CN103740669A - Method for improving beta-cyclodextrin production capability of cyclodextrin glycosyltransferase by calcium ion binding site amino acid residue mutation - Google Patents

Abstract

The invention provides a mutant of cyclodextrin glycosyltransferase (CGT enzyme for short) with capability for high-yield of beta-cyclodextrin and mutating method thereof, and belongs to the fields of gene engineering and enzyme engineering. The invention employs a site-directed mutagenesis method for improving beta-cyclodextrin production capability of CGT enzyme, and provides a mutating scheme for improving beta-cyclodextrin production capability of Bacillus circulans STB01CGT, that is alanine at the 315th position of the calcium ion binding site in CGT enzyme is changed into arginine (Arg) or aspartic acid (Asp), and the mutants A315R and A315D are obtained. Compared with the wild CGT enzyme, the two mutants have higher beta-cyclodextrin production capability, and are suitable for industrial production of beta-cyclodextrin.

Description

The amino-acid residue sudden change of calcium binding site improves the method for cyclomaltodextrin glucanotransferase product beta-cyclodextrin ability

Technical field

There is mutant and the method thereof of the cyclomaltodextrin glucanotransferase of high yield beta-cyclodextrin ability, the invention belongs to genetically engineered and enzyme engineering field.The present invention specifically utilizes site-directed mutagenesis technique to improve the product specificity of cyclomaltodextrin glucanotransferase.

Background technology

Because the ring-type hollow conic structure of cyclodextrin has interior hydrophobic, outer hydrophilic characteristic, can form inclusion compound with various hydrophobic material, therefore, in field extensive application such as medicine, chemical industry, food, material and analytical chemistry, wherein α-, β-and γ-cyclodextrin is the most common.

At present, cyclodextrin is synthesized by cyclomaltodextrin glucanotransferase (being called for short CGT enzyme) effect starch conventionally.CGT enzyme is a kind of multifunctional type enzyme, energy catalytic cyclization reaction, coupled reaction, disproportionation reaction and hydrolysis reaction, and wherein cyclization is the basis of its industrial application.The product of known wild CGT enzyme cyclization be all α-, β-and the mixture of γ-cyclodextrin, be unfavorable for the separation and purification of product, increased the production cost of cyclodextrin, therefore, be necessary to improve its product specificity.Research discovery, calcium binding site is prevalent in CGT enzyme, and may be closely related with the product specificity of CGT enzyme.

The present invention suddenlys change deriving from calcium binding site the 315th amino acids residue in the CGT enzyme of Bacillus circulans (Bacillus circulans) STB01, to improve the product specificity of CGT enzyme.

Summary of the invention

The object of this invention is to provide the sudden change scheme that improves B.circulans STB01CGT enzyme product beta-cyclodextrin ability.

Another object of the present invention is mutant A315R and A315D and the construction process thereof that proposes to have higher beta-cyclodextrin throughput.

Technical scheme of the present invention:

Deriving from mutant A315R and the A315D of the CGT enzyme of B.circulans STB01, is that the 315th Ala of calcium binding site in CGT enzyme sported respectively to Arg and Asp.Than wild CGT enzyme, mutant A315R and A315D have higher beta-cyclodextrin throughput.

Described mutant A315R and the preparation method of A315D, according to B.circulans STB01CGT enzyme gene order, design respectively the primer in corresponding mutational site, CGT enzyme gene is suddenlyd change, and identify Ala315 codon and become the mutator gene of Arg315, Asp315 codon, and express in subtilis (Bacillus subtilis) WB600.

(1) rite-directed mutagenesis

Utilize fast PCR technology, take the expression vector pST/cgt containing wild CGT enzyme gene as template, carry out rite-directed mutagenesis.

The mutant primer of introducing Arg315 codon is:

Forward primer: 5 '-GCAGCCGATTAC cGCcAGGTGGATG-3 ', underscore is mutating alkali yl,

Reverse primer: 5 '-GTCATCCACCTG gCGgTAATCGGCTG-3 ', underscore is mutating alkali yl;

The mutant primer of introducing Asp315 codon is:

Forward primer: 5 '-GCAGCCGATTAC gACcAGGTGGATG-3 ', underscore is mutating alkali yl.

Reverse primer: 5 '-GTCATCCACCTG gTCgTAATCGGCTG-3 ', underscore is mutating alkali yl.

PCR reaction system is: 5 × PrimeSTAR Buffer (Mg ²⁺plus) 10 μ L, dNTPs (each 2.5mM) 4 μ L, forward primer (10 μ M) 1 μ L, reverse primer (10 μ M) 1 μ L, template DNA 1 μ L, PrimeSTAR HS DNA Polymerase (2.5U/ μ L) 0.5 μ L, adds distilled water 32.5 μ L.

PCR reaction amplification condition is: pcr amplification condition is: 98 ℃ of denaturation 4min; 98 ℃ of 10s subsequently, 55 ℃ of 15s, 72 ℃ of 8min carry out 35 circulations; Last 72 ℃ of insulation 10min.

By PCR product after DpnI digestion 2h, proceed in intestinal bacteria (Escherichia coli) JM109 competent cell, be applied to overnight incubation in the LB solid medium that contains agar, picking list bacterium colony extracts plasmid and carries out sequence verification in LB liquid nutrient medium after overnight incubation.Mutant plasmid is proceeded in expressive host B.subtilis WB600 competent cell.In each substratum, all add 5 μ g/mL sulphuric acid kanamycins and 10 μ g/mL Plant hormones regulators,gibberellins.

(2) expression and purification of mutant

Picking in LB substratum, is cultivated 8～12h containing the mono-clonal of the expressive host B.subtilis WB600 of mutant plasmid under 37 ℃, 200r/min, is inoculated in TB substratum, at 37 ℃, 200r/min bottom fermentation 48h with 4% (v/v) inoculum size.By fermented liquid in 4 ℃, the centrifugal 20min of 10000rpm to remove thalline, collect supernatant liquor purifying, obtain respectively mutant A315R and A315D enzyme preparation.In each substratum, add 5 μ g/mL kantlex and 10 μ g/mL Plant hormones regulators,gibberellins.

Final effect of the present invention: built mutant A315R and A315D, all realized the specific raising of beta-cyclodextrin product, be more conducive to the suitability for industrialized production of beta-cyclodextrin than wild-type CGT enzyme.

Accompanying drawing explanation

The wild CGT enzyme of Fig. 1 and mutant thereof act on 5% maltodextrin solution and produce cyclodextrin situation at pH6.0,50 ℃.A: wild CGT enzyme, B: mutant A315R, C: mutant A315D; ■, alpha-cylodextrin, ●, beta-cyclodextrin, ▲, γ-cyclodextrin

Embodiment

Embodiment 1: this example illustrates the preparation of mutant A315R and A315D

(1) rite-directed mutagenesis

Utilize fast PCR technology, take the expression vector pST/cgt containing wild CGT enzyme gene as template, carry out rite-directed mutagenesis.

The mutant primer of introducing Arg315 codon is:

Forward primer: 5 '-GCAGCCGATTAC cGCcAGGTGGATG-3 ', underscore is mutating alkali yl,

Reverse primer: 5 '-GTCATCCACCTG gCGgTAATCGGCTG-3 ', underscore is mutating alkali yl;

The mutant primer of introducing Asp315 codon is:

Forward primer: 5 '-GCAGCCGATTAC gACcAGGTGGATG-3 ', underscore is mutating alkali yl.

Reverse primer: 5 '-GTCATCCACCTG gTCgTAATCGGCTG-3 ', underscore is mutating alkali yl.

PCR reaction system is: 5 × PrimeSTAR Buffer (Mg ²⁺plus) 10 μ L, dNTPs (each 2.5mM) 4 μ L, forward primer (10 μ M) 1 μ L, reverse primer (10 μ M) 1 μ L, template DNA 1 μ L, PrimeSTAR HS DNA Polymerase (2.5U/ μ L) 0.5 μ L, adds distilled water 32.5 μ L.

PCR reaction amplification condition is: pcr amplification condition is: 98 ℃ of denaturation 4min; 98 ℃ of 10s subsequently, 55 ℃ of 15s, 72 ℃ of 8min carry out 35 circulations; Last 72 ℃ of insulation 10min.

By PCR product after DpnI digestion 2h, proceed in intestinal bacteria (Escherichia coli) JM109 competent cell, be applied to overnight incubation in the LB solid medium that contains agar, picking list bacterium colony extracts plasmid and carries out sequence verification in LB liquid nutrient medium after overnight incubation.Mutant plasmid is proceeded in expressive host B.subtilis WB600 competent cell.In each substratum, all add 5 μ g/mL sulphuric acid kanamycins and 10 μ g/mL Plant hormones regulators,gibberellins.

(2) expression and purification of mutant

Picking proceeds to the mono-clonal of expressive host B.subtilis WB600 in LB substratum, under 37 ℃, 200r/min, cultivates 8～12h, is inoculated in TB substratum, at 37 ℃, 200r/min bottom fermentation 48h with 4% (v/v) inoculum size.By fermented liquid in 4 ℃, the centrifugal 20min of 10000rpm to remove thalline, collect supernatant liquor purifying, obtain respectively mutant A315R and A315D enzyme preparation.In each substratum, add 5 μ g/mL kantlex and 10 μ g/mL Plant hormones regulators,gibberellins.

Supernatant liquor precipitates and spends the night in 70% ammoniumsulphate soln.Throw out is through appropriate Start buffer (20mmol/L Tris-HCl, pH8.0) dissolve, HiTrap ANX FF (high sub, 5mL) loading after Start buffer balance for post, elutriant is the Start buffer that contains 1mol/L NaCl, distribute and collect, obtain purified mutant body A315R and A315D.

Embodiment 2: this example illustrates enzyme activity analysis

(1) mensuration of enzyme activity

Tropeolin-D method is measured the method for α-cyclisation vigor: the enzyme liquid 0.1mL that gets suitable dilution, add and be equipped with in 1% (w/v) Zulkovsky starch solution that 0.9mL uses 50mM phosphoric acid buffer (pH6.5) preparation in advance, at 40 ℃, react after 10min, add the hydrochloric acid stopped reaction of 1.0mL1.0N, add again the 0.1mM methyl orange solution of 1.0mL 50mM phosphoric acid buffer preparation, at 16 ℃, be incubated 20min, under 505nm, measure absorbancy.An enzyme unit definition alive is that per minute generates the required enzyme amount of 1 μ mol alpha-cylodextrin under these conditions.

Phenolphthalein method is measured the method for β-cyclisation vigor: the enzyme liquid 0.1mL that gets suitable dilution, add and be equipped with in the test tube of 1% (w/v) Zulkovsky starch solution that 0.9mL uses 50mM phosphoric acid buffer (pH6.5) preparation in advance, at 40 ℃, react after 10min, add 3.5mL30mM NaOH and 0.5mL by 5mM Na ₂cO ₃0.02% (w/v) phenolphthalein solution stopped reaction of solution preparation, is at room temperature incubated 20min, under 550nm, measures absorbancy.An enzyme unit definition alive is that per minute generates the required enzyme amount of 1 μ mol beta-cyclodextrin under these conditions.

Bromine cresols chlorine method is measured the method for γ-cyclisation vigor: the enzyme liquid 0.1mL that gets suitable dilution, add and be equipped with in the test tube of 1% (w/v) Zulkovsky starch solution that 0.9mL uses 50mM phosphoric acid buffer (pH6.5) preparation in advance, at 40 ℃, react after 10min, add the hydrochloric acid stopped reaction of 50 μ L1.0N, add again 2mL0.2M citrate buffer solution (pH4.2) and 100 μ L5mM bromine cresols chlorine solution, at room temperature be incubated 20min, under 630nm, measure absorbancy.An enzyme unit definition alive is that per minute generates the required enzyme amount of 1 μ mol γ-cyclodextrin under these conditions.

(2) enzyme activity comparison

Experimental result is listed in table 1, found that, wild CGT enzyme has higher β-cyclisation vigor; Compared with wild enzyme, α-cyclisation vigor of mutant A315R has reduced by 86%, β-cyclisation vigor and has increased by 12%, γ-cyclisation vigor increase by 107%; α-cyclisation vigor of mutant A315D has reduced by 71%, β-cyclisation vigor and has increased by 10%, γ-cyclisation vigor increase by 72%.Total cyclisation vigor of mutant A315R and A315D remains unchanged substantially.And the ratio that β-cyclisation vigor of mutant A315R and A315D accounts for total cyclisation vigor is higher than wild enzyme.

Table 1

Embodiment 3: this example illustrates utilizes HPLC to measure cyclodextrin growing amount

To prepare 5% (wet basis, water content 8%, w/v) maltodextrin (DE3) solution is as substrate, and 5g maltodextrin (DE3) is dissolved in 90mL sodium phosphate buffer (pH6.0), be settled to 100mL, in boiling water, boil 30min.Add respectively a certain amount of wild CGT enzyme, mutant A315R and A315D that enzyme in reaction system is lived as 1U/mL, be placed at 50 ℃ and react 9h, interval samples 600 μ L, boils the enzyme 10min that goes out, the centrifugal 10min of 12000rpm, get supernatant 500 μ L, add 5 μ L saccharifying enzyme (70U/mL), at 30 ℃ of saccharification 1h, 10min boils deactivation, the centrifugal 30min of 12000rpm, gets supernatant and after 0.45 μ m ultrafiltration membrance filter, gets HPLC analysis on 20 μ L.

HPLC condition determination is: Waters600 high performance liquid chromatograph (joining differential refraction detector), chromatographic column Lichrosorb NH ₂(4.6mm × 150mm), moving phase adopts 68% acetonitrile solution, and column temperature is 30 ℃, and flow velocity is 1mL/min.

Experimental result as shown in Fig. 1 and table 2, wild CGT enzyme main product beta-cyclodextrin; Than wild enzyme, mutant A315R and A315D have higher beta-cyclodextrin throughput, are more suitable for the production of beta-cyclodextrin.After cultivating by 9h, compared with wild-type, the beta-cyclodextrin output of these two mutant has increased respectively 15.1% and 10.8%, and γ-cyclodextrin output also has increase in various degree, and alpha-cylodextrin output has reduced respectively 56.4% and 38.5%.

Table 2

Claims (3)

1. the mutant A315R and the A315D that derive from the cyclomaltodextrin glucanotransferase (being called for short CGT enzyme) of Bacillus circulans (Bacillus circulans) STB01, is characterized in that: the 315th L-Ala of calcium binding site in CGT enzyme (Ala) sported respectively to arginine (Arg) or aspartic acid (Asp); Than wild CGT enzyme, mutant A315R and A315D have higher beta-cyclodextrin throughput.

2. the preparation method of mutant A315R and A315D described in claims 1, it is characterized in that according to B.circulans STB01CGT enzyme gene order, design respectively the primer in corresponding mutational site, CGT enzyme gene is suddenlyd change, after sequence verification, mutator gene is proceeded in subtilis (Bacillus subtilis) WB600 and express.

3. the preparation of mutant A315R and A315D described in claims 2:

(1) rite-directed mutagenesis

Utilize fast PCR technology, take the expression vector pST/cgt containing wild CGT enzyme gene as template, carry out rite-directed mutagenesis.

The mutant primer of introducing Arg315 codon is:

Forward primer: 5 '-GCAGCCGATTAC cGCcAGGTGGATG-3 ', underscore is mutating alkali yl,

Reverse primer: 5 '-GTCATCCACCTG gCGgTAATCGGCTG-3 ', underscore is mutating alkali yl;

The mutant primer of introducing Asp315 codon is:

Forward primer: 5 '-GCAGCCGATTAC gACcAGGTGGATG-3 ', underscore is mutating alkali yl.

Reverse primer: 5 '-GTCATCCACCTG gTCgTAATCGGCTG-3 ', underscore is mutating alkali yl.

PCR reaction system is: 5 × PrimeSTAR Buffer (Mg ²⁺plus) 10 μ L, dNTPs (each 2.5mM) 4 μ L, forward primer (10 μ M) 1 μ L, reverse primer (10 μ M) 1 μ L, template DNA 1 μ L, PrimeSTAR HS DNA Polymerase (2.5U/ μ L) 0.5 μ L, adds distilled water 32.5 μ L.

PCR reaction amplification condition is: pcr amplification condition is: 98 ℃ of denaturation 4min; 98 ℃ of 10s subsequently, 55 ℃ of 15s, 72 ℃ of 8min carry out 35 circulations; Last 72 ℃ of insulation 10min.

By PCR product after DpnI digestion 2h, proceed in intestinal bacteria (Escherichia coli) JM109 competent cell, be applied to overnight incubation in the LB solid medium that contains agar, picking list bacterium colony extracts plasmid and carries out sequence verification in LB liquid nutrient medium after overnight incubation.Mutant plasmid is proceeded in expressive host B.subtilis WB600 competent cell.In each substratum, all add 5 μ g/mL sulphuric acid kanamycins and 10 μ g/mL Plant hormones regulators,gibberellins.

(2) expression of mutant A315R and A315D

Picking in LB substratum, is cultivated 8～12h containing the mono-clonal of the expressive host B.subtilis WB600 of mutant plasmid under 37 ℃, 200r/min, is inoculated in TB substratum, at 37 ℃, 200r/min bottom fermentation 48h with 4% (v/v) inoculum size.By fermented liquid in 4 ℃, the centrifugal 20min of 10000rpm to remove thalline, collect supernatant liquor purifying, obtain respectively mutant A315R and A315D enzyme preparation.In each substratum, add 5 μ g/mL kantlex and 10 μ g/mL Plant hormones regulators,gibberellins.

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