Pullulanase mutant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a pullulanase mutant as well as a preparation method and application thereof. According to the pullulanase mutant and the preparation method and application thereof, the pullulanase mutant has high enzyme activity within the pH range of 4.5-5.0 and at the high temperature of 65 DEG C, meets the relevant temperature and pH conditions for achieving the maximum catalytic efficiency of saccharifying enzyme and the like and reducing the probability of bacterial contamination in industrial production, and has the advantages that the pullulanase mutant can be used for preparing the pullulanase mutant; the industrial application is facilitated.

Description

Technical Field

[0001] The present application belongs to the field of enzyme engineering technology, and specifically relates to a pullulanase mutant and a preparation method and application thereof. Background Art

[0002] Most plants, such as rice, corn, sorghum, barley, peas, and potatoes, contain 60%-90% amylopectin, with glutinous rice varieties exceeding 90%. On average, amylopectin accounts for approximately 75%-85% of the total starch content, of which 4%-5% are α-1,6-glucosidic bonds. Efficient hydrolysis of both α-1,4-glucosidic and α-1,6-glucosidic bonds in starch is crucial for the production of products such as dextrin, maltose, glucose, and alcohol. However, saccharifying enzymes, such as α-amylase and β-amylase, are only highly efficient at hydrolyzing α-1,4-glucosidic bonds, while catalyzing α-1,6-glucosidic bonds is inefficient, sometimes even unusable, leading to significant waste of starch resources.

[0003] Pullulanase (EC.3.2.1.41) is a type of starch debranching enzyme that can specifically cut the α-1,6-glycosidic bonds in pullulan, amylopectin and glucan to form amylose. Among them, pullulanase is known in the art. In order to maximize the synergistic effect between pullulanase and saccharifying enzyme or β-amylase, pullulanase and saccharifying enzyme, β-amylase need to have the same action temperature. For example, in industrial production, in order to achieve the maximum catalytic efficiency of saccharifying enzymes and reduce the chance of contamination, the saccharification process temperature and pH are generally controlled at 50-60 ° C and pH 4.5-5.0, and the reaction time is more than 48 hours. However, the pullulanase in traditional technology cannot have high enzyme activity within this temperature and pH range.

[0004] Therefore, it is necessary to further explore pullulanase suitable for saccharification process in industrial production. Summary of the Invention

[0005] Based on this, one embodiment of the present application provides a pullulanase mutant and a preparation method and application thereof.

[0006] One or more embodiments of the present application provide a pullulanase mutant and its encoding nucleic acid, vectors and cells containing the encoding nucleic acid, products containing the same, and applications thereof, including the following technical solutions:

[0007] One or more embodiments of the present application provide a pullulanase mutant. Relative to the alkaline protease with an amino acid sequence as shown in SEQ ID NO. 1, the pullulanase mutant has the following mutation: V760A.

[0008] One or more embodiments of the present application also provide a nucleic acid encoding the pullulanase mutant.

[0009] One or more embodiments of the present application also provide a vector comprising the nucleic acid.

[0010] One or more embodiments of the present application further provide a cell, wherein the cell expresses the pullulanase mutant, or comprises the nucleic acid, or comprises the vector.

[0011] One or more embodiments of the present application further provide a method for producing the pullulanase mutant, comprising the steps of culturing the cells and isolating the pullulanase mutant from the obtained culture.

[0012] One or more embodiments of the present application also provide a use of the pullulanase mutant in preparing an enzyme-containing product.

[0013] One or more embodiments of the present application further provide an enzyme-containing product, wherein the enzyme-containing product includes the pullulanase mutant.

[0014] One or more embodiments of the present application further provide a method for using a pullulanase mutant, comprising contacting the pullulanase mutant or the enzyme-containing product with a target containing a substrate to generate an enzyme-catalyzed reaction.

[0015] The present application provides a pullulanase mutant, a preparation method, and an application thereof. The pullulanase mutant provided in the present application has higher enzyme activity than the parent G1P at pH 4.5; it still has high enzyme activity within the pH range of 4.5-5.0 and after high-temperature treatment at 65°C for 5 minutes, which meets the relevant temperature and pH conditions in industrial production to achieve maximum catalytic efficiency of saccharifying enzymes and reduce the chance of contamination, and is conducive to industrial application. DETAILED DESCRIPTION

[0016] Below in conjunction with embodiment and example, the application is described in further detail.Should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description hereinafter, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0018] the term

[0019] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0020] The terms "and, or", "or, and", and "and, or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and, or", "or, and", and "and, or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and also undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and, or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and, or, B, and, or, C, and, or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0021] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0022] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0023] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0024] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0025] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0026] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0027] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0028] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0029] In this application, % (w, w) and wt% all refer to weight percentage, % (v, v) refers to volume percentage, and % (w, v) refers to mass volume percentage.

[0030] All documents mentioned in this application are cited as references in this application, just as each document is cited as a reference individually. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0031] This application proposes a pullulanase mutant, its preparation method, and application. The pullulanase mutant provided in this application exhibits higher enzymatic activity at pH 4.5 than its parent, G1P. It also maintains high enzymatic activity within a pH range of 4.5-5.0 and after high-temperature treatment at 65°C for 5 minutes, making it suitable for industrial applications.

[0032] In one aspect, the present application provides a pullulanase mutant having V760A relative to the pullulanase with the amino acid sequence set forth in SEQ ID NO. 1. All of the V760A-containing mutations described herein exhibit higher enzyme activity than the parent G1P at pH 4.5. They also maintain high enzyme activity within a pH range of 4.5-5.0 and after high-temperature treatment at 65°C for 5 minutes, facilitating industrial applications.

[0033] The term "parent pullulanase" or "parent" refers to a starch debranching enzyme having pullulan 6-glucan-hydrolase activity (EC 3.2.1.41), and refers to a pullulanase expressed by a naturally occurring microorganism (e.g., a bacterium, yeast, or filamentous fungus found in nature). The present application particularly refers to the wild-type pullulanase (G1P) obtained from Bacillus deramificans, whose amino acid sequence is shown in SEQ ID NO: 1.

[0034] SEQ ID NO.1:

[0035] EKDAEDAAKPAVSNAYLDASNQVLVKLSQPLTLGEGASGFTVHDDTANKDIPVTSVKDASLGQDVTAVLAGTFQHIFGGSDWAPDNHSTLLKKVTNNLYQFSGDLPEGNYQYKVALNDSWNNPSYPSDNINLTVPAGGAHVTFSYIPSTHAVYDTINNPNADLQVESGVKTDLVTVTLGEDPDVSHTLSIQTDGYQAKQVIPRNVLNSSQYYYSGDDLGNTYTQKATTFKVWAPTSTQVNVLLYDSATGSVTKIVPMTASGHGVWEATVNQNLENWYYMYEVTGQGSTRTAVDPYATAIAPNGTRGMIVDLAKTDPAGWNSDKHITPKNIEDEVIYEMDVRDFSIDPNSGMKNKGKYLALTEKGTKGPDNVKTGIDSLKQLGITHVQLMPVFASNSVDETDPTQDNWGYDPRNYD VPEGQYATNANGNARIKEFKEMVLSLHREHIGVNMDVVYNHTFATQISDFDKIVPEYYRTDDAGNYTNGSGTGNEIAAERPMVQKFIIDSLKYWVNEYHIDGFRFDLMALLGKDTMSKAASELHAINPGIALYGEPWTGGTSALPDDQLLTKGAQKGMGVAVFNDNLRNALDGNVFDSSAQGFATGATGLTDAIKNGVEGSINDFT SSPGETINYVTSHDNYTLWDKIALSNPNDSEADRIKMDELAQAVVMTSQGVPFMQGGEEMLRTKGDNSYNAGDAVNEFDWSRKAQYPDVFNYYSGLIHLRLDHPAFRMTTANEINSHLQFLNSPENTVAYELTDHVNKDKWGNIIVVYNPNKTVATINLPSGKWAINATSGKVGESTLGQAEGSVQVPGISMMILHQEVSPDHGKK

[0036] Specifically, the pullulanase mutants of the present application have improved properties. For example, in some specific embodiments, at 60°C and pH 4.5-5.0, the pullulanase mutants of the present application have increased activity relative to the parent pullulanase G1P. In some specific embodiments, after heat treatment at 65°C for 5 minutes, the pullulanase mutants of the present application have increased residual activity relative to the parent pullulanase G1P, i.e., have higher thermal stability.

[0037] In describing the mutants of the present application, the nomenclature described below is used for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0038] Substitution: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine at position 429 with alanine is represented as "Ala429Thr" or "A429T." Multiple mutations are separated by a forward slash (" / "), for example, "Ala429Thr / Ala535Ile" or "A429T / A535I" indicates that alanine (A) at position 429 and isoleucine (I) at position 535 are substituted, respectively.

[0039] Deletions: For amino acid deletions, the following nomenclature is used: original amino acid, position, *. For example, a deletion of alanine at position 429 would be represented as "Ala429*" or "A429*." Multiple deletions are separated by a forward slash (" / "), for example, "Ala429* / Ala535*" or "A429* / A535*."

[0040] Insertion: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. For example, the insertion of lysine after the alanine at position 429 is represented as "Ala429AlaLys" or "A429AK." Multiple amino acid insertions are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, the insertion of lysine and alanine after the alanine at position 429 is represented as "Ala429AlaLysAla" or "A429AKA."

[0041] In the case where different changes can be introduced at one position, the different changes are separated by commas, and the different changes or optional substitutions can be indicated by brackets, such as S236[A,D,N,T], which means that S at position 236 can be replaced by A, D, N or T.

[0042] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0043] For the purpose of the present application, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Genetics Trend] 16:276-277) (preferred 5.0.0 version or later). The parameters used are gap opening penalty 10, gap extension penalty 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output (obtained using the non-simplified (-nobrief) option) labeled "longest consistency" is used as percent consistency and is calculated as follows: (consistent residue x 100) / (total number of gaps in the alignment length-alignment).

[0044] In one aspect, the present application provides a pullulanase mutant comprising a substitution at position 760 corresponding to SEQ ID NO: 1, and optionally at positions 7, 11, 37, 93, 102, 111, 178, 179, 204, 207, 208, 224, 225, 230, 236, 241, 247, 250, 254, 264, 270, 271, 272, 279, 283, 284, 297, 315, 317, 320, 330, 331 , 333, 339, 347, 348, 360, 372, 377, 380, 394, 398, 400, 403, 405, 415, 424, 425, 426, 428, 429, 438, 439, 443, 444, 445, 446, 460, 461, 462, 471, 473, 478, 479, 481, 492, 494, 500, 50 7, 509, 514, 516, 535, 537, 538, 539, 543, 547, 557, 559, 561, 562, 563, 565, 567, 570, 572, 574, 576, 577, 580, 586, 590, 591, 594, 595, 597, 606, 609, 623, 638, 646, 666, 667, 673, The mutant comprises a substitution at one or more of positions 682, 687, 690, 696, 698, 705, 711, 754, 757, 759, 764, 766, 767, 769, 771, 778, 779, 789, 791, 792, 818, 820, 826, and 827, wherein the mutant has pullulanase activity and increased activity compared to the parent pullulanase G1P.

[0045] In some embodiments, the mutant has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence of the parent pullulanase. In some specific embodiments, the number of substitutions in the present application's pullulanase mutant sites is 1 to 41, such as 3, 4, 5, 6, 7, 8, 9, 10, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, such as 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41.

[0046] In a specific embodiment, the present application provides a pullulanase mutant comprising a substitution at position 760 corresponding to SEQ ID NO: 1, and optionally at positions 7, 11, 37, 93, 102, 111, 178, 179, 204, 207, 208, 224, 225, 230, 236, 241, 247, 250, 254, 264, 270, 271, 272, 279, 283, 284, 297, 315, 317, 320, 330, 331, 333, 339, 347, 348, 360, 372 , 377, 380, 394, 398, 400, 403, 405, 415, 424, 425, 426, 428, 429, 438, 439, 443, 444, 445, 446, 460, 461, 462, 471, 473, 478, 479, 481, 492, 494, 500, 507, 509, 514, 516, 535, 537, 538, 539, 543, 547, 557, 559 , 561, 562, 563, 565, 567, 570, 572, 574, 576, 577, 580, 586, 590, 591, 594, 595, 597, 606, 609, 623, 638, 646, 666, 667, 673, 682, 687, 690, 696, 698, 705, 711, 754, 757, 759, 764, 766, 767, 769, 771, 778, 779 , 789, 791, 792, 818, 820, 826 and 827, wherein the mutant has pullulanase activity; the mutant has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence of the parent pullulanase; and the mutant has increased activity compared to the parent pullulanase G1P, in particular, at least 1.1 times the activity of the parent pullulanase G1P measured at 60°C and pH 4.5-5.0.

[0047] In another specific embodiment, the present application provides a pullulanase mutant comprising a substitution at position 760 corresponding to SEQ ID NO: 1, and optionally at positions 7, 11, 37, 93, 102, 111, 178, 179, 204, 207, 208, 224, 225, 230, 236, 241, 247, 250, 254, 264, 270, 271, 272, 279, 283, 284, 297, 315, 317, 320, 330, 331, 333, 339, 347, 348, 360, 372 , 377, 380, 394, 398, 400, 403, 405, 415, 424, 425, 426, 428, 429, 438, 439, 443, 444, 445, 446, 460, 461, 462, 471, 473, 478, 479, 481, 492, 494, 500, 507, 509, 514, 516, 535, 537, 538, 539, 543, 547, 557, 559 , 561, 562, 563, 565, 567, 570, 572, 574, 576, 577, 580, 586, 590, 591, 594, 595, 597, 606, 609, 623, 638, 646, 666, 667, 673, 682, 687, 690, 696, 698, 705, 711, 754, 757, 759, 764, 766, 767, 769, 771, 778, 779 A pullulanase mutant comprising substitutions at one or more positions 789, 791, 792, 818, 820, 826, and 827, wherein the mutant has pullulanase activity; the mutant has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence of the parent pullulanase; and the mutant has increased residual activity compared to the parent pullulanase G1P, i.e., has higher thermal stability. In particular, after heat treatment at 65°C for 5 minutes, the pullulanase mutant of the present application has higher enzymatic activity and higher residual activity relative to the parent pullulanase G1P.

[0048] In a specific embodiment, the present application provides a pullulanase mutant, which comprises a mutant corresponding to SEQ ID NO:1, position V760A, and optionally, A7V, A11T, A37T, K93M, S102L, Q111R, L178Q, G179V, N204K, N207D, S208L, Q224K, K225N, K230R, S236[A,D,N,T], V241L, A247E, S250[A,P], I254T, V264[P,S,T], N270[H,P], Q271G, N272D, M279[L,R,S,T], T283K, G284V, T297[A,L,M,R,V,Y], D315N, A317[P,T] , N320E, I330P, E331V, E333A, D339[H,S], P347S, N348K, L360F, K372N, S377Y, Q380E, S394F, D398N, T400[L,N], T403S, D405[C,G,R,S,Y], D415 N, N424[D,L,R,T], A425P, N426H, N428T, A429[C,E,G,L,P,R,T], V438I, L439[I,Q], R443Q, E444D, H445G, I446T, T460[K,V], Q461[E,G,H,N,S,T ,V], I462T, E471[I,Q,T], Y473H, D478[A,N,S,Y], A479[C,S], N481[D, F,I,T], I492[L,V], A494[D,G], Q500[C,R], L507V, Y509F, Y514S, I516V ,A535[C,I,L,N,W],S537T,E538Q,L539H,N543D,A547L,T557[E,H,I,K, L,P,Q,R,S,V], A559S, P561T, D562[A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S, T,V,Y], D563S, L565Q, T567[F,I,K,V,Y], A570[C,G,H,K,M,R], K572R,M 574L, V576[C,I,M,P], A577[G,I], N580D, A586G, N590S, V591I, S594P, S 595[A,D,F,H,K,M,R,T,Y], Q597R, L606Y, A609E, S623A, Y638L, L646Q, V 666I, V667F, V673[F,I,L], M682F, G687Y, D690[C,S], G696V, A698[L,Q,The invention relates to a method for preparing a pullulanase comprising: preparing a pullulanase comprising: selecting one or more of the group consisting of: S705K, P711T, Y754[F,R,S], H759Y, V760[A,P], K764T, G766K, N767D, I769V, V771I, V778[A,D,L,S], A779E, A789[E,L,R,S], N791V, A792V, I818V, H820Y, D826S and H827D, wherein the mutant has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the amino acid sequence of the parent pullulanase; and the mutant has pullulanase activity and has increased activity compared to the parent pullulanase G1P, particularly at 60°C, pH 8. Measured at 4.5-5.0, it is at least 1.1 times the activity of the parent pullulanase G1P.

[0049] In another specific embodiment, the present application provides a pullulanase mutant, which comprises a mutant corresponding to SEQ ID NO:1, position V760A, and optionally, A7V, A11T, A37T, K93M, S102L, Q111R, L178Q, G179V, N204K, N207D, S208L, Q224K, K225N, K230R, S236[A,D,N,T], V241L, A247E, S250[A,P], I254T, V264[P,S,T], N270[H,P], Q271G, N272D, M279[L,R,S,T], T283K, G284V, T297[A,L,M,R,V,Y], D315N, A317[P,T] , N320E, I330P, E331V, E333A, D339[H,S], P347S, N348K, L360F, K372N, S377Y, Q380E, S394F, D398N, T400[L,N], T403S, D405[C,G,R,S,Y], D415 N, N424[D,L,R,T], A425P, N426H, N428T, A429[C,E,G,L,P,R,T], V438I, L439[I,Q], R443Q, E444D, H445G, I446T, T460[K,V], Q461[E,G,H,N,S,T ,V], I462T, E471[I,Q,T], Y473H, D478[A,N,S,Y], A479[C,S], N481[D, F,I,T], I492[L,V], A494[D,G], Q500[C,R], L507V, Y509F, Y514S, I516V ,A535[C,I,L,N,W],S537T,E538Q,L539H,N543D,A547L,T557[E,H,I,K, L,P,Q,R,S,V], A559S, P561T, D562[A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S, T,V,Y], D563S, L565Q, T567[F,I,K,V,Y], A570[C,G,H,K,M,R], K572R,M 574L, V576[C,I,M,P], A577[G,I], N580D, A586G, N590S, V591I, S594P, S 595[A,D,F,H,K,M,R,T,Y], Q597R, L606Y, A609E, S623A, Y638L, L646Q, V 666I, V667F, V673[F,I,L], M682F, G687Y, D690[C,S], G696V, A698[L,Q,S, T, V], S705K, P711T, Y754[F, R, S], H759Y, V760[A, P], K764T, G766K, N767D, I769V, V771I, V778[A, D, L, S], A779E, A789[E, L, R, S], N791V, A792V, I818V, H820Y, D826S and H827D, wherein the mutant has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence of the parent pullulanase; and the mutant has pullulanase activity and increased residual activity compared to the parent pullulanase G1P, i.e., has higher thermostability. In particular, after heat treatment at 65°C for 5 minutes, the pullulanase mutant of the present application has higher enzyme activity and higher residual activity than the parent pullulanase G1P.

[0050] In a specific embodiment, the present application provides a pullulanase mutant comprising a substitution at position V760A corresponding to SEQ ID NO: 1, wherein the mutant has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence of the parent pullulanase, and, specifically, the mutant comprises one of the substitution combinations described above.

[0051] On the other hand, the present application provides a nucleic acid encoding the pullulanase mutant.

[0052] The present application relates to polynucleotides encoding the mutants of the present application. The present application also relates to nucleic acid constructs comprising polynucleotides encoding the mutants of the present application, operably linked to one or more control sequences, which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0053] The control sequence can be a promoter containing transcriptional control sequences that mediate expression of the mutant. Some examples of suitable promoters for directing transcription of the nucleic acid construct of the present application in a bacterial host cell include: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Escherichia coli lac operon, Escherichia coli trc promoter and tac promoter.

[0054] The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3'-end of the polynucleotide encoding the mutant. Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL) and Escherichia coli ribosomal RNA (rrnB).

[0055] The control sequence may also be a signal peptide that directs the mutant into the cell's secretory pathway.The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the mutant.

[0056] On the other hand, the present application provides a vector comprising the nucleic acid.

[0057] The application further relates to the recombinant expression vector of the polynucleotide, promotor and transcription and translation termination signal that comprise the mutant of coding this application.Various Nucleotide and control sequence can link together to produce recombinant expression vector, and this recombinant expression vector can comprise one or more suitable restriction sites to allow to insert or replace the polynucleotide of coding mutant at such site.Alternately, can express this polynucleotide by polynucleotide or the nucleic acid construct that comprises this polynucleotide being inserted in the appropriate vector.When producing expression vector, coding sequence is positioned in the carrier, makes coding sequence operably connected with the appropriate control sequence that is used to express like this.

[0058] The recombinant expression vector preferably contains one or more selectable markers that allow easy selection of transformed cells, transfected cells, transduced cells, etc. Selectable markers are genes whose products provide biocide resistance or viral resistance, heavy metal resistance, prototrophy of auxotrophs, etc. Examples of bacterial selectable markers are the dal genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance.

[0059] One or more copies of the polynucleotide of the present application can be inserted into the host cell to increase the production of the mutant.

[0060] The procedures for ligating the above-mentioned elements to construct the recombinant expression vector of the present application are well known to those skilled in the art.

[0061] On the other hand, the present application provides a cell, which expresses the pullulanase mutant, or includes the nucleic acid, or includes the vector.

[0062] The present application also relates to a recombinant host cell comprising a polynucleotide encoding the mutant of the present application and operably linked to one or more control sequences.

[0063] Host cell can be any cell useful in the recombinant production of mutant, for example prokaryotic cell. Prokaryotic host cell includes but is not limited to Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include but are not limited to Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0064] On the other hand, the present application provides a method for constructing the cell, which includes the step of introducing the nucleic acid or the vector into the cell to be transformed.

[0065] On the other hand, the present application provides a method for preparing the pullulanase mutant, characterized in that the production method includes the steps of culturing the cells and isolating the pullulanase mutant from the obtained culture.

[0066] The present application also relates to a method for producing a mutant, comprising: (a) culturing the host cell of the present application under conditions suitable for expressing the mutant; and (b) recovering the mutant.

[0067] Use methods known in the art to cultivate host cells in a nutrient medium that is suitable for producing mutants. For example, can be by shake flask culture, or in applicable substratum and under the condition that allows mutant expression and / or separation, carry out small-scale or large-scale fermentation (comprising continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) and cultivate cells in laboratory or industrial fermentor tank.If mutant is secreted in the nutrient medium, then mutant can directly reclaim from substratum.If mutant does not secrete, then it can reclaim from cell pyrolysis liquid.

[0068] Mutants can be recovered using methods known in the art. For example, mutants can be recovered from the nutrient medium by a variety of conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0069] Mutants can be purified by a variety of procedures known in the art to obtain substantially pure mutants, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE extraction.

[0070] On the other hand, the present application provides use of the pullulanase mutant in preparing enzyme-containing products.

[0071] On the other hand, the present application provides an enzyme-containing product, which includes the pullulanase mutant.

[0072] On the other hand, the present application provides a method for using a pullulanase mutant, which comprises contacting the pullulanase mutant or the enzyme-containing product with a target containing a substrate to cause an enzyme-catalyzed reaction.

[0073] The present application also relates to a composition comprising the pullulanase mutant of the present application. Alternatively, the composition may comprise multiple enzyme activities, such as one or more enzymes selected from the group consisting of α-amylase, glucoamylase, β-amylase, protease.

[0074] The present application also relates to methods for using the pullulanase mutants of the present application in various industrial applications. Specifically, methods for processing or producing fermentation products from starch-containing materials. In a specific embodiment, the present application relates to the use of the pullulanase mutants according to the present application for producing syrups and / or fermentation products from starch-containing materials.

[0075] In a specific embodiment, the present application relates to use of the pullulanase mutant according to the present application for producing ethanol from starch-containing materials, such as fuel ethanol, drinking ethanol and industrial ethanol.

[0076] The pullulanase mutants of the present application can be used in starch processes, in particular starch liquefaction processes, where the starch material may be gelatinized. It is also contemplated that an enzyme composition for starch conversion purposes may comprise, in addition to the pullulanase mutants of the present application, α-amylases, glucoamylases, β-amylases, proteases, and the like.

[0077] Any suitable starch-containing starting material can be used in the method of the present application. Examples of starch-containing materials suitable for use in the method of the present application include barley, beans, cassava, cereals, corn, peas, potatoes, rice, rye, sago, sorghum, sweet potato, cassava, wheat and whole grains or any mixture thereof. In a preferred embodiment, the starch-containing material is corn. In a preferred embodiment, the starch-containing material is wheat.

[0078] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0079] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0080] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0081] Example 1

[0082] Unless otherwise specified, the biological materials, reagents, and devices used in the following examples can be obtained from conventional commercial sources or by known methods. Molecular biology experimental procedures not specifically described in the following examples were performed according to the methods outlined in J. Sambrook's Molecular Cloning: A Laboratory Manual (3rd edition), or according to the kits and product instructions.

[0083] 1. Design and construct a pullulanase sequence collection

[0084] Wild-type pullulanase (G1P) from Bacillus deramificans was obtained using gene synthesis technology from Twist Bioscience. To further improve activity, thermostability, and activity under low / high pH conditions, multiple mutant libraries were designed based on sequence and structural analysis. Each mutant was designed to contain one to multiple specific mutation sites. These mutant libraries were then constructed using standard site-directed mutagenesis methods and cloned into the pET-22b(+) vector (Sigma-Aldrich, catalog number #69744-3).

[0085] 2. Preparation of E. coli-produced pullulanase in a high-throughput platform (HTP)

[0086] The recombinant pullulanase encoding gene obtained from a single colony of Escherichia coli BL21 (DE3) (New England Biolabs: catalog number # C2527H) was inoculated into 180 μL LB medium containing 1% glucose and 100 μg / mL ampicillin, cultured in a 96-well plate, and sealed with a breathable sterile sealing film. After inoculation, the plate was incubated overnight at 30°C, 85% humidity, and shaken at 200 rpm. The OD600 value of the overnight culture was measured, and all cultures were diluted to a final OD600 value of 0.05 and transferred to a fresh 96-well plate containing TB medium containing 100 μg / mL ampicillin. The plate was incubated at 37°C, 85% humidity, and shaken at 250 rpm. The OD600 value was monitored, and when it reached OD600 = 0.8, the plate was induced with IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 0.15 mM. The plate was then incubated at 25°C, 85% humidity, and shaking at 250 rpm for 24 hours. After growth, the cells on the plate were centrifuged at 4000 rpm for 15 minutes and cooled to 4°C. The supernatant was discarded, and the plate containing the centrifuged cell pellet was frozen at -80°C overnight.

[0087] To lyse the cell pellet, remove the plate from the -80°C freezer and thaw at 37°C for 15 minutes. After thawing, add 200 μL of B-PER Complete Lysis Buffer (ThermoFisher Scientific, catalog #89821) to each well of the plate and shake on a benchtop shaker at room temperature for 90 minutes. Following lysis, centrifuge at 4000 rpm at 4°C for 15 minutes to pellet the cell debris. The lysate was transferred to a new plate and stored at -20°C for later use in enzymatic assays.

[0088] 3. Determination of the activity of wild-type and mutant pullulanase produced by E. coli at pH 4.5 or pH 5.0 at 60°C in a high-throughput platform (HTP)

[0089] Thaw the pullulanase wild-type and / or mutant plates produced in step 2. Dilute the supernatant 700 to 9200-fold with 0.2 M sodium acetate buffer (pH 4.5) for activity determination at pH 4.5. Dilute the supernatant 600 to 9200-fold (depending on the plate) with 0.2 M sodium acetate buffer (pH 5.0) for pullulanase activity determination at pH 5.0.

[0090] Prepare a 1.5% pullulan solution by dissolving 1.50 g pullulan in 80 mL of 0.2 M sodium acetate buffer, pH 4.5 or pH 5.0, and stirring at room temperature until dissolved. Once dissolved, adjust the pH if necessary and bring the volume to 100 mL.

[0091] For both assays (pH 4.5 and pH 5.0), the corresponding 1.5% pullulan substrate solution was added to a 96-well PCR plate (30 μL per well). The reaction was initiated by adding 30 μL of diluted enzyme to a plate containing a 1.5% pullulan solution at pH 4.5 or pH 5.0, each diluted in 0.2 M sodium acetate buffer at pH 4.5 or pH 5.0. Subsequently, the plate was sealed and incubated at 60° C. with a shaking of 650 rpm for 1.5 hours. After incubation, 90 μL of DNS (dinitrosalicylic acid) reagent was added to terminate the reaction.

[0092] The DNS reagent is prepared as follows: 6.30 g of 3,5-dinitrosalicylic acid is added to 500 mL of water, followed by 21.0 g of sodium hydroxide and stirring. The solution is heated to 50°C until all added solids are completely dissolved. While the first solution is dissolving, 182.0 g of sodium thiosulfate is added to 300 mL of deionized water and dissolved under gentle heating. After all solids have dissolved, the sodium thiosulfate solution is added to the first solution. 5.00 g of phenol and 5.00 g of anhydrous sodium sulfite are added to the stirring solution and stirring is continued until the solids are dissolved. The solution is cooled to room temperature, then the volume is adjusted to 1000 mL, filtered, and stored in an opaque bottle. It must stand for 7 days before use.

[0093] After adding the DNS reagent, the plate was sealed, heated to 95°C for 7 minutes using a thermal cycler, cooled to 4°C for 2 minutes, mixed by turning upside down 5-6 times, and then centrifuged at 1000rpm for 30 seconds. The assay plate was unsealed, 65μL was transferred from each well to a new transparent bottom plate containing 130μL of water, and mixed thoroughly. The absorbance of each well was measured at 550nm, and the absorbance value represented the enzyme activity in each well. The activity of the mutant enzyme at pH 4.5 or pH 5.0 was compared with the activity of the wild-type (G1P) enzyme at the same pH to determine the increase in activity at each pH. The first generation (G1) results shown in Table 1 showed increased activity compared to G1P, where the activity of the positive control (G1P) was set to 1.0 (corresponding to an enzyme activity of 35U / mL).

[0094] The results of the second generation (G2) are shown in Table 1. The activity was calculated as follows: for pH 4.5, activity relative to G1P = 1.7× (measured activity relative to G2P); for pH 5.0, activity relative to G1P = 1.6× (measured activity relative to G2P).

[0095] The results for generation 3-1 (G3-1) in Table 1 show that the increase in activity compared to G1P was calculated as follows: for pH 4.5, activity relative to G1P = 3.7 x (measured activity relative to G3P-1).

[0096] For the mutants of generation 3-2 (G3-2), the increase in activity compared to G1P was calculated as follows: for pH 4.5, activity relative to G1P = 5.4× (relative to the measured activity of G3P-2); for pH 5.0, activity relative to G1P = 4.3× (relative to the measured activity of G3P-2).

[0097] 4. Determination of the thermal stability of wild-type and mutant pullulanase produced by E. coli at 65°C for 5 minutes in a high-throughput platform (HTP)

[0098] The plates of pullulanase wild type and / or mutants produced according to step 2 were thawed and diluted to approximately 1 U / mL in 0.2 M pH 4.5 sodium acetate buffer in a 96-well PCR plate. A control reaction (according to step 3, pH 4.5) was performed using the same initial dilution plate without pre-incubation at 65°C. The remaining enzyme was incubated at approximately 1 U / mL at 65°C for 5 minutes, followed by cooling to 4°C in a thermal cycler for 2 minutes. The heat-challenged enzyme was further diluted 10-fold in 0.2 M pH 4.5 sodium acetate buffer in a new 96-well plate.

[0099] The diluted enzyme (30 μL) was added to a 96-well PC plate containing 30 μL of 1.5% pullulan substrate (pH 4.5), prepared as described in step 3. The plate was then sealed and incubated at 60°C at 650 rpm for 1.5 hours. Following incubation, the reaction was terminated by adding 90 μL of DNS reagent (as described in step 3).

[0100] After adding DNS, the plate was sealed, heated to 95°C for 7 minutes, cooled to 4°C for 2 minutes, mixed by inverting 5-6 times, and then centrifuged at 1000 rpm for 30 seconds. The absorbance of each well was measured at 550 nm to indicate enzyme activity.

[0101] The enzyme activities of the mutants after thermostability challenge were compared with those of the wild type (G1P).

[0102] The first generation (G1) results shown in Table 1 show the increased activity compared to G1P, where the activity of the positive control (G1P) was set to 1.0. The second generation (G2) results shown in Table 1 show that the increased thermal stability compared to G1P was calculated as follows:

[0103] Activity relative to G1P = 2.2 x (measured activity relative to G2P).

[0104] The results for Generation 3-2 (G3-2) in Table 1 show that the improvement in thermal stability compared to G1P is calculated as follows:

[0105] Activity relative to G1P = 9.0 x (measured activity relative to G3P-2).

[0106] Table 1

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] 5. Determination of the specific activity of wild-type and mutant pullulanase produced by E. coli

[0134] Pullulanase wild-type and mutant samples were generated according to step 2.

[0135] For specific activity and validation assays, multiple wells of the same wild-type or mutant enzyme were incubated, lysed, and then pooled to provide sufficient volume for manipulation. Following pullulanase production, samples were desalted using Zeba 96-well desalting plates (ThermoFisher Scientific, catalog number 87774) according to the manufacturer's instructions.

[0136] Subsequently, the desalted samples were tested for activity according to the Chinese national standard GB1886.174-2016A.9.1.2. Briefly, a 0.5% (w / v) pullulan solution was prepared using 0.13 M acetate buffer at pH 4.5, while the wild-type pullulanase or mutant enzyme was diluted to the appropriate range using 0.13 M acetate buffer at pH 4.5. A glass tube (one for each sample) containing 1 mL of 0.5% (w / v) pullulan solution was incubated in a 60°C water bath for 10 minutes. The diluted enzyme samples were also pre-incubated in glass tubes at 60°C for 5 minutes. After pre-incubation, 1 mL of enzyme solution from each sample was added to the corresponding glass tube containing 0.5% (w / v) pullulan. The reaction was incubated at 60°C for 30 minutes, followed by termination with 3 mL of DNS reagent (see step 3 for the preparation of DNS reagent), and then each sample was mixed. All samples were then transferred to a boiling water bath and the color development reaction was allowed to proceed for 7 minutes. After removing the reaction tubes from the boiling water bath, they were cooled, and 300 μL of the complete reaction was added to a cuvette containing 600 μL of deionized water. The absorbance was measured at 550 nm and used to calculate the activity (U / mL) of each sample. Each desalted sample should be repeated at least 3 times to calculate the specific activity.

[0137] In order to determine the titer of pullulanase, a BSA standard curve was created. 2mg / mL BSA standard (Bio-Rad Laboratories, Inc., catalog number (Cat. No.) 5000206) was diluted to prepare the standard of 0.2mg / mL, 0.1mg / mL, 0.05mg / mL and 0.025mg / mL. According to expression level, the desalination enzyme sample was diluted to an appropriate range. Each BSA standard and pullulanase sample were prepared with loading buffer and reducing agent. Prepared sample was loaded onto a standard SDS-PAGE gel and run at 170V for 25 minutes. The gel obtained was stained and developed and then imaged. A standard curve was created from the BSA standard and the titer (mg / mL) of the wild-type and mutant pullulanase for calculating the desalination sample.

[0138] The activity values ​​(U / mL) obtained from the pullulanase assay and the titer (mg / mL) of the desalted samples were used to calculate the specific activity of each wild-type and pullulanase mutant in U / mg. The calculated specific activity values ​​compared to G1P are shown in Table 2.

[0139] 6. Verification: Determination of the activity of wild-type and mutant pullulanase produced by E. coli at pH 4.0, pH 4.5 or pH 5.0 at 60°C. The wild-type and mutant pullulanase samples were generated according to step 2.

[0140] For relative pH activity validation assays, multiple wells of the same wild-type or mutant enzyme were incubated, lysed, and then pooled to allow for sufficient volume to work with.

[0141] Relative pH activity was determined at pH 4.5 (0.13 M acetate buffer), pH 4.0 (0.29 M acetate buffer), and pH 5.0 (0.13 M acetate buffer). 0.5% (w / v) pullulan substrates at pH 4.0, 4.5, and 5.0 were prepared by dissolving the substrates in the corresponding buffers.

[0142] Wild-type and mutant pullulanases were diluted to the desired concentrations in the corresponding acetate buffers at pH 4.0, 4.5, and 5.0. To measure enzyme activity at different pH values, the Chinese National Standard protocol GB1886.174-2016A.9.1.2 was followed as described in step 5, except that the pH values ​​of the substrate and buffer were varied.

[0143] After obtaining the U / mL activity of each wild-type and mutant pullulanase at different pH values, the percentage relative activity was calculated using pH 4.5 as the optimal pH (100% relative activity). The results of the pH relative activity assay are shown in Table 2.

[0144] 7. Validation of the thermal stability of wild-type and mutant pullulanase produced by E. coli at 65°C for 5 minutes

[0145] Pullulanase wild-type and mutant samples were generated according to step 2. For the thermostability validation assay, multiple wells of the same wild-type or mutant enzyme were incubated, lysed, and then combined to allow for sufficient volume to be processed. The activity levels (U / mL) of the wild-type and mutant enzymes were tested according to step 6, and activity at pH 4.5 was determined.

[0146] Based on the enzyme activity at pH 4.5, wild-type and mutant pullulanase samples were diluted to 1 U / mL in pH 4.5 (0.13 M acetic acid) buffer. The diluted samples (150 μL) were added to the wells of the PCR plate and incubated at 65°C in a PCR instrument for 5 minutes. After the heat challenge incubation, the sample was further diluted to the required assay concentration. To measure the enzyme activity after the heat challenge incubation, the Chinese National Standard Protocol GB1886.174-2016A.9.1.2 described in step 5 was followed. The percentage residual activity after 5 minutes at 65°C was calculated compared to the sample that was not heat challenged. The results of the thermal stability assay are shown in Table 2.

[0147] Table 2

[0148]

[0149] The amino acid sequence of G2P is shown in SEQ ID NO: 2, the amino acid sequence of G3P-1 is shown in SEQ ID NO: 3, and the amino acid sequence of G3P-2 is shown in SEQ ID NO: 4, wherein Pullulanase is pullulanase.

[0150] Pullulanase G2P protein SEQ ID NO:2

[0151] 。

[0152] Pullulanase G3P-1 protein SEQ ID NO:3

[0153] 。

[0154] Pullulanase G3P-2 protein SEQ ID NO:4

[0155] 。

[0156] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A pullulanase mutant, characterized in that Compared with the pullulanase with an amino acid sequence as shown in SEQ ID NO. 1, the pullulanase mutant has V760A.

2. The pullulanase mutant according to claim 1, characterized in that The pullulanase mutant has the following mutation combination: V760A and V576I; Optionally, the pullulanase mutant further has one or more of M279L, V264T and A535I.

3. The pullulanase mutant according to any one of claims 1 to 2, characterized in that The pullulanase mutant further has one or more of the following mutations: A7V、A11T、A37T、K93M、S102L、Q111R、L178Q、G179V、N204K、N207D、S208L、Q224K、K225N、K230R、S236[A,D,N,T]、V241L、A247E、S250[A,P]、I254T、V264[P,S,T]、N270[H,P]、Q271G、N272D、M279[L,R,S,T]、T283K、G284V、T297[A,L,M,R,V,Y]、D315N、A317[P,T]、N320E、I330P、E331V、E333A、D339[H,S]、P347S、N348K、L360F、K372N、S377Y、Q380E、S394F、D398N、T400[L,N]、T403S、D405[C,G,R,S,Y]、D415N、N424[D,L,R,T]、A425P、N426H、N428T、A429[C,E,G,L,P,R,T]、V438I、L439[I,Q]、R443Q、E444D、H445G、I446T、T460[K,V]、Q461[E,G,H,N,S,T,V]、I462T、E471[I,Q,T]、Y473H、D478[A,N,S,Y]、A479[C,S]、N481[D,F,I,T]、I492[L,V]、A494[D,G]、Q500[C,R]、L507V、Y509F、Y514S、I516V、A535[C,I,L,N,W]、S537T、E538Q、L539H、N543D、A547L、T557[E,H,I,K,L,P,Q,R,S,V]、A559S、P561T、D562[A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,Y]、D563S、L565Q、T567[F,I,K,V,Y]、A570[C,G,H,K,M,R]、K572R、M574L、V576[C,I,M,P]、A577[G,I]、N580D、A586G、N590S、V591I、S594P、S595[A,D,F,H,K,M,R,T,Y]、Q597R、L606Y、A609E、S623A、Y638L、L646Q、V666I、V667F、V673[F,I,L]、M682F、G687Y、D690[C,S]、G696V、A698[L,Q,S,T,V]、S705K、P711T、Y754[F,R,S]、H759Y、V760[A,P], K764T, G766K, N767D, I769V, V771I, V778[A,D,L,S], A779E, A789[E,L,R,S], N791V, A792V, I818V, H820Y, D826S, and H827D; Optionally, the pullulanase mutant has any one of the following mutation combinations:

4. A nucleic acid, characterized in that The nucleic acid encodes the pullulanase mutant according to any one of claims 1 to 3.

5. A carrier, characterized in that The vector comprises the nucleic acid of claim 4.

6. A cell, characterized in that The cell expresses the pullulanase mutant according to any one of claims 1 to 3, or comprises the nucleic acid according to claim 4, or comprises the vector according to claim 5.

7. A method for constructing the cell according to claim 6, characterized in that: The construction method comprises the step of introducing the nucleic acid according to claim 4 or the vector according to claim 5 into the cell to be transformed.

8. An enzyme-containing product, characterized in that The enzyme-containing product comprises the pullulanase mutant according to any one of claims 1 to 3; Optionally, the enzyme-containing product comprises one or more of the following enzymes: glucoamylase, α-amylase, β-amylase and protease.

9. Use of the pullulanase mutant according to any one of claims 1 to 3 for producing syrups and / or fermentation products from starch-containing materials.

10. A method for using a pullulanase mutant, characterized in that: The method comprises contacting the pullulanase mutant according to any one of claims 1 to 3 or the enzyme-containing product according to claim 8 with a target substance containing a substrate to cause an enzyme-catalyzed reaction.

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