Mutant of Cyclodextrin Glycosyltransferase

Abstract

The present invention discloses a mutant of cyclodextrin glycosyltransferase and belongs to the fields of gene engineering and enzyme engineering. According to the present invention, a mutant having higher disproportionation activity of cyclodextrin glycosyltransferase is obtained by mutating the cyclodextrin glycosyltransferase. The disproportionation activity of enzymes of mutants V6D, S90G, T168A, T171A, T383A, G608A and V6D/S90G/T168A/T171A/T383A/G608A is respectively 1.89 times, 1.21 times, 1.21 times, 1.22 times, 1.32 times, 2.03 times and 3.16 times that of the wild enzyme in shake flask fermentations. The present invention has certain significance for the industrial production of cyclodextrin glycosyltransferase, and improves the application potential of the enzyme in food, medicine and chemical industries.

Claims

1. A mutant of cyclodextrin glycosyltransferase, comprising one or more mutations of valine (Val) at site 6, serine (Ser) at site 90, threonine (Thr) at site 168, threonine (Thr) at site 171, threonine (Thr) at site 383 and glycine (Gly) at site 608 in cyclodextrin glycosyltransferase derived from Bacillus circulans.

2. The mutant of claim 1, wherein the amino acid sequence of the cyclodextrin glycosyltransferase derived from Bacillus circulans is set forth in SEQ ID NO. 2.

3. The mutant of claim 1, comprising: (a) a mutation of the valine (V) at site 6 to aspartic acid (V6D) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (b) a mutation of the serine (S) at site 90 to glycine (S90G) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (c) a mutation of the threonine (T) at site 168 (T168A) to alanine in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (d) a mutation of the threonine (T) at site 171 (T171A) to alanine in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (e) a mutation of the threonine (T) at site 383 (T383A) to alanine in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (f) a mutation of the glycine (G) at site 608 (G608A) to alanine in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (g) a mutation of the valine (V) at site 6 to the aspartic acid, a mutation of the serine (S) at site 90 to glycine, a mutation of the threonine (T) at site 168 to alanine, a mutation of the threonine (T) at site 171 to alanine, a mutation of the threonine (T) at site 383 to alanine, and a mutation of the glycine (G) at site 608 to alanine (V6D/S90G/T168A/T171A/T383A/G608A) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2.

4. A method for constructing the mutant of claim 1, comprising: (1) designing a site-directed mutagenesis mutant primer according to the determined mutation site, and performing site-directed mutagenesis using a vector carrying a cyclodextrin glycosyltransferase gene as a template; constructing a plasmid vector comprising a gene encoding the mutant; (2) transforming the mutant plasmid into a host cell; (3) selecting positive clones for fermentation culture, and centrifuging the supernatant to obtain a crude enzyme solution of the cyclodextrin glycosyltransferase mutant.

5. A method for constructing the mutant of claim 2, comprising: (1) designing a site-directed mutagenesis mutant primer according to the determined mutation site, and performing site-directed mutagenesis using a vector carrying a cyclodextrin glycosyltransferase gene as a template; constructing a plasmid vector comprising a gene encoding the mutant; (2) transforming the mutant plasmid into a host cell; (3) selecting positive clones for fermentation culture, and centrifuging the supernatant to obtain a crude enzyme solution of the cyclodextrin glycosyltransferase mutant.

6. A method for constructing the mutant of claim 3, comprising: (1) designing a site-directed mutagenesis mutant primer according to the determined mutation site, and performing site-directed mutagenesis using a vector carrying a cyclodextrin glycosyltransferase gene as a template; constructing a plasmid vector comprising a gene encoding the mutant; (2) transforming the mutant plasmid into a host cell; (3) selecting positive clones for fermentation culture, and centrifuging the supernatant to obtain a crude enzyme solution of the cyclodextrin glycosyltransferase mutant.

7. The method of claim 4, wherein the plasmid vector is any one of pUC series, pET series, or pGEX.

8. The method of claim 4, wherein the plasmid vector is any one of pUC series, pET series, or pGEX.

9. The method of claim 4, wherein the plasmid vector is any one of pUC series, pET series, or pGEX.

10. The method of claim 4, wherein the plasmid is cgt/pET20b(+).

11. The method of claim 6, wherein the plasmid is cgt/pET20b(+).

12. The method of claim 4, wherein the host cell is a bacterial or fungal cell.

13. The method of claim 6, wherein the host cell is a bacterial or fungal cell.

14. A method to use the mutant of claim 1 in the fields of food, medicine and chemical industry, comprising adding the mutant as an enzyme to a preparation of a food, a medicine or a chemical.

Description

DETAILED DESCRIPTION

[0023] The examples of the present invention are merely illustrative of the present invention and are not intended to limit the content or scope of the present invention.

[0024] The media and detection methods involved in the following examples are as follows:

[0025] LB medium (g.Math.L.sup.1): Tryptone 10, yeast extract5, sodium chloride 10.

[0026] TB medium (g.Math.L.sup.1): Tryptone 12, yeast extract 24, glycerol 5, KH.sub.2PO.sub.4 2.31, K.sub.2HPO.sub.4.Math.3H.sub.2O 16.43, glycine 7.5.

[0027] Method for determining the activity of cyclodextrin glycosyltransferase in catalyzing disproportionation reaction: A 50 mmol/L phosphate buffer solution with a 5.5 pH value is taken as a solvent, 12 mM EPS (4,6-ethylidene-p-nitrophenyl--D-maltoheptaoside) and a 20 mM maltose solution are respectively prepared, 300 L of the 12 mM EPS and 300 L of the 20 mM maltose solution are mixed and preheated in a 50 C. water bath, and 100 L of a diluted enzyme solution is added; after 10 min of precise reaction, immediate boiling is performed for 10 min to terminate the reaction; after cooling, 100 L of -glucosidase and 100 L of deionized water are added, mixing is performed well and reacting is performed in a water bath at 60 C. for 60 min or more, 100 L of 1M Na.sub.2CO.sub.3 solution is added, mixing is performed well, and finally the absorbance is determined at 400 nm. The activity of the cyclodextrin glycosyltransferase in catalyzing disproportionation reaction is defined as the amount of enzyme that converts one micromole of EPS per minute. (See van der Veen B A, Leemhuis H, Kralj S, et al. Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase[J]. Journal of Biological Chemistry, 2001, 276(48): 44557-44562 for the method for determining disproportionation activity.)

EXAMPLE 1

Expression of Wild Type Cyclodextrin Glycosyltransferase

[0028] Glycerol tubes preserved in an earlier stage of a laboratory are inoculated with Cgt/pET20b(+)/BL21(DE3) (Yang Yulu, Wang Lei, Chen Sheng, et al. Optimization of process conditions for the production of -cyclodextrin by recombinant -cyclodextrin glycosyltransferase [J]. Biotechnology Bulletin, 2014, 8: 175-181.); and is cultured in an LB liquid medium (containing 100 mg/L ampicillin) for 8 h, and the seed solution is inoculated into a TB liquid fermentation medium (containing 100 mg/L ampicillin) according to an inoculum size of 5%. Escherichia coli by shake cultivation at 25 C. is fermented for 48 h, and a certain volume of fermentation broth is centrifuged at 4 C., 12000 rpm for 15 min, and the fermentation supernatant is taken as the crude enzyme solution of wild enzyme.

EXAMPLE 2

Preparation and Expression of Cyclodextrin Glycosyltransferase Single Mutant

[0029] (1) Single Mutation Preparation of Cyclodextrin Glycosyltransferase

[0030] According to the gene sequence of Bacillus circulans cyclodextrin glycosyltransferase, primers for introducing single mutations are respectively designed and synthesized, site-directed mutagenesis on the cyclodextrin glycosyltransferase gene Cgt is performed, and whether the encoding genes of the cyclodextrin glycosyltransferase mutants are correct is respectively confirmed by sequencing; the vector carrying the mutant gene is introduced into Escherichia coli for expression to obtain single mutant cyclodextrin glycosyltransferase.

[0031] PCR amplification of site-directed mutant encoding genes: By the rapid PCR technique, the expression vector Cgt/pET-20b(+) carrying the gene encoding the wild-type cyclodextrin glycosyltransferase is used as a template.

[0032] Site-directed mutagenesis primers introducing V6D mutation are:

[0033] Forward primer with nucleotide sequence shown in SEQ ID NO. 3:

TABLE-US-00001 5-CCGGATACCAGCGATAGCAACAAGCAG-3 (theunderlinedisamutatedbase)

[0034] Reverse primer with nucleotide sequence shown in SEQ ID NO. 4:

TABLE-US-00002 5-CTGCTTGTTGCTATCGCTGGTATCCGG-3 (theunderlinedisamutatedbase)

[0035] Site-directed mutagenesis primers introducing S90G mutation are:

[0036] Forward primer with nucleotide sequence shown in SEQ ID NO. 5:

TABLE-US-00003 5-CTATAGCATTATCAACTACGGCGGTGTGAATAATACGG-3 (theunderlinedisamutatedbase)

[0037] Reverse primer with nucleotide sequence shown in SEQ ID NO. 6:

TABLE-US-00004 5-CCGTATTATTCACACCGCCGTAGTTGATAATGCTATAG-3 (theunderlinedisamutatedbase)

[0038] Site-directed mutagenesis primers introducing T168A mutation are:

[0039] Forward primer with nucleotide sequence shown in SEQ ID NO. 7:

TABLE-US-00005 5-CTGGGCGGTTATGCCAATGACACCC-3 (theunderlinedisamutatedbase)

[0040] Reverse primer with nucleotide sequence shown in SEQ ID NO. 8:

TABLE-US-00006 5-CTGGGCGGTTATGCCAATGACACCC-3 (theunderlinedisamutatedbase)

[0041] Site-directed mutagenesis primers introducing T171A mutation are:

[0042] Forward primer with nucleotide sequence shown in SEQ ID NO. 9:

TABLE-US-00007 5-CGGTTATACCAATGACGCCCAAAATCTGTTTC-3 (theunderlinedisamutatedbase)

[0043] Reverse primer with nucleotide sequence shown in SEQ ID NO. 10:

TABLE-US-00008 5-GAAACAGATTTTGGGCGTCATTGGTATAACCG-3 (theunderlinedisamutatedbase)

[0044] Site-directed mutagenesis primers introducing T383A mutation are:

[0045] Forward primer with nucleotide sequence shown in SEQ ID NO. 11:

TABLE-US-00009 5-CCAAGTTTTAGCGCGAGCACGACGG-3 (theunderlinedisamutatedbase)

[0046] Reverse primer with nucleotide sequence shown in SEQ ID NO. 12:

TABLE-US-00010 5-CCGTCGTGCTCGCGCTAAAACTTGG-3 (theunderlinedisamutatedbase)

[0047] Site-directed mutagenesis primers introducing G608A mutation are:

[0048] Forward primer with nucleotide sequence shown in SEQ ID NO. 13:

TABLE-US-00011 5-CAAAATGTGTATCTGACGGCCAGCGTGAGCGAACTGGG-3 (theunderlinedisamutatedbase)

[0049] Reverse primer with nucleotide sequence shown in SEQ ID NO. 14:

TABLE-US-00012 5-CCCAGTTCGCTCACGCTGGCCGTCAGATACACATTTTG-3 (theunderlinedisamutatedbase)

[0050] PCR reaction systems: 0.5 L of 20 M forward primers and 0.5 L of 20 M reverse primers, 4 L of dNTPs Mix, 10 L of 5*PS Buffer, 0.5 L of 2.5 U/L PrimeStar polymerase, 0.5 L of template, and the balance of double distilled water filled 50 L.

[0051] PCR conditions: Pre-denaturation at 94 C. for 4 min; followed by 25 cycles (94 C. for 10 s, 55 C. for 5 s, 72 C. for 7 min 50 s), extension at 72 C. for 10 min; finally, heat preservation at 4 C. The PCR product is detected by 1% agarose gel electrophoresis.

[0052] The above verified correct PCR product is digested with Dpn I and transferred into Escherichia coli JM 109 competent cells; an LB agar plate containing 100 mg/L ampicillin is coated with the transformed product; after culturing at 37 C. for overnight, two single colonies are picked from the agar plate and an LB liquid medium is inoculated with the two single colonies; after 8 h, extracting the plasmid and sequencing it to confirm that the result is correct. The correctly sequenced plasmid is transferred into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli expressing a single mutant.

[0053] (2) Expression of Mutants

[0054] The LB liquid medium (containing 100 mg/L ampicillin) is respectively inoculated with the recombinant Escherichia coli expressing the single mutant prepared in step (1) of the present example, and culturing is performed for 8 h, and a TB liquid fermentation medium (containing 100 mg/L ampicillin) are inoculated with the seeds according to an inoculum size of 5%. The Escherichia coli by shake cultivation at 25 C. is fermented for 48 h, a certain volume of fermentation broth is centrifuged at 4 C., 12000 rpm for 15 min, and the fermentation supernatant is taken as the crude enzyme solution of the single mutant.

EXAMPLE 3

Preparation and Expression of Cyclodextrin Glycosyltransferase Six-Mutant

[0055] (1) Six-Mutation Preparation of Cyclodextrin Glycosyltransferase

[0056] A plasmid carrying the gene encoding the mutant V6D constructed in Example 2 is used as a template for the six-mutation, and according to the primers for 590G, T168A, T383A and G608A site-directed mutagenesis designed in Example 2, site-directed mutagenesis is performed on the plasmid carrying the gene encoding the mutant V6D by the rapid PCR technique to obtain a cyclodextrin glycosyltransferase V6D/S90G/T168A/T383A/G608A five-mutant. Then, a new primer is designed by using the plasmid of the V6D/S90G/T168A/T383A/G608A five-mutant as a template, and T171A mutation is introduced to construct a cyclodextrin glycosyltransferase V6D/S90G/T168A/T171A/T383A/G608A six-mutant.

[0057] The site-directed mutagenesis primers introducing T171A mutation are changed to:

[0058] Forward primer with nucleotide sequence shown in SEQ ID NO. 15:

TABLE-US-00013 5-CGGTTATGCCAATGACGCCCAAAATCTGTTTC-3 (theunderlinedisamutatedbase)

[0059] Reverse primer with nucleotide sequence shown in SEQ ID NO. 16:

TABLE-US-00014 5-GAAACAGATTTTGGGCGTCATTGGCATAACCG-3 (theunderlinedisamutatedbase)

[0060] (2) Expression of Mutants

[0061] An LB liquid medium (containing 100 mg/L ampicillin) is inoculated with the recombinant Escherichia coli expressing the mutant prepared in step (1) of the present example, and culturing is performed for 8 h; a TB liquid fermentation medium (containing 100 mg/L ampicillin) is inoculated with the seeds according to an inoculum size of 5%. The Escherichia coli by shake cultivation at 25 C. is fermented for 48 h, a certain volume of fermentation broth is centrifuged at 4 C., 12000 rpm for 15 min, and the fermentation supernatant is taken as the crude enzyme solution of the six-mutant.

EXAMPLE 4

Analysis of Disproportionation Activity of Cyclodextrin Glycosyltransferase

[0062] The disproportionation activity of fermentation supernatants obtained in Example 1, Example 2 and Example 3 is respectively determined. The OD.sub.600nm and the enzyme disproportionation activity of wild-type cyclodextrin glycosyltransferase (WT) and mutants subjected to shake flask cultivation for 48 h are shown in Table 1. The results show that the enzyme disproportionation activity of all mutants is higher than that of the wild type. The disproportionation activity of enzyme of mutants V6D, S90G, T168A, T171A, T383A, G608A and V6D/S90G/T168A/T171A/T383A/G608A is respectively 1.89 times, 1.21 times, 1.21 times, 1.22 times, 1.32 times, 2.03 times and 3.16 times of the wild enzyme in shake flask fermentations.

TABLE-US-00015 TABLE 1 OD.sub.600 nm and enzyme disproportionation activity of wild and mutant enzymes of cyclodextrin glycosyltransferase in shake flask fermentation Enzyme activity Enzyme OD.sub.600 nm (U/mL) WT 11.2 44.1 V6D 7.1 83.3 S90G 8.8 53.4 T168A 10.1 53.4 T171A 10.3 53.8 T383A 9.9 58.2 G608A 8.9 89.7 V6D/S90G/T168A/T171A/T383A/G608A 6.9 139.2