Puramidase mutants and their use in the production of mannose
By performing site-directed mutagenesis on pullulanase, its thermal stability was improved, solving the problem of poor thermal stability of pullulanase in mannose production, thus achieving more efficient mannose production and reduced costs.
Patent Information
- Application Number
- CN202611543210.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-10-08
- Publication Date
- 2026-11-06
AI Technical Summary
The poor thermal stability of existing pullulanases limits their application in mannose production, resulting in high production costs and low efficiency.
By performing site-directed mutagenesis on pullulanase derived from Pullulanibacillus naganoensis, the mutant PLL-M(7) was obtained, which improved its thermal stability and optimized its use in multi-enzyme systems.
The optimal reaction temperature of pullulanase was increased to 60°C, the amount of pullulanase used was reduced, which significantly improved the production efficiency of mannose and reduced the production cost.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, and more specifically to pullulanase mutants and their application in the production of mannose. Background Technology
[0002] Pullulanase (EC 3.2.1.41, Pullulanase; abbreviated as Pul) belongs to the α-amylase family and is a commonly used industrial amylase debranching enzyme. It specifically hydrolyzes the α-1,6 glycosidic bonds of amylopectin, dextrin, pullulanose, etc., shortening saccharification reaction time and increasing yield and conversion rate. Based on the different mechanisms of glycosidic bond hydrolysis and the differences in the products generated, pullulanases are divided into type I and type II pullulanases. Type I pullulanases are responsible for hydrolyzing α-1,6 glycosidic bonds, producing maltotriose and some linear oligosaccharides. Type II pullulanases are bifunctional, not only hydrolyzing α-1,6 glycosidic bonds but also randomly hydrolyzing α-1,4 glycosidic bonds on the starch linear chain, generating reducing ends, with glucose, maltose, and maltotriose as the main products. During saccharification, pullulanase is often combined with other amylases such as saccharifying enzymes, α-amylases, β-amylases, or cyclodextrin glucosyltransferases to rapidly decompose starch. In addition, pullulanase is also widely used in the production of fructose syrup, modified starch, ethanol fuel, beer and other fields.
[0003] Biosynthesis of mannose utilizes microbial fermentation or enzymatic catalysis to convert monosaccharides or polysaccharides into D-mannose. Biosynthesis offers mild reaction conditions and low cost. D-mannose 2-epimerase is currently the most studied catalytic enzyme for D-mannose preparation; its substrate is fructose. However, separating fructose and mannose presents certain difficulties, making it less suitable for industrial production.
[0004] Existing technologies utilize a multi-enzyme system to convert maltodextrin into mannose, employing starch debranching enzymes, α-glucan phosphorylase (α-GP), glucose phosphate mutase (PGM), bifunctional enzymes glucose phosphate isomerase / mannose 6-phosphate isomerase (PGI / MPI), and 6-phosphate mannose phosphatase (MPP). Pullulanase, a type of starch debranching enzyme, specifically hydrolyzes the α-1,6 glycosidic bonds in substrates such as amylopectin, glycogen, and maltodextrin, converting branched polysaccharides into linear polysaccharides. It provides sufficient substrate for glucan phosphorylase (aGP) and subsequent enzymatic catalysis in the multi-enzyme system. The optimal reaction temperature for existing pullulanases is 55°C, and their stability is relatively poor. Improving the thermal stability of pullulanase can reduce the production cost of mannose, which is of great significance for the preparation and industrial application of mannose.
[0005] Therefore, providing pullulanase mutants and their application in mannose production is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the present invention provides pullulanase mutants and their application in the production of mannose.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] This invention addresses the poor thermostability of pullulanase in multi-enzyme systems by rationally designing and molecularly modifying pullulanase.
[0009] This invention first involves site-directed mutagenesis of pullulanase derived from *Pullulanibacillus naganoensis* (Sequence ID: AEV53626.1), with the amino acid sequence SEQ ID NO.1. The nucleotide sequence of SEQ ID NO.2 is then constructed into the pET28a vector. Using the constructed plasmid as a template, site-directed amino acid mutagenesis is performed. The mutant plasmid is then transformed into *Escherichia coli* BL21(DE3) to obtain the mutant engineered bacterium. After induction of expression, the mutant pullulanase protein is obtained.
[0010] Pullulanase mutant, based on the amino acid sequence shown in SEQ ID NO.1, undergoes the following mutations: simultaneous mutations in D150I, A212V, N468I, H483C, I544K, N710S and G864L, abbreviated as PLL-M(7).
[0011] Furthermore, the gene encoding the pullulanase mutant.
[0012] Furthermore, an expression vector containing the coding gene of the pullulanase mutant described above.
[0013] Furthermore, recombinant cells containing the coding gene of the pullulanase mutant described above.
[0014] Furthermore, the application of the pullulanase mutant in the production of mannose.
[0015] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses pullulanase mutants and their application in the production of mannose, which has the following beneficial effects: (1) The present invention obtains pullulanase mutant with improved thermal stability. The optimal reaction temperature of the mutant is increased to 60℃, which is suitable for the reaction temperature of the transformation system.
[0016] (2) By performing site-directed mutagenesis on pullulanase, this invention obtained a pullulanase mutant strain PLL-M(7) with improved thermostability. Compared with the wild type, when used in combination with whole cells of α-glucan phosphorylase, glucose phosphate mutase, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase and 6-phosphate mannose phosphatase, the amount of pullulanase mutant PLL-M(7) used is reduced to 25% of that used in the wild type, which greatly improves the production efficiency of mannose and reduces costs. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0018] Figure 1 The thermal stability of pullulanase wild-type and mutant PLL-M(7) was measured. Detailed Implementation
[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0020] The amino acid sequence of pullulanase is shown in SEQ ID NO.1.
[0021] MDGNTTTIIVHYFRPSGDYTDWNLWMWPENGDGAEYDFNQPTDSYGEVASVDIPGNPSQVGIIVRKGNWDAKDIDSDRYIDLSKGHEIWLVQGNSQIFYSEKDAEAAAQPAVSNAYLDASNQVLVKLSQPFTLGEGSSGFTVHDDTANKDIPVTSVSDANQVTAVLAGTFQHIFGGSDWAPDNHNTLLKKVNSNLYQFSGNLPEGNYQYKVALNDSWNNPSYPSDNINLTVPAGGAHVTFSYIPSTHAVYDTINNPNADLQVDSSGVKTDLVAVTLGENPDVSHTLSIQTEDYQAGQVIPRKVLDSSQYYYSGDDLGNTYTKNATTFKVWAPTSTQVNVLLYNSATGAVTKTVPMTASGHGVWEATVNQDLENWYYMYEVTGQGSTRTAVDPYATAIAPNGTRGMIVDLAKTDPAGWESDKHITPKNIEDEVIYEMDVRDFSIDSNSGMKNKGKYLALTEKGTKGPDNVKTGVDSLKQLGITHVQLQPVFAFNSVNENDPTQYNWGYDPRNYNVPEGQYATNANGTTRIKEFKEMVLSLHQDHIGVNMDVVYNHTFATQISDFDKIVPEYYYRTDDAGNYTNGSGTGNEIAAERPMVQKFIIDSLKFWVNEYHVDGFRFDLMALLGKDTMSKAATQLHAIDPGIALYGEPWTGGTSALPADQLLTKGAQKGMGVAVFNDNLRNGLDGSVFDSSAQGFATGATGLTDAIKNGVEGSINDFTASPGETINYVTSHDNYTLWDKIAQSNPNDSEADRIKMDELAQAIVMTSQGIPFMQGGEEMLRTKGGNDNSYNAGDVVNEFDWSRKAQYPDVFNYYSGLIHLRLDHPAFRMTTANEINSHLQFLNSPENTVAYELSDHANKDTWGNIVVIYNPNKTAETINLPSGKWEINATSGKVGESTLGQAEGSVQVPGISMMILHQEVSPSDGK; SEQ ID NO.1.
[0022] The nucleotide sequence of pullulanase is shown in SEQ ID NO.2.
[0023]
[0024] The amino acid sequence of glucan phosphorylase is shown in SEQ ID NO.3.
[0025] MLEKLPENLKELESLAYNLWWSWSRPAQRLWRMIDSEKWEEHRNPVKILREVSKERLEELSKDEDFIALYELTLERFTDYMEREDTWFNVNYPEWDEKIVYMCMEYGLTKALPIYSGGLGILAGDHLKSASDLGLPLIAVGLLYKHGYFTQQIDSDGRQIEIFPEYDIEGLPMKPLRDEDGNQVIVEVPIDNDTVKARVFEVQVGRVKLYLLDTDFEENEDRFRKIADYLYNPEPDVRVSQEILLGIGGMKLLKTLKIKPGVIHLNEGHPAFSSLERIKSYMEEGYSFTEALEIVRQTTVFTTHTPVPAGHDRFPFDFVEKKLTKFFEGFESKELLMNLGKDEDGNFNMTYLALRTSSFINGVSKLHADVSRRMFKNVWKGVPVEEIPIEGITNGVHMGTWINREMRKLFDRYLGRVWREHTDLEGIWYGVDRIPDEELWEAHLNAKKRFIDYIRESIKRRNERLGINEPLPEISENVLIIGFARRFATYKRAVLLFSDLERLKRIVNNSERPVYIVYAGKAHPRDEGGKEFLRRIYEVSQMPDFKNKIIVLENYDIGMARLMVSGVDVWLNNPRRPMEASGTSGMKAAANGVLNASVYDGWWVEGYNGRNGWVIGDESVLPETEADDPKDAEALYELLENEIIPTYYENREKWIFMMKESIKSVAPKFSTTRMLKEYTEKFYIKGLVNREWLERRENVEKIGAWKERILKNWENVSIERIVLEDSKSVEVTVKLGDLTPNDVIVELVAGRGEGMEDLEVWKVIHIRRYRKENDLFVYTYTNGVLGHLGSPGWFYAVRVIPYHPRLPIKFLPEVPVVWKKVLE; SEQ ID NO.3.
[0026] The nucleotide sequence of glucan phosphorylase is shown in SEQ ID NO.4.
[0027]
[0028] The amino acid sequence of glucose phosphate mutase is shown in SEQ ID NO.5.
[0029] MGKLFGTFGVRGIANEEITPEFALKIGMAFGTLLKREGRERPLVVVGRDTRVSGEMLKDALISGLLSTGCDVIDVGIAPTPAIQWATNHFNADGGAVITASHNPPEYNGIKLLEP NGMGLKKEREAIVEELFFSEDFHRAKWNEIGELRKEDIIKPYIEAIKNRVDVEAIKKRRPFVVVDTSNGAGSLTLPYLLRELGCKVVSVNAHPDGHFPARNPEPNEENLKGFMEI VKALGADFGVAQDGDADRAVFIDENGRFIQGDKTFALVADAVLRENGGGLLVTTIATSNLLDDIAKRNGAKVMRTKVGDLIVARALLENNGTIGGEENGGVIFPDFVLGRDGAMT SEQ ID NO.5.
[0030] The nucleotide sequence of glucose phosphate mutase is shown in SEQ ID NO.6.
[0031]
[0032] The amino acid sequence of glucose phosphate isomerase / mannose-6-phosphate isomerase is shown in SEQ ID NO.7.
[0033] MHHHHHHVEKLDQIEEVKKLDPKDMYSAVYNLPEQIEKAAEVAYKAEIKNISDKIDKVLVLGMGGSAIGGDVLQSVLFKEAKFPVFVNRDYDLPNWVDERTLVFAVSYSGHTVETVNSAKLSYSRGASVVAVTSGGVLKEFAKEKNLTLVEIPGGMAPRAAIGYVTIPSFAILERLGLISPKR SEQ IDNO.7.
[0034] The nucleotide sequence of glucose phosphate isomerase / mannose-6-phosphate isomerase is shown in SEQ ID NO.8.
[0035]
[0036] The amino acid sequence of mannose-6-phosphate phosphatase is shown in SEQ ID NO.9.
[0037] MYRVFVFDLDGTLLNDNKEISEKDRKVIERLSRKCHVVFASGRMLVSVLNIEKKYFGRTFPTIAYNGAMVYIPEEGVILNEKISPEVAKDIVEYIKPFNVHWQAYIDDVLYSERDNEEIRGYAKHSSVEYRVEPNL SEQ ID NO.9.
[0038] The nucleotide sequence of mannose-6-phosphate phosphatase is shown in SEQ ID NO.10.
[0039] atgtaccgtgttttcgttttcgacctggacggtaccctgctgaacgacaacaaagaaatctctgaaaaagaccgtaaagttatcgaacgtctgtctcgtaaatgccacgttgttttcgcttctggtcgtatgctggtttctgttctgaacatcgaaaaaaaatacttcggtcgtaccttcccgaccatcgcttacaacggtgctatggtttacatcccggaagaaggtgttatcctgaacgaaaaaatctctccggaagttgctaaagacatcgttgaatacatcaaaccgttcaacgttcactggcaggcttacatcgacgacgttctgtactctgaacgtgacaacgaagaaatccgtggttacgctaaacactcttctgttgaataccgtgttgaaccgaacctgctggaactggtttctaaagttggtaccaccaaaatcctgctgatcgacaccccggaacgtctggacgaactgaaaaaaatcctgtctgaaaaattcaacgacgttgttaaagttttcaaatctttcccgacctacctggaaatcgttccggaaaacgttgacaaaggtaaagctctgcgtttcctgcgtgaacgtatgggttggaaaaaagaagaaatcgttgttttcggtgacaacgaaaacgacctgttcatgttcgaagaagctggtctgcgtgttgctatgggtaacgctatcgacaaagttaaagaagctgctgacgttgttaccctgaccaacaacgactctggtgtttcttacgttctggaactgatctctaccgactgcctggacggt; SEQ ID NO.10.
[0040] The amino acid sequence of the pullulanase mutant (simultaneous mutations of D150I, A212V, N468I, H483C, I544K, N710S and G864L) is shown in SEQ ID NO.11.
[0041] MDGNTTTIIVHYFRPSGDYTDWNLWMWPENGDGAEYDFNQPTDSYGEVASVDIPGNPSQVGIIVRKGNWDAKDIDSDRYIDLSKGHEIWLVQGNSQIFYSEKDAEAAAQPAVSNAYLDASNQVLVKLSQPFTLGEGSSGFTVHDDTANKIIPVTSVSDANQVTAVLAGTFQHIFGGSDWAPDNHNTLLKKVNSNLYQFSGNLPEGNYQYKVVLNDSWNNPSYPSDNINLTVPAGGAHVTFSYIPSTHAVYDTINNPNADLQVDSSGVKTDLVAVTLGENPDVSHTLSIQTEDYQAGQVIPRKVLDSSQYYYSGDDLGNTYTKNATTFKVWAPTSTQVNVLLYNSATGAVTKTVPMTASGHGVWEATVNQDLENWYYMYEVTGQGSTRTAVDPYATAIAPNGTRGMIVDLAKTDPAGWESDKHITPKNIEDEVIYEMDVRDFSIDSNSGMKNKGKYLALTEKGTKGPDIVKTGVDSLKQLGITCVQLQPVFAFNSVNENDPTQYNWGYDPRNYNVPEGQYATNANGTTRIKEFKEMVLSLHQDHKGVNMDVVYNHTFATQISDFDKIVPEYYYRTDDAGNYTNGSGTGNEIAAERPMVQKFIIDSLKFWVNEYHVDGFRFDLMALLGKDTMSKAATQLHAIDPGIALYGEPWTGGTSALPADQLLTKGAQKGMGVAVFNDNLRNGLDGSVFDSSAQGFATGATGLTDAIKSGVEGSINDFTASPGETINYVTSHDNYTLWDKIAQSNPNDSEADRIKMDELAQAIVMTSQGIPFMQGGEEMLRTKGGNDNSYNAGDVVNEFDWSRKAQYPDVFNYYSGLIHLRLDHPAFRMTTANEINSHLQFLNSPENTVAYELSDHANKDTWLNIVVIYNPNKTAETINLPSGKWEINATSGKVGESTLGQAEGSVQVPGISMMILHQEVSPSDGK;SEQ ID NO.11。
[0042] The nucleotide sequence of pullulanase mutants (simultaneous mutations of D150I, A212V, N468I, H483C, I544K, N710S and G864L) is shown in SEQ ID NO.12.
[0043]
[0044] LB medium (g / L): peptone 10, yeast extract 5, sodium chloride 10.
[0045] The molecular cloning kit is a product of TransGen.
[0046] Example 1: Construction of pullulanase expression strain The pullulanase used in this invention is named PLL, derived from Pullulanibacillus naganoensis (NCBI accession number AEV53626.1), with an amino acid sequence as shown in SEQ ID NO.1 and a nucleotide sequence encoding the polypeptide as shown in SEQ ID NO.2. The wild-type pullulanase sequence was cloned between the NcoI and XhoI sequences of the pET28a vector to form the pET28a-PLL expression vector.
[0047] The wild-type pullulanase sequence was subjected to site-directed mutagenesis (simultaneous mutations of D150I, A212V, N468I, H483C, I544K, N710S, and G864L). The amino acid sequence of the mutated pullulanase is shown in SEQ ID NO.11, and the nucleotide sequence encoding the polypeptide is shown in SEQ ID NO.12. The mutated pullulanase sequence was cloned into the pET28a vector sequence between NcoI and XhoI to form the pET28a-PLL-M(7) expression vector.
[0048] The expression vectors pET28a-PLL and pET28a-PLL-M(7) were transformed into BL21(DE3) and plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 37 °C. Single colonies were randomly selected for sequencing analysis to obtain wild-type PLL and mutant PLL-M(7) strains.
[0049] Example 2 Expression of pullulanase Wild-type PLL and mutant PLL-M(7) strains were inoculated into 4 mL of LB medium (50 μg / mL kanamycin) and cultured overnight at 37°C and 200 rpm. 1% of the inoculum was then transferred to 200 mL of fresh LB medium. The culture was continued at 37°C until the OD600 reached 0.8. IPTG was then added to a final concentration of 0.1 mM and the culture was induced for expression at 16°C for 20 h. The bacterial cells were collected and dissolved in 20 mM PBS (pH 6.5).
[0050] Example 3 Enzyme activity assay of pullulanase Take 1 mL of substrate (1.0% pullulan solution) and 0.9 mL of 0.1 mol / L pH 4.5 acetate buffer into separate test tubes, and preheat in a water bath at the enzyme activity detection temperature for approximately 10 min. Add 0.1 mL of diluted enzyme sample, vortex to mix, and react for 10 min. Add 2 mL of DNS to terminate the reaction, incubate in a boiling water bath for 7 min, and then cool in ice water. Add 10 mL of distilled water to the above reaction system, mix well, and measure the absorbance at 540 nm.
[0051] Enzyme activity is defined as the amount of glucose produced per minute by 1 mL of enzyme solution under given reaction conditions. This is called one unit of enzyme activity, denoted by U.
[0052] Example 4: Determination of optimal operating temperature and thermal stability of pullulanase Enzyme activity was measured at different temperatures (40, 45, 50, 55, 60, 65 and 70°C) according to the standard enzyme activity detection method in Example 3, and the temperature at which the highest enzyme activity was obtained was determined as the optimal reaction temperature of the enzyme. The optimal temperature for wild-type pullulanase was 55°C, and the optimal reaction temperature for PLL-M(7) mutant was 60°C.
[0053] The original enzyme solution was incubated at 60℃, and samples were taken every 30 minutes to determine the remaining enzyme activity. The enzyme thermostability was determined by taking the enzyme activity of the untreated sample as 100%. The thermostability results of the two enzymes are shown below. Figure 1 .
[0054] Example 5: Application of pullulanase mutant in the preparation of mannose The amino acid sequence of glucan phosphorylase is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4; the amino acid sequence of glucose phosphate mutase is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6; the amino acid sequence of glucose phosphate isomerase / mannose-6-phosphate isomerase is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8; the amino acid sequence of mannose-6-phosphate phosphatase is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10.
[0055] A method for preparing pullulanase wild-type, pullulanase mutant, dextran phosphorylase αGP, glucose phosphate mutase PGM, glucose phosphate isomerase / mannose-6-phosphate isomerase PGI / MPI, and mannose-6-phosphate phosphatase M6PP, comprising the following steps: ① The sequences shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.10 were cloned into the NcoI and XhoI of the pET28a vector (commercial grade) to form pET28a-αGP plasmid, pET28a-PGM plasmid, pET28a-PGI / MPI plasmid, and pET28a-M6PP plasmid, respectively.
[0056] ② The pET28a-αGP, pET28a-PGM, pET28a-PGI / MPI, and pET28a-M6PP plasmids were transformed into Escherichia coli BL21(DE3), and after recovery at 37°C for 1 hour, they were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C with the plates inverted.
[0057] ③ Select single colonies and transfer them to test tubes containing 4 mL of LB liquid medium (50 μg / mL kanamycin). Incubate overnight at 37°C and 200 rpm on a shaker. Transfer 1% of the inoculum to a shake flask containing 200 mL of LB medium and continue incubation at 37°C until the OD600 of the solution reaches 0.8. Add 0.1 mM IPTG to the final concentration, adjust the shaker temperature to 16°C, and induce expression for 20 h.
[0058] ④ Collect bacterial cells (including pullulanase wild-type and mutant) into 50mL centrifuge tubes, add 20 mM PBS buffer to resuspend, and then place them in a 70℃ water bath shaker for 30 minutes to increase cell membrane permeability, thereby obtaining whole-cell catalysts for PLL, PLL-M(7), αGP, PGM, PGI / MPI, and M6PP.
[0059] In a 1 mL reaction system, maltodextrin to a final concentration of 150 g / L, 20 mM sodium phosphate buffer (pH 7.5), 0.5 M magnesium sulfate solution, and heat-treated whole cells were added, respectively. The OD of wild-type pullulanase was... 600 The OD values of 1, α-glucan phosphorylase, glucose phosphate mutase, bifunctional enzymes glucose phosphate isomerase / mannose-6-phosphate isomerase, and mannose-6-phosphate phosphatase are... 600 The concentrations were 14, 4, 4, and 30, respectively. After catalytic reaction at 60℃ for 24 h, the mannose conversion rate was 56.55%. Adding pullulanase mutants to the enzyme-catalyzed reaction resulted in a mannose conversion rate of 65.85%. The addition amounts of wild-type and mutant PLL-M(7) were reduced to OD... 600=0.25, the yields of mannose were 64.8 g / L and 93.375 g / L, respectively, with conversion rates of 43.2% and 62.25%. Compared with the wild-type control, the amount of pullulanase mutant used was reduced by 75%, but the conversion rate was basically unaffected, still reaching 60%. The mutant PLL-M(7) can effectively reduce the amount of pullulanase used in the preparation of mannose.
[0060] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A pullulanase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the following mutations were performed simultaneously in D150I, A212V, N468I, H483C, I544K, N710S, and G864L.
2. The encoding gene of the pullulanase mutant according to claim 1.
3. An expression vector containing the encoding gene of the pullulanase mutant as described in claim 2.
4. Recombinant cells containing the encoding gene of the pullulanase mutant as described in claim 2.
5. The use of the pullulanase mutant according to claim 1 in the production of mannose.