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Current Applications of Cold Actives Enzymes in Molecular BiologyBY: Shekhar C Bisht | Category: Applications | Submitted: 2011-03-07 10:40:43
Current Applications of Cold Actives Enzymes in Molecular biology High catalytic efficiency at low and moderate temperatures of cold-active enzyme offer a number of advantages for biotechnology processes, such as the shortening of process times, saving of energy costs, prevention of the loss of volatile compounds, performance of reactions that involve thermosensitive compounds, and reduced risk of contamination. The first cold-adapted enzymes from Antarctic bacteria that have been cloned, sequenced and expressed in a recombinant form were lipases, subtilisins and α-amylase, i.e. well-known representatives of industrial enzymes. This illustrates that besides the fundamental research on biocatalysis in the cold, the immense biotechnological potential of psychrophilic enzymes. Alkaline phosphatases are mainly used in molecular biology for the dephosphorylation of DNA vectors prior to cloning to prevent recircularization, for the dephosphorylation of 5'-nucleic acid termini before 5'-end labelling by polynucleotide kinase or for removal of dNTPs and pyrophosphate from PCR reactions. However, the phosphatase has to be carefully removed after dephosphorylation to avoid interferences with the subsequent steps. Furthermore, E. coli and calf intestinal alkaline phosphatase (that was the preferred enzyme for these applications) are heat-stable and require detergent addition for inactivation. It follows that heat-labile alkaline phosphatases are excellent alternatives as they are inactivated by moderate heat treatment allowing one to perform the subsequent steps in the same test tube and minimizing nucleic acid losses. The heat-labile alkaline phosphatase from Antarctic bacterium as a new tool in molecular biology, this interesting finding is now well established and expressed in E. coli. This heat-labile alkaline phosphatase sold as Antarctic phosphatase and now proposed to market by New England Biolabs (USA). In the same context, the heat-labile alkaline phosphatase from the Arctic shrimp Pandalus borealis is also available for instance from Biotec Pharmacon ASA (Norway) or GE Healthcare Life Sciences (UK). Two other psychrophilic enzymes are also marketed for molecular biology applications taking advantage of the heat-labile property. Shrimp nuclease selectively degrades double stranded DNA: for instance, it is used for the removal of carry-over contaminants in PCR mixtures, and then it is heat-inactivated prior to addition of the template. This enzyme is produced in recombinant form in Pichia pastoris and is available from Biotec Pharmacon ASA (Norway), USB Corporation (USA) or Thermo Scientific (UK). Heat-labile uracil- DNA N-glycosylase from Atlantic cod (Gadus morhua), that presents typical cold adaptation features, is also used to remove DNA contaminants in sequential PCR reactions (Leiros et al. 2003). When PCR is performed with dUTP instead of dTTP, PCR products become distinguishable from target DNA, and can be selectively degraded by uracil-DNA N-glycosylase. Following degradation of contaminants, the enzyme is completely and irreversibly inactivated after heat treatment. Heatlabile uracil-DNA N-glycosylase, produced in recombinant form in E. coli, is available from Biotec Pharmacon ASA (Norway). However, recent developments based on cold-adapted organisms and on their biomolecules, such as those mentioned above, have clearly demonstrated the huge biotechnological potential. This potential appears to be even larger than other extremophiles when considering both the broader psychrophilic biodiversity that encompasses microorganisms, plants and animals, and the broader fields of application. Most biotechnological applications of psychrophiles are environmentally friendly and contribute to energy saving, both aspects being of increasing significance. Article Source: http://www.biotecharticles.com/ About Author / Additional Info: Comments on this article: (0 comments so far)
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