A Cyclodextrin Glucosyltransferase Mutant with Increased AA‑2G Conversion

Abstract

The invention discloses a cyclodextrin glucosyltransferase mutant for improving AA-2G conversion rate and belongs to the field of genetic engineering and enzyme engineering. According to the cyclodextrin glucosyltransferase mutant, the 228th lysine near the active center of the cyclodextrin glucosyltransferase of thermophilic fat bacillus (Bacillus stearotherm opilus NO2) is mutated into arginine to obtain a mutant K228R, the 367th aspartic acid is mutated into serine to obtain a mutant D367S and amphimutation is carried out on the basis to obtain a mutant D367S / K228R. The mutant can be used for realizing the improvement of the AA-2G conversion rate and has relatively high industrial value.

Description

technical field

[0001] The invention relates to a mutant of cyclodextrin glucosyltransferase which improves the conversion rate of AA-2G, and belongs to the fields of genetic engineering and enzyme engineering. Background technique

[0002] Vitamin C (VC) is a water-soluble vitamin that cannot be synthesized by the human body itself. It participates in many physiological activities in the body, such as promoting the conversion of cholesterol into bile acids, promoting the synthesis of adrenal cortex hormones, participating in the metabolism of aromatic amino acids, promoting iron absorption and Participate in various redox reactions in the body and play an important role in maintaining and promoting human health. In addition, vitamin C can also promote the synthesis of collagen, restore the formed melanin, and play a certain role in maintaining skin elasticity, whitening, and wrinkle removal. Widely used in food, medicine, cosmetics and other fields. However, the hydroxyl ...

Examples

Embodiment 1

[0022] Embodiment 1: Recombinant bacteria construction

[0023] According to the cgt gene sequence registered on NCBI (NCBI sequence number X59042.1), the cyclodextrin glucosyltransferase gene sequence cgt was synthesized by chemical synthesis. The plasmid used for the construction of E. coli was pET20b(+) with T7 promoter. The pET20b(+) plasmid and the plasmid containing the cgt gene were digested with Nco Ⅰ and Hind Ⅲ, respectively, and the digested products were recovered by tapping the rubber, and then ligated with T4 ligase, and the ligated products were transformed into E.coli JM109 competent cells, and after 37 Cultivate at ℃ for 8 hours, pick the transformant and shake culture in LB medium containing 100 mg / L ampicillin liquid, extract the plasmid, and verify by enzyme digestion to obtain the expression plasmid cgt / pET20b(+).

[0024] Transform the plasmid cgt / pET20b(+) into E.coli BL21(DE3) host bacteria, smear the LB plate containing ampicillin (100mg / L), culture at...

Embodiment 2

[0025] Embodiment 2: the preparation of mutant

[0026] (1) Single mutation

[0027] Two mutant enzymes K228R and D367S of cyclodextrin glucosyltransferase derived from B. stearothermophilus NO2:

[0028] According to the gene sequence of B. stearothermophilus NO2 cyclodextrin glucosyltransferase (NCBI sequence number X59042.1), primers for introducing K228R and D367S mutations were designed and synthesized respectively, and the cyclodextrin glucosyltransferase gene was subjected to site-directed mutation. In the DNA coding sequence, it was identified that the Lys codon at position 228 was changed to an Arg codon, and the Asp codon at position 367 was changed to a Ser codon. The mutant gene is placed in an appropriate expression vector and introduced into Bacillus subtilis, Escherichia coli or Bacillus pumilus for expression to obtain a single mutant cyclodextrin glucosyltransferase. Site-directed mutation of single mutations K228R and D367S: Using rapid PCR technology, usin...

Embodiment 3

[0048] Embodiment 3: the concentration of mutant crude enzyme liquid

[0049] Slowly add ammonium sulfate with a concentration of 26% relative to the mass fraction of the enzyme liquid to the enzyme liquid obtained in Example 2 while stirring, stir until the ammonium sulfate is dissolved, and stand at 4°C for 8-10 hours to precipitate protein. The mixture was centrifuged (8000rpm, 10min) to collect the precipitate, and the minimum volume of 50mM KH 2 PO 4 -Na 2 HPO 4 Buffer solution (pH 6.0) was redissolved, and after reconstitution, the solid matter was removed by centrifugation again, and the supernatant was collected and dialyzed to obtain a concentrated enzyme solution.

technical field

[0001] The invention relates to a mutant of cyclodextrin glucosyltransferase which improves the conversion rate of AA-2G, and belongs to the fields of genetic engineering and enzyme engineering. Background technique

[0002] Vitamin C (VC) is a water-soluble vitamin that cannot be synthesized by the human body itself. It participates in many physiological activities in the body, such as promoting the conversion of cholesterol into bile acids, promoting the synthesis of adrenal cortex hormones, participating in the metabolism of aromatic amino acids, promoting iron absorption and Participate in various redox reactions in the body and play an important role in maintaining and promoting human health. In addition, vitamin C can also promote the synthesis of collagen, restore the formed melanin, and play a certain role in maintaining skin elasticity, whitening, and wrinkle removal. Widely used in food, medicine, cosmetics and other fields. However, the hydroxyl ...

Examples

Embodiment 1

[0022] Embodiment 1: Recombinant bacteria construction

[0023] According to the cgt gene sequence registered on NCBI (NCBI sequence number X59042.1), the cyclodextrin glucosyltransferase gene sequence cgt was synthesized by chemical synthesis. The plasmid used for the construction of E. coli was pET20b(+) with T7 promoter. The pET20b(+) plasmid and the plasmid containing the cgt gene were digested with Nco Ⅰ and Hind Ⅲ, respectively, and the digested products were recovered by tapping the rubber, and then ligated with T4 ligase, and the ligated products were transformed into E.coli JM109 competent cells, and after 37 Cultivate at ℃ for 8 hours, pick the transformant and shake culture in LB medium containing 100 mg / L ampicillin liquid, extract the plasmid, and verify by enzyme digestion to obtain the expression plasmid cgt / pET20b(+).

[0024] Transform the plasmid cgt / pET20b(+) into E.coli BL21(DE3) host bacteria, smear the LB plate containing ampicillin (100mg / L), culture at...

Embodiment 2

[0025] Embodiment 2: the preparation of mutant

[0026] (1) Single mutation

[0027] Two mutant enzymes K228R and D367S of cyclodextrin glucosyltransferase derived from B. stearothermophilus NO2:

[0028] According to the gene sequence of B. stearothermophilus NO2 cyclodextrin glucosyltransferase (NCBI sequence number X59042.1), primers for introducing K228R and D367S mutations were designed and synthesized respectively, and the cyclodextrin glucosyltransferase gene was subjected to site-directed mutation. In the DNA coding sequence, it was identified that the Lys codon at position 228 was changed to an Arg codon, and the Asp codon at position 367 was changed to a Ser codon. The mutant gene is placed in an appropriate expression vector and introduced into Bacillus subtilis, Escherichia coli or Bacillus pumilus for expression to obtain a single mutant cyclodextrin glucosyltransferase. Site-directed mutation of single mutations K228R and D367S: Using rapid PCR technology, usin...

Embodiment 3

[0048] Embodiment 3: the concentration of mutant crude enzyme liquid

[0049] Slowly add ammonium sulfate with a concentration of 26% relative to the mass fraction of the enzyme liquid to the enzyme liquid obtained in Example 2 while stirring, stir until the ammonium sulfate is dissolved, and stand at 4°C for 8-10 hours to precipitate protein. The mixture was centrifuged (8000rpm, 10min) to collect the precipitate, and the minimum volume of 50mM KH 2 PO 4 -Na 2 HPO 4 Buffer solution (pH 6.0) was redissolved, and after reconstitution, the solid matter was removed by centrifugation again, and the supernatant was collected and dialyzed to obtain a concentrated enzyme solution.

Images