Hexulose phosphate isomerase and gene coding said isomerase

A technology of hexanone phosphate and isomerase, which is applied in the field of DNA and ketohexose phosphate isomerase, and can solve the problems of unreported enzyme purification

Inactive Publication Date: 2002-08-07
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, not only the structure of the enzyme protein and its gene, but also the purificat...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: the ketohexose phosphate synthase (HPS) that purification brevis bacillus S1 bacterial strain produces

[0062] First, to purify HPS, Bacillus brevis S1 strain (NCIMB12524) was added to a volume of 1 liter in a 2 liter flask containing 500 ml of medium A of the following composition, and shake cultured at 45°C for 16 hours.

[0063] [Composition of medium A]

[0064] Methanol 2ml / L

[0065] Dipotassium hydrogen phosphate 4.65 g / L

[0066] Sodium hydrogen phosphate monohydrate 1.5 g / l

[0067] Ammonium sulfate 1.5 g / L

[0068] Magnesium sulfate heptahydrate 0.2 g / L

[0069] Yeast Extract 0.5 g / L

[0070] Peptone 0.5 g / L

[0071] Casamino acids 0.5 g / L

[0072] vitamin solution * 1 1ml / L

[0073] trace metal solution * 2 0.2ml / L

[0074] (pH7)

[0075] * 1: In 100ml, contains 10mg pantothenic acid, 10mg riboflavin, 1mg vitamin B 12 , 1 mg lipoic acid, 1 mg folic acid, 10 mg biotin, 10 mg thiamine, 10 mg nicotinamide, 2 mg ...

Embodiment 2

[0081] Example 2: Partial structure of ketohexose phosphate synthase (HPS) produced by Bacillus brevis S1 strain

[0082] Subsequently, the partial amino acid sequence of the HPS obtained in Example 1 was determined. The protein band of HPS developed by SDS-PAGE was replicated onto a PVDF (polyvinyl fluoride) membrane by a conventional method, and the band was excised. Then, the N-terminal amino acid sequence of the protein was analyzed by Edman degradation method. As a result, it was found that it was MQLQLALDLVNIEEAKQVVAEVQEYVDIVE (SEQ ID NO: 4). In addition, for the internal amino acid sequence of the protein, the protein was partially degraded with V8 protease, subjected to SDS-PAGE, and copied onto a PVDF membrane as well. Then, all the detectable peptide fragment bands were excised, and their amino acid sequences were analyzed by Edman degradation method. As a result, the sequences were determined as VAKAAEHGADIVTILAAAEDVSIKGAVEEAKKLGXK (SEQ ID NO: 5) and MGVDYIXVHAGYD...

Embodiment 3

[0083] Embodiment 3: obtain the genomic DNA of brevis bacillus S1 bacterial strain

[0084] Brevibacillus strain S1 was inoculated in 5 ml of medium B (CM129 medium (OXOID company)), and cultured overnight at 45°C. This culture was inoculated into 500 ml of medium B at a rate of 1% until the OD (610 nm) of the culture reached 1.0, and then the culture broth was centrifuged to collect cells. Wash the cells with a salt-EDTA solution (composition: 0.15 mol / liter NaCl, 0.01 mol / liter EDTA, pH8.0), then suspend in 500 milliliters of the same solution, add 80 mg of lysozyme to the suspension, and Hold at 37°C for 3 hours.

[0085] Then, 2 ml of 25% SDS and 10 ml of proteinase K (10 mg / ml) were added to the suspension, and shaken overnight at 37°C. Then, the suspension was treated at 60° C. for 20 minutes, 14 ml of 5 mol / l sodium perchlorate and 30 ml of a chloroform / isoamyl alcohol mixture (mixing ratio: 24:1) were added and stirred gently for 30 minutes. The suspension was centr...

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PUM

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Abstract

There are provided a DNA coding for phosphohexuloisomerase, which is a protein defined in the following (A) or (B), and a method for producing the enzyme: (A) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing, (B) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing including substitution, deletion, insertion or addition of one or several amino acid residues and having phosphohexulose isomerase activity.

Description

technical field [0001] The present invention relates to phosphoketohexose isomerase and DNA encoding it. More precisely, the present invention relates to phosphoketohexose isomerase originating from the thermotolerant bacterium Brevibacillus brevis and the DNA encoding it. Background technique [0002] In methylotrophic organisms that can utilize single carbon (C1) compounds such as methane and methanol as carbon sources, it is known that there is a ribulose monophosphate pathway (RuMP) that metabolizes these compounds. The important key enzyme of this pathway is the catalyzed Phosphohexulose-6-phosphate synthase (HPS, 3-hexulose-6-phosphate synthase), which initiates the ribulose monophosphate pathway, and phosphohexulose isomerase (PHI, phospho- 3-Hexulose isomerase). [0003] In addition, the specific position of the target compound molecule uses a stable isotope, carbon 13 ( 13 C) Labeled biochemical substances can be used to study biological metabolic pathways. More...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K14/32C12N1/15C12N1/19C12N1/21C12N5/10C12N9/90C12N15/61
CPCC12N9/90
Inventor 加藤畅夫安枝寿
Owner AJINOMOTO CO INC
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