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Novel enzyme protein, process for production of enzyme protein, and gene encoding enzyme protein

A coding and protein technology, applied in the direction of biochemical equipment and methods, enzymes, transferases, etc., can solve the problems of glycoproteins and glycolipids in reaction results, high and limited specificity of sugar receptor substrates, etc., to achieve easy-to-operate effect

Inactive Publication Date: 2013-04-17
JAPAN TOBACCO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these animal-derived enzymes have extremely high substrate specificity for sugar acceptors, and the sugar chain structures that can transfer sialic acid in vivo are limited.
That is, the reaction result of sialyltransferase and the produced glycoprotein and glycolipid are extremely limited

Method used

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  • Novel enzyme protein, process for production of enzyme protein, and gene encoding enzyme protein
  • Novel enzyme protein, process for production of enzyme protein, and gene encoding enzyme protein
  • Novel enzyme protein, process for production of enzyme protein, and gene encoding enzyme protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1: Production of mutant β-galactoside-α2,3-sialyltransferase

[0146] (1) Production of ISH467-N2C0 mutant

[0147] For β-galactoside-α2,3-sialyltransferase N2C0 derived from Photobacterium phosphoreum JT-ISH-467 strain (Patent Document 3: WO2006 / 112253), mutants were produced in which amino acids were substituted. PCR (1st PCR) was performed using ISH467-N2C0 / pTrc99A (Patent Document 3: WO2006 / 112253) prepared by the inventors as a template. The primer sequences and their combinations used in the reaction are shown in Tables 1 and 2. The 50 μl reaction solution contained plasmid, 5 μl of 10×PyroBest buffer, 4 μl of 2.5 mM dNTP, 5 picomoles of each primer, and 0.5 μl of PyroBest DNA Polymerase (manufactured by TaKaRa Co., Ltd.). , once at 96°C for 3 minutes, 15 times at 96°C for 1 minute, at 55°C for 1 minute, at 72°C for 2 minutes, and once at 72°C for 1 minute (1st PCR). The primer combinations used in PCR are shown in Table 2. These PCR products were s...

Embodiment 2

[0177] Example 2: Activity determination of mutant β-galactoside-α2,3-sialyltransferase

[0178] (1) Determination of sialyltransferase activity of mutant N2C0 containing His×6-Tag

[0179] Regarding the mutant N2CO protein containing His×6-Tag purified in Example 1-(2) (four mutants of F318A, E319A, F318AE319A and S337A), β-galactoside-α2,3-sialic acid Transferase activity assay. The sialic acid transfer activity was determined by the method described in J. Biochem., 120, 104-110 (1996), which is incorporated herein by reference in its entirety. Specifically, the sugar donor substrate CMP-NeuAc (containing 70nmol, 14 C The NeuAc-labeled CMP-NeuAc was 25000 cpm, which was 356 cpm / nmol. NeuAc stands for N-acetylneuraminic acid), lactose (1.25 μmol) as a sugar acceptor substrate, NaCl was added to a concentration of 0.5M, and the reaction solution (30 μl) containing the enzyme prepared by the method described above was used for the enzyme reaction. The enzyme reaction w...

Embodiment 3

[0185] Example 3: Properties of modified β-galactoside-α2,3-sialyltransferase

[0186] Using the modified N2C0 protein without His × 6-Tag purified in Example 1-(4) (two kinds of E319A, F318AE319A) and the wild-type N2C0 protein without His × 6-Tag, calculate their relative lactose and CMP-sialic acid rate constants (Table 3). Km and V of ISH467-N2C0 without His×6-Tag relative to lactose and CMP-sialic acid max They are 8.4mM and 0.72mM respectively. Km and V of ISH467-N2C0 mutants E319A and F318AE319A without His×6-Tag relative to lactose and CMP-sialic acid maxThey are 22mM, 0.53mM and 85mM, 1.59mM respectively.

[0187] [Table 3] Table 3 Modified and wild-type N2C0 protein relative to the rate constant of lactose and CMP-sialic acid

[0188] lactose

[0189]

K m (mM)

V max (nmol / min)

k cat ( / Second)

N2CO enzyme

8.4

3.9

11.2

E319A mutant enzyme

22

12

99.7

F318AE319A mutant enzyme

85

7.2...

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Abstract

The present invention relates to: a protein which does not have a neuraminidase activity substantially and has a beta-galactoside-alpha 2,3-sialyltransferase activity; a nucleic acid which encodes the enzyme protein; and a process for producing the enzyme using a microorganism transformed with a gene encoding the protein. This protein is a modified enzyme protein produced by mutating a specific amino acid residue in a beta-galactoside-alpha 2,3-sialyltransferase protein derived from a microorganism belonging to the family Vibrio, the genus Photobacterium to substantially inactive the neuraminidase activity of the protein.

Description

technical field [0001] The present invention relates to a protein substantially not having neuraminidase activity but having β-galactoside-α2,3-sialyltransferase activity, a nucleic acid encoding the enzyme protein, and a microorganism transformed with a gene encoding the protein A method of making the enzyme. Background technique [0002] The sugar chain refers to a compound composed of various sugars, such as monosaccharides such as galactose or N-acetylglucosamine. Sugar chains such as glycoproteins and glycolipids (hereinafter also referred to as complex sugar chains) have very important functions in living bodies. According to the research so far, it is considered that among the sugar chains, especially the sugar chain containing a monosaccharide called sialic acid exhibits an important function. For example, mainly in mammalian cells, sialic acid-containing sugar chains are important molecules that function as signal transduction between cells and cell-extracellular ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N9/10
CPCC12N9/1081C12Y204/99004
Inventor 锋利喜山本岳片山桵子
Owner JAPAN TOBACCO INC
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