products, lowered waste treatment costs, and under mild temperature and pressure conditions . Accordingly, the use of lipases holds a great promise for green and economical process chemistry [8,9]. However, performance of a lipase is not always sufficient for an industrial application  and most enzymes have sub-optimal properties for processing conditions . In fact, there are still disproportionally few examples of commercial scale applications of such biocatalysts in the manufacture of fine chemicals. In order to improve enzyme-mediated process efficiency, two different pathways can be followed: i) fitting the process to the available biocatalyst by medium engineering or modification of the manufacturing system to suit the sensitivities of the biocatalyst , or ii) obtaining better biocatalysts through different strategies that can be run in parallel . These strategies (Figure 1) include the exploration of biodiversity to expand the sources and number of new biocatalysts, immobilization of existing enzymes, reaction conditions modification [12,13], or the proper modification of these biocatalysts to get the most suitable variant for a defined industrial process . In this case the use of rational protein design to improve enzymes for which the 3D structure has been elucidated or homology-modelled , or the use of directed evolution can provide optimal biocatalysts .09/2012; 2(3):e201209005. DOI:10.5936/csbj.201209005
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ABSTRACT: Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters that can potentially replace certain plastics derived from petroleum. PHAs can be produced using a combination of renewable feedstocks and biological methods. Native and recombinant microorganisms have been generally used for making PHAs via fermentation processes. As much as 90 % of the microbial dry mass may accumulate as PHAs. A range of PHAs has been produced using fermentation methods, including copolymers and block copolymers. Alternative production schemes based on genetically modified plants are becoming established and may become the preferred route for producing certain PHAs. Production in plants is likely to be inexpensive compared to production by fermentation, but it does not appear to be as versatile as microbial synthesis in terms of the range of products that may be generated. Cell-free enzymatic production of PHAs in vitro is receiving increasing attention and may become the preferred route to some specialty products. This review discusses the recent advances in production of polyhydroxyalkanoates by the various methods. Methods of recovering the polymer from microbial biomass are reviewed. Established and emerging applications of PHAs are discussed.Journal of Polymers and the Environment 06/2012; 21(2). DOI:10.1007/s10924-012-0527-1 · 1.63 Impact Factor