阅读说明：本技术 一种环糊精酶在制备麦芽七糖中的应用 (Application of cyclodextrin enzyme in preparation of maltoheptaose ) 是由 黄燕 吴敬 王蕾 武权 陈晟 宿玲恰 于 2020-06-24 设计创作，主要内容包括：本发明公开了一种环糊精酶在制备麦芽七糖中的应用,属于生物技术领域。本发明提供了一种重组枯草芽孢杆菌,该重组枯草芽孢杆菌以枯草芽孢杆菌为宿主,表达热球菌Thermococcus sp.Strain B1001来源的环糊精酶。本发明通过将热球菌来源的CDase在食品准入的枯草芽孢杆菌中进行了重组表达,酶活为5.9U/mL,应用该重组CDase生产麦芽七糖,底物浓度80g/Lβ-环糊精,转化率高达82.33％。本发明的CDase在高底物浓度下的转化率和产物纯度高,可有效降低高纯度麦芽七糖的生产加工成本,具有很好的应用价值和商业开发意义。(The invention discloses an application of cyclodextrin in preparation of maltoheptaose, belonging to the technical field of biology. The invention provides a recombinant bacillus subtilis, which takes the bacillus subtilis as a host and expresses a cyclodextrin enzyme from a Thermococcus sp. According to the invention, CDase from Thermococcus is subjected to recombinant expression in food-accessible Bacillus subtilis, the enzyme activity is 5.9U/mL, the recombinant CDase is used for producing maltoheptaose, the substrate concentration is 80g/L beta-cyclodextrin, and the conversion rate is up to 82.33%. The CDase has high conversion rate and product purity under high substrate concentration, can effectively reduce the production and processing cost of high-purity maltoheptaose, and has good application value and commercial development significance.)

1. The application of the cyclodextrin enzyme in the preparation of maltoheptaose or products containing maltoheptaose is characterized in that the amino acid sequence of the cyclodextrin enzyme is shown as SEQ ID NO. 2.

2. Use of a cyclodextrin enzyme according to claim 1 for the preparation of maltoheptaose or a maltoheptaose-containing product, wherein the nucleotide sequence encoding the cyclodextrin enzyme is as shown in SEQ ID No. 1.

3. A method for preparing maltoheptaose is characterized in that the maltoheptaose is prepared by catalysis by taking cyclodextrin enzyme with an amino acid sequence shown as SEQ ID NO.2 as a catalyst and beta-cyclodextrin as a substrate.

4. The method for producing maltoheptaose according to claim 3, wherein the cyclodextrin enzyme is added in an amount of not less than 15U/g.

5. The method for producing maltoheptaose according to claim 3 or 4, wherein the concentration of the substrate is 40 to 120 g/L.

6. The method for preparing maltoheptaose according to any one of claims 3 to 5, wherein the substrate concentration is 100-120 g/L.

7. The process for producing maltoheptaose according to any one of claims 3 to 6, wherein the reaction temperature in the reaction system is 80 to 90 ℃, the pH is 5.0 to 6.0, and the reaction time is 2 to 8 hours.

8. Use of a recombinant plasmid comprising a gene encoding a cyclodextrin enzyme having the nucleotide sequence shown in SEQ ID No.1, a host cell carrying a gene encoding a cyclodextrin enzyme having the nucleotide sequence shown in SEQ ID No.1 or a method according to any of claims 4-7 for the preparation of maltoheptaose or a product containing maltoheptaose.

9. The use of claim 8, wherein the recombinant plasmid is a pUB110, pHT43 or pMK3 plasmid.

10. The use of claim 8, wherein the host cell is bacillus subtilis.

Technical Field

The invention relates to an application of cyclodextrin in preparation of maltoheptaose, belonging to the technical field of biology.

Background

The linear chain malto-oligosaccharide, also commonly called malto-oligosaccharide, is a functional oligosaccharide formed by connecting 3-10 glucose units through alpha-1, 4 glycosidic bonds, has good processing adaptability and unique physiological effects, and is widely applied to the fields of food, medicine, feed, cosmetics and the like internationally, such as fillers, humectants, fat substitutes, energy supply and the like.

The malto-oligosaccharide with different polymerization degrees has obvious difference in performance, and along with the increase of the polymerization degree, the osmotic pressure is reduced, the viscosity is increased, the moisturizing effect is increased, the heat, acid and alkali stability is enhanced, the film forming property is enhanced, and the like. However, oligosaccharides with high degree of polymerization, such as maltooctaose and maltononaose, are easy to age due to high degree of polymerization, and have poor stability of aqueous solution and poor application performance; the maltoheptaose is the maltooligosaccharide with the highest polymerization degree, which is stable in aqueous solution, in the maltooligosaccharide, has lower osmotic pressure, higher viscosity, better moisturizing effect and stronger film-forming property compared with the maltooligosaccharide with low polymerization degree, and has modified reducing and non-reducing ends, and the maltoheptaose is used as a preferable substrate for measuring the activity of alpha-amylase in human serum and urine in clinical experiments, so that the demand for the maltoheptaose in actual production is increasing day by day.

The preparation method of the maltoheptaose at home and abroad mainly comprises a chemical preparation method and an enzymatic hydrolysis method, wherein when the maltoheptaose is prepared by the chemical method, an organic solvent (p-xylene, ethanol and the like) is required, so that the requirement on equipment is high and the cost is high. The enzymatic preparation process is mild, the molecular weight distribution is relatively easy to control, and the product has high safety, so that the method is an ideal method for preparing the maltoheptaose. Currently, in the industrial enzymatic production of maltoheptaose, starch is mostly used as a substrate, a plurality of glucose units are cut by hydrolysis of an amylose maltooligosaccharide generating enzyme, and the subsequent purification steps are complicated, so that maltoheptaose with high purity is difficult to obtain, the cost is high, and the industrial production cannot be directly expanded.

Hangyan Ji et al teach that maltoheptaose can be prepared in a one-step enzymatic reaction process in which the yield of maltoheptaose increases with increasing β -CD concentration, with the peak maltoheptaose content occurring at β -CD concentrations between 8% and 10% (w/v). However, when the concentration of β -CD is as high as 12% (w/v), the enzyme reaction is inhibited. This may be due to the fact that the high concentration of the matrix reduces the effective concentration of water and reduces the diffusivity of the molecules. Furthermore, an excess of β -cyclodextrin may inhibit the activity of PpCD enzyme, as β -cyclodextrin may be a competitive inhibitor of certain hydrolases; the amount of maltoheptaose in the preparation process first increased for 30 minutes and then decreased as β -CD gradually degraded; when the concentration of beta-CD was 8% (w/v) and the reaction time was 180 minutes, the proportion of maltoheptaose to all products (maltooligosaccharide and beta-CD) was only 43.36%. (see in particular the paper "Preparation of macro-oligosaccharides with specific purification of polymerization by a novel cyclic exotherapy from Palaeococcus pathogens", Hangyan Ji et al, Carbohydrate Polymers 210(2019) 64-72).

Therefore, the method for efficiently producing the maltoheptaose under the condition of high substrate concentration is obtained, and has good application value and commercial development significance.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of a cyclodextrin enzyme (CDase) with an amino acid sequence shown as SEQ ID NO.2 in preparation of maltoheptaose or products containing the maltoheptaose.

In one embodiment of the present invention, the above-mentioned cyclodextrin (CDase) is derived from pyrococcusp.

In one embodiment of the invention, the nucleotide sequence encoding the cyclodextrin enzyme is shown in SEQ ID NO. 1.

The invention also provides a method for preparing maltoheptaose, which takes the cyclodextrin enzyme as a catalyst and takes beta-cyclodextrin as a substrate to prepare maltoheptaose in a catalytic manner.

In one embodiment of the present invention, the cyclodextrin enzyme is added in an amount of not less than 15U/g.

In one embodiment of the invention, the substrate concentration is 40-120 g/L.

In one embodiment of the invention, the substrate concentration is 100-120 g/L.

In one embodiment of the invention, in the reaction system, the reaction temperature is 80-90 ℃, the pH is 5.0-6.0, and the reaction time is 2-8 h.

The invention also provides a recombinant plasmid containing the gene of the cyclodextrin with the coding nucleotide sequence shown as SEQ ID NO.1, a host cell carrying the gene of the cyclodextrin with the coding nucleotide sequence shown as SEQ ID NO.1 or application of the method for preparing the maltoheptaose in preparing the maltoheptaose or products containing the maltoheptaose.

In one embodiment of the present invention, the vector of the recombinant plasmid is a pUB110, pHT43 or pMK3 plasmid.

In one embodiment of the present invention, the vector of the recombinant plasmid is a pUB110 plasmid.

In one embodiment of the invention, the host cell is Bacillus subtilis.

In one embodiment of the invention, the host cell is Bacillus subtilis cctcc M2016536.

[ advantageous effects ]

(1) The invention provides a method for preparing maltoheptaose, which is characterized in that cyclodextrin enzyme from Thermococcus B1001 is used as a catalyst, and beta-cyclodextrin is used as a substrate to catalyze and prepare maltoheptaose; compared with other methods for preparing maltoheptaose, the method provided by the invention does not need to adjust the pH and the temperature, does not need other synergistic enzymes for multi-step reaction, and has the advantages of simple and convenient process and production cost saving.

(2) Compared with other preparation methods using starch as a raw material, the method for preparing the maltoheptaose provided by the invention has the advantages that the substrate is beta-cyclodextrin, the substrate beta-cyclodextrin is single in component, side reactions in the reaction process are few, and the product maltoheptaose obtained by the production method is simple in extraction process and high in purity.

(3) By adopting the preparation method of maltoheptaose provided by the invention, the conversion rate and the product purity are high under the condition of high substrate concentration, and by adopting the method, the substrate conversion rate can reach 92.17% at most, and the purity of the obtained product can reach 97.61% at most; can effectively reduce the production and processing cost of the high-purity maltoheptaose, and has good application value and commercial development significance.

(4) Compared with other methods for preparing the maltoheptaose, the preparation method provided by the invention has the advantages that under the condition of obtaining the same yield, the equipment utilization rate is further improved, the reaction time is reduced, the production cost is saved, and the substrate input times are reduced, so that the labor cost is saved.

(5) By adopting the method for preparing the maltoheptaose, the adopted cyclodextrin enzyme is expressed by the bacillus subtilis which is allowed to be admitted into food, and the food safety is good.

Drawings

FIG. 1: the flow chart of the construction of the recombinant plasmid pUB 110-tscd.

FIG. 2: a nucleic acid electrophoresis chart of the POE-PCR amplification product; wherein, M: DL 5000 DNA marker; 1: POE-PCR product.

FIG. 3: double enzyme digestion verification results of the recombinant plasmid pUB 110-TSBCDase; wherein, M: DL 5000 DNAmarker; 1,2: the band after cleavage of pUB 110-tscd.

FIG. 4: SDS-PAGE electrophoresis result of fermentation liquor obtained by fermentation of the recombinant strain pUB 110-TSBCDase; wherein, M: a standard protein marker; 1: extracellular supernatant; 2: breaking cell wall and supernatant; 3: breaking the wall and precipitating.

FIG. 5: HPLC chromatogram of maltoheptaose in the reaction solution.

Detailed Description

The invention is further illustrated with reference to specific examples.

The pET-24a (+) vector referred to in the examples below was purchased from novagen, the pUB110 plasmid was purchased from Biovector plasmid vector strain cell Gene Collection, and the SCK6 competent cells were purchased from Shanghai Chengning Biotech, Inc.

The media involved in the following examples are as follows:

LB medium (g/L): peptone 10, yeast extract 5, NaCl 10.

TB medium (g/L): peptone 10, yeast powder 24, glycerol 5, K₂HPO₄·3H₂O 16.43，KH₂PO₄2.31。

Hypertonic liquid medium (g/L): yeast powder 5, tryptone 10, NaCl10, sorbitol 91.

Electroporation Medium (g/L): sorbitol 91, mannitol 91, glycerol 100.

RM medium (g/L): yeast powder 5, tryptone 10, NaCl10, sorbitol 91, mannitol 69.

The detection methods referred to in the following examples are as follows:

enzyme activity determination of cyclodextrin: cyclodextrin enzyme activity the amount of reducing end exposed by hydrolysis of cyclodextrin was determined by DNS. The reaction system is as follows: 1.9mL of 2% (w/v) beta-cyclodextrin (now ready for use), the enzyme solution was diluted to the appropriate concentration and added to the final volume of 2 mL. After reaction at 90 ℃ for 10min, 3mL of 3, 5-dinitrosalicylic acid (DNS) was added to terminate the reaction. After boiling the mixture in boiling water for 7min, it was immediately cooled on ice, then deionized water was added to a final volume of 15mL, and finally the absorbance was measured at 540 nm.

The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1. mu. mol of reducing end per minute.

The enzyme activity calculation formula is as follows: the enzyme activity (U/mL) is the dilution factor × amount of maltoheptaose produced by conversion (μmol)/conversion time.

Detection of maltoheptaose yield: the diluted sample and pure acetonitrile were mixed at a ratio of 1: 1 volume mixing, coprecipitation for 2h, then 12000r min^-1Centrifuging for 10min, filtering the supernatant, detecting the converted product by HPLC, and calculating the yield of maltoheptaose by HPLC analysis with amino column APS-2HYPERSIL (250 × 4.6.6): mobile phase, acetonitrile: water (75: 25); column temperature, 30 ℃; flow rate, 0.8 mL. min^-1(ii) a The detector is a differential refractometer detector (RID).

Detection of all malto-oligosaccharides:

the diluted sample and pure acetonitrile were mixed at a ratio of 1: 1 volume mixing, coprecipitation for 2h, then 12000r min^-1Centrifuging for 10min, filtering the supernatant, detecting the converted product by HPLC, and calculating the yield of all maltooligosaccharide, and high performance liquid chromatography adopts amino column APS-2HYPERSIL (250 × 4.6.6) as detection conditions, such as mobile phase, acetonitrile, water (75:25), column temperature of 30 deg.C, flow rate of 0.8 mL/min^-1(ii) a The detector is a differential refractometer detector (RID).