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Thermostable, Solvent-Tolerant Biocatalyst for Extreme Conditions

詳細技術說明

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*Abstract

Haloarchaeal Inorganic Pyrophosphatase Operates in a Variety of Solvents to Aid DNA Sequencing, PCR, SNP Genotyping, and RNA Synthesis

This biocatalyst, or enzyme, accelerates organic synthesis reactions in extreme conditions that available biocatalysts do not tolerate. This thermostable inorganic pyrophosphatase (PPA) developed by University of Florida researchers thermodynamically favors the hydrolysis of one mole of pyrophosphate (PPi) into two moles of orthophosphate (Pi), even in extreme conditions such as buffers with two to three moles of salt and 25% (v/v) organic solvent. Enzymes play an increasingly important role as biocatalysts in the synthesis of key intermediates for the biotechnology industry. The global market for biopharmaceuticals, $160 billion in 2011, was expected to exceed $200 billion by 2016. PPAs that are easily purified, thermostable, and solvent-tolerant are desirable for biotechnologies. However, PPAs that retain catalytic activity in high salt solutions or organic solvents are not commercially available. The haloarchaeal inorganic pyrophosphatase developed at UF is thermostable and operates in a variety of solvents, including solvents with low water activity, such as high salt solutions and organic solvents. This biocatalyst increases the solubility of hydrophobic substrates, allows for new synthetic chemistry, alters substrate specificity, eases product recovery, and reduces microbial contamination for many biotechnological applications.

Application

Biocatalyst for biotechnological applications that is easily purified, thermostable, readily soluble, and active in conditions of low water activity.

Advantages

• Soluble, thermostable, and active in conditions of low water activity, enabling application in a broad range of reactions
• Rapidly hydrolyzes PPi into 2 moles of Pi, which is readily detected by colorimetric assay
• Removes contaminant PPi prior to single-base-extension to increase the yield of RNA synthesis
• Prevents the accumulation of PPi during DNA sequencing and PCR reactions, providing a considerable amount of energy that can drive unfavorable reactions to completion

Technology

Inorganic pyrophosphatases (PPAs) are valuable for the biotechnology industry because they catalyze the hydrolysis of pyrophosphate (PPi), a by-product of the biosynthesis of DNA, RNA, proteins, lipids, cellulose, starch, and many other compounds. The hydrolysis of PPi releases a considerable amount of energy that can push unfavorable biochemical transformations to completion. University of Florida researchers identified a novel subset of Class A Type archeal PPAs, haloarcheals, which were modeled by Haloferax volcanii PPA (HvPPA). HvPPA was then purified 357-fold by immobilized metal ion affinity chromatography and size exclusion chromatography to isolate the hexameric form of HvPPA, which is ultimately used in the hydrolysis of PPi. This haloarchaeal PPA is thermostable and beneficial for accelerating biosynthetic reactions in the forward direction in extreme conditions, such as buffers with two to three molesof salt and 25% (v/v) organic solvent.

*Principal Investigation

Name: Julie A Maupin

Department:

Name: Lana McMillan

Department:

Name: Nathaniel Hepowit

Department:

其他

國家/地區

美國