CN103555685A - Mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase - Google Patents

Abstract

The invention relates to a mutation method for enhancing the ability of beta-cyclodextrin glucosyltransferase (abbreviated as beta-CGTase) to produce beta-cyclodextrin, belonging to the fields of genetic engineering and enzyme engineering. The present invention adopts a site-directed mutation method to improve the β-cyclodextrin-producing ability of a CGTase, and provides a mutation scheme for enhancing the β-cyclodextrin-producing ability of a β-CGTase derived from Bacillus circulans STB01. The 31st CGTase Alanine was mutated to arginine (Arg), proline (Pro) or threonine (Thr) to obtain mutants A31R, A31P and A31T. Compared with the wild CGTase, the ability of the mutant to produce β-cyclodextrin is obviously enhanced, and it is more suitable for the industrial production of β-cyclodextrin.

Description

Mutation method for enhancing the ability of β-cyclodextrin glucosyltransferase to produce β-cyclodextrin

technical field

The invention relates to a mutation method for enhancing the ability of beta-cyclodextrin glucosyltransferase to produce beta-cyclodextrin, which belongs to the fields of genetic engineering and enzyme engineering. The invention is a technique for enhancing product specificity of cyclodextrin glucosyltransferase by site-directed mutation method.

Background technique

Cyclodextrin is composed of D-glucopyranose connected by α-1,4-glycosidic bonds, and has a ring-shaped hollow conical structure, in which α-, β- and Gamma-cyclodextrin is the most common. Due to its external hydrophilic and internal hydrophobic properties, cyclodextrin can form inclusion complexes with many hydrophobic guest molecules, thereby changing the physical and chemical properties of the guest molecules. Therefore, it is widely used in food, medicine and other industrial fields.

The industrial production of cyclodextrin adopts enzymatic process, that is, it is synthesized by converting starch through cyclization reaction under the catalysis of cyclodextrin glucosyltransferase (CGTase). Since wild CGT enzymes act on starch, the products obtained are all mixtures of α-, β- and γ-cyclodextrins, which brings a lot of inconvenience to the separation and purification of a single type of cyclodextrin products. At present, the industrial separation method of β-cyclodextrin is usually selective crystallization or organic solvent complexation.

The β-CGT enzyme derived from Bacillus circulans STB01 used in the present invention is mainly to generate β-cyclodextrin, but α- and γ-cyclodextrin still occupy a large amount in the cyclization product. Therefore, further improving the ability of the enzyme to produce β-cyclodextrin will be more beneficial to the industrial production of β-cyclodextrin.

Contents of the invention

The purpose of the present invention is to provide a mutation scheme for further improving the ability of B.circulans STB01CGT enzyme to produce β-cyclodextrin.

Another object of the present invention is to propose mutants A31R, A31P and A31T that enhance the ability of β-CGTase to produce β-cyclodextrin, and their construction methods.

Technical scheme of the present invention:

Three mutants A31R, A31P and A31T derived from the CGTase of B.circulans STB01, the 31st alanine in the CGTase was mutated to arginine (Arg), proline (Pro) or threonine, respectively acid (Thr), which have a higher ability to produce β-cyclodextrins than wild-type CGTase.

According to the preparation method of the three mutants A31R, A31P and A31T, according to the gene sequence of the B.circulans STB01CGT enzyme, respectively design and synthesize mutation primers introducing Arg31, Pro31 and Thr31 codons, perform site-directed mutation on the gene, and measure the DNA Sequences were used to identify mutant genes in which Ala31 codon was changed to Arg31 codon, Pro31 codon and Thr31 codon, and expressed in Bacillus subtilis WB600.

(1) Site-directed mutation

Using rapid PCR technology, site-directed mutagenesis was carried out with the expression vector pST/cgt containing the wild CGTase gene as a template.

Primers for site-directed mutagenesis introducing the Arg31 codon:

Forward primer: 5'-GACGGCAATCCC CGC AACAATCC-3', the underline is the mutant base,

Reverse primer: 5'-GGATTGTT GCG GGGATTGCCGTC-3', the underline is the mutant base;

Primers for site-directed mutagenesis introducing the Pro31 codon:

Forward primer: 5'-GACGGCAATCCC CCC AACAATCC-3', the underline is the mutant base,

Reverse primer: 5'-GGATTGTT GGG GGGATTGCCGTC-3', the underline is the mutant base;

Primers for site-directed mutagenesis introducing the Thr31 codon:

Forward primer: 5'-GACGGCAATCCC ACC AACAATCC-3', the underline is the mutant base,

Reverse primer: 5'-GGATTGTT GGT GGGATTGCCGTC-3', the underline is the mutated base.

The PCR reaction system is: 5×PrimeSTAR Buffer (Mg ²⁺ Plus) 10 μL, dNTPs (2.5 mM each) 4 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL, PrimeSTARHS DNA Polymerase (2.5 U/μL) 0.5 μL, add 32.5 μL of double distilled water.

The PCR amplification conditions are: PCR amplification conditions are: pre-denaturation at 98°C for 4min; followed by 35 cycles of 98°C for 10s, 55°C for 15s, and 72°C for 8min; and finally, incubation at 72°C for 10min.

After the PCR product was digested by DpnI for 2 hours, it was transferred into Escherichia coli (Escherichia coli) JM109 competent cells, spread to LB solid medium containing agar and cultured overnight, and a single colony was picked and cultured overnight in LB liquid medium Plasmids were extracted and verified by sequencing. The mutant plasmid was transformed into the expression host B. subtilis WB600 competent cells. 5 μg/mL kanamycin sulfate and 10 μg/mL gibberellin were added to each medium.

(2) Expression and purification of mutants

Pick the single clone of the expression host B.subtilis WB600 containing the mutant plasmid in LB medium, culture it at 37°C and 200r/min for 8-12h, and inoculate it into TB medium with 4% (v/v) inoculum , fermented at 37°C and 200r/min for 48h. The fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 minutes to remove bacteria, and the supernatant was collected and purified to obtain mutant A31R, A31P, and A31T enzyme products, respectively. 5 μg/mL kanamycin and 10 μg/mL gibberellin were added to each medium.

Beneficial effects of the present invention: Three meaningful mutants A31R, A31P and A31T have been constructed, all of which have improved the specificity of β-cyclodextrin products, and are more conducive to the industrial production of β-cyclodextrin than wild-type CGT enzymes .

Description of drawings

Fig. 1 Cyclodextrin produced by wild CGTase and its mutants acting on 5% (moisture basis, w/v) maltodextrin at pH 6.0 and 50°C. A, wild CGTase; B, mutant A31R; C, mutant A31P; D, mutant A31T; ■, α-cyclodextrin; ●, β-cyclodextrin; ▲, γ-cyclodextrin

Detailed ways

Example 1: This example illustrates the preparation of mutants A31R, A31P and A31T.

(1) Site-directed mutation

Using rapid PCR technology, site-directed mutagenesis was carried out with the expression vector pST/cgt containing the wild CGTase gene as a template.

Primers for site-directed mutagenesis introducing the Arg31 codon:

Forward primer: 5'-GACGGCAATCCC CGC AACAATCC-3', the underline is the mutant base,

Reverse primer: 5'-GGATTGTT GCG GGGATTGCCGTC-3', the underline is the mutant base;

Primers for site-directed mutagenesis introducing the Pro31 codon:

Forward primer: 5'-GACGGCAATCCC CCC AACAATCC-3', the underline is the mutant base,

Reverse primer: 5'-GGATTGTT GGG GGGATTGCCGTC-3', the underline is the mutant base;

Primers for site-directed mutagenesis introducing the Thr31 codon:

Forward primer: 5'-GACGGCAATCCC ACC AACAATCC-3', the underline is the mutant base,

Reverse primer: 5'-GGATTGTT GGT GGGATTGCCGTC-3', the underline is the mutated base.

The PCR reaction system is: 5×PrimeSTAR Buffer (Mg ²⁺ Plus) 10 μL, dNTPs (2.5 mM each) 4 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL, PrimeSTARHS DNA Polymerase (2.5 U/μL) 0.5 μL, add 32.5 μL of double distilled water.

The PCR amplification conditions are: PCR amplification conditions are: pre-denaturation at 98°C for 4min; followed by 35 cycles of 98°C for 10s, 55°C for 15s, and 72°C for 8min; and finally, incubation at 72°C for 10min.

After the PCR product was digested by DpnI for 2 hours, it was transferred into Escherichia coli (Escherichia coli) JM109 competent cells, spread to LB solid medium containing agar and cultured overnight, and a single colony was picked and cultured overnight in LB liquid medium Plasmids were extracted and verified by sequencing. The mutant plasmid was transformed into the expression host B. subtilis WB600 competent cells. 5 μg/mL kanamycin sulfate and 10 μg/mL gibberellin were added to each medium.

(2) Expression and purification of mutants

Pick the single clone of the expression host B. subtilis WB600 containing the mutant plasmid in a 50mL LB medium Erlenmeyer flask, culture it at 37°C and 200r/min for 8-12h, and inoculate it with 4% (v/v) inoculum In TB medium, ferment for 48 hours at 37°C and 200r/min. The fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 min to remove bacteria, and the supernatant was collected. 5 μg/mL kanamycin and 10 μg/mL gibberellin were added to each medium.

The supernatant was precipitated overnight in 70% ammonium sulfate solution. The precipitate was dissolved with an appropriate amount of Start buffer (20mmol/LTris-HCl, pH8.0). The HiTrap ANX FF (high sub, 5mL) column was equilibrated with Start buffer and then loaded. The eluent was Start buffer containing 1mol/L NaCl and collected step by step to obtain purified mutants A31R, A31P and A31T.

Example 2: This example illustrates an enzyme activity assay.

(1) Determination of enzyme activity

Determination of α-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution and add it to a test tube containing 0.9 mL of 1% (w/v) soluble starch solution prepared in advance with 50 mM phosphate buffer (pH 6.0). After reacting at 50°C for 10min, add 1.0mL of 1.0N hydrochloric acid to stop the reaction, then add 1.0mL of 0.1mM methyl orange solution prepared with 50mM phosphate buffer solution and incubate at 20°C for 15min, and measure the absorbance at 505nm. Using the inactivated enzyme as a blank, the content of α-cyclodextrin was determined corresponding to the α-cyclodextrin standard curve. One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol of cyclodextrin per minute under the above conditions.

Determination of β-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution and add it to a test tube containing 0.9 mL of 1% (w/v) soluble starch solution prepared in advance with 50 mM phosphate buffer (pH 6.0). After reacting at 50°C for 10 min, add 3.5 mL of 30 mM NaOH and 0.5 mL of 0.02% (w/v) phenolphthalein solution prepared from 5 mM Na ₂ CO ₃ solution for reaction, keep warm at room temperature for 15 min, and measure the absorbance at 550 nm. The inactivated enzyme was used as a blank. One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol β-cyclodextrin per minute under the above conditions.

Determination of γ-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution and add it to a test tube containing 0.9 mL of 1% (w/v) soluble starch solution prepared in advance with 50 mM phosphate buffer (pH 6.0). After reacting at 50°C for 10 min, add 50 μL of 1.0 N hydrochloric acid to stop the reaction, then add 2 mL of 0.2M citric acid buffer (pH 4.2) and 100 μL of 5 mM bromocresol green solution, incubate at room temperature for 15 min, and measure the absorbance at 615 nm. The inactivated enzyme was used as a blank. One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol γ-cyclodextrin per minute under the above conditions.

(2) Enzyme activity comparison

The experimental results are listed in Table 1. It was found that, compared with the wild CGTase, the α-cyclization activity of the mutant A31R was reduced by 70%, the β-cyclization activity was increased by 25%, and the γ-cyclization activity was increased by 21%; The α-cyclization activity of the mutant A31P was reduced by 41%, and both the β- and γ-cyclization activities were increased by 12%. The α-cyclization activity of the mutant A31T was reduced by 20%, and the β-cyclization activity was increased by 6%. - Cyclization activity increased by 7%. The total cyclization activity of mutants A31R, A31P and A31T remained basically unchanged. Moreover, the ratio of the β-cyclization activity to the total cyclization activity of the mutants A31R, A31P and A31T was higher than that of the wild enzyme.

Table 1

Example 3: This example illustrates the use of HPLC to analyze the amount of cyclodextrin produced

To prepare 5% (wet basis, water content 8%, w/v) maltodextrin (DE3) solution as substrate, 5g maltodextrin (DE3) was dissolved in 90mL sodium phosphate buffer (pH6.0), fixed Make up to 100mL, boil in boiling water for 30min. Add a certain amount of wild CGTase, mutants A31R, A31P, and A31T to make the enzyme activity in the reaction system 1U/mL, place it at 50°C for 9 hours, sample 600 μL at intervals, boil the enzyme for 10 minutes, centrifuge at 12000rpm for 10 minutes, and take Add 5 μL of glucoamylase (70 U/mL) to 500 μL of supernatant, saccharify at 30°C for 1 hour, boil for 10 minutes to inactivate, centrifuge at 12,000 rpm for 30 minutes, and take 20 μL of the supernatant for HPLC analysis after filtering through a 0.45 μm ultrafiltration membrane.

The HPLC measurement conditions are: Waters600 high performance liquid chromatography (equipped with differential refractive index detector), chromatographic column Lichrosorb NH ₂ (4.6mm × 150mm), mobile phase adopts 68% acetonitrile aqueous solution, column temperature is 30 ℃, flow rate is 1mL/ min.

The experimental results are shown in Figure 1 and Table 2. Compared with the wild CGTase, the mutants A31R, A31P and A31T have higher β-cyclodextrin production capacity and are more suitable for the production of β-cyclodextrin. After 9 hours of cultivation, compared with the wild type, the β-cyclodextrin production of these three mutants increased by 25.8%, 16.1%, 9.7%, respectively, while the α-cyclodextrin production decreased by 41.0%, 25.6% and 15.4%, the yield of γ-cyclodextrin changed relatively little.

Table 2

Claims (3)

1. three kinds of mutants A31R, A31P and A31T derived from the cyclodextrin glucosyltransferase of Bacillus circulans STB01, referred to as CGTase, are characterized in that: the 31st alanine in the CGTase Acid (Ala) was substituted with arginine (Arg), proline (Pro) or threonine (Thr), respectively, and named A31R, A31P and A31T; and A31T have higher ability to produce β-cyclodextrin. 2. The preparation method of three mutants A31R, A31P and A31T in claim 1 is to design mutation primers respectively according to the CGT enzyme gene sequence of B.circulans STB01, and after performing site-directed mutation and sequencing verification of the CGT enzyme gene, the The mutant gene was transformed into Bacillus subtilis WB600 for expression. 3. the preparation of mutant A31R, A31P and A31T described in claim 2: (1) Site-directed mutation Using rapid PCR technology, site-directed mutagenesis was carried out with the expression vector pST/cgt containing the wild CGTase gene as a template. Primers for site-directed mutagenesis introducing the Arg31 codon: Forward primer: 5'-GACGGCAATCCC CGC AACAATCC-3', the underline is the mutant base, Reverse primer: 5'-GGATTGTT GCG GGGATTGCCGTC-3', the underline is the mutant base; Primers for site-directed mutagenesis introducing the Pro31 codon: Forward primer: 5'-GACGGCAATCCC CCC AACAATCC-3', the underline is the mutant base, Reverse primer: 5'-GGATTGTT GGG GGGATTGCCGTC-3', the underline is the mutant base; Primers for site-directed mutagenesis introducing the Thr31 codon: Forward primer: 5'-GACGGCAATCCC ACC AACAATCC-3', the underline is the mutant base, Reverse primer: 5'-GGATTGTT GGT GGGATTGCCGTC-3', the underline is the mutated base. The PCR reaction system is: 5×PrimeSTAR Buffer (Mg ²⁺ Plus) 10 μL, dNTPs (2.5 mM each) 4 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL, PrimeSTARHS DNA Polymerase (2.5 U/μL) 0.5 μL, add 32.5 μL of double distilled water. The PCR amplification conditions are: PCR amplification conditions are: pre-denaturation at 98°C for 4min; followed by 35 cycles of 98°C for 10s, 55°C for 15s, and 72°C for 8min; and finally, incubation at 72°C for 10min. After the PCR product was digested by DpnI for 2 hours, it was transferred into Escherichia coli (Escherichia coli) JM109 competent cells, spread to LB solid medium containing agar and cultured overnight, and a single colony was picked and cultured overnight in LB liquid medium Plasmids were extracted and verified by sequencing. The mutant plasmid was transformed into the expression host B. subtilis WB600 competent cells. 5 μg/mL kanamycin sulfate and 10 μg/mL gibberellin were added to each medium. (2) Expression of mutants Pick the single clone of the expression host B.subtilis WB600 containing the mutant plasmid in LB medium, culture it at 37°C and 200r/min for 8-12h, and inoculate it into TB medium with 4% (v/v) inoculum , fermented at 37°C and 200r/min for 48h. The fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 minutes to remove bacteria, and the supernatant was collected and purified to obtain mutant A31R, A31P, and A31T enzyme products, respectively. 5 μg/mL kanamycin and 10 μg/mL gibberellin were added to each medium.

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