A kind of sucrose isomerase mutant and its application

Abstract

The invention discloses a sucrose isomerase mutant and application thereof. The present invention provides a protein named as PRSi-QRY protein, as shown in sequence 3 of the sequence listing. The gene encoding PRSi-QRY protein also belongs to the protection scope of the present invention. The present invention also protects the application of PRSi-QRY protein as sucrose isomerase. The invention also protects a recombinant bacterium, which is a recombinant bacterium obtained by introducing the recombinant plasmid pPicZa-PRSi-QRY into Pichia pastoris X33; the recombinant plasmid pPicZa-PRSi-QRY is a recombinant plasmid obtained by inserting the gene into the pPICZA vector. The invention also protects a method for producing sucrose isomerase, comprising the following steps: fermenting the recombinant bacteria to obtain sucrose isomerase. The invention has very huge application prospect and industrial value.

Description

technical field

[0001] The invention relates to a sucrose isomerase mutant and its application. Background technique

[0002] Isomaltulose occurs naturally in honey at levels up to 1%, and in sugarcane juice. Isomaltulose is a functional sweetener with natural and pure taste, non-hygroscopic, non-deliquescent, non-cariogenic, and has multiple health effects. The health characteristics of isomaltulose are mainly in the following aspects: (1) It is safe and reliable, and can be eaten in large quantities; the safety of isomaltulose has been fully recognized by the Japanese Ministry of Health, Welfare and Welfare, the EU Food Safety Authority, and the US FDA; (2) ) will not form dental plaque, and can inhibit the formation of tooth decay; the U.S. Food and Drug Administration (FDA) approved isomaltulose as a non-cariogenic sugar source; (3) Provide energy continuously, slowly, and in a balanced manner, improving Anti-fatigue ability; (4) stabilize the influence of sucrose, glu...

Examples

Embodiment 1

[0073] Embodiment 1, discovery of mutein

[0074] On the basis of the sucrose isomerase shown in sequence 1 in the sequence listing, a mutant protein library (including single mutation, multiple mutation, etc., consisting of hundreds of thousands of proteins with specific sequences) is constructed. The enzymatic activity of each protein in the mutant protein library as a sucrose isomerase is detected, and mutant proteins with significantly increased enzyme activity are discovered, and one of the mutant proteins is shown in sequence 3 of the sequence listing.

[0075] The protein shown in sequence 1 of the sequence listing is named PRSi protein, and its coding gene is shown in sequence 2 of the sequence listing. The protein shown in sequence 3 of the sequence listing is named as PRSi-QRY protein, and its coding gene is shown in sequence 4 of the sequence listing. Compared with PRSi protein, PRSi-QRY protein has three point mutations, which are K132Q, K171R and D329Y.

Embodiment 2

[0076] Embodiment 2, the acquisition of recombinant yeast

[0077] 1. Obtaining recombinant yeast PRSi

[0078] 1. Insert the double-stranded DNA molecule shown in the sequence 2 of the sequence listing from the 1st to the 1800th nucleotide at the 5' end forwardly between the XhoI and NotI restriction sites of the pPICZA vector to obtain the recombinant plasmid pPicZa-PRSi.

[0079] 2. Digest the recombinant plasmid pPicZa-PRSi with the restriction endonuclease SacI to obtain a linearized plasmid.

[0080] 3. Introducing the linearized plasmid obtained in step 2 into Pichia pastoris X33 to obtain recombinant yeast PRSi.

[0081] 2. Obtaining recombinant yeast PRSi-QRY

[0082] 1. Insert the double-stranded DNA molecule shown in the sequence 4 of the sequence listing from the 1st to the 1800th nucleotide at the 5' end in the forward direction between the XhoI and NotI restriction sites of the pPICZA vector to obtain the recombinant plasmid pPicZa-PRSi- QRY. In the recombina...

Embodiment 3

[0085] Example 3, the preparation of protein and the enzymatic properties of protein as sucrose isomerase

[0086] 1. Protein preparation

[0087] BMGY liquid medium: the solvent is PBS buffer solution with pH 6.0 and 0.1M; the solute and its concentration are as follows: yeast extract 10g / L, tryptone 20g / L, glycerol 10g / L, YNB 13.4g / L, biotin 4×10 -4 g / L.

[0088] BMMY liquid culture medium: solvent is the PBS damping fluid of pH6.0, 0.1M; Solute and its concentration are as follows: yeast extract 10g / L, tryptone 20g / L, methanol 0.5% (volume percentage), YNB 13.4g / L, biotin 4×10 -4 g / L.

[0089] Solution I: the solvent is Tris-HCl buffer solution with pH 8.0 and 20 mM; the solute and its concentration are as follows: 20 mM imidazole, 300 mM NaCl.

[0090] Solution II: The solvent is Tris-HCl buffer solution with pH 8.0 and 20mM; the solute and its concentration are as follows: 250mM imidazole, 300mM NaCl.

[0091] The recombinant yeast is: recombinant yeast PRSi or rec...

technical field

[0001] The invention relates to a sucrose isomerase mutant and its application. Background technique

[0002] Isomaltulose occurs naturally in honey at levels up to 1%, and in sugarcane juice. Isomaltulose is a functional sweetener with natural and pure taste, non-hygroscopic, non-deliquescent, non-cariogenic, and has multiple health effects. The health characteristics of isomaltulose are mainly in the following aspects: (1) It is safe and reliable, and can be eaten in large quantities; the safety of isomaltulose has been fully recognized by the Japanese Ministry of Health, Welfare and Welfare, the EU Food Safety Authority, and the US FDA; (2) ) will not form dental plaque, and can inhibit the formation of tooth decay; the U.S. Food and Drug Administration (FDA) approved isomaltulose as a non-cariogenic sugar source; (3) Provide energy continuously, slowly, and in a balanced manner, improving Anti-fatigue ability; (4) stabilize the influence of sucrose, glu...

Examples

Embodiment 1

[0073] Embodiment 1, discovery of mutein

[0074] On the basis of the sucrose isomerase shown in sequence 1 in the sequence listing, a mutant protein library (including single mutation, multiple mutation, etc., consisting of hundreds of thousands of proteins with specific sequences) is constructed. The enzymatic activity of each protein in the mutant protein library as a sucrose isomerase is detected, and mutant proteins with significantly increased enzyme activity are discovered, and one of the mutant proteins is shown in sequence 3 of the sequence listing.

[0075] The protein shown in sequence 1 of the sequence listing is named PRSi protein, and its coding gene is shown in sequence 2 of the sequence listing. The protein shown in sequence 3 of the sequence listing is named as PRSi-QRY protein, and its coding gene is shown in sequence 4 of the sequence listing. Compared with PRSi protein, PRSi-QRY protein has three point mutations, which are K132Q, K171R and D329Y.

Embodiment 2

[0076] Embodiment 2, the acquisition of recombinant yeast

[0077] 1. Obtaining recombinant yeast PRSi

[0078] 1. Insert the double-stranded DNA molecule shown in the sequence 2 of the sequence listing from the 1st to the 1800th nucleotide at the 5' end forwardly between the XhoI and NotI restriction sites of the pPICZA vector to obtain the recombinant plasmid pPicZa-PRSi.

[0079] 2. Digest the recombinant plasmid pPicZa-PRSi with the restriction endonuclease SacI to obtain a linearized plasmid.

[0080] 3. Introducing the linearized plasmid obtained in step 2 into Pichia pastoris X33 to obtain recombinant yeast PRSi.

[0081] 2. Obtaining recombinant yeast PRSi-QRY

[0082] 1. Insert the double-stranded DNA molecule shown in the sequence 4 of the sequence listing from the 1st to the 1800th nucleotide at the 5' end in the forward direction between the XhoI and NotI restriction sites of the pPICZA vector to obtain the recombinant plasmid pPicZa-PRSi- QRY. In the recombina...

Embodiment 3

[0085] Example 3, the preparation of protein and the enzymatic properties of protein as sucrose isomerase

[0086] 1. Protein preparation

[0087] BMGY liquid medium: the solvent is PBS buffer solution with pH 6.0 and 0.1M; the solute and its concentration are as follows: yeast extract 10g / L, tryptone 20g / L, glycerol 10g / L, YNB 13.4g / L, biotin 4×10 -4 g / L.

[0088] BMMY liquid culture medium: solvent is the PBS damping fluid of pH6.0, 0.1M; Solute and its concentration are as follows: yeast extract 10g / L, tryptone 20g / L, methanol 0.5% (volume percentage), YNB 13.4g / L, biotin 4×10 -4 g / L.

[0089] Solution I: the solvent is Tris-HCl buffer solution with pH 8.0 and 20 mM; the solute and its concentration are as follows: 20 mM imidazole, 300 mM NaCl.

[0090] Solution II: The solvent is Tris-HCl buffer solution with pH 8.0 and 20mM; the solute and its concentration are as follows: 250mM imidazole, 300mM NaCl.

[0091] The recombinant yeast is: recombinant yeast PRSi or rec...

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