CN113430156B

CN113430156B - Genetically engineered bacterium for expressing dextrin debranching enzyme and application thereof

Info

Publication number: CN113430156B
Application number: CN202110629171.0A
Authority: CN
Inventors: 李兆丰; 王亚梅; 班宵逢; 李才明; 顾正彪; 洪雁; 程力
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-06-03
Filing date: 2021-06-03
Publication date: 2023-04-28
Anticipated expiration: 2041-06-03
Also published as: CN113430156A

Abstract

The invention discloses a genetically engineered bacterium for expressing dextrin debranching enzyme and application thereof, belonging to the technical field of food and biology. The recombinant expression vector containing the coding gene is successfully constructed, so that a genetic engineering strain is obtained, and the novel dextrin debranching enzyme can be efficiently expressed.

Description

Genetically engineered bacterium for expressing dextrin debranching enzyme and application thereof

Technical Field

The invention relates to a genetically engineered bacterium for expressing dextrin debranching enzyme and application thereof, belonging to the technical field of food and biology.

Background

Currently, starch debranching enzymes are mainly divided into two classes: including pullulanase (ec 3.2.1.41) and isoamylase (ec 3.2.1.68). Pullulanase and isoamylase are widely distributed and are closely related to the hydrolysis of starch. They can catalyze and hydrolyze alpha-1, 6-glycosidic bond in pullulan and pullulan exclusively and efficiently, thus being widely applied in industry, but their substrate specificityThe pullulanase has remarkable hydrolytic activity on pullulan and beta-limit dextrin, can hydrolyze pullulan polysaccharide with high efficiency, but has lower hydrolytic activity on high molecular weight amylopectin, and has little hydrolytic action on glycogen with very dense components. The isoamylase has high hydrolysis activity on amylopectin and glycogen, has low hydrolysis activity on branched dextrin with small molecular weight, and cannot hydrolyze pullulan. Kainuma et al compared the specificity of Pseudomonas isoamylase and Klebsiella pullulanase for the substrate structure. The most suitable substrate of the Pseudomonas isoamylase is found to be high molecular weight glucan, such as potato amylopectin, oyster glycogen and the like; klebsiella pullulanase has remarkable hydrolysis effect on two maltotriose groups or branched oligosaccharides connected by alpha-1, 6 glycosidic bond between maltotriose groups and maltotetraose groups, such as pullulan and 6 ³ -O-alpha-maltotriosyl-maltotriose, 6 ³ -O-alpha-maltotriosyl-maltotetraose and the like. The biggest difference between two debranching enzymes is that pullulanase can only hydrolyze the optimal substrate of isoamylase at a very slow rate, while isoamylase can hardly hydrolyze the optimal substrate of pullulanase.

The enzymes used in the traditional maltose syrup production process at present are alpha-amylase and beta-amylase, and the alpha-amylase and the beta-amylase cannot hydrolyze dextrin generated in the production process, so pullulanase or isoamylase and the beta-amylase are required to be hydrolyzed cooperatively to realize sufficient debranching of a substrate, and the yield of maltose is obviously improved.

In recent years, along with the rapid development of the starch sugar industry in China, the application of preparing the starch sugar by compounding saccharifying enzyme and debranching enzyme is very wide. In the process of producing starch sugar by the holoenzyme method, the total conversion rate of the reaction often does not reach the production expected value because the hydrolysis rate of alpha-1, 6-glycosidic bonds by saccharifying enzyme is limited. Theoretically, increasing the amount of saccharifying enzyme or decreasing the concentration of starch slurry can increase the conversion rate, but the former increases the cost of production; the latter needs to increase the volume of the saccharification tank, which leads to increased evaporation cost and bacteria contamination risk, affecting the enterprise benefit. Therefore, adding debranching enzyme in saccharification stage can reduce the dosage of saccharifying enzyme, increase hydrolysis speed of starch, save saccharification time and increase substrate concentration to save subsequent evaporation cost.

The starch saccharification usually adopts a reaction temperature of 60-65 ℃, so that the requirements on the stability and heat resistance of debranching enzyme are higher, and the novel debranching enzyme with high enzyme activity and better heat stability is screened, so that the method has much important application significance. The viscosity and molecular weight of the liquefied starch solution are greatly reduced to form dextrin solutions with different hydrolysis degrees, and the most widely used debranching enzyme is pullulanase in the debranching enzyme compounded with saccharifying enzyme at present, but the optimal reaction temperature (45 ℃) of the debranching enzyme is lower than the saccharification temperature (60-65 ℃) of starch, and the pullulanase and the saccharifying enzyme are required to be added separately, so that in the saccharification process, temperature change control at different stages is required to be carried out in order to improve the utilization rate of a substrate and increase the conversion rate, and the production cost and the production difficulty are increased.

The problem of low yield and high production cost of a plurality of microorganism-derived debranching enzymes such as pullulanase and isoamylase reported at present causes the limitation of commercial application, and the two enzymes have production limitations and have been monopoly in foreign countries, so that the debranching enzymes with different substrate specificities are screened out, and the expression quantity of the debranching enzymes is improved to adapt to wider industrial requirements, thereby having important significance and commercial value.

Disclosure of Invention

In order to solve the problems of low stability and low product specificity of debranching enzyme in the prior art, the invention provides a genetically engineered bacterium, which takes escherichia coli as an expression host and expresses dextrin debranching enzyme with an amino acid sequence shown as SEQ ID NO. 2.

In one embodiment of the present invention, pET-20b (+), pET-15b or pET-28a is used as an expression vector.

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

In one embodiment of the present invention, E.coli BL21 (DE 3) is used as the expression host.

The invention also provides a preparation method of the dextrin debranching enzyme, which is prepared by adopting the genetically engineered bacterium for fermentation.

In one embodiment of the invention, the genetically engineered bacteria are added into a seed culture medium to prepare seed liquid, the seed liquid is transferred into a fermentation culture medium, and the dextrin debranching enzyme is prepared by fermentation.

In one embodiment of the invention, the seed liquid is prepared: single colonies were picked into LB medium containing 50mL kanamycin at a concentration of 2% (v/v), 37℃and 200rpm, and shake cultured for 12 hours.

In one embodiment of the invention, the prepared seed solution is transferred to a TB medium containing kanamycin according to an inoculum size of 4% (v/v), and IPTG is added when the culture is carried out until the OD600 = 0.6; fermenting the shaking table for 72 hours at 25 ℃ and 200rpm to obtain fermentation liquor; centrifuging the prepared fermentation liquor at 4 ℃ and 10000rpm for 20min, collecting thalli, carrying out ultrasonic crushing, and centrifuging to obtain supernatant, namely the dextrin debranching enzyme crude enzyme liquid.

The invention also provides a method for hydrolyzing dextrin, polysaccharide or starch, which comprises the steps of adding the genetically engineered bacterium or dextrin debranching enzyme with an amino acid sequence shown as SEQ ID NO.2 into a reaction system containing dextrin, polysaccharide or starch for reaction.

In one embodiment of the invention, the dextrins are maltodextrins having a DE value of 2, 4, 6 or 7 to 9.

In one embodiment of the invention, the starch comprises: one or more of corn amylopectin, potato amylopectin, wheat starch, rice starch, common corn starch, tapioca starch, and waxy corn starch.

In one embodiment of the invention, the polysaccharide comprises pullulan or glycogen.

In one embodiment of the invention, the substrate dextrin, polysaccharide or starch is added in an amount of at least 10g/L.

In one embodiment of the invention, the dextrin debranching enzyme is added in an amount of at least 50U/g in the reaction system.

In one embodiment of the present invention, the reaction conditions in the reaction system are: the reaction was carried out at 70℃and pH4.0 for 24h.

The invention also provides the application of the genetically engineered bacterium or the dextrin debranching enzyme with the amino acid sequence shown as SEQ ID NO.2 in the aspects of starch deep processing, sugar industry and resistant starch production.

Advantageous effects

(1) The invention provides a genetically engineered bacterium for expressing novel dextrin debranching enzyme derived from thermophilic cocci, which is suitable for expressing the dextrin debranching enzyme gene, and the enzyme activity of recombinant expression protein obtained by induction at the optimal induction temperature (25 ℃) is 683U/mL.

(2) The dextrin debranching enzyme has the optimal temperature of 70 ℃ and the optimal pH of 4.0, has good heat stability and pH tolerance, and meets the requirements on heat resistance and acid resistance in the starch sugar production process.

(3) The invention also provides a new application of the novel dextrin debranching enzyme, the novel dextrin debranching enzyme can hydrolyze dextrin, polysaccharide and starch simultaneously, and the optimal reaction substrate of the novel dextrin debranching enzyme is dextrin, the optimal debranching chain length is DP 12-24, so that the gap in the aspect of the specificity of the existing commonly used debranching enzyme substrate is filled, and the novel dextrin debranching enzyme has a higher potential application prospect in the industrial fields of starch sugar, resistant starch, beer production, ethanol fuel and the like.

(4) The dextrin debranching enzyme obtained by screening has high enzyme activity and good thermal stability, the temperature change control is not needed by adopting the compound use of the dextrin debranching enzyme and the saccharifying enzyme, the dextrin debranching enzyme still has higher catalytic hydrolysis capability under the condition of 60-65 ℃, and the saccharifying enzyme and the dextrin debranching enzyme can be added together for reaction, so that the difficulty and the production cost of a production process are greatly reduced, the dosage of the saccharifying enzyme is reduced, and the saccharifying time is saved.

Drawings

Fig. 1: selection of different expression vectors in the production of novel dextrin debranching enzymes: (A) Novel dextrin debranching enzyme SDS-PAGE protein analysis, wherein M is a protein Marker; pET-20b (+); pET-15b; pET-28a; purifying enzyme by using a pET-28a nickel column; (B) Influence of different expression vectors on the activity of the novel dextrin debranching enzyme.

Fig. 2: and (3) analyzing the optimal reaction temperature and thermal stability of the novel dextrin debranching enzyme: (A) Analysis of the optimum reaction temperature (determination at pH 4.0); (B) thermal stability analysis: the residual activities (pH 4.0) were measured after cooling by sampling at regular intervals by incubating at 50℃at 60℃at 70℃at 80℃and at 90℃for 150min, respectively.

Fig. 3: analysis of pH and pH tolerance of the novel dextrin debranching enzyme: (A) optimal reaction pH analysis (measured at 70 ℃ C.); (B) pH tolerance analysis: residual activity (measured at 70 ℃) was measured after incubation at 4℃for 3 h.

Fig. 4: novel dextrin debranching enzyme treatment DE6 maltodextrin front and rear chain length distribution change.

Fig. 5: the molecular weight of the product after the novel dextrin debranching enzyme is used for treating different substrates is changed, (A) DE6 maltodextrin is used as a substrate; (B) tapioca starch is used as a substrate; and (C) taking waxy corn starch as a substrate.

Fig. 6: product analysis of maltodextrin co-processed by novel dextrin debranching enzyme and saccharifying enzyme, wherein G1 represents glucose; g4 represents maltotetraose.

Detailed Description

In order to more clearly describe the technical contents of the present invention, the following is further described in connection with specific embodiments.

The test methods described in the following examples, unless otherwise specified, are all conventional; the reagents or consumables, unless otherwise specified, are commercially available. Escherichia coli JM109 and E.coli BL21 (DE 3) referred to in the examples below are available from Qiagen, U.S.A.; plasmid pET-28a was purchased from Novagen, USA.

The extraction of the genome referred to in the examples below is referred to the kit instructions for rapid extraction of genomic DNA of the Genaray bacterium.

The following examples relate to the following media:

LB liquid medium 10g/L tryptone, 10g/L sodium chloride and 5g/L yeast powder.

LB solid medium: 1.5% (w/v) agar powder was added on the basis of LB liquid medium.

TB liquid medium: 12g/L tryptone, 24g/L yeast powder5g/L glycerol, KH ₂ PO ₄ 2.3135g/L，K2HPO4·3H2O 16.4318g/L。

The following examples relate to the methods for enzyme activity detection as follows:

1.00g of amylopectin is weighed, a certain amount of deionized water is added for boiling water bath gelatinization for 30min, and after cooling, the volume is fixed to 100mL to prepare 1% (w/w) of amylopectin; absorbing 700 mu L of amylopectin solution into a centrifuge tube, adding 150 mu L of 500mM sodium acetate buffer solution (pH 4.0), uniformly mixing, placing in a 70 ℃ water bath kettle, keeping the temperature for 10min, and adding 150 mu L of dextrin debranching enzyme into a substrate for 15min; after the reaction is finished, the reaction product is boiled in water for 15min to terminate the reaction, 100 mu L of reaction liquid is sucked into a centrifuge tube after cooling, and 100 mu L of 0.01. 0.01M I is added ₂ And (3) standing and developing the color for 15min by 0.1M KI, adding 4.8mL of deionized water, sufficiently shaking and uniformly mixing, and measuring the absorbance value under the condition of 610 nm. The crude enzyme solution with high-temperature enzyme deactivation was used as a blank. Definition at A _610nm At the absorbance value, 1% (w/w) of pullulan was used as a substrate and increased by 0.1 per hour as 1 enzyme activity unit.

Wherein 5.0 = global system (mL); 4 = time (min), 15min reaction time converted to 1h;0.1 =a _610nm The absorbance value raised per hour; 0.150 Amount of added enzyme (mL); 10 1mL of the reaction system, 100. Mu.L of the reaction system was aspirated for color reaction.

Sample polymerization degree analysis:

adding the crude enzyme solution of the dextrin debranching enzyme into 10mL of 1% substrate solution, regulating the pH of the solution to 4.0, carrying out water bath reaction for 24 hours at 70 ℃, boiling for 30 minutes to inactivate the enzyme, centrifuging at 10000rpm for 1 minute to remove protein, diluting a sample to 5mg/mL, removing impurities by a 0.22 mu L filter membrane, and analyzing the polymerization degree distribution of the sample by using high-efficiency anion chromatography under the following analysis conditions: chromatographic column CarboPac PA200×250mm; mobile phase 250mM NaOH, 1.0M NaAc and ultrapure water; pulse ampere detector, flow rate 0.5mL/min, column temperature 35 ℃.

Sample molecular weight analysis:

adding the crude enzyme solution of the dextrin debranching enzyme into 10mL of 1% (w/w) substrate solution, regulating the pH of the solution to 4.0, performing water bath reaction at 70 ℃ for 24 hours, inactivating the enzyme by boiling the reaction sample in boiling water for 30 minutes, centrifuging at 10000rpm for 1 minute, removing protein, freeze-drying supernatant into powder, dissolving the sample to a concentration of 5mg/mL by using DMSO solution, heating in a water bath for 12 hours, removing impurities by using a 0.22 mu L filter membrane, and analyzing the molecular weight distribution of the sample by using gel permeation chromatography.

Example 1: construction of genetically engineered bacteria

The method comprises the following specific steps:

(1) Amplification of thermophilic cocci dextrin debranching enzyme Gene

Using Thermococcus gammatolerans EJ genome DNA as a template, designing two-end primers respectively containing Nco I and Xho I sites according to Thermococcus gammatolerans dextrin debranching enzyme gene sequence (nucleotide sequence is shown as SEQ ID No. 1) in NCBI database, wherein:

5' end primer: 5' -GATGGCCATGGGCAGAACCATCTTAGCCGGCAACG；

3' -terminal primer: 5' -TGGTGCTCGAGTAGGGGTTCAACGCTAACACCTC；

PCR amplification reaction: using the extracted genome as a template, 50. Mu.L of an amplification system: buffer 10. Mu.L, dNTPs 4. Mu.L, 5 '-end primer 1. Mu.L, 3' -end primer 1. Mu.L, template DNA 1. Mu.L, template ligase 1. Mu.L, ddH ₂ O 32μL。

PCR amplification conditions: pre-denaturation at 98℃for 3min, denaturation at 98℃for 10s, annealing at 60℃for 15s, elongation at 68℃for 2.5min, 30 cycles, and finally incubation at 68℃for 10min.

The PCR product obtained by amplification is verified by agarose gel electrophoresis, and after gel recovery, the PCR product is cloned to a plasmid pMD18-T simple Vector to obtain a cloning Vector containing a target gene, E.coli JM109 is transformed, the transformed recipient bacterium is coated on an LB solid plate containing 20 mug/mL kanamycin, a single colony is picked up to 50mL of LB liquid medium containing 2% (v/v) kanamycin, the culture is carried out for 12 hours at 37 ℃, the plasmid is extracted, enzyme digestion electrophoresis and sequencing identification are adopted, and sequence determination is completed by Shanghai Jin Weizhi Limited company.

Comparing the sequencing result with NCBI and analyzing the result, the obtained dextrin debranching enzyme gene DNA consists of 1863 nucleotides, and the sequence is shown as SEQ ID NO. 1. The DNA codes 620 amino acids, and the sequence of the DNA is shown as SEQ ID NO. 2.

(2) Construction of recombinant expression vector of E.coli containing dextrin debranching enzyme

Cloning vectors pMD18-T simple/dde and pET-28a are subjected to double digestion by using Nco I and Xho I respectively, a target fragment and a vector plasmid containing the same cohesive end are recovered by cutting glue, the target fragment and the vector plasmid are connected by using T4 DNA ligase, then the connected product is transformed into E.coli JM109 to obtain a transformant, the transformant is coated on LB solid medium containing 20 mug/mL kanamycin, single colony is picked up to 50mL of LB liquid medium containing 2% (v/v) kanamycin, the culture is carried out for 12 hours at 37 ℃, plasmid extraction and enzyme cutting electrophoresis and sequencing identification are carried out, finally the expression vector pET-28a-dde containing the correct sequence is obtained, and E.coli BL21 (DE 3) is transformed, thus obtaining genetically engineered bacterium E.coli BL21 (DE 3)/pET-28 a-dde.

Control strain:

the specific steps are the same as above, and the difference is that pET-20b (+) or pET-15b is adopted as an expression vector to respectively prepare and obtain the genetic engineering bacteria: e.coli BL21 (DE 3)/pET-20 b (+) -dde, E.coli BL21 (DE 3)/pET-15 b-dde.

Example 2: expression and purification of recombinant dextrin debranching enzyme

The method comprises the following specific steps:

1. expression of recombinant dextrin debranching enzyme

(1) Scribing a plate: respectively dipping the bacterial solutions prepared in the example 1 by using an inoculating loop, streaking the bacterial solutions on LB solid medium containing 20 mug/mL kanamycin, and then placing the bacterial solutions in a 37 ℃ incubator for culturing for 12 hours;

(2) Activating: picking single colonies respectively into LB medium containing 50mL kanamycin with the concentration of 2% (v/v), and performing shake culture at 37 ℃ and 200rpm for 12 hours; respectively preparing seed liquid;

(3) Fermentation: inoculating the prepared seed solution to TB liquid culture medium containing kanamycin with the concentration of 2% (v/v), adding IPTG (adding amount: 20 mu L/50mL culture medium, final concentration of 0.01 mM) when the inoculation amount is 4% (v/v) and OD600 = 0.6; 25 ℃ (200 rpm) of fermentation shaking table for 72 hours; obtaining fermentation liquor, centrifuging the obtained fermentation liquor at 4 ℃ and 10000rpm for 20min, collecting thalli, adding Tris-HCl (pH 7.0) buffer solution in equal proportion to re-suspend thalli, carrying out ultrasonic crushing, and collecting supernatant by high-speed centrifugation to respectively obtain crude enzyme solutions containing recombinant dextrin debranching enzyme, and respectively detecting enzyme activity data of the crude enzyme solutions, wherein the results are shown in Table 1:

table 1: enzyme activity data of recombinant dextrin debranching enzyme prepared by different recombinant strains

As shown in Table 1, the crude enzyme prepared from the genetically engineered bacterium E.coli BL21 (DE 3)/pET-28 a-dde has the highest enzyme activity data, so the crude enzyme prepared from the strain is used for subsequent study.

2. Separation and purification of recombinant dextrin debranching enzyme

And (3) centrifuging the fermentation broth prepared by fermenting the genetically engineered bacterium E.coli BL21 (DE 3)/pET-28 a-dde in the step (1) at a high speed for 20min, discarding the supernatant, re-suspending the collected thalli by using an equal volume of loading buffer solution (500mM NaCl,50mM Tris-HCl,20mM imidazole), carrying out ultrasonic cell disruption, centrifuging at a high speed, collecting the supernatant, passing through a 45 mu m water-based film, and loading the sample to a nickel column for purification.

The purification process is carried out according to the following steps: (1) The nickel column is balanced by 20mL of loading buffer, then loaded at a speed of 1.5mL/min, and the nickel column is balanced by the loading buffer after loading is completed. (2) Nickel column elution: eluting the sample by using a gradient elution mode eluent B (500mM NaCl,50mM Tris-HCl,500mM imidazole), collecting peak eluent to obtain eluting protein, performing SDS-PAGE protein electrophoresis verification (shown in figure 1) on the eluent, and detecting enzyme activity data, wherein the enzyme activity of pure enzyme is detected as follows: 533U/mg.

Example 3: enzymatic characterization of dextrin debranching enzymes

In the detection of the enzymatic properties (steps 1 to 3) in the following examples, the pure enzyme solution prepared in step 2 of example 2 was used.

1. Determination of optimum reaction temperature and thermal stability

(1) The activity of the dextrin debranching enzyme is measured at different temperatures (30-100 ℃), and the highest enzyme activity is 100%. The results are shown in Table 2:

table 2: enzyme activity data at different reaction temperatures

(2) The recombinant enzyme is respectively incubated at 50, 60, 70, 80 and 90 ℃, sampled at different time points, rapidly cooled to 0 ℃, and the residual activity of the enzyme is measured, wherein the activity of the enzyme liquid which is not incubated is 100%. The results are shown in FIG. 2 and Table 3:

table 3: residual enzyme activity data of recombinase at different temperatures for different times

As shown in figure 2, the optimal reaction temperature of the dextrin debranching enzyme is 70 ℃, and the dextrin debranching enzyme still has activity of more than 80% in the temperature range of 50-80 ℃, has higher optimal temperature and belongs to a heat-resistant debranching enzyme. The dextrin debranching enzyme is kept at 70 ℃ for 90min, and the residual enzyme activity is more than 50%, so that the enzyme has good thermal stability, meets the requirements of industrial application, and has great application potential.

2. Determination of optimum pH and pH tolerance

(1) 100mM disodium hydrogen phosphate-citric acid buffer solutions with different pH values (pH 3.0-10.0) are prepared, the activity of dextrin debranching enzyme is measured at 70 ℃, and the optimal pH of the recombinase is examined.

To determine the pH tolerance of the recombinase, 100mM disodium hydrogen phosphate-citric acid buffer with different pH values (pH 3.0 to 8.0) was mixed with the recombinase, and the remaining enzyme activity of the recombinase was determined at 4℃for 3 hours, defined as the highest enzyme activity. The results are shown in FIG. 3 and Table 4:

table 4: the recombinase is kept for 3 hours under buffer solutions with different pH values for residual enzyme activity data

As is clear from FIG. 3 and Table 4, the dextrin debranching enzyme has an optimum pH of 4.0, maintains 80% or more of the enzyme activity at a pH of 3.5 to 5.0, and has excellent acid resistance. The dextrin debranching enzyme is kept for 3 hours within the pH range of 3.0-6.5, and the enzyme activity is still more than 80%, which shows that the enzyme has good catalytic activity and good pH tolerance in an acidic environment.

3. Influence of Metal ions and chemical Agents on enzyme Activity

Different kinds of metal ions (final concentration of 1 mM) and chemical reagents (1% or 5%, v/v) were added to a standard system for dextrin debranching enzyme activity assay to give 100% activity without adding metal ions and chemical reagents. The results are shown in Table 5:

table 5: influence of Metal ions and chemical reagents on the Activity of novel dextrin debranching enzymes

As shown in Table 5, 5mM EDTA has obvious inhibition effect on the activity of the dextrin debranching enzyme, the inhibition degree is about 50%, which indicates that the activity of the dextrin debranching enzyme depends on the metal ion cofactor;

pb at 1mM ²⁺ 、Ba ²⁺ The activity of the recombinant enzyme is obviously activated, and the activation is respectively increased by about 91 percent and 116 percent;

1mM Ca ²⁺ has slight activation effect on the activity of the recombinant enzyme, 1mM Mn ²⁺ 、Cu ²⁺ 、Mg ²⁺ And Zn ²⁺ Has moderate inhibition effect on the activity of the recombinant enzyme, and 1mM Fe ³⁺ And Co ²⁺ Completely inhibit the activity of the recombinant enzyme.

Polyethylene glycol, glycerol and DMSO are added to have certain activation effect on the activity of the recombinant enzyme, the activation effect is respectively increased by 20%, 25% and 25%, and Tween-80 and triton X-100 have certain inhibition effect on the activity of the enzyme.

4. Substrate specificity analysis

(1) Respectively preparing DE2 maltodextrin, DE4 maltodextrin, DE6 maltodextrin, DE 7-9 maltodextrin glycogen, corn amylopectin, potato amylopectin, wheat starch, rice starch, common corn starch, tapioca starch and waxy corn starch with the concentration of 1% (w/w);

(2) And (2) respectively taking 0.8mL of the substrate solution in the step (1) into a 10mL centrifuge tube, adding 100 mu L of 500mM sodium acetate buffer solution (pH 4.0), uniformly mixing, placing in a 70 ℃ water bath kettle, preserving heat for 10min, then respectively adding 100 mu L of pure enzyme solution, reacting for 15min at the temperature of 70 ℃, and measuring the hydrolase activity of dextrin debranching enzyme on different substrates by using a DNS reducing sugar method. The ability to hydrolyze polysaccharides such as starch and glycogen was calculated using the enzyme activity of the substrate reaction with the highest enzyme activity as defined as 100%; the results are shown in Table 6:

table 6: data on the hydrolytic Activity of the recombinant enzymes on different substrates

According to the invention, the conditions of different substrates are analyzed by the dextrin debranching enzyme, as shown in Table 6, the dextrin debranching enzyme has relatively strong hydrolysis capability on dextrin substrates, the relative activity of DE6 maltodextrin is highest, when the enzyme activity of the reaction taking DE6 maltodextrin as the substrate is defined as 100%, the relative activities of DE2 maltodextrin, DE4 maltodextrin, wheat starch and glycogen are found to be more than 50%, and the relative activities of corn amylopectin, potato amylopectin, corn starch, waxy corn starch, rice starch, tapioca starch and pullulan are all below 50%. Indicating that the enzyme tends to hydrolyze dextrins of relatively small molecular weight, whereas starches of larger molecular weight do not exhibit significant hydrolytic activity.

5. Product specificity analysis

In order to identify the product specificity of the recombinant expression of the dextrin debranching enzyme, the present invention uses two methods to analyze the products of the hydrolysis of DE6 maltodextrin, tapioca starch and waxy corn starch by the dextrin debranching enzyme.

5.1 analysis of product specificity Using high Performance anion chromatography

(1) 1g of DE6 maltodextrin, tapioca starch and waxy corn starch are respectively weighed, deionized water is added to prepare a substrate solution with the concentration of 1% (w/w), the substrate solution is cooled to room temperature after being gelatinized in a boiling water bath for 30min, and the pH is regulated to 4.0, so that the treated substrate solution is prepared;

(2) Adding the dextrin debranching enzyme crude enzyme solution into a substrate solution according to the enzyme adding amount of 50U/g, reacting for 24 hours at the optimal temperature of 70 ℃, inactivating enzyme in a boiling water bath for 30 minutes after the reaction is finished, and freeze-drying a reaction product to obtain sample powder;

(3) 10mg of sample powder is weighed and thoroughly dissolved in ultrapure water, diluted to a concentration of 5mg/mL, and after centrifugation through a membrane, the chain length distribution in the reaction solution can be analyzed by using HPAEC-PAD.

The products of the hydrolysis of DE6 maltodextrin, tapioca starch and waxy corn starch were analyzed by high performance anion chromatography and the results are shown in Table 7:

table 7: recombinant enzyme treatment of DE6 maltodextrin front and rear chain length distribution data

According to the invention, the chain length distribution situation of the enzymatic hydrolysis substrate of the dextrin debranching enzyme is analyzed, as shown in Table 7 and FIG. 4, DE6 maltodextrin is used as a substrate, the distribution rule of the chain length of the maltodextrin and the product after debranching have obvious differences, the ratio of DP <6 small molecule sugar to A chain (DP 6-12) in the product is reduced in the early stage of the reaction, the ratio of B1 chain (DP 13-24) to B2 chain (DP 24-36) is increased to the peak value in the middle stage of the reaction, the ratio of B1 chain (DP 13-24) to B2 chain (DP 24-36) is reduced in the late stage of the reaction, and the ratio of DP <6 small molecule sugar to A chain (DP 6-12) in the product is greatly increased.

When tapioca starch and waxy corn starch are used as substrates, no debranching signal is detected by HPAEC-PAD after dextrin debranching enzyme hydrolysis, and the chain length distribution of the products is not changed significantly (not shown in the figure). Thus, the most suitable substrate for the dextrin debranching enzyme is dextrin, and preferentially hydrolyzes the branching point of the B1 chain (DP 13-24), and shows preference for branching of the small molecule saccharides DP <6 and A chain (DP 6-12) as the reaction proceeds.

5.2 gel permeation chromatography GPC

Step (1) and step (2) are the same as 5.1, and step (3) is as follows: 10mg of sample powder is weighed into a sample bottle, 2mL of DMSO with a film is added, a rotor is sealed by a sealing film and tinfoil, and then the mixture is stirred magnetically and heated in a boiling water bath overnight, and after passing through the film, the molecular weight change in the reaction solution can be analyzed by GPC.

The products of the hydrolysis of DE6 maltodextrin, tapioca starch and waxy corn starch were analyzed by gel permeation chromatography and the results are shown in Table 8:

table 8: molecular weight data in the products after treatment of different substrates with recombinant enzymes

Note that: mn is the number average molecular weight; mw is weight average molecular weight, peak1 is amylopectin; peak2 is amylose.

The molecular weight distribution of the substrate for the enzymolysis of the dextrin debranching enzyme is analyzed, as shown in table 8 and fig. 5: when DE6 maltodextrin is used as a substrate, detecting 2 components, namely an amylopectin component and an amylose component, by gel permeation chromatography, wherein after debranching treatment by dextrin debranching enzyme, the detection signal of the peak position of polymerized amylopectin is reduced, and the signal of the peak position of amylose is enhanced; when tapioca starch and waxy corn starch are used as substrates, 2-3 components, namely a polymeric amylopectin peak, an intermediate component (macromolecular linear glucan and branched glucan) and an amylose peak (small molecular linear glucan and small molecular sugar) are detected by gel permeation chromatography, the area of a large molecular weight elution peak gradually decreases and the area of an intermediate molecular weight elution peak gradually increases along with the change of time, which indicates that the polymeric amylopectin is gradually decomposed, and macromolecular linear glucan and branched glucan are generated, but the peaks of small molecular linear glucan and small molecular oligosaccharide are not detected, and the result corresponds to the chain length distribution result.

The debranching treatment of the dextrin debranching enzyme can hydrolyze most of the polymerized amylopectin to generate macromolecular linear glucan, branched glucan and micromolecular linear glucan, thereby further illustrating that the optimal reaction substrate of the dextrin debranching enzyme is dextrin.

Example 4: application of dextrin debranching enzyme in maltodextrin saccharification

The method comprises the following specific steps:

(1) DE 7-9 maltodextrin (purchased from Shandong Baolin biological Co., ltd.) is prepared into 20% (w/w) emulsion, and the emulsion is gelatinized at 100deg.C for 20min with magnetic stirring to obtain gelatinized liquid;

(2) The temperature of the gelatinized liquid is regulated to 55 ℃, 50U/g of the crude enzyme solution of the dextrin debranching enzyme and 15U/g of saccharifying enzyme (GenBank: CAA 34708.1) with enzyme are added for reaction for 24 hours, the reaction product is taken out, the enzyme is boiled for deactivation, and the reaction product is detected by HPAEC-PAD after the reaction product is diluted and subjected to membrane filtration.

As a control, 15U/g of enzyme-added saccharifying enzyme was added, the reaction was carried out for 24 hours, the reaction product was taken out, the enzyme was boiled off, diluted and then the reaction product was detected by HPAEC-PAD, and the results are shown in Table 9.

Table 9: maltodextrin product analysis data of synergistic treatment of dextrin debranching enzyme and saccharifying enzyme

The maltodextrin product treated by the synergistic action of the dextrin debranching enzyme and the saccharifying enzyme is analyzed in the invention, as shown in table 9 and fig. 6: the alpha-1, 6-glycosidic bond in the maltodextrin substrate is hydrolyzed by using the dextrin debranching enzyme, so that more amylopectin in the substrate is hydrolyzed into amylose, a mode of synchronous debranching and hydrolysis is realized, and the total conversion rate of the reaction and the ratio of a main product (G4) are improved by increasing the molecular number of the substrate acted by the saccharifying enzyme, so that the following is known: the synergistic treatment of maltodextrin with dextrin debranching enzyme and saccharifying enzyme can raise the conversion rate of reaction and the ratio of main product, reduce reaction time and saccharifying enzyme consumption.

In conclusion, the present invention successfully obtains dextrin debranching enzyme gene and amino acid sequence from thermophilic coccus. The recombinant host cell obtained by the invention is suitable for the expression of the dextrin debranching enzyme gene, and the enzyme activity of the recombinant expression protein obtained by induction at the optimal induction temperature (25 ℃) is 683U/mL.

The dextrin debranching enzyme prepared by the method has the optimal temperature of 70 ℃, still has the activity of more than 80% in the temperature range of 50-80 ℃, is kept at the temperature of 70 ℃ for 90min, has the residual enzyme activity of more than 50%, has the optimal pH of 4.0, keeps the enzyme activity of more than 80% at the pH of 3.5-5.0, keeps the temperature for 3h in the pH range of 3.0-6.5, has the enzyme activity of still more than 80%, has good heat stability and pH tolerance, and meets the requirements on heat resistance and acid resistance in the starch sugar production process.

The optimal reaction substrate of the enzyme is identified to be dextrin by high-efficiency anion chromatography (HPAEC-PAD) and Gel Permeation Chromatography (GPC) analysis, the optimal debranching chain length is DP 12-24, the blank of the existing commonly used debranching enzyme substrate specificity is filled, and the enzyme has higher potential application prospect in the industrial fields of starch sugar, resistant starch, beer production, ethanol fuel and the like.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of Jiangnan

<120> a genetically engineered bacterium expressing dextrin debranching enzyme and application thereof

<130> BAA210367A

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1863

<212> DNA

<213> artificial sequence

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gaagtttctc cggaaccgga cttcatcggg gcatcctcaa ccttcacccg ggcggtttca 180

cacttctctc tcggcgagcg gggaatcctg gtgaggctaa gaacgctgga cggggtttac 240

gaggaaaagc tctccttcta caacacgtcg gaagaatcgc tcggtgtcaa ggtcaggtac 300

tcctacgagg cccccattga ggacatattc caggtcaggg gcttcatggg gctgaagagc 360

ggaaaggcga tagccccagc cggaggaacg cacgtcaaag agagcccctc cggaaggagg 420

agtctctcca tcgagaccaa catggaaagg gaggggagtc tcttaagggc ggaacttgag 480

atacctcccc tcggaaaggc agtcctctac gtccgcttca tccccaaaat cgaggggagt 540

atctcggaga tactcgggga gaaaaggaaa acgataaaaa acgtcgcctt caccggctca 600

cccgccatcg acgggatatt tgagagggcg gttgagaaca taaacgccct gacgctattc 660

acgcgcttcg ggcctgttcc ccttgcgggg attccgtact tcgcctgtcc cttcggaagg 720

gacgcgataa tagcctcact ctttctcctg ccgtattacc ccgaatacgc cgccggcacg 780

ctgaggctct tcgggcggct ccaggggaag agaaccaacc cgaagaacga ggaagaacct 840

ggaaagatac cacatgagtt ccgtctcggg gagctcgcgc agtcggggaa ggttcccttc 900

gcgccctact acggcacggt ggatgccact ccactctacg tggcattagc cggtgagtac 960

ctgcgctgga cgggggacag aaaattaatc gaggaactga gaccgaacct gacggccgcc 1020

gtcgagtgga tactcaaaaa gctcgatgat gggtacataa cctacgttcc ggggatactc 1080

ggcaacaagg ggtggaagga ttcgagggat gggataattg acgaggaagg gaaaattcca 1140

aagccgccga tagcgctcgt tgaggtgcag ggctacacct actgggcgct taaacttgca 1200

ggggagctca gcctgacaga cctcgatgaa aaaacccttc tcgcagaggc ggaaaagttg 1260

aagaaaaggt tcaaccgcga cttctggctc ggctcctact acgccctcgc gcttgatggg 1320

gagggaaggc cgcttagagt cgtctcctcc aacatgggac acctactcct gacgggtata 1380

gccgagcacg aggaggaact cgcggagagg cttttccggc cggacatgtt ctcccggtac 1440

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ccgcaggcct ggagctcggc gagcgttttc gccttcgtta ccgcctcgct cggaatggaa 1740

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tga 1863

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Arg Gly Asp Met Pro Ser His Tyr Asp Gly Phe Tyr Phe Leu Asp Thr

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Arg Phe Val Arg Lys Ala Arg Leu Glu Val Ser Pro Glu Pro Asp Phe

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Ile Gly Ala Ser Ser Thr Phe Thr Arg Ala Val Ser His Phe Ser Leu

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Gly Glu Arg Gly Ile Leu Val Arg Leu Arg Thr Leu Asp Gly Val Tyr

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Glu Glu Lys Leu Ser Phe Tyr Asn Thr Ser Glu Glu Ser Leu Gly Val

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Lys Val Arg Tyr Ser Tyr Glu Ala Pro Ile Glu Asp Ile Phe Gln Val

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Arg Gly Phe Met Gly Leu Lys Ser Gly Lys Ala Ile Ala Pro Ala Gly

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Gly Thr His Val Lys Glu Ser Pro Ser Gly Arg Arg Ser Leu Ser Ile

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Glu Thr Asn Met Glu Arg Glu Gly Ser Leu Leu Arg Ala Glu Leu Glu

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Ile Pro Pro Leu Gly Lys Ala Val Leu Tyr Val Arg Phe Ile Pro Lys

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Ile Glu Gly Ser Ile Ser Glu Ile Leu Gly Glu Lys Arg Lys Thr Ile

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Lys Asn Val Ala Phe Thr Gly Ser Pro Ala Ile Asp Gly Ile Phe Glu

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Arg Ala Val Glu Asn Ile Asn Ala Leu Thr Leu Phe Thr Arg Phe Gly

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Pro Val Pro Leu Ala Gly Ile Pro Tyr Phe Ala Cys Pro Phe Gly Arg

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Asp Ala Ile Ile Ala Ser Leu Phe Leu Leu Pro Tyr Tyr Pro Glu Tyr

245 250 255

Ala Ala Gly Thr Leu Arg Leu Phe Gly Arg Leu Gln Gly Lys Arg Thr

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Asn Pro Lys Asn Glu Glu Glu Pro Gly Lys Ile Pro His Glu Phe Arg

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Leu Gly Glu Leu Ala Gln Ser Gly Lys Val Pro Phe Ala Pro Tyr Tyr

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Gly Thr Val Asp Ala Thr Pro Leu Tyr Val Ala Leu Ala Gly Glu Tyr

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Leu Arg Trp Thr Gly Asp Arg Lys Leu Ile Glu Glu Leu Arg Pro Asn

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Leu Thr Ala Ala Val Glu Trp Ile Leu Lys Lys Leu Asp Asp Gly Tyr

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Ile Thr Tyr Val Pro Gly Ile Leu Gly Asn Lys Gly Trp Lys Asp Ser

355 360 365

Arg Asp Gly Ile Ile Asp Glu Glu Gly Lys Ile Pro Lys Pro Pro Ile

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Ala Leu Val Glu Val Gln Gly Tyr Thr Tyr Trp Ala Leu Lys Leu Ala

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Gly Glu Leu Ser Leu Thr Asp Leu Asp Glu Lys Thr Leu Leu Ala Glu

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Ala Glu Lys Leu Lys Lys Arg Phe Asn Arg Asp Phe Trp Leu Gly Ser

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Tyr Tyr Ala Leu Ala Leu Asp Gly Glu Gly Arg Pro Leu Arg Val Val

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Ala Val Phe Asp Ala Ala Lys Leu Leu Pro Glu Arg Glu Leu Pro Glu

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Leu Tyr Ser Gly Leu Asn Glu Leu Val Pro Val Pro Arg Ala Asn Ser

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Pro Gln Ala Trp Ser Ser Ala Ser Val Phe Ala Phe Val Thr Ala Ser

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Claims

1. A genetically engineered bacterium is characterized by using escherichia coliE.coliBL21 (DE 3) is an expression host, pET-28a is an expression vector, and dextrin debranching enzyme with an amino acid sequence shown as SEQ ID NO.2 is expressed.

2. The genetically engineered bacterium of claim 1, wherein the dextrin debranching enzyme has a nucleotide sequence shown in SEQ ID No. 1.

3. A method of hydrolyzing maltodextrin, comprising the steps of:

(1) Preparing maltodextrin gelatinization liquid;

(2) Adding the genetically engineered bacterium described in the claims 1 or 2 or dextrin debranching enzyme and saccharifying enzyme with the amino acid sequence shown in SEQ ID NO.2 into the gelatinized liquid for reaction.

4. A method for hydrolyzing dextrin, polysaccharide or starch is characterized in that the genetically engineered bacterium of claim 1 or 2, or dextrin debranching enzyme with an amino acid sequence shown as SEQ ID NO.2 is added into a reaction system containing dextrin, pullulan or starch for reaction.

5. The method of claim 4, wherein the dextrin is maltodextrin having a DE value of 2, 4, 6 or 7-9.

6. The method of claim 5, wherein the starch comprises: one or more of corn amylopectin, potato amylopectin, wheat starch, rice starch, common corn starch, tapioca starch, and waxy corn starch.

7. The genetically engineered bacterium of claim 1 or 2, or the use of dextrin debranching enzyme with an amino acid sequence shown as SEQ ID NO.2 in starch deep processing, sugar industry and resistant starch production.