CN104611284A - Strain for production of cyclodextrin glucosyltransferase and application of strain - Google Patents

Abstract

The invention discloses a gene engineering strain for recombinant expression of Bacillus stearothermophilus alpha/beta-CGT enzyme and application of the gene engineering strain, belonging to the fields of gene engineering and enzyme engineering. According to the invention, the recombinant plasmid p SVEB-alpha/beta-CGTase is constructed, and Bacillus pumilus Brevibacillus brevis (CCTCC AB 94025) is transformed, so that recombinant Bacillus pumilus is obtained; based on the recombinant Bacillus pumilus as the strain, fermentation is carried to produce an alpha/beta-CGT enzyme which is applied to preparation of beta-cylodextrin and has good effect. The gene engineering strain for recombinant expression of Bacillus stearothermophilus alpha/beta-CGT enzyme disclosed by the invention has the advantages that the food-safe Bacillus pumilus is adopted as an expression host to recombine and produce alpha/beta-CGT enzyme, the recombinant strain is high in enzyme production, the ferment materials are wide in resources, and the production cost is relatively low.

Description

A kind of cyclomaltodextrin glucanotransferase produces bacterial strain and application thereof

Technical field

The invention discloses a kind of recombinant expressed Bacillus stearothermophilus α/β-CGT enzyme engineering strain and application thereof, belong to genetically engineered and enzyme engineering field.CGT enzyme gene is connected into bacillus pumilus expression vector pSVEB, then Brevibacillus brevis is transformed with recombinant vectors, achieve the high expression of cyclomaltodextrin glucanotransferase (α/β-CGTase) in bacillus pumilus (Brevibacillus brevis), belong to enzyme engineering and genetically engineered field.

Background technology

CGT enzyme is the enzyme of a multifunctional type, the reaction that its energy catalysis four kinds is different: three kinds turn glycosylation (disproportionation reaction, cyclization and coupled reaction) and hydrolysis reaction.CGT enzyme can utilize Starch Production cyclodextrin by cyclization, and this is the basis of its industrial application.Except producing cyclodextrin by cyclization, also use CGT enzyme catalysis coupling and disproportionation reaction recently, transfer donator, if the oligose on starch or cyclodextrin is on various acceptor molecule, obtains various special oligose.By the glycosylation of catalysis different acceptor molecule, functional, water-soluble, the stability to chemical substance that glycosylation can significantly improve acceptor molecule is carried out to such as stevioside, Hesperidin, violaguercitrin, vitamins C, rhamnosyl, xitix etc.In addition, the application of CGT enzyme limit dextrin is also found, and it can be applicable to top sizing in paper industry and coating, can improve the writing quality of paper and has smooth and be suitable for the surface of printing.CGT enzyme also can be used to process dough/pasta, by the disproportionation reaction of CGT enzyme, can increase the volume of the grilled product made by this dough.

The microbe species of production CGT enzyme is numerous, has genus bacillus, actinomycetes, micrococci, tyrothricin, aspergillus, archeobacteria etc.The CGT enzyme that present industrial production cyclodextrin uses is mainly from bacillus.According to cyclization initial period to generate the main Types of cyclodextrin different, CGT enzyme can be divided into α, β and γ type CGT enzyme, i.e. α-, β-and γ-CGT enzyme.Major part CGT enzyme is β type.

At present, many CGT enzyme obtain purifying, and the encoding gene (cgt gene) of corresponding CGT enzyme obtains clone.Because the throughput secreting the wild strain of CGT enzyme is general lower, in order to overcome the underproductivity of wild strain, recombination bacillus coli obtains great attention abroad in suitability for industrialized production CGT enzyme.But many recombinant C GT enzymes accumulate at cell interior as a kind of inclusion body in intestinal bacteria, the activation of this inclusion body needs the critical process such as complicated sex change and refolding, therefore, is unfavorable for suitability for industrialized production.At home, most of investigator is mainly devoted to the production of enzyme improving original strain.

Bacillus pumilus has the following advantages as expression system: (1) having the protein direct secretion of function to outer (2) non-virulent of born of the same parents, can be applied to foodstuffs industry.In picture intestinal bacteria, due to comparatively far away with many gram-positive microorganism sibships, so may correction be difficult to the albumen that some positive bacterias are originated.And bacillus pumilus is as a kind of gram-positive microorganism, can be good at the protein of expressing positive bacteria source, simultaneously can also can be correct for the protein with quaternary structure folding expression.And bacillus pumilus exocytosis is good, intracellular protein content is few, is suitable as very much the expressive host of CGT enzyme.

Cyclodextrin is the oligose irreducibility series of compounds with the hydrophobic conical structure of ring-type be formed by connecting by more than six glucose, wherein the most frequently used be α-, β-, γ-cyclodextrin, they are made up of 6,7,8 glucose units respectively.At present, the suitability for industrialized production of cyclodextrin all adopts enzymatic clarification, namely under CGT enzyme katalysis, by cyclization converted starch and relevant matrix synthesis cyclodextrin.Because cyclodextrin can form inclusion compound with many objects, thus the physics and chemistry character of change guest molecule is as solubleness, stability etc., therefore has a wide range of applications in fields such as food, medicine, agricultural, weaving, environmental protection, makeup, biotechnology and analytical chemistry.At present, the annual growth of cyclodextrin reaches 20%-30%.The application percentage of cyclodextrin in each industrial circle is roughly: foodstuffs industry 80%, medicine 10%, agricultural chemicals 5%-10%.Beta-cyclodextrin is low because of its solubleness, more easily precipitates, in 3 kinds of cyclodextrin, the most easily realize large-scale industrial production.

Summary of the invention

The present invention's first object is to provide a kind of restructuring bacillus pumilus of α/β-CGT enzyme, is the genetic engineering bacterium obtained by the coding α/β deriving from bacstearothermophilus-CGT enzyme channel genes bacillus pumilus.

Described coding α/β-CGT enzyme gene nucleotide series is as shown in SEQ ID NO.1.

Described bacillus pumilus is Brevibacillus brevis (CCTCC AB 94025).

The present invention's second object is to provide the construction process of the restructuring bacillus pumilus of a kind of secreting, expressing α/β-CGT enzyme.

The construction process of described restructuring bacillus pumilus is as follows:

1) according to the gene design primer of bacstearothermophilus α/β-CGT enzyme, with original bacteria genome for template, the goal gene containing coded signal peptide sequence is cloned.

2) the goal gene SEQ ID NO.1 cloned is connected on carrier pSVEB, Transformed E .coli JM109, the LB that coating has resistance is dull and stereotyped, PCR, double digestion checking positive transformant, obtain recombinant plasmid pSVEB-α/β-CGTase, the recombinant plasmid after qualification is carried out sequencing analysis.

3) enter in bacillus pumilus by recombinant plasmid pSVEB-α/β-CGTase by electricity conversion, the TM that coating has resistance is dull and stereotyped, and PCR, double digestion checking positive transformant, waits to do to express and verify.

3rd object of the present invention is to provide a kind of method utilizing described bacillus pumilus production α/β-CGTase.

The described concrete grammar of restructuring bacillus pumilus production α/β-CGT enzyme that utilizes is as follows:

From the upper picking list bacterium colony of the Liu Suanyan NEOMYCIN SULPHATE TM flat board (TM substratum: polyprotein peptone 1%, beef extract 0.5%, yeast powder 0.2%, glucose 1%) containing Liu Suanyan NEOMYCIN SULPHATE 10mg/mL in TM substratum, in 30 DEG C, 200r/min cultivates 48 hours.The centrifugal 5min of fermented liquid 8000rpm obtained is removed thalline, and the fermented supernatant fluid of acquisition is crude enzyme liquid, and the fermented supernatant fluid enzyme work obtained is at 85U/mL; When 3L fermentor tank is produced, be inoculated in 3L fermentor tank after 1 grade, 2 grades kinds spread cultivation, substratum is yeast powder substratum, and end in 32 hours of fermenting, final enzyme work is at 403.5U/mL.

4th object of the present invention is to provide a kind of method utilizing restructuring α/β-CGTase converted starch to prepare beta-cyclodextrin.

The described concrete grammar utilizing restructuring α/β-CGT enzyme converted starch to prepare beta-cyclodextrin is as follows:

Drop into the yam starch of 50-300g/L in the reactor, add enzyme liquid described in 150U in 70 DEG C of liquefaction, the NaOH of post liquefaction 5M regulates pH to 6.5, add the enzyme liquid that 50mL hexanaphthene adds 750-2250U again, at 30-60 DEG C, react 24 hours in the shaking bath of 150rpm, after reaction terminates, reaction solution is boiled 15 minutes by enzyme-deactivating.By this method, the final transformation efficiency of cyclodextrin can reach 70-81.6%.

Accompanying drawing explanation

Fig. 1 recombinant bacterium shake flask fermentation produces enzyme curve

The outer supernatant SDS-PAGE electrophorogram of Fig. 2 recombinant bacterium shake flask fermentation born of the same parents

Fig. 3 is 3L ferment tank thalli growth and produces enzyme curve

The outer supernatant liquor SDS-PAGE electrophorogram of Fig. 4 recombinant bacterium 3L ferment tank born of the same parents

Embodiment

Embodiment 1: the structure of the Cloning and Expression carrier of bacstearothermophilus α/β-CGT enzyme encoding gene

Bacstearothermophilus (deposit number is CCTCC M 2013413) is cultivated 2 days in LB liquid nutrient medium, 10000rpm collected by centrifugation thalline, sterilized water washs, collecting precipitation is suspended in 500uL Tris-EDTA damping fluid, add 15uL N,O-Diacetylmuramidase, 30min is incubated at 37 DEG C, add 5uL RNA enzyme again, 30min is incubated at 37 DEG C, add 30 μ L 10% sodium lauryl sulphate and 15 μ L Proteinase Ks, 60min is incubated at 37 DEG C, add NaCl 100uL and the cetyl trimethylammonium bromide 80 μ L of 5M, 20min is incubated at 65 DEG C, phenol with 700 μ L: chloroform: primary isoamyl alcohol volume ratio is the mixed-solvent extraction of 25:24:1, 10000rpm is centrifugal, the supernatant liquor chloroform of 700 μ L: primary isoamyl alcohol volume ratio is the mixed-solvent extraction of 24:1, 10000rpm is centrifugal, the supernatant liquor ice primary isoamyl alcohol of 1400 μ L volumes mixes,-20 DEG C of precipitation 30min, 10000rpm is centrifugal, precipitation adds the ethanol purge of 200 μ L70% concentration, 10000rpm is centrifugal, precipitation uses Tris-EDTA buffer solution, be bacstearothermophilus STb gene,

α/β-CGTase gene design primer P1, P2 according in database:

P1：5’-ggATCCgCgggCAACCTgAACAAAgTgAAC-3’

P2：5’-AAgCTTTTAgTTCTgCCAgTCAACgATAAT-3’

With original bacteria genome for template, clone is containing the goal gene of coded signal peptide sequence, PCR system is: 10 μMs of each 1 μ L of primer P1 and P2,2mM dNTPS 5 μ L, 10 × KOD-Plus-NeoBuffer 5 μ L, the PrimeStar polysaccharase 1uL of 1U/ μ L, template 0.5 μ L, adds distilled water polishing 50 μ L.PCR condition: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min 30s, 30 circulations.PCR primer and carrier pSVEB are carried out double digestion respectively, and glue reclaims, spend the night in 16 DEG C of connections, Transformed E .coli JM109, the LB of coating containing ammonia benzyl (100 μ g/mL) resistance is dull and stereotyped, cultivates 10-12h for 37 DEG C, picking 3 transformants, extract recombinant plasmid to go forward side by side performing PCR and double digestion checking, then DNA sequence dna is measured, positive colony and pSVEB-α/β-CGTase to the correct recombinant plasmid of checking.

Embodiment 2: the conversion of bacillus pumilus

1) fresh TM flat board (TM substratum: polyprotein peptone 1%, beef extract 0.5%, yeast powder 0.2%, glucose 1%) choose bacillus pumilus be Brevibacillus brevis (CCTCC AB 94025) single colony inoculation in 10ml TM substratum, incubated overnight.

2) measure OD in shaking flask, control inoculum size well, make the OD of substratum after inoculation ₆₀₀between 0.19-0.2.Substratum is 50mL GM (peptone 0.5%, soy peptone 0.5%, beef extract 0.5%, yeast extract 0.25%, glucose 0.5%, α-Sodium Glycerophosphate 1.9%, vitamins C 0.05%, MgSO ₄0.012%).37 DEG C, 200rpm is cultured to OD ₆₀₀=1.0 (about 3-4 hour) left and right.

3) whole bacterium liquid ice-water bath 10min, then 5000rpm, 8min is got, 4 DEG C of collected by centrifugation thalline.

4) turn damping fluid ETM (sorbyl alcohol 9%, N.F,USP MANNITOL 9%, glycerol 10%) with the electricity of 40ml precooling and wash thalline, 5000rpm, 8min, 4 DEG C centrifugal removes supernatant, rinsing like this 3 times.

5) thalline after washing is resuspended in etc. in the ETM of 500 μ L, often pipe packing 60 μ L.

6) add 1-6 μ L plasmid by 60 μ l competent cells, ice bath hatches 5min, adds in the electric revolving cup (1mm) of precooling, and electric shock once.Electroporation is arranged: 2.0kv, 25uF 200 Ω, 1mm, shock by electricity 1 time (between time length 4.5ms-5ms).

7) shock by electricity and completely add 1mL recovery medium TM immediately, 37 DEG C, 200rpm, after recovery 3h, coated plate.37 DEG C, incubated overnight, picking colony, in TM Liu Suanyan NEOMYCIN SULPHATE substratum, is verified, carries out next step and produce enzyme after correct.

Embodiment 3: shake flask fermentation produces enzyme

1) fermentation culture

By screening the product cyclomaltodextrin glucanotransferase inoculation that obtains in embodiment 1 in TM substratum, with 5% inoculum size be forwarded in TM fermention medium after cultivating 12h at 30 DEG C, 30 DEG C of constant temperature culture 44 ~ 48h produce enzymes.After fermentation ends, collected by centrifugation supernatant liquor is crude enzyme liquid.

TM substratum (g/L): polyprotein peptone 10, beef powder 5, yeast powder 2, glucose 10, pH7.0

2) enzyme activity determination

Take Zulkovsky starch as the enzyme work that substrate utilization tropeolin-D method measures this enzyme.Enzyme activity determination system is: and 2.5mL (be the Zulkovsky starch of 2% containing final concentration, 50mM KH ₂pO ₄-Na ₂hPO ₄damping fluid, pH 6.0), add the enzyme liquid reaction 10min that 100 μ L suitably dilute at 50 DEG C after, add 3M hydrochloric acid soln 200 μ l termination reaction, at 16 DEG C, add 0.44mM tropeolin-D 200 μ L develop the color, at spectrophotometer 505nm place mensuration light absorption value.Under this condition, the per minute enzyme amount generated needed for 1 μm of ol alpha-cylodextrin is defined as a Ge Meihuo unit (U).

Enzyme is lived and is extended in time and increase gradually as seen from Figure 1, and reach stable at 48 hours, maximum enzyme vigor is about 85U/ml, and protein electrophoresis result is presented at 66.2kDa place a band (Fig. 2) consistent with theoretical molecular.

3L tank enzymatic production experiment in embodiment 4:TM substratum

1) preparation of one-level kind: single for bacillus pumilus bacterium colony is chosen 30 DEG C, 220rpm incubated overnight in TM liquid nutrient medium, and gained bacterial classification is one-level kind.

2) preparation of secondary kind: one-level kind is inoculated in 200mL TM substratum, is cultured to OD ₆₀₀be about 3 (cultivating 11-12 hour).

3) 3L ferment tank: secondary kind be inoculated in 3L fermentor tank, TM substratum (supplements KH ₂pO ₄1.4%, (NH ₄) ₂hPO ₄0.5%, citric acid 0.2%, MgSO ₄0.68g/L), 30 DEG C, ventilate and stir, dissolved oxygen controls at 20-30%, and with citric acid, pH is at 6-8 in NaOH control, cultivates about 32 hours.Fermented liquid is through 10000g bactofugation, and supernatant is α/β-CGT enzyme stoste, and final enzyme is lived as 246.8U/mL.

Embodiment 5: 3L tank enzymatic production experiment in beef extract substratum

1) preparation of one-level kind: single for bacillus pumilus bacterium colony is chosen 30 DEG C, 220rpm incubated overnight in TM liquid nutrient medium, and gained bacterial classification is one-level kind.

2) preparation of secondary kind: one-level kind is inoculated in 200mL TM substratum, is cultured to OD ₆₀₀be about 3 (cultivating 11-12 hour).

3) 3L ferment tank: secondary kind is inoculated in 3L fermentor tank, beef extract substratum (beef extract 2%, yeast powder 0.2%, KH ₂pO ₄1.4%, (NH ₄) ₂hPO ₄0.5%, citric acid 0.2%, MgSO ₄0.68g/L, glucose 1%), 25-30 DEG C, ventilate and stir, dissolved oxygen controls at 20-30%, and with citric acid, pH is at 6-8 in NaOH control, cultivates about 32 hours.Fermented liquid is through 10000g bactofugation, and supernatant is α/β-CGT enzyme stoste, and final enzyme is lived as 321.5U/mL.

Embodiment 6: 3L tank enzymatic production experiment in yeast powder substratum

1) preparation of one-level kind: single for bacillus pumilus bacterium colony is chosen 30 DEG C, 220rpm incubated overnight in TM liquid nutrient medium, and gained bacterial classification is one-level kind.

2) preparation of secondary kind: one-level kind is inoculated in 200mL TM substratum, is cultured to OD ₆₀₀be about 3 (cultivating 11-12 hour).

3) 3L ferment tank: secondary kind is inoculated in 3L fermentor tank, yeast powder substratum (yeast powder 3%, KH ₂pO ₄1.4%, (NH ₄) ₂hPO ₄0.5%, citric acid 0.2%, MgSO ₄0.68g/L, glucose 1%), 25-30 DEG C, ventilate and stir, dissolved oxygen controls at 20-30%, and with citric acid, pH is at 6-8 in NaOH control, cultivates about 32 hours.Fermented liquid is through 10000g bactofugation, and supernatant is α/β-CGT enzyme stoste, and final enzyme is lived as 403.5U/mL (Fig. 3), is the highest level that this enzyme is reported at present.In upper tank fermenting process, the SDS-PAGE protein electrophoresis result of different time fermented supernatant fluid sample as indicated at 4.

Embodiment 7: the impact that enzyme concentration is produced CD

Enzymatic production process: the yam starch dropping into 150g/L in the reactor, add enzyme liquid described in 150U in 70 DEG C of liquefaction, the NaOH of post liquefaction 5M regulates pH to 6.5, add the enzyme liquid that 50mL hexanaphthene adds 750-2250U (see table 1) again, at 40 DEG C, react 24 hours in the shaking bath of 150rpm, after reaction terminates, reaction solution is boiled 15 minutes by enzyme-deactivating.

HPLC testing conditions is: Agilent1200HPLC chromatographic instrument, Agilent automatic sampler, Agilent Composition distribution; Chromatographic column Zorbax NH ₂(4.6mm × 150mm), column temperature 40 DEG C; The ratio (v:v) of moving phase is acetonitrile: water is 70:30, flow velocity 0.8mL × min ^-1.Adopt external standard method, determine the concentration of corresponding CD according to retention time and peak area.

The results are shown in Table 1, under different enzyme concentration, β-CD is to the mass transitions rate of substrate yam starch.Can find out, when enzyme concentration is 1950U, the total conversion rate of CD is the highest, therefore 1950U/L transforms the best enzyme concentration producing β-CD, the content of the beta-cyclodextrin now obtained reaches 120.08g/L, and the transformation efficiency of beta-cyclodextrin is 80.5%, accounts for 98.7% of all cyclodextrin product.

The conversion situation of β-CD under the different enzyme concentration of table 1

Embodiment 8: the impact that temperature is produced beta-cyclodextrin

Enzymatic production process: the yam starch dropping into 150g/L in the reactor, add enzyme liquid described in 150U in 70 DEG C of liquefaction, the NaOH of post liquefaction 5M regulates pH to 6.5, add the enzyme liquid that 50mL hexanaphthene adds 750U again, at different temperatures (see table 2), react 24 hours in the shaking bath of 150rpm, after reaction terminates, reaction solution is boiled 15 minutes by enzyme-deactivating.

HPLC detection method is with embodiment 8

The results are shown in Table 2, under differing temps, β-CD is to the mass transitions rate of substrate yam starch.Can find out, 50 DEG C time, the total conversion rate of CD is the highest, in addition, because the specificity of high temperature to product is favourable, so the β-CD proportion generated is also the highest, therefore 50 DEG C is transform the optimum temps of producing β-CD, the content of the beta-cyclodextrin now obtained reaches 118.8g/L, and the transformation efficiency of beta-cyclodextrin is 79.2%, accounts for 96.6% of all cyclodextrin product.

The conversion situation of β-CD under table 2 differing temps

Claims (6)

1. the bacillus pumilus genetic engineering bacterium of a plant height efficient expression Bacillus stearothermophilus α/β-CGT enzyme.

2. α/β-the CGT enzyme of genetic engineering bacterium production according to claim 1, its parent amino acid sequence and accession number in ncbi database are that the bacstearothermophilus CGT enzyme of P31797.1 is consistent.

3. recombination engineering bacteria according to claim 1, it is characterized in that, the used carrier building its expression plasmid is pSVEB, and host strain used is bacillus pumilus Brevibacillus brevis (CCTCC AB 94025), builds recombinant bacterium step as follows:

1) upgrading grain from Laboratories Accession bacterial strain, PCR, enzyme are cut and are obtained α/β-CGT enzyme gene;

2) connect expression vector pSVEB, build the recombinant plasmid containing α/β-CGT enzyme gene, be converted in E.coli JM109 and increase;

3) recombinant plasmid electricity after digestion verification is gone in Brevibacillus brevis, in resistant panel, select transformant.

4. recombination engineering bacteria described in claim 1, its zymotechnique is as follows:

1) preparation of one-level kind: single for described restructuring bacillus pumilus bacterium colony is chosen in 30 DEG C, 220rpm in TM liquid nutrient medium, cultivates 8-10 hour;

2) preparation of secondary kind: one-level kind is inoculated in 200mL TM substratum, is cultured to OD ₆₀₀be about 3 (cultivating 11-12 hour);

3) 3L ferment tank: secondary kind is inoculated in and is equipped with in the 3L fermentor tank of sterilization fermentation substratum, control temperature 25-37 DEG C, ventilate and stir, dissolved oxygen controls at 20-30%, with citric acid, pH is at 6-8 in NaOH control, cultivates about 32 hours, fermented liquid is through 10000g bactofugation, and supernatant is α/β-CGT enzyme fermented liquid.

5. fermention medium is according to claim 4 TM substratum, beef extract substratum or yeast powder substratum.

6. α/β-CGT enzyme according to claim 4 is preparing the application in beta-cyclodextrin:

1) by the gelatinization at 60-90 DEG C of 50 ~ 300g/L yam starch, α/β-CGT enzyme liquid is added according to the amount of every gram of dry starch 0.5U, at 60-80 DEG C of heated and stirred 10-30 minute;

2) add α/β-CGT enzyme liquid and the 50ml/L hexanaphthene of 250-1000U/L, at 30-50 DEG C, react 12-24h;

3) distillation removing hexanaphthene, namely obtains the solution of β-CD.