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Enzymatic production of glycolic acid

A glycolic acid and glycolonitrile technology, applied in the field of microbiology and molecular biology, can solve the problem of no nitrilase activity and other problems

Active Publication Date: 2012-12-12
HIGH PURITY TECH & SCI LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Brevibacterium R312 is known to have nitrile hydratase and amidase activities, but no nitrilase activity (Toumeix et al., Antonie van Leeuwenhoek, 52:173-182 (1986))

Method used

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  • Enzymatic production of glycolic acid
  • Enzymatic production of glycolic acid
  • Enzymatic production of glycolic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0242] Construction of High Copy Nitrilase Expression Plasmid

[0243] Synthetic oligonucleotide primers

[0244] 165

[0245] (5′-CGA CTGCAG TAAGGAGGAATAGGACATGGTTTCGTATAACAGCAAGTTC-3'; SEQ ID NO: 1) and

[0246] 166

[0247] (5′-TGA TCTAGA GCTTGGAGAATAAAGGGGAAGACCAGAGATG-3′; SEQ ID NO: 2) (which incorporated PstI and XbaI restriction sites (underlined) respectively) was used for PCR amplification of genomic DNA from Acidovorax facilis 72W (ATCC 55746) (SEQ ID NO :5) the nitrilase gene.

[0248] Typical PCR parameters are as follows:

[0249] Step 1: 5 minutes at 95°C

[0250] Step 2: 0.5 minutes at 95°C (denaturation)

[0251] Step 3: 0.5 minutes at 55°C (annealing)

[0252] Step 4: 1 minute at 74°C (extension)

[0253] Repeat steps 2-4 for 25 cycles.

[0254] PCR reagents were provided by Roche Diagnostics Corporation (Indianapolis, IN) and used as recommended.

[0255] The only change from the native A. facilis 72W sequence was a change in the first nucleot...

Embodiment 2

[0257] Expression of Active Nitrilase in E. coli

[0258] Plasmid pSW138 was used to transform E. coli MG1655 (ATCC 47076) and E. coli FM5 (ATCC 53911 ) to generate two strains, called (1) MG1655 / pSW138 and (2) FM5 / pSW138, respectively. Each strain was grown, induced, harvested and assayed for nitrilase activity (conversion of glycolonitrile to glycolic acid) as previously described. Each strain was repeated 6 times.

[0259] 1. Bacterial culture

[0260] The strain inoculum was grown in LB medium supplemented with ampicillin (50 mg / L) at 37°C with shaking (200 rpm) for 16-18 hours.

[0261] 2. Induction of nitrilase expression

[0262]Sufficient inoculum was added to fresh LB medium supplemented with ampicillin (50 mg / L) and IPTG (1 mM) to give a starting OD (600 nm) of approximately 0.1. The culture was incubated at 37°C with shaking (200 rpm) for about 6-8 hours.

[0263] 3. Bacteria harvest

[0264] Bacterial cells were harvested by centrifugation, removing a...

Embodiment 3

[0270] Construction of random mutagenesis library of nitrilase from Acidovorax facilis 72W by error-prone polymerase chain reaction

[0271] According to the manufacturer's instructions (Gentra Systems, Minneapolis, MN), use DNA Isolation Kit Genomic DNA was prepared from Acidovorax facilis 72W (ATCC 55746). according to Instructions for use provided by the PCR mutagenesis kit (Stratagene, La Jolla, CA), carry out error-prone PCR on the Acidovorax facilis 72W nitrilase gene (coding sequence; SEQ ID NO: 5), identified as SEQ ID NO: Primers for 3 (5'-GCGCATATGGTTTCGTATAACAGCAAGTTCC-3') and SEQ ID NO: 4 (5'-ATAGGATCCTTATGGCTACTTTGCTGGGACCG-3'). Use reaction conditions recommended to yield low mutation frequencies (0-3 mutations / kb) and intermediate mutation frequencies (3-7 mutations / kb). According to pTrcHis2 According to the instruction provided by TA expression kit (Invitrogen, Carlsbad, CA), 10% of the 1.1 kb PCR product was ligated into the expression vector pTrcHis...

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Abstract

Various methods are provided for the enzymatic production of glycolic acid from glycolonitrile. These methods include: 1) use of Acidovorax facilis 72W nitrilase mutants having improved nitrilase activity for converting glycolonitrile to glycolic acid, and 2) methods to improve catalyst stability and / or productivity. The methods to improve catalyst stability / productivity include use of reaction stabilizers, running the reactions under substantially oxygen free conditions, and controlling the concentration of substrate in the reaction mixture.

Description

[0001] This application is a divisional application of Chinese patent application 200580048285.5 "Enzymatic Production of Glycolic Acid" with a filing date of December 21, 2005. [0002] This application claims the benefit of US Provisional Application Nos. 60 / 638,176 and 60 / 638,127, both filed December 22,2004. technical field [0003] The present invention relates to the fields of microbiology and molecular biology. More specifically, a method for the enzymatic production of glycolic acid from glycolonitrile using a mutated nitrilase having improved nitrilase activity is provided. Background technique [0004] Glycolic acid (HOCH 2 COOH; CAS Registry No. 79-14-1) is the simplest member of the alpha-hydroxy acid family of carboxylic acids. Its properties make it ideal for a wide range of consumer and industrial uses, including in water well restoration, the leather industry, the oil and gas industry, the laundry and textile industries, as a monomer in the preparation of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12R1/19
CPCC12N9/78C12P7/42
Inventor R.迪科西莫M.S.佩恩A.帕诺瓦D.P.奥基夫J.S.汤普森
Owner HIGH PURITY TECH & SCI LLC
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