阅读说明：本技术 一种具有广泛pH值适应性的淀粉普鲁兰酶及应用 (Starch pullulanase with wide pH value adaptability and application thereof ) 是由 柏玉香 李晓晓 金征宇 于 2020-07-22 设计创作，主要内容包括：本发明公开了一种具有广泛pH值适应性的淀粉普鲁兰酶及应用,属于酶工程及生物改性淀粉技术领域。本发明提供了一种新的淀粉普鲁兰酶,并将其用于淀粉液化,以期为后续淀粉深加工提供合适底物。该淀粉普鲁兰酶在pH5.5和pH8.5条件下具有等同的活性,且在pH4.5-10.0之间保持超过一半的稳定的酶活。该酶的最适温度为75-80℃,且半衰期约为8h。此酶的热稳定性、广泛的pH作用范围,使其可单独或与多种酶类复配,应用于淀粉深加工,提高淀粉液化效率及淀粉底物的利用率,为淀粉加工提供了新的思路,有着巨大潜力和应用前景。(The invention discloses a starch pullulanase with wide pH value adaptability and application thereof, belonging to the technical field of enzyme engineering and biological modified starch. The invention provides a novel starch pullulanase, which is used for starch liquefaction so as to provide a proper substrate for subsequent starch deep processing. The starch pullulanase has equivalent activity under the conditions of pH5.5 and pH8.5, and more than half of stable enzyme activity is kept between pH4.5 and pH 10.0. The optimal temperature of the enzyme is 75-80 ℃, and the half life period is about 8 h. The enzyme has thermal stability and wide pH action range, can be independently compounded with various enzymes or compounded with various enzymes, is applied to deep processing of starch, improves the starch liquefaction efficiency and the utilization rate of a starch substrate, provides a new idea for starch processing, and has huge potential and application prospect.)

1. A method for promoting starch to be rapidly liquefied and viscosity to be reduced comprises the steps of hydrolyzing starch milk by using starch pullulanase shown in SEQ ID No. 1; the dosage of the starch pullulanase is 10-40U/g starch; the mass fraction of the starch milk is 1-40%, and the temperature is controlled at 68-95 ℃ in the hydrolysis process.

2. The method of claim 1, wherein the pH of the starch milk is 4.5-10.0.

3. The method according to claim 1 or 2, wherein the liquefaction time is between 0.5 and 8 hours.

4. The method according to any one of claims 1 to 3, wherein the amylopullulanase is produced by the following method:

(1) connecting a gene which is shown in SEQ ID NO.2 and used for coding the starch pullulanase with a plasmid to construct an expression vector;

(2) transferring the expression vector constructed in the step (1) into host bacteria, selecting positive monoclonal fermentation culture, and collecting starch pullulanase crude enzyme liquid.

5. The method according to claim 4, wherein the plasmid is any one of PMC series, pET series or pGEX series.

6. The method according to claim 4, wherein the step (2) is carried out by using Escherichia coli, Bacillus subtilis, yeast or Aspergillus niger as a host.

7. The method according to claim 4, wherein the crude enzyme liquid of the starch pullulanase is further subjected to separation and purification; the separation and purification method comprises affinity chromatography, hydrophobic chromatography, ultrafiltration chromatography or gel filtration chromatography.

8. The method according to any one of claims 1 to 7, wherein the starch is one or more of soluble starch, potato starch, tapioca starch, sweet potato starch, wheat starch, corn starch, rice starch, pea starch, mung bean starch, sorghum starch and waxy corn starch.

9. The method of any one of claims 1 to 8, applied to the preparation of starch hydrolysate or downstream products thereof in the fields of food, chemical industry and medicine.

The application of the starch pullulanase shown in SEQ ID NO.1 in the aspect of liquefying starch.

Technical Field

The invention relates to a starch pullulanase with wide pH value adaptability and application thereof, belonging to the technical field of enzyme engineering and biological modified starch.

Background

Currently, the amount of starch used for industrial conversion worldwide is up to 5 million tons per year, and 1 million tons per year of starch are used in China for converting glucose, sugar for fermentation or sugar for alcohol production. The close relation between sugar and the national civilization plays an important role in national economy, and the sugar is widely applied to the fields of food, beverage, fermentation and the like. At present, the starch sugar making process is mainly an enzyme method and comprises two stages of liquefaction and saccharification. The liquefaction stage is the most important stage of the whole process for producing glucose by starch conversion, and the result has great influence on both economic indexes and quality indexes of the final product glucose. The pH value of liquefaction in the conventional process is generally 6.0-6.5, and the pH value is not set according to the optimum reaction pH for converting starch into glucose, but is set according to the optimum reaction conditions of alpha-amylase. When the liquefaction reaction is carried out under the pH condition, glucose molecules at the reducing end of starch molecules are isomerized into fructose molecules under the action of higher pH and heat. In the subsequent saccharification reaction, the saccharifying enzyme cannot hydrolyze the alpha-1, 4-glycosidic bond between fructose and glucose, and maltulose by-product exists in the product after the saccharification is finished, and the by-product can influence the utilization of subsequent glucose syrup. In addition, the optimal reaction conditions for the saccharifying enzyme are about pH4.5-5.0, and thus it is necessary to adjust the pH of the liquefied product before the saccharification step, resulting in acid and base consumption. Due to the progress of the industrial technology of the enzyme preparation, different enzyme preparations are utilized to ensure that the liquefied pH value is closer to the direction of high yield of glucose, and the pH value of the slurry mixing is close to the pH value during saccharification, so that the consumption of acid and alkali is reduced, the process steps are simplified, and the ion exchange and resin regeneration cost during the post-treatment of the saccharified liquid is reduced.

Disclosure of Invention

The inventor provides the starch pullulanase with wide pH application range through a large amount of screening, the starch pullulanase has wide pH adaptability and good thermal stability, can realize the quick liquefaction and viscosity reduction of starch under different conditions, avoids the problem that the pH needs to be adjusted for the second time when the starch pullulanase is compounded with other enzymes, and can be used for the starch processing industry.

The first purpose of the invention is to provide the application of the starch pullulanase shown in SEQ ID NO.1 in the aspect of liquefying starch.

The second object of the present invention is to provide a gene encoding pullulanase; the gene contains a nucleotide sequence shown in SEQ ID NO. 2.

The third purpose of the invention is to provide a vector carrying the gene.

In one embodiment of the invention, the plasmid is one of PMC series, pET series or pGEX series.

The fourth purpose of the invention is to provide a genetically engineered bacterium for expressing the amylopullulanase shown in SEQ ID NO. 1.

In one embodiment, the genetically engineered bacteria are host bacteria selected from the group consisting of E.coli, Bacillus subtilis, yeast, and Aspergillus niger.

The fifth purpose of the invention is to provide a method for promoting starch to be rapidly liquefied and reduced in viscosity, wherein starch milk is hydrolyzed by starch pullulanase shown in SEQ ID No. 1; the dosage of the starch pullulanase is 10-40U/g starch; the mass fraction of the starch milk is 1-40%, and the temperature is controlled at 68-95 ℃ in the hydrolysis process.

In one embodiment, the pH of the starch milk is 4.5 to 10.0.

In one embodiment, the liquefaction time is from 0.5 to 8 hours.

In one embodiment, the method specifically comprises: starch is mixed according to a certain concentration, starch pullulanase is added for heating liquefaction, and the enzyme is boiled and killed.

In one embodiment, the starch pullulanase is produced by the following specific process:

(1) the gene SEQ ID NO.2 for coding the starch pullulanase is accessed into a plasmid to construct an expression vector, so as to prepare the plasmid carrying the starch pullulanase gene;

(2) and (3) transferring the plasmid into host bacteria, selecting positive monoclonal fermentation culture to obtain a crude starch pullulanase, and separating and purifying to obtain the pure starch pullulanase.

In one embodiment, the separation and purification method is affinity chromatography, hydrophobic chromatography, ultrafiltration chromatography or gel filtration chromatography.

In one embodiment, the starch is soluble starch obtained by preliminary acidolysis of a starch raw material, or one or more of potato starch, tapioca starch, sweet potato starch, wheat starch, corn starch, rice starch, pea starch, mung bean starch, sorghum starch and waxy corn starch.

The invention also claims the application of the method in the fields of food, chemical industry and medicine.

Has the advantages that: the invention provides a novel starch pullulanase and application thereof in starch liquefaction and viscosity reduction. The starch pullulanase can simultaneously hydrolyze alpha-1, 4 and alpha-1, 6 glycosidic bonds in starch, and has wide pH adaptability, so that the starch pullulanase can be used for starch liquefaction pretreatment, is convenient for later compounding with other enzymes, avoids the problems that the starch pullulanase is difficult to stir due to overhigh viscosity and the pH of different enzymes needs to be secondarily adjusted, and provides convenience for starch processing and production.

Drawings

FIG. 1: an intracellular supernatant component of a fermentation liquid of a recombinant bacterium Amypul-pET-28a (+)/E.coli BL21(DE3), an intracellular supernatant component heat-treated for 10min at 75 ℃, and an SDS-PAGE electrophoresis result of nickel affinity chromatography purification; wherein, M: protein molecular weight standards; 1: an intracellular supernatant fraction; 2: heat treating the intracellular supernatant component at 75 deg.C for 10 min; 3: the pure enzyme after two-step purification.

FIG. 2: the enzyme activity of the starch pullulanase of the invention is changed under different pH conditions.

FIG. 3: the pullulanase of the invention has the enzyme activity change after being preserved for 24 hours under different pH conditions.

Detailed Description

And (3) enzyme activity determination of starch pullulanase: 1.0g of soluble starch was weighed, dispersed in 50mM pH5.5 acetate buffer and pH8.5 phosphate buffer, stirred at 100 ℃ for 30min to sufficiently gelatinize the starch sample, and then incubated at 75 ℃. Taking 0.9mL soluble starch (1%) substrate, adding 0.1mL pure enzyme, keeping the temperature at 75 ℃ for 10min, adding 1mL DNS solution to stop the reaction, boiling in a water bath for 5min, placing in an ice water bath to cool, and measuring the light absorption value at 540 nm. The reaction system with the inactivated enzyme solution added under the same conditions was used as a blank control.

The enzymatic activity (U) of amylopullulanase is defined as: under the above analysis conditions, the amount of enzyme that catalyzes the production of reducing power equivalent to 1. mu. moL of glucose equivalents per minute is defined as one unit of activity.

And (3) determining the DE value of the starch hydrolysate:

DE value ═ reducing sugar content (%)/dry matter content (%) × 100

Wherein, the dry matter content is determined by an Abbe refractometer, the reducing sugar content is determined by a Fehling reagent method, and the specific test method comprises the following steps:

(1) and (3) calibrating a Fehling reagent: 5ml of each of the feilin reagents A and B are sucked and placed in a 250ml triangular flask, 10ml of distilled water is added, and 0.2% standard glucose solution is added into a plurality of milliliters from a burette, wherein the amount of the standard glucose solution is controlled at the time of the later titration (0.5-1.0 ml of 0.2% standard glucose is consumed). Shaking, heating on electric furnace to boil, maintaining slight boiling for 2min, adding 2 drops of 1% methylene blue solution, and continuously titrating with 0.2% standard glucose solution until blue disappears. The titration was completed within 1min and the volume of 0.2% standard glucose solution consumed was recorded as V₀And (4) milliliters.

(2) Sugar determination preparation test: 5ml of each of the Fehling solution A and the Fehling solution B is sucked and placed in a 250ml triangular flask, 10ml of sample sugar solution is accurately added, and the mixture is shaken up on an electric furnace and heated to boil. 2 drops of 1% methylene blue solution were added and the blue colour was titrated with 0.2% standard glucose solution until it disappeared. Consumption of standard glucose solution V₁And (4) milliliters.

(3) Determination of reducing sugars in the samples: accurately sucking 5ml of each of the Filin 'A' and the Filin 'B' solution, placing the Filin 'A' and the Filin 'B' solution into a 250ml triangular flask, accurately adding 10ml of sample sugar solution, and supplementing (V)₀-V₁) Ml of distilled water, and (V) is added from the burette₁-1) ml of 0.2% standard glucose solution. Shaking, heating on electric furnace to boil, maintaining slight boiling for 2min, adding 2 drops of 1% methylene blue solution, and continuously titrating with 0.2% standard glucose until the blue color disappears. This operation was completed within 1 min. The total volume of the standard glucose solution consumed was recorded as V ml.

Reducing sugar content (g/mL, calculated as glucose) ═ V₀-V)×0.2×0.1×n

Wherein: v₀-a fihlin reagent calibration value; v-sample sugar solution measurement value; 0.2-standard glucose liquid concentration; 10-sample sugar liquid volume; n-sample dilution factor.