# US 5,707,846 A · N-acetylglucosaminyl transferase gene coding therefor and process for production thereof

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- Number: US 5,707,846 A
- Document ID: US5707846A
- Status: Granted
- Kind: A
- Application number: 08/405,230
- Filed: 1995-03-16
- Granted: 1998-01-13
- Earliest claimed: 1992-08-24
- Assignees: Suntory Limited; Naoyuki Taniguchi
- Inventors: Naoyuki Taniguchi; Atsushi Nishikawa; Nozomi Yamaguchi
- Web page: https://patentgrep.com/patent/US5707846A

## CPC

- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A .beta.1,6-N-acetylglucosaminyl transferase having the following properties: (1) Action: it transfers N-acetylglucosamine from UDP-N-acetylglucosamine to .alpha.-6-D-mannoside; (2) Substrate specificity: it shows a reactivity of about 79% for GnGnF-bi-PA, about 125% for GnGnGn-tri-PA and about 66% for GnM-Pa, when taking a reactivity for GnGn-bi-PA as 100%; (3) Optimum pH: 6.2 to 6.3; (4) Inhibition, Activation and Stability: Mn.sup.2+ is not necessary for expression of activity, and the activity is not inhibited in the presence of 20 mM EDTA; (5) Molecular weight: about 73,000 as determined by SDS-PAGE in the absence of reducing agent; and about 73,000 and about 60,000 as determined in the presence of a reducing agent; (6) Km value: 133 .mu.M and 3.5 mM for acceptor GnGn-bi-PA and donor UDP-GlcNAc, respectively; and (7) It includes the following peptide fragments: (SEQ ID NO.1) Thr-Pro-Trp-Gly-Lys (SEQ ID NO.2) Asn-Ile-Pro-Ser-Tyr-Val (SEQ ID NO.3) Val-Leu-Asp-Ser-Phe-Gly-Thr-Glu-Pro-Glu-Phe-Asn-His-Ala-Asn-Tyr-Ala (SEQ ID NO.4) Asp-Leu-Gln-Phe-Leu-Leu (SEQ ID NO.5) Asn-Thr-Asp-Phe-Phe-Ile-Gly, and gene coding for said enzyme, and a process for production of the enzyme.

## Claims

### Claim 1

An isolated .beta.1,6-N-acetylglucosaminyl transferase having the following properties:
(1) Action: it transfers N-acetylglucosaminyl from uridine-5'-diphospho-N-acetylglucosamine to .alpha.-6-D-mannoside;
(2) Substrate specificity: it shows a reactivity of about 79% for GnGnF-bi-PA (GlcNAc.beta.1-2Man.alpha.1-3-(GlcNAc.beta.1-2Man.alpha.1-6)Man.beta.1-4Gl cNAc.beta.1-4GlcNAc(Fuc.alpha.1-6)-2-aminopyridine), about 125% for GnGnGn-tri-PA (GlcNAc.beta.1-2) (GlcNAc.beta.1-4)Man.alpha.1-3(GlcNAc.beta.1-2Man.alpha.1-6)-Man.beta.1-4G lcNAc.beta.1-4GlcNAc-2-aminopyridine) and about 66% for GnM-PA (Man.alpha.1-3(GlcNAc.beta.1-2Man.alpha.1-6)Man.beta.1-4 GlcNAc.beta.1-4GlcNAc-2-aminopyridine), with the reactivity for GnGn-bi-PA (GlcNAc.beta.1-2Man.alpha.1-3-(GlcNAc.beta.1-2Man.alpha.1-6)Man.beta.1-4Gl cNAc.beta.1-4GlcNAc-2-aminopyridine) being defined as 100%;
(3) Optimum pH: 6.2 to 6.3;
(4) Inhibition, Activation and Stability: Mn.sup.2+ is not necessary for expression of activity, and the activity is not inhibited in the presence of 20 mM EDTA;
(5) Molecular weight: about 73,000 as determined by SDS-PAGE in the absence of reducing agent; and about 73,000 and about 60,000 as determined in the presence of a reducing agent;
(6) Km value: 133 .mu.M and 3.5 mM for acceptor GnGn-bi-PA (GlcNAc.beta.1-2Man.alpha.1-3-(GlcNAc.beta.1-2Man.alpha.1-6)Man.beta.1-4Gl cNA.beta.1-4GlcNAc-2-aminopyridine) and donor UDP-GlcNAc (uridine-5'-diphospho-acetylglucosamine), respectively; and
(7) It includes the following peptide fragments:
______________________________________ (SEQ ID NO. 1) Thr--Pro--Trp--Gly--Lys (SEQ ID NO. 2) Asn--I1e--Pro--Ser--Tyr--Val (SEQ ID NO. 3) Val--Leu--Asp--Ser--Phe--Gly--Thr--Glu--Pro Glu--Phe--Asn--His--Ala--Asn--Tyr--Ala (SEQ ID NO. 4) Asp--Leu--Gln--Phe--Leu--Leu (SEQ ID NO. 5) Asn--Thr--Asp--Phe--Phe--Ile--Gly. ______________________________________

### Claim 2

An isolated .beta.1,6-N-acetylglucosaminyl transferase encoded by the nucleotide sequence of SEQ ID No: 8.

### Claim 3

An isolated .beta.1,6-N-acetylglucosaminyl transferase having a nucleotide sequence having one or more nucleotide modifications in the nucleotide sequence of SEQ ID No: 8.

### Claim 4 (depends on 3)

The isolated .beta.1,6-N-acetylglucosaminyl transferase according to claim 3 wherein the modification is an addition, deletion, replacement with other nucleotides, or a combination thereof.

### Claim 5 (depends on 1)

The .beta.1,6-N-acetylglucosaminyl transferase of claim 1, wherein the transferase is of human origin.

### Claim 6 (depends on 1)

The .beta.1,6-N-acetylglucosaminyl transferase of claim 1, wherein the transferase is derived from lung carcinoma cells.

### Claim 7 (depends on 5)

The .beta.1,6-N-acetylglucosaminyl transferase of claim 5, wherein the transferase is derived from human lung carcinoma cells.

### Claim 8 (depends on 7)

The .beta.1,6-N-acetylglucosaminyl transferase of claim 7, wherein the transferase is derived from human lung carcinoma SBM331 (FERM BP-3967).

### Claim 9 (depends on 1)

The .beta.1,6-N-acetylglucosaminyl transferase of claim 1, wherein the transferase is purified from a protein-free culture.

### Claim 10 (depends on 1)

The .beta.1,6-N-acetylglucosaminyl transferase of claim 1, wherein the transferase is extracellularly secreted.