A mutant of a type iii pullulan hydrolase and a method for catalyzing synthesis of panose thereof

Abstract

The application discloses a type III pullulan hydrolase mutant and a method for catalyzing synthesis of panose, and relates to the technical fields of genetic engineering and enzyme engineering. The amino acid sequence of the type III pullulan hydrolase mutant is shown as SEQ ID NO. 3. The type III pullulan hydrolase mutant is obtained by mutating the 410th amino acid of type III pullulan hydrolase from a thermophilic archaeon Thermococcus kodakarensis into histidine. The specific enzyme activity of the type III pullulan hydrolase mutant to pullulan is greatly improved, and the type III pullulan hydrolase mutant can efficiently convert pullulan into panose. The conversion rate of the type III pullulan hydrolase mutant for catalyzing synthesis of panose from 80 mg / mL pullulan as a substrate is up to 91%. The type III pullulan hydrolase mutant has a very wide application prospect in the biological enzyme catalysis synthesis of panose.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, and in particular to a type III pullulan hydrolase mutant and a method for catalyzing the synthesis of panose. Background Technology

[0002] Panose is a functional oligosaccharide, a trisaccharide composed of a maltose molecule linked to a glucose molecule via an α-1,6-glycosidic bond. Panose possesses various biological activities, such as promoting the growth and metabolism of probiotics, enhancing immunity, and maintaining gut health. Panose can be used in the food industry as a functional food and prebiotic. Digestive enzymes in the human gastrointestinal tract (such as amylase and sucrase) have difficulty hydrolyzing the α-1,6-glycosidic bond in panose molecules. Therefore, panose is almost not digested and absorbed by the human body, produces no calories, and does not cause a rise in blood sugar, making it ideal for diabetics and obese individuals. Panose can reach the intestines undigested, selectively promoting the proliferation of beneficial bacteria such as Bifidobacteria and Lactobacillus while inhibiting the growth of harmful bacteria (such as Salmonella). This "high selectivity" is superior to many other prebiotics, meaning that only a small amount is needed to achieve excellent probiotic effects, improve gut microbiota, and enhance immunity. Panose and its derivatives have good film-forming properties, water solubility, and biocompatibility, and are not easily metabolized by the human body. Panose can be used in the pharmaceutical field for encapsulating drugs, preparing sustained-release formulations, or as a stabilizer for protein drugs. Furthermore, the presence of multiple hydrophilic hydroxyl groups in the panose molecule gives it excellent hygroscopic and moisturizing properties, making it suitable as a natural moisturizing ingredient in cosmetics.

[0003] Pullulan is an extracellular, water-soluble, mucopolysaccharide produced by the fermentation of *Brachystomata*. Its structure consists of a polymer formed by repeating maltotriose (three glucose molecules linked by α-1,4 glycosidic bonds) linked by α-1,6 glycosidic bonds. The thermophilic acidic type III pullulan hydrolase TK-PUL, derived from the thermophilic archaea *Thermococcus kodakarensis*, can hydrolyze the α-1,4- and α-1,6-glycosidic bonds in pullulan. TK-PUL can directly catalyze the synthesis of panose from pullulan; the catalytic reaction system is simple, and the reaction conditions are mild, making it a typical green biomanufacturing process. However, currently, the application of TK-PUL to the enzymatic preparation of panose suffers from low conversion efficiency of the resulting panose product. Summary of the Invention

[0004] The purpose of this invention is to provide a type III pullulan hydrolase mutant and a method for catalyzing the synthesis of panose, thereby solving the problems existing in the prior art. This type III pullulan hydrolase mutant can efficiently catalyze the synthesis of panose using pullulan as a substrate.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a type III pullulan hydrolase mutant, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also provides a coding gene for the above-mentioned pullulan hydrolase mutant of type III.

[0008] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0009] The present invention also provides a recombinant vector comprising the above-described encoding gene.

[0010] The present invention also provides a recombinant microbial strain comprising the above-described recombinant vector.

[0011] The present invention also provides the application of the above-mentioned coding gene, recombinant vector or recombinant microbial strain in the preparation of the above-mentioned pullulan hydrolase mutant.

[0012] The present invention also provides a method for preparing the above-mentioned type III pullulan hydrolase mutant, comprising the steps of inducing expression culture of the above-mentioned recombinant microbial strain, and then extracting and purifying to obtain the type III pullulan hydrolase mutant.

[0013] The present invention also provides the application of the above-mentioned type III pullulan hydrolase mutant in the catalytic synthesis of panose using pullulan as a substrate.

[0014] The present invention also provides a method for catalytic synthesis of panose, comprising the steps of using pullulanose as a substrate and the aforementioned type III pullulan hydrolase mutant as an enzyme catalyst to catalytically synthesize panose.

[0015] The present invention also provides a method for improving the enzyme activity of type III pullulan hydrolase TK-PUL in catalyzing the synthesis of pullulanose into panose, wherein the amino acid sequence of type III pullulan hydrolase TK-PUL is shown in SEQ ID NO.1;

[0016] The method includes the step of mutating the 410th amino acid of the type III pullulan hydrolase TK-PUL—serine (S)—to histidine (H).

[0017] The present invention discloses the following technical effects:

[0018] The type III pullulan hydrolase mutant provided by this invention exhibits a 0.18-fold increase in the specific enzyme activity of pullulanose from the control (before mutation) of 123.78 U / mg to 146.23 U / mg; and a 0.26-fold increase in its catalytic efficiency for pullulanose from the control (before mutation) of 89.82 mL / (mg·s) to 113.37 mL / (mg·s). Using 80 mg / mL pullulanose as a substrate, the addition of 0.5 U / mL of the type III pullulan hydrolase mutant, reacting at 80℃ for 6 h, catalyzed the synthesis of 72.7 mg / mL panose, achieving a panose conversion rate as high as 91%. The conversion rate of panose synthesized by the mutant S410H was 51.4% higher than that of TK-PUL. The type III pullulan hydrolase mutant can efficiently catalyze the synthesis of panose using pullulanose as a substrate, showing potential application in the preparation of high-purity panose. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 SDS-PAGE images of pullulan hydrolase type III TK-PUL and its mutant S410H;

[0021] Figure 2 TLC results of the products synthesized by pullulan hydrolase type III TK-PUL and mutant S410H using 80 mg / mL pullulanose as a substrate. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] The main experimental materials involved in the following examples are as follows:

[0028] 1. Strains and vectors

[0029] Escherichia coli JM109 (purchased from Takara), Bacillus subtilis WB600 (purchased from Takara), and Bacillus subtilis expression vector pSTOP1622 (purchased from MoBiTec).

[0030] 2. Enzymes and other biochemical reagents

[0031] The gene site-directed mutagenesis kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; DNA polymerase, DNA restriction endonuclease, T4 DNA ligase, DNA gel extraction kit, and plasmid extraction kit were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; Chelating Sepharose™ Fast Flow was purchased from GE Healthcare, USA; pullulanose was purchased from Beijing Bio-Rad Biotechnology Co., Ltd.; and other chemical reagents were domestically produced or imported analytical grade.

[0032] 3. Culture medium

[0033] LB medium: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl, pH 7.0. LB medium containing 100 μg / mL ampicillin was used for screening.

[0034] The molecular cloning and protein detection techniques used in this invention are conventional techniques in the field. Techniques not described in detail in the following examples were performed according to the relevant sections of the following laboratory manual: Green MR, Sambrook J. Molecular cloning: a laboratory manual [M]. New York: ColdSpring Harbor Laboratory Press, 2012.

[0035] 4. Amino acid and nucleotide sequences

[0036] The amino acid sequence of pullulan hydrolase type III TK-PUL is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2. The amino acid sequence of mutant S410H is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4.

[0037] SEQ ID NO.1:

[0038] MKKGGLLILLILVSIAEKGCPSGRVPVKFTYNPGNKTVKSVSLRGSFNNWGEWPMELKNGTWETTVCLRPGRYEYKYFINGQWVKDMSDDGTGRPYDPDADAYAPDGYGGKNAVRVVEGREAFYVEFDPRDPAYLSIADKRTVVRFEAKRDTVESAVLVTDHGNYTMKLQVWWDFGETWRAEMPVEPADYYILVTSSDGGKFA VLNTSESPFFHFDGVEGFPQLEWVSNGITYQIFPDRFNNGNKSNDALALDHDELILNQVNPGQPILSNWSDPITPLHCCHQYFGGDIKGITEKLDYLQSLGV TIIYINPIFLSGSAHGYDTYDYYRLDPKFGTEDELREFLDEAHRRGMRVIFDFVPNHCGIGNPAFLDVWEKGNESPYWDWFFVKKWPFKLGDGSAYVGWWGFG SLPKLNTANQEVREYLIGAALHWIEFGFDGIRVDVPNEVLDPGTFPELRKAVKEKNPDAYLVGEIWTESPEWVKGDRFDSLMNYALGRDILLNYAKGLLSGESAMKMMGRYYASYGENVVAMGFNLVDSHDTSRVLTDLGGKLGDTPSNESIQRLKLLSTLLYALPGTPVTFQGDERGLLGDKGHYDEQRYPIQWDTVNEDVLNHYRALAELRKRVPALRSSAMRFYTAKGGVMAFFRGHHDEVLVVANSWKKPALLELPEGEWKVIWPEDFSPELLRGTVEVPAIGIIILERGHHHHH.

[0039] SEQ ID NO.2:

[0040]

[0041] SEQ ID NO.3:

[0042] MKGGGLLLILLILVSIAEKGCPSGRVPVKFTYNPGNKTVKSVSLRGSFNNWGEWPMELKNGTWETTVCLRPGRYEYKYFINGQWVKDMSDDGTGRPYDPDADAYAPDGYGGKNAVRVVEGREAFYVEFDPRDPAYLSIADKRTVVRFEAKRDTVESAVLVTDHGNYTMKLQVWWDFGETWRAEMPVEPADYYILVTSSDGGKFAVLNTSESPFFHDGVEGFPQLEWVSNGITYQIFPDRFNNGNKSNDALADHDELILNQVNPGQPILSNWSDPITPLHREHQYFGGDIKGITEKLDYLQSLGVTIIYINPIFLSGSAHGYDTYDYYRLDPKFGTEDELREFLDEAHRRGMRVIFDFVPNHCGIGNPAFLDVWEKGNESPYWDWFFVKKWPFKLGDGSAYVGWWGFG H LPKLNTANQEVREYLIGAALHWIEFGFDGIRVDVPNEVLDPGTFPELRKAVKEKNPDAYLVGEIWTESPEWVKGDRFDSLMNYALGRDILLNYAKGLLSGESAMKMMGRYYASYGENVVAMGFNLVDSHDTSRVLTDLGGKLGDTPSNESIQRLKLLSTLLYALPGTPVTFQGDERGLLGDKGHYDEQRYPIQWDTVNEDVLNHYRALAELRKRVPALRSSAMRFYTAKGGVMAFFRGHHDEVLVVANSWKKPALLELPEGEWKVIWPEDFSPELLRGTVEVPAIGIIILERGHHHHH.

[0043] SEQ ID NO.4:

[0044]

[0045] Example 1 Construction of recombinant strains

[0046] (1) Construction of expression vector pSTOP1622-tkpul

[0047] Using the nucleotide sequence shown in SEQ ID NO.4 as a template and primers P1 and P2 (Table 1) as primers, PCR amplification was performed. The PCR amplification conditions were: 98℃ for 5 min; 98℃ for 15 sec, 60℃ for 20 sec, 72℃ for 20 sec, 30 cycles; 72℃ for 5 min. The amplification product was double-digested with Spe I and BamHI and ligated into the vector pSTOP1622 to construct the recombinant plasmid pSTOP1622-tkpul.

[0048] Table 1 Primers used to construct recombinant plasmids

[0049]

[0050] Note: The underlined parts are restriction enzyme cleavage sites.

[0051] (2) Construction of expression vector pSTOP1622-tkpulS410H

[0052] Using the recombinant plasmid pSTOP1622-tkpul as a template and primers S410H-F and S410H-R (Table 1), a site-directed mutagenesis kit was used to mutate serine at position 410 of TK-PUL to histidine. PCR amplification conditions were: 98℃ for 5 min; 98℃ for 15 sec, 60℃ for 20 sec, 72℃ for 3 min, 30 cycles; 72℃ for 5 min. The amplified product was treated with Dpn I enzyme and then electroporated into *E. coli* JM109 competent cells. The cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37℃. Single transformants were picked from the LB plates, and the recombinant plasmid was extracted. Using the recombinant plasmid as a template and primers P1 and P2, PCR amplification was performed. The amplified DNA fragment was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to identify whether it was the mutant gene tkpulS410H. The correctly identified recombinant plasmid was named pSTOP1622-tkpulS410H.

[0053] (3) Construction of recombinant strains B. subtilis WB600 / pSTOP1622-tkpul and B. subtilis WB600 / pSTOP1622-tkpulS410H

[0054] The recombinant plasmid pSTOP1622-tkpul was transformed into Bacillus subtilis WB600 to obtain the recombinant strain B. subtilis WB600 / pSTOP1622-tkpul. The recombinant plasmid pSTOP1622-tkpulS410H was then transformed into Bacillus subtilis WB600 to obtain the recombinant strain B. subtilis WB600 / pSTOP1622-tkpulS410H.

[0055] Example 2: Induction and purification of recombinase

[0056] Single colonies of the two recombinant strains were inoculated separately into 20 mL LB broth containing 20 μg / mL tetracycline and cultured overnight at 37°C with rapid shaking. The overnight cultures were then transferred at a 1% inoculum to 50 mL LB broth containing 20 μg / mL tetracycline and cultured at 37°C with rapid shaking until the bacterial OD reached the target value. 600nm The concentration reached approximately 0.8. Xylose was added to a final concentration of 0.5%, and the mixture was incubated at 37°C for 30 h. The fermentation supernatant was then collected by centrifugation at 12000 r / min for 10 min. Ni was used... 2+ The target protein in the fermentation supernatant was purified using affinity chromatography, and eluted with 200 mmol / L imidazole elution buffer to obtain the purified recombinase. The purity of the recombinase was detected by SDS-PAGE, and the concentration was determined using the Bradford method. The SDS-PAGE results of type III pullulan hydrolase TK-PUL and the mutant S410H are shown below. Figure 1 As shown.

[0057] Example 3: Determination of specific enzyme activity and kinetic parameters of recombinant enzyme

[0058] Specific enzyme activity assay of recombinant enzymes: The specific enzyme activities of recombinant enzyme TK-PUL and mutant S410H were determined using pullulan as a substrate. 10 μL of enzyme solution (enzyme concentration 50 μg / mL) was mixed with 490 μL of 50 mmol / L LMES buffer (pH 4.5) containing 1% (m / v) pullulan. The mixture was reacted at 100℃ for 30 min, and then the reaction was quickly terminated by placing the mixture in an ice-water bath. The amount of reducing sugar in the reaction system was then determined using the 3,5-dinitrosalicylic acid method. The generated reducing sugar was converted to maltose mass using a maltose standard curve. Enzyme activity unit (U) definition: Under certain reaction conditions, the amount of enzyme that catalyzes the production of 1 μmol of maltose per minute is defined as one enzyme activity unit (U). The results of the specific enzyme activity assay of recombinant enzymes to pullulan are shown in Table 2. The pullulan hydrolase type III, TK-PUL, exhibited a pullulan-specific activity of 123.78 ± 3.11 U / mg, while the mutant S410H showed a pullulan-specific activity of 146.23 ± 4.21 U / mg. Compared to TK-PUL, the pullulan-specific activity of the mutant S410H was increased by 0.18 times.

[0059] Table 2 Results of specific enzyme activity and kinetic parameters of recombinant enzymes

[0060]

[0061] Pullulan was used as a substrate, and different concentrations of substrate were prepared using 50 mmol / L MES buffer at pH 4.5. Equal volumes of enzyme solution were added to each substrate concentration, and the specific enzyme activity with pullulan as the substrate was measured. Using the double reciprocal plot method, the Michaelis constant K was calculated by plotting the reciprocal of substrate concentration on the x-axis and the reciprocal of enzyme specific activity on the y-axis. m Reaction constant k cat and catalytic efficiency k cat / K m The pullulan concentration gradient was set sequentially to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 mg / mL. The kinetic parameters of the recombinase were determined as shown in Table 2. The catalytic efficiency k of TK-PUL using pullulan as a substrate was... cat / K m The catalytic efficiency k of mutant S410H is 89.82 mL / (mg·s). cat / K m The concentration was 113.37 mL / (mg·s), which was 0.26 times higher than that of TK-PUL.

[0062] Example 4: Recombinase catalyzes the synthesis of panose using pullulanose as a substrate.

[0063] 100 mL of pullulanose solutions of different mass concentrations (50 mmol / L MES, pH 4.5) were prepared. Recombinant enzymes (TK-PUL and mutant S410H prepared in Example 2) were added at a concentration of 0.5 U / mg pullulanose. This mixture was incubated at 80°C with a stirring rate of 400 r / min for 6 h, and then the reaction was stopped. The pullulanose mass concentration gradient was set sequentially to 10, 40, 80, and 100 mg / mL. After the reaction, the mixture was treated at 100°C for 10 min, centrifuged at 12000 ×g for 10 min, and the supernatant was collected. 1 mL of the supernatant was freeze-dried to obtain the dried product, which was then weighed. 1 mL of the supernatant was diluted 10000 times and filtered through a 0.22 μm aqueous filter membrane to obtain the product to be analyzed. The panose content of the product was determined by HPLC, and the panose conversion rate was calculated.

[0064] The formula for calculating the conversion rate of panose is:

[0065] Panose conversion rate = mass concentration of product panose / mass concentration of substrate pullulanose × 100%.

[0066] The HPLC detection conditions were as follows: Instrumentation: Thermo Fisher ICS5000 ion chromatograph; Column: Dionex Carbopac™ PA200 (3×150 mm); Mobile phase: A: H2O, B: 130 mmol / L NaOH, C: 65 mmol / L NaOH and 1025 mmol / L NaOAC; Flow rate: 0.3 mL / min; Injection volume: 25 μL; Column temperature: 30℃; Detector: Electrochemical detector.

[0067] The conversion rates of pullulan hydrolase type III TK-PUL to panose using pullulan as a substrate are shown in Table 3, and those of mutant S410H are shown in Table 4. The conversion rates of panose catalyzed by mutant S410H using pullulan as a substrate are both higher than those of TK-PUL. Mutant S410H can efficiently convert pullulan to panose; with 80 mg / mL pullulan as a substrate, the mass concentration of the product panose is 73.21 mg / mL, and the conversion rate is as high as 91.5%. Under the same reaction conditions, the mass concentration of the product panose synthesized by TK-PUL is 32.08 mg / mL, and the conversion rate is 40.1%. That is, with 80 mg / mL pullulan as a substrate, the conversion rate of panose synthesized by mutant S410H is 51.4% higher than that of TK-PUL, which is 1.28 times higher.

[0068] Table 3. Conversion rate of panose synthesized by pullulanose-based pullulanase TK-PUL (Type III pullulan hydrolase)

[0069]

[0070] Table 4. Conversion rate of panose synthesized by mutant S410H using pullulanose as a substrate

[0071]

[0072] The reaction products of type III pullulan hydrolase TK-PUL and its mutant S410H using 80 mg / mL pullulanose as a substrate were detected by TLC. Recombinant enzyme was added at a concentration of 0.5 U / mg pullulanose, and the mixture was incubated at 80 °C with a stirring rate of 400 r / min for 6 h before the reaction was stopped. After the reaction, the mixture was heated at 100 °C for 10 min, centrifuged at 12000 ×g for 10 min, and the supernatant was collected for TLC analysis. The TLC results of the products synthesized by TK-PUL and its mutant S410H using 80 mg / mL pullulanose as a substrate are shown below. Figure 2 As shown, the yield and purity of panose synthesized by mutant S410H are superior to those of TK-PUL; moreover, the panose synthesized by mutant S410H has higher purity and does not require a complex purification process.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type III pullulan hydrolase mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO.

3.

2. The encoding gene of the type III pullulan hydrolase mutant as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

4. A recombinant vector, characterized in that, Includes the coding gene as described in claim 2 or 3.

5. A recombinant microbial strain, characterized in that, Includes the recombinant vector as described in claim 4.

6. The use of the encoding gene as described in claim 2 or 3, the recombinant vector as described in claim 4, or the recombinant microbial strain as described in claim 5 in the preparation of the type III pullulan hydrolase mutant as described in claim 1.

7. A method for preparing the type III pullulan hydrolase mutant as described in claim 1, characterized in that, The method includes the steps of inducing expression culture of the recombinant microbial strain described in claim 5, and then extracting and purifying it to obtain the type III pullulan hydrolase mutant.

8. The application of the type III pullulan hydrolase mutant as described in claim 1 in the catalytic synthesis of panose using pullulan as a substrate.

9. A method for catalytic synthesis of panose, characterized in that, The method includes the step of catalyzing the synthesis of panose using pullulanose as a substrate and the pullulanose type III hydrolase mutant as described in claim 1 as an enzyme catalyst.

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