A raw starch hydrolyzing enzyme mutant, expression strain and application thereof

Abstract

The application discloses a raw starch hydrolysis enzyme mutant, an expression strain thereof and application. Pontibacillus The alpha-amylase AmyZ1 of sp.ZY is used as a starting enzyme, a substrate binding sub-site is determined through molecular docking, a site-directed mutation is performed on a residue at a +1 site 199, and a mutant enzyme L199F with higher glucose yield is obtained. When the mutant enzyme is used in an industrial sugar production process and corn raw starch is used as a substrate, the DE value of the corn raw starch after liquefaction for 2 hours can reach 35%, which is 1.44 times that of the starting enzyme; the DE value can reach 99% after saccharification for 18 hours, which is 5 hours earlier than that of the starting enzyme. Therefore, the mutant enzyme has high potential application value in the field of corn raw starch-based sugar production industry.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a raw starch hydrolase mutant, its expression strain, and its applications. Background Technology

[0002] Starch hydrolases are enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. Currently, of all discovered α-amylases, only about 10% are capable of degrading raw starch, and these are found in bacteria, fungi, and animals. These α-amylases can hydrolyze raw starch from potatoes, wheat, corn, and other crops.

[0003] In industrial applications, the starch processing industry commonly uses 20-30% (w / v) starch slurry as raw material, and research on enzymatic degradation of raw starch at room temperature has received widespread attention. Currently, research on low-temperature hydrolysis of high-concentration raw starch using raw starch hydrolysants is limited. Traditional starch-to-saccharification processes require two steps: liquefaction and saccharification. Raw starch needs to be gelatinized sequentially at 100°C, liquefied with thermophilic α-amylase at approximately 95°C, and saccharified with glucosylamylase at 50-60°C. Clearly, existing starch processing technologies consume a large amount of energy, thus necessitating the development of more efficient and energy-saving processes. Raw starch degrading enzymes (RSDEs) can act directly on raw starch granules below the starch gelatinization temperature. Therefore, RSDEs can significantly reduce energy requirements and simplify the starch processing flow. The stability of amylases is also crucial in starch-to-saccharification processes; many amylases, due to their poor stability, cannot sustain their hydrolytic activity during the hydrolysis process. Therefore, utilizing protein engineering technology to obtain raw starch hydrolysants with good thermal stability is of significant value for the application of hydrolyzing high-concentration corn raw starch for saccharification. Summary of the Invention

[0004] This invention provides a raw starch hydrolase mutant, its expression strain, and its applications. This invention utilizes bacteria derived from seabed sediments in the South China Sea. Pontibacillus Based on the high specific activity of the starch hydrolase AmyZ1, sp.ZY was developed through site-directed mutagenesis after identifying key substrate-binding subsites via molecular docking. This resulted in a mutant with increased glucose production. Using rice and corn starch as substrates, the mutant enzyme produced twice the glucose of the original enzyme; at 35°C and pH 7.0, its half-life was 7.5 times that of the original enzyme. When used in the high-concentration corn starch saccharification process, the DE (glucose dilution) reached 36% after 2 hours of liquefaction and exceeded 99% after 18 hours of saccharification. This mutant enzyme has potential application value in the high-concentration corn starch saccharification industry.

[0005] The present invention relates to a raw starch hydrolase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1, wherein the leucine at position 199 of the amino acid sequence of the raw starch hydrolase is mutated to phenylalanine.

[0006] The coding gene for the raw starch hydrolase mutant of the present invention has the nucleotide sequence shown in SEQ ID NO: 2.

[0007] The expression strain of the raw starch hydrolase mutant of this invention is classified and named as follows: Bacillus subtilis WB600 / pBHSSs142-AmyZ1(L199F) has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242140, on September 30, 2024, at Wuhan University, Wuhan, China.

[0008] The method for constructing the expression strain of the raw starch hydrolase mutant of the present invention includes the following steps:

[0009] Firstly, from Bacillus licheniformis Using the α-amylase BLA structure as a template, the structure of the starch hydrolase AmyZ1 was homologously modeled using Swiss-Model. AutoDock was used to dock G3-G8 with the enzyme. Both macromolecules and small molecules underwent pretreatment (dehydration, hydrogenation, and charge addition); 264 Glu residues were set as semi-flexible residues; a docking parameter file was prepared with the grid size set to X, Y, Z: 40, 42, 40, and the grid center set to -41.16, 26.186, -6.347 (x, y, z); vina docking was then performed. The optimal substrate for this enzyme was determined to be G7, thus possessing seven subsites. These subsites were named with the reducing end of the substrate as positive and the non-reducing end as negative. The subsites are -5, -4, -3, -2, -1, +1, and +2. According to the literature, the amino acid at the +1 site affects the formation of glucose products. Mutating the amino acid at the +1 site into a hydrophobic amino acid will increase the affinity with the substrate. Therefore, leucine at position 199 of the +1 site was selected to be mutated into phenylalanine.

[0010] Based on the gene sequence of the starch hydrolase AmyZ1, mutant primers were designed and synthesized. Using a recombinant plasmid containing the AmyZ1 gene as a template and the synthesized mutant primers (Table 1), site-directed mutagenesis was performed using overlap extension PCR to obtain the mutant gene of a glucose product-specific starch hydrolase. The mutant gene was then coupled with the vector pBHS using POE-PCR. S142 Ligation is performed to obtain the ligation product; the ligation product is then transformed into the host bacteria. Bacillus subtilis WB600, on plates with Kanamycin resistance, was used to screen positive clones containing the mutant gene of this invention by plasmid PCR screening and sequencing.

[0011] The expression plasmid vector in the construction method comprises pBHS S142 etc.

[0012] The host bacteria in the construction method comprise Bacillus subtilis WB600, etc.

[0013] The raw starch hydrolytic enzyme mutant of the application can be obtained by fermentation of the engineering strain.

[0014] The raw starch hydrolytic enzyme mutant of the application is used in starch hydrolysis for sugar production.

[0015] The starch concentration is 20%-30% (W / V).

[0016] The starch comprises rice, corn raw starch, etc.

[0017] When 20% (W / V) rice, corn raw starch is used as the substrate, the glucose yield of the raw starch hydrolytic enzyme mutant of the application is 2 times that of the original enzyme; under the condition of 35℃ and pH 7.0, the half-life is 7.5 times that of the original enzyme. The raw starch hydrolytic enzyme mutant of the application is used in the high-concentration corn raw starch sugar production process, and after liquefaction for 2h, DE can reach 36%; after saccharification for 18h, DE is more than 99%. The mutant enzyme has potential application value in the industry based on high-concentration corn raw starch hydrolysis for sugar production. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The maltose standard curve.

[0019] Figure 2 , 3 The electrophoretogram of the PCR amplification product of the application.

[0020] Figure 4 The SDS-PAGE pattern of the purified mutant enzyme and the original enzyme AmyZ1: the mutant enzyme is eluted out under the concentration of 200mM imidazole.

[0021] Figure 5 The stability of the mutant enzyme and the original enzyme under the condition of 35℃ and pH 7.0.

[0022] Figure 6 A is the TLC pattern of the reaction product of the original enzyme and malt oligosaccharide, and B is the TLC pattern of the reaction product of the mutant enzyme and malt oligosaccharide.

[0023] Figure 7 The DE change graph of the mutant enzyme and the original enzyme used in the corn raw starch sugar production process. DETAILED DESCRIPTION

[0024] The implementation methods in the following examples are all conventional methods unless otherwise specified.

[0025] (I) Construction of expression strains containing the mutant gene of raw starch hydrolyzing enzyme of the present application

[0026] 1. Selection of mutation sites of raw starch hydrolyzing enzyme gene

[0027] Based on sequence alignment, the raw starch hydrolyzing enzyme AmyZl has 71% amino acid sequence identity with the α-amylase BLA from Bacillus licheniformis. Using the structure of BLA as a template, the structure of the raw starch hydrolyzing enzyme AmyZl was homologously modeled using Swiss-Model (http: / / swissmodel.expasy.org / ; Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T. The SWISS-MODEL Repository and associated resources. Nucleic Acids Research. 2009, 37, D387-392.). Bacillus licheniformis

[0028] According to the simulated structure, G3-G8 were subjected to molecular docking with the enzyme using AutoDock (O. Trott, A. J. Olson: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, Journal of Computational Chemistry 31 (2010), 455-461). The macromolecule and small molecule were pre-processed to remove water, add hydrogen, and add charges; 264 Glu was set as a semi-flexible residue; the docking parameter file was prepared by setting the size of the grid to X, Y, Z: 40, 42, 40, and then setting the center of the grid to -41.16, 26.186, -6.347 (x, y, z); vina docking was performed. It was determined that the optimal substrate for the enzyme was G7, and therefore the enzyme has seven sub-sites. The substrate was named with the reducing end as positive and the non-reducing end as negative. The sub-sites were -5, -4, -3, -2, -1, +1, and +2, respectively. According to the literature, the amino acid at the +1 site affects the production of glucose product, and mutating the amino acid at the +1 site to a hydrophobic amino acid increases the affinity between the substrate and the enzyme. Therefore, the leucine at position 199 in the +1 site was mutated to phenylalanine.

[0029] 2. Construction of mutant gene engineering strains of raw starch hydrolyzing enzyme

[0030] ​According to the gene sequence of raw starch hydrolyzing enzyme AmyZ1 and the designed mutation L199F, the following 6 site-directed mutation primers are designed (Table 1).

[0031] The recombinant plasmid containing the AmyZ1 gene is used as the template plasmid, and the target fragment is obtained by overlap extension PCR amplification; then the pBHS-F and pBHS-R amplification vectors are used for POE-PCR connection. 142 The target fragment and the vector are used as templates and primers for POE-PCR connection. The connection product is transformed into Bacillus subtilis by chemical transformation, and the correct transformants are obtained by plasmid PCR screening bands and sequencing on a Kanamycin-resistant plate to obtain the mutant gene engineering strain of the application Bacillus subtilis WB600 / pBHSSs142-AmyZ1(L199F).

[0032] The expression strain of the raw starch hydrolyzing enzyme mutant of the application is classified and named as Bacillus subtilis WB600 / pBHSSs142-AmyZ1(L199F), which has been preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M 20242140, a preservation time of September 30, 2024, and a preservation address of Wuhan, China.

[0033]

[0034] (II) Expression and protein purification of the raw starch hydrolyzing enzyme mutant gene engineering bacteria containing the raw starch hydrolyzing enzyme mutant gene of the application

[0035] The constructed mutant strain is inoculated into a 5mL LB medium containing Kana in a small volume, and cultured in a 37℃, 200rpm shaker for 12h. The bacterial liquid cultured for 12h is used as a seed liquid, and 5mL seed liquid is inoculated into 100mL YT liquid medium containing 30mg / mL Kana, and fermented in a 30℃, 200rpm shaker for 48-60h. The fermentation liquid cultured for 48h is centrifuged at 8000xg in a large centrifuge for 30min, and the obtained supernatant is the crude enzyme liquid.

[0036] The crude enzyme liquid is purified by Ni-NTA column chromatography. The imidazole concentration in the eluate is 200mM, and elution is performed for 3 column volumes. The obtained protein is detected by SDS-PAGE, and the purity reaches more than 95%.

[0037] When corn raw starch is used as the substrate, the optimal temperature of the mutant enzyme is 35℃, the optimal pH is 7.0, and the catalytic activity is more than 80% of the highest enzyme activity in the pH range of 5.5-7.5.

[0038] (III) Detection of the specific enzyme activity of the raw starch hydrolyzing enzyme mutant of the application (DNS method)

[0039] 1. Enzyme activity definition

[0040] 1 U is the amount of protein needed to produce 1 µM maltose per minute.

[0041] 2. Maltose standard curve drawing

[0042] In the EP tube containing the enzyme activity measuring buffer, maltose was added to make the final concentration 55.55, 66.66, 77.77, 88.88, 100.00, 111.11, 122.22, 133.33, 144.44, 155.55, 166.66, 177.77, 188.88, 199.99, 200.00, 211.11 and 222.22 µM, respectively, with a total volume of 600 µL. 300 µL of DNS was added and mixed well by vortex, boiled in boiling water for 15 min, and then the EP tube was taken out and placed on ice to quickly cool to room temperature. The absorbance value was measured at 540 nm, and the standard curve was drawn. As shown in Figure 1 , the maltose standard curve is: y = 0.0016x + 0.0466, R² = 0.999.

[0043] 3. Specific enzyme activity determination

[0044] According to the reaction system and procedure in Table 2, the absorbance value at 540 nm was obtained, the amount of reducing sugar was calculated according to the maltose standard curve formula, and then the specific enzyme activity was calculated according to the enzyme activity definition.

[0045]

[0046] (Four) Detection of the stability of the raw starch hydrolyzing enzyme containing the mutant of the application

[0047] Under the conditions of 35℃ and pH 7.0, the starting enzyme AmyZ1 and the mutant were subjected to heat treatment, and samples were taken every half hour or one hour, taking the initial enzyme activity as 100%, and the residual rate of enzyme activity after heat treatment for a certain time was calculated.

[0048] (Five) Reaction of the mutant of the raw starch hydrolyzing enzyme containing the mutant of the application with malt oligosaccharide

[0049] 1% maltose-maltoheptaose (G2-G8) as substrate, 5U / mg of starch hydrolytic enzyme, 300 μL substrate, 270 μL Na2HPO4-KH2PO4 buffer (50 mM, pH 7.0), incubation at 35°C for 1 h, boiling for 15 min to deactivate the enzyme. Qualitative and quantitative detection of the product by TLC and HPLC. TLC development agent is water: chloroform: acetic acid = 1:4:9; color developing agent is ethanol: sulfuric acid = 19:1; HPLC uses evaporative light detector, chromatographic column is TSKgel Amide-80 5 μm, mobile phase is 55% acetonitrile, column temperature is 25°C, evaporator temperature is 90°C, atomizer temperature is 80°C, flow rate is 0.5 ml / min. Prepare 0.1 mg / ml-1.5 mg / ml maltose-maltoheptaose to draw standard curve.

[0050] (VI) Determination of reaction product of rice and corn raw starch containing the mutant of the raw starch hydrolytic enzyme of the application

[0051] 20% (W / V) of rice or corn raw starch is dissolved in Na2HPO4-KH2PO4 (50 mM, pH 7.0) buffer with a final concentration of 1 mM CaCl2. 0.5 U / mg of raw starch hydrolytic enzyme is added, and the total system is 10 mL. Hydrolysis reaction is carried out at 35°C in a 200 rpm shaking water bath. After 4 h of reaction, the reaction enters a plateau phase, and the product is analyzed qualitatively and quantitatively by HPLC.

[0052] (VII) Application of high-concentration corn raw starch containing the mutant of the raw starch hydrolytic enzyme of the application to sugar production

[0053] The starch processing industry usually uses starch slurry with a concentration as high as 20-30% as a starting concentration, and α-amylase is added in the liquefaction stage to hydrolyze it to generate a series of malt oligosaccharides, and then saccharifying enzyme is added to cut the α-1,4 glycosidic bond, which is hydrolyzed to generate glucose. In this process, 20% (W / V) of corn raw starch is used as substrate, and the total system is 30 mL. After 30 min of pretreatment at 70°C, 0.4 U / mg of the raw starch hydrolytic enzyme containing the mutant of the application is added, and the liquefaction is carried out at 30°C in a 200 rpm shaking water bath for 2 h. Then 100 U / g of saccharifying enzyme is added, and the saccharification is carried out at 30°C in a 200 rpm shaking water bath for 48 h.

[0054]

[0055]

[0056] As can be seen from Table 3, when malt oligosaccharide is used as substrate, the glucose produced by the mutant enzyme is 2 times that of the starting enzyme; as can be seen from Table 4, the glucose produced by the mutant enzyme in the hydrolysis of high-concentration corn and rice raw starch is 2 times that of the starting enzyme.

Claims

1. A raw starch hydrolyzing enzyme mutant, whose amino acid sequence is shown in SEQ ID NO:

1. 2.A gene encoding the raw starch hydrolyzing enzyme mutant of claim 1, whose nucleotide sequence is shown in SEQ ID NO:

2. 3.An expression strain of the raw starch hydrolyzing enzyme mutant of claim 1, characterized in that: the taxonomic name of the expression strain is Bacillus subtilis WB600 / pBHSSs142-AmyZ1 (L199F), which has been deposited with the China Center for Type Culture Collection (CCTCC) on September 30, 2024, and the deposit number is CCTCC NO: M 20242140, and the deposit address is Wuhan, China, Wuhan University. 4.The raw starch hydrolyzing enzyme mutant of claim 1 in the application of starch hydrolysis to produce sugar, characterized in that: the starch is one or more of rice starch and corn raw starch, and the sugar is glucose and maltotetraose. 5.The application of claim 4, characterized in that: the concentration of starch in the system is 20%-30%. 6.The application of claim 4, characterized in that: the temperature of the system is 30-35℃, and the pH is 7.

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