Thermostable haloarchaeal inorganic pyrophosphatase

Patent metadata

Field	Value
Publication number	US-11466260-B2
Application number	US-201615766447-A
Country	US
Kind code	B2
Filing date	Oct 19, 2016
Priority date	Oct 19, 2015
Publication date	Oct 11, 2022
Grant date	Oct 11, 2022

Abstract

The invention pertains to a PPA from a microorganism belonging to the family Halobacteriaceae (HPPA), for example, a PPA from Haloferax volcanii . The HPPA provided by the invention is soluble, thermostable and active at high concentrations of salt and/or organic solvent. An embodiment of the invention provides a method of increasing the rate of a reaction by adding an HPPA to the reaction mixture, wherein the reaction produces PPi, for example, an enzymatic reaction, and wherein the reaction is carried out at moderately high temperature and/or low water activity. Further embodiments of the invention provide an assay to detect the PPi released during a reaction which produces PPi by adding an HPPA to convert the PPi in to Pi and measuring the resultant Pi. The invention further pertains to an assay to monitor a reaction which produces PPi in the presence or the absence of an HPPA.

First claim

We claim:

1. A method comprising:
conducting a first reaction to produce inorganic pyrophosphates (PPi) in a reaction mixture comprising: 10% to 50% v/v of one or more of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol or ethanol; and
adding an inorganic pyrophosphatase from a microorganism belonging to family Halobacteriaceae (HPPA) comprising SEQ ID NO:2, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2, to the reaction mixture to catalyze hydrolysis of PPi to form inorganic phosphates (Pi),
wherein the HPPA catalyzes the hydrolysis of PPi and removal of PPi from the reaction mixture and drives the first reaction in a direction toward PPi production.

2. The method of claim 1 , wherein the first reaction is an enzymatic reaction.

3. The method of claim 2 , wherein the enzymatic reaction is performed under high temperature from about 50° C. to about 100° C.

4. The method of claim 2 , wherein the enzymatic reaction is performed under low water activity, wherein low water activity comprises high salt concentration.

5. The method of claim 2 , wherein the enzymatic reaction is performed under moderately high temperature from about 40° C. to about 50° C.

6. The method of claim 4 , wherein the high salt concentration comprises the concentration of about 1M to about 5M.

7. The method of claim 3 , wherein the HPPA is thermostable at high temperature of about 50° C. to about 100° C.

8. A composition comprising a reaction mixture that removes inorganic pyrophosphates (PPi) from a prior reaction and produces inorganic phosphates (Pi), the reaction mixture comprising (1) an inorganic pyrophosphatase from a microorganism belonging to family Halobacteriaceae (HPPA) of SEQ ID NO: 2 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, and (2) 10 to 50% v/v of one or more of DMSO, DMF, methanol or ethanol, wherein the inorganic pyrophosphatase catalyzes the hydrolysis of PPi to Pi.

Assignees

• Univ Florida

Inventors

• Maupin-Furlow Julie A
• Hepowit Nathaniel L
• Mcmillan Lana

Classifications

• C12N9/14 Primary

  Hydrolases (3) · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12Y306/01001

  Inorganic diphosphatase (3.6.1.1) · CPC title

• C12Q1/42

  involving phosphatase · CPC title