Mannanase mutant with improved thermal stability and specific activity, and use thereof in industrial production

Abstract

Provided in the present invention are a mannanase mutant with improved thermal stability and a specific activity, and the use thereof in industrial production. The amino acid sequence of the mutant has at least 90% sequence identity to a parent β-mannanase as shown in SEQ ID NO: 1, and contains the following amino acid mutation combination: K316P, D319N and G321H.

Description

Mannanase mutants with improved thermal stability and specific activity and their application in industrial production

[0001] The present application claims priority to the prior application filed with the China National Intellectual Property Office on August 6, 2024, with the patent application number 2024110713899, and the title of “Mannanase mutants with improved thermal stability and specific activity and their application in industrial production”. The entire contents of the prior application are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of protein engineering. More specifically, it relates to a mannase mutant with improved thermal stability and specific activity. BACKGROUND

[0003] Mannan is the second most abundant hemicellulose in nature after xylan. Complete degradation of mannans requires the concerted action of mannolytic enzymes. Among them, β-mannanase (endo-β-1,4-D-mannanase, EC 3.2.1.78) is a key enzyme because it can cleave the internal β-1,4-D-mannose bonds of the mannans backbone, producing mannooligosaccharides (Chauhan PS, Puri N, Sharma P, Gupta N (2012) Mannanases: microbial sources, production, properties and potential biotechnological applications. Appl Microbiol Biotechnol 93: 1817-1830). Based on primary structure alignment and hydrophobic cluster analysis, almost all known β-mannanases are classified into glycoside hydrolase (GH) families 5, 26 and 113. Analysis of the three-dimensional (3-D) structure shows that all β-mannanases in the three families belong to the glycoside hydrolase superfamily A, and their catalytic domain consists of an (β / α)8-TIM (triosephosphate isomerase) barrel structure composed of eight β-strands and eight α-helices arranged alternately. Due to the complex and symmetrical three-dimensional structure of β-mannanases, the study of their structure and function has been lagging behind (van Zyl WH, Rose SH, Trollope K, JF (2010) Fungal β-mannanases: mannan hydrolysis, heterologous production and biotechnological applications. Process Biochem 45: 1203-1213).

[0004] With the development of natural hemicellulose resources and the discovery of the medicinal value of mannose oligosaccharides, β-mannanase has been widely used in feed, medicine, papermaking, textile printing and dyeing, oil exploitation and other aspects. For example, in the feed aspect, β-mannanase can effectively hydrolyze the anti-nutritional factor mannose in feed, reduce the viscosity of chyme, and promote the absorption of nutrients. In the paper industry, β-mannanase and other hemicellulose-degrading enzymes such as β-xylanase are used synergistically to effectively remove hemicellulose in paper pulp. In the oil industry, β-mannanase is a high-quality biological breaker with the advantages of high efficiency, low cost and small damage to the formation. Although β-mannanase has great potential in industrial processes, the wide application of most commercial β-mannanases is limited by their low catalytic activity, weak substrate affinity and / or poor tolerance to extreme environments (Li J, Wei X, Tang C, Wang J, Zhao M, Pang Q, Wu M (2014) Directed modification of the Aspergillus usamii β-mannanase to improve its ubstrate affinity by in silico design and site-directed mutagenesis. J Ind Microbiol Biotechnol 41: 693-700). How to screen and modify enzymes with excellent performance and strong tolerance and apply them in industrial production has become the biggest challenge for the application of β-mannanase.

[0005] In order to meet the growing demand for β-mannanases with superior performance, more and more people pay attention to modifying the primary and / or three-dimensional structure of β-mannanases through directed evolution and rational design. Although there are some literatures and patents about the cloning and expression of β-mannanase genes, there are few reports about the directed modification of β-mannanase molecules, especially the rational design of β-mannanase molecules based on computational biology.

[0006] SUMMARY

[0007] The present application provides a β-mannanase mutant, which has significantly improved thermal stability and specific activity compared to wild-type β-mannanase, which is beneficial to the application of the enzyme in industrial production.

[0008] The present application is achieved by a beta-mannanase mutant having at least 90% sequence identity to a parent beta-mannanase represented by SEQ ID NO: 1 and comprising the following amino acid mutation combination: K316P, D319N and G321H, wherein the positions are numbered with reference to SEQ ID NO: 1, wherein the variant has beta-1,4-D-mannoside bond hydrolysis activity.

[0009] Another object of the present application is to provide a gene encoding the above-mentioned beta-mannanase mutant with improved thermostability and specific activity.

[0010] Another object of the present application is to provide a recombinant vector comprising the above-mentioned gene encoding the beta-mannanase mutant.

[0011] Another object of the present application is to provide a recombinant strain comprising the above-mentioned gene encoding the beta-mannanase mutant.

[0012] Another object of the present application is to provide a method for preparing the above-mentioned beta-mannanase mutant with improved thermostability and specific activity.

[0013] Another object of the present application is to provide the use of the above-mentioned beta-mannanase mutant with improved thermostability and specific activity.

[0014] Beneficial technical effects

[0015] The beta-mannanase mutants ManA-mut1 to ManA-mut9 provided by the present application have significantly improved thermostability and specific activity compared with the parent beta-mannanase represented by SEQ ID NO: 1. Specifically, the specific activity of the mutants ManA-mut1 to ManA-mut9 is at least 189% higher than that of the parent beta-mannanase. In terms of heat resistance, the residual enzyme activity of the parent beta-mannanase is about 50% after 3 minutes of water bath at 85°C, while the residual enzyme activity of the beta-mannanase mutants ManA-mut1 to ManA-mut9 is greater than 67% after 3 minutes of water bath at 85°C and greater than 45% after 3 minutes of water bath at 90°C, which is significantly better than the parent beta-mannanase. The mutants ManA-mut1 to ManA-mut9 have higher acid resistance and can better adapt to the acidic environment of the gastrointestinal tract of animals. In addition, the mutants ManA-mut1 to ManA-mut9 have better performance in hydrolyzing palm kernel meal than the parent beta-mannanase. The beta-mannanase mutants provided by the present application exhibit excellent performance and are particularly suitable for use in industrial production, for example, in the fields of food, chemical industry, medicine, agriculture and feed. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 shows a protein electrophoresis gel of purified samples of the mutants ManA-mutl to ManA-mut9 and the parent beta-mannanase ManA of the application (lane 1 : ManA-mutl, lane 2: ManA-mut2, lane 3: ManA-mut3, lane 4: ManA-mut4, lane 5: ManA-mut5, lane 6: ManA-mut6, lane 7: ManA-mut7, lane 8: ManA-mut8, lane 9: ManA-mut9, lane 10: ManA, M: protein Marker)

[0017] DETAILED DESCRIPTION

[0018] The beta-mannanase mutants of the application having improved thermostability and specific activity are designed based on the beta-mannanase ManA of the parent Aspergillus niger CBS 513.88 (UniProtKB / Swiss-Prot: A2QKT4.1) by molecular dynamics simulations and structure-based protein sequence generation models.

[0019] The present application provides a beta-mannanase mutant having at least 90% sequence identity to the parent beta-mannanase of SEQ ID NO: 1 and comprising the following combination of amino acid mutations: K316P, D319N and G321H, the positions being numbered with reference to SEQ ID NO: 1, wherein said variant has beta-1,4-D-mannosidic bond hydrolytic activity.

[0020] Preferably, the mutant of the application has at least 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.4%, 97.5%, 98%, 98.5% or 99% sequence identity to the parent beta-mannanase of SEQ ID NO: 1.

[0021] Preferably, the mutant further comprises at least one, two, three, four or more of the amino acid mutations S24A, S155K, S173G, G192S, A195P, G256Q, T298E, G301A or N322F.

[0022] Preferably, the mutant comprises the following combinations:

[0023] 1) K316P, D319N, G321H and N322F;

[0024] 2) S173G, G192S, A195P, T298E, K316P, D319N, G321H and N322F;

[0025] 3) S155K, A195P, K316P, D319N, G321H and N322F;

[0026] 4) G192S, A195P, K316P, D319N, G321H and N322F;

[0027] 5) S173G, A195P, T298E, K316P, D319N, G321H and N322F;

[0028] 6) S173G, T298E, G301A, K316P, D319N, G321H and N322F;

[0029] 7) S24A, S173G, G256Q, G192S, A195P, K316P, D319N, G321H and N322F;

[0030] 8) S155K, G192S, A195P, G301A, K316P, D319N, G321H and N322F;

[0031] 9) G192S, A195P, T298E, G301A, K316P, D319N, G321H and N322F.

[0032] Preferably, the amino acid sequences corresponding to the mutants are as shown in SEQ ID NO: 2-SEQ ID NO: 10, respectively.

[0033] In certain embodiments, the mutant further comprises a site known in the prior art to improve the expression, specific activity, thermal stability or acid tolerance of SEQ ID NO: 1 or its natural variants, for example Y25H (see US 8999695), Y216W (see US 8865444), V279I and T280Y (see CN111117987A), the positions being numbered with reference to SEQ ID NO: 1.

[0034] In certain embodiments, the mutant further comprises a mutation site derived from a natural variant of SEQ ID NO: 1, wherein the natural variant of SEQ ID NO: 1 refers to a β-mannanase variant derived from the same species or the same genus and having a high sequence identity, for example greater than 99%, with SEQ ID NO: 1.

[0035] Preferably, the mutant further comprises one, two, three or more of the following amino acid mutations in positions Q74E, D75N, D101G, Q144H, P169L, C174R, G231D, D251N, E275Q, S289A, W290C, D320N, D320G, T328A, V339L, G343D, the positions being numbered with reference to SEQ ID NO: 1.

[0036] Preferably, the mutant comprises one, two, three or more of the following mutations in positions:

[0037] 1) Q74E and D75N (based on XP_026629027.1);

[0038] 2) G231D and S289A (based on GKZ97895.1 or ACJ06979.1, see also US 8999695 and US 8865444);

[0039] 3) D101G and S289A (based on GLA40945.1);

[0040] 4) C174R and D320N (based on ADK88903.1);

[0041] 5) G343D (based on AJK28605.1, see also CN111117987A);

[0042] 6) D320G and G343D (based on ADZ99027.1);

[0043] 7) Q73E, D74N, D101G and D251N (based on GKZ85842.1);

[0044] 8) P169L, T328A and V339L (based on AKC98250.1);

[0045] 9) Q144H, E275Q and W290C (based on AEY76082.1).

[0046] In certain embodiments, the mutant further comprises one or more additional changes, e.g., one or more conservative amino acid substitutions or insertions; small deletions of 1 to 30 amino acids; or small extensions at the amino or carboxyl terminus; e.g., an amino terminal methionine residue; a small linker peptide of up to 20 to 25 residues; or a small extension to facilitate purification by altering net charge or another function, e.g., a histidine tag, an antigenic epitope, or a binding domain. These additional amino acid changes can be of a trivial nature, but do not significantly affect the folding and / or activity of the mutant.

[0047] Conservative amino acid substitutions include substitutions of an amino acid residue with an amino acid residue having a similar side chain, e.g., substitutions made between residues that are physicochemically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Common amino acid substitutions that do not alter enzymatic activity, for example: Ala / Ser (A / S), Val / Ile (V / I), Asp / Glu (D / E), Thr / Ser (T / S), Ala / Gly (A / G), Ala / Thr (A / T), Ser / Asn (S / N), Ala / Val (A / V), Ser / Gly (S / G), Tyr / Phe (W / F), Ala / Pro (A / P), Lys / Arg (K / R), Asp / Asn (D / N), Leu / Ile (L / I), Leu / Val (L / V), Ala / Glu (A / E), and Asp / Gly (D / G).

[0048] Furthermore, the present application also provides a polynucleotide encoding the beta-mannanase mutant according to any of the above, including the complementary strand thereof. The polynucleotide of the present application is a recombinant molecule containing a genetically engineered non-naturally occurring sequence. As used herein, "polynucleotide" refers to both RNA and DNA, and it can be single-stranded or double-stranded. It can also be complementary DNA (cDNA). cDNA means a DNA molecule synthesized from a messenger RNA template obtained from a eukaryotic or prokaryotic organism. Furthermore, the polynucleotide can be degenerate to any of the sequences as defined above as a result of the genetic code. This means that different codons can code for the same amino acid.

[0049] The present application also provides a recombinant expression vector comprising the above-mentioned polynucleotide encoding the β-mannanase mutant operably linked to a control sequence capable of directing expression of the gene encoding the β-mannanase variant in a suitable host. The control sequence can be derived from the host organism or from another organism. The expression vector can also comprise a marker gene for selection of transformant strains, or the selection marker can be introduced into the host by co-transformation in another vector construct.

[0050] The present application provides a host cell comprising the gene encoding the β-mannanase mutant or the recombinant expression vector, which is a bacterium or a fungus, preferably a filamentous fungus or a yeast, more preferably Aspergillus niger, Trichoderma reesei or Pichia pastoris.

[0051] The present application also provides a method for constructing the host cell, comprising the following steps:

[0052] 1) constructing a recombinant expression vector comprising the gene encoding the β-mannanase mutant;

[0053] Preferably, the vector is pPICZαA;

[0054] 2) transforming the recombinant expression vector constructed in step 1) into a host cell;

[0055] Preferably, the host cell is Pichia pastoris; preferably, the Pichia pastoris is Pichia pastoris X-33.

[0056] The present application also relates to a method for producing the β-mannanase variant with improved thermostability and specific activity, comprising the steps of transforming a host cell with an expression vector encoding the polypeptide, culturing the host cell under conditions capable of producing the polypeptide, and optionally recovering and purifying the polypeptide. The production medium can be a medium suitable for growing the host organism and containing an inducer for efficient gene expression.

[0057] In some embodiments of the application, the beta-mannanase is secreted extracellularly. Cultures of the expression organism are prepared in appropriate volumes according to standard fermentation procedures. In preferred embodiments, the strains are grown in fermentors and growth conditions such as pH, temperature, oxygen and / or nutrient supply are controlled. The first step in purification involves separation of the bacterial cells from the supernatant using one or more of several techniques such as sedimentation, flocculation or centrifugation. In preferred embodiments, the method of choice is centrifugation. Further purification of the protein from the supernatant or concentrated supernatant can be carried out using one or more of several methods including extraction or fractionation methods such as ammonium sulfate or ethanol or acid precipitation, or chromatographic methods including, but not limited to, ion exchange, hydrophobic interaction, hydroxyapatite, size exclusion by gel filtration, phosphocellulose or lectin chromatography and affinity chromatography, or any combination thereof. If expressed intracellularly, the cells are subjected to a disruption process to release the product of interest from the cells. The disruption process can include, for example, pressurization, osmotic shock, freezing, sonication or other processes to produce a cell lysate which can be subjected to further purification.

[0058] In further embodiments of the application, the fermentation cell suspension comprising the expressed beta-mannanase is dried as a whole using methods such as, but not limited to, fluid bed drying, spray drying or drum drying or any combination thereof.

[0059] The present application provides enzyme preparations comprising the beta-mannanase variants of the present application. As used in the context of the present application, "enzyme preparation" means any enzyme product or composition comprising at least one of the novel beta-mannanase variants described herein. Such enzyme preparation can be a spent culture medium or filtrate containing one or more beta-mannanase variants or one or more beta-mannanase variants and one or more other enzymes. Spent culture medium means the culture medium of the host comprising the produced enzymes. Preferably, the host cells are separated from the culture medium after production. The enzyme preparation or composition can be "whole broth" obtained optionally after inactivation of the production host or microorganism without any biomass separation, downstream processing or purification of the desired cellulolytic enzymes, since the beta-mannanase variants can be secreted into the culture medium and they exhibit activity in the ambient conditions of the spent culture medium.

[0060] In some embodiments of the application, the enzyme preparation further comprises at least one enzyme selected from the group consisting of cellulases, amylases, lipases, proteases, hemicellulases, ligninases, pectinases, xylanases, beta-glucanases, alpha-galactosidases and glucose oxidases.

[0061] The enzyme preparation can contain the enzymes in at least partially purified and isolated form. It can even consist essentially of the desired enzyme(s). If desired, the enzyme preparation can be dried, spray-dried or freeze-dried, granulated, or the enzyme activity can be otherwise concentrated and / or stabilized for storage. If desired, the desired enzyme can be crystallized or isolated or purified according to conventional methods, e.g., filtration, extraction, precipitation, chromatography, affinity chromatography, electrophoresis, etc.

[0062] In still another aspect of the present application, there is provided a use of the beta-mannanase mutant in the field of daily chemical, food, chemical industry, medicine, agriculture or feed.

[0063] In a further embodiment, the present application provides a detergent comprising the beta-mannanase mutant or an enzyme preparation thereof. The detergent can further comprise one or more other enzymes in addition to the one or more beta-mannanase variants of the present application, which can be, for example, cellulases, amylases, lipases, proteases, hemicellulases, ligninases, pectinases, and / or oxidases. More specifically, the enzyme preparation can comprise at least one other enzyme selected from the group consisting of cellobiohydrolases, endoglucanases, beta-glucanases, beta-glucosidases, serine proteases, xylanases, beta-xylosidases, endopectin lyases, pectate lyases, pectin esterases, laccases, cutinases, peroxidases, and copper- dependent lytic polysaccharide monooxygenases, i.e., enzymes of glycosyl hydrolase family 61. The detergent of the present application can also comprise one or more suitable additives selected from the group consisting of surfactants or surface-active preparations, buffers, corrosion inhibitors, stabilizers, bleaching agents, mediators, builders, corrosion and abrasion agents, optical brighteners, anti-redeposition agents, dyes, pigments, fragrances, etc. The detergent can be provided as a liquid or a solid, e.g., as a dry powder or granulate, in particular a non-dusting granulate, a stabilized liquid, a tablet, a crystal or a crystal suspension.

[0064] In a further embodiment, the present application provides a feed additive comprising the beta-mannanase mutant or an enzyme preparation thereof. The feed additive can further comprise alpha-galactosidase, cellulase and pectinase, and optionally a nutritional ingredient or a fungus, etc. The feed additive of the present application can efficiently degrade hemicellulose and function in the acidic environment of the gastrointestinal tract of animals.

[0065] In a further embodiment, the present application provides a yeast processing application comprising the beta-mannanase mutant or an enzyme preparation thereof, such as the production of yeast cream, nucleic acids or yeast cell wall polysaccharides. The beta-mannanase mutant of the present application or an enzyme preparation thereof is used in combination with a beta-glucanase to break down the yeast cell wall.

[0066] In further embodiments, the present application provides the use of the β-mannanase mutant or enzyme preparation thereof in papermaking, the β-mannanase mutant or enzyme preparation thereof of the present application is used in cooperation with hemicellulose-degrading enzymes such as β- xylanase, which can effectively remove hemicellulose in pulp and significantly improve paper quality.

[0067] In further embodiments, the present application provides the use of the β-mannanase mutant or enzyme preparation thereof in the degradation of hemicellulose in natural fibers, such as in the degumming process of ramie.

[0068] In further embodiments, the present application provides the use of the β-mannanase mutant or enzyme preparation thereof as a biological breaker in the petroleum industry.

[0069] Definitions of terms

[0070] β-mannanase mutant, mutant or mutated β-mannanase: refers to a polypeptide having β-mannanase activity comprising an alteration, i.e. substitution, insertion and / or deletion, of one or more (several) amino acid residues at one or more (several) positions. Substitution refers to replacing an amino acid occupying a certain position with a different amino acid; deletion refers to removing an amino acid occupying a certain position; and insertion refers to adding 1-5 amino acids adjacent to and after the amino acid occupying a certain position. Mutating a parent β-mannanase also refers to substitution, insertion and / or deletion of an amino acid at at least one position compared to the parent β-mannanase, preferably refers to substitution of an amino acid at at least one position, such as "S173G", i.e. substitution of serine at position 173 of the parent β-mannanase with glycine.

[0071] Parent: refers to a beta-mannanase that can produce the mutant of the present application after the mutation described in the present application. The parent can be a naturally occurring (wild-type) phytase or a mutant thereof prepared by a suitable method, and the parent can also be an allelic variant. In a specific embodiment of the present application, the parent is beta-mannanase ManA (UniProtKB / Swiss-Prot: A2QKT4.1) derived from Aspergillus niger CBS 513.88; in another specific embodiment of the present application, the parent is XP_026629027.1 (positions 39-383), GKZ97895.1 (positions 39-383), or ACJ06979.1 (positions 1-345), GLA40945.1 (positions 39-383), ADK88903.1 (positions 38-382), AJK28605.1 (positions 18-362), ADZ99027.1 (positions 39-383), GKZ85842.1 (positions 39-383), AKC98250.1 (positions 39-383), and AEY76082.1 (positions 39-383) having more than 99% sequence identity to beta-mannanase ManA.

[0072] Thermostability: refers to the beta-mannanase mutant of the present application that still maintains a certain amount of enzyme activity after a given period of time at a specific temperature. When referring to a property such as thermostability, an improved thermostability refers to a higher enzyme activity after a period of time compared to other beta-mannanase variants and / or the parent beta-mannanase. In the context of the present application, "a specific temperature" means a temperature from 70°C to 120°C, preferably from 80°C to 100°C, more preferably from 80°C to 90°C.

[0073] Acid resistance: refers to the beta-mannanase mutant of the present application that still maintains a certain amount of enzyme activity after a given period of time at a specific pH. When referring to a property such as acid resistance, an improved acid resistance refers to a higher enzyme activity after a period of time compared to other beta-mannanase variants and / or the parent beta-mannanase. In the context of the present application, "a specific pH" means a pH value from 2.0 to 4.0, preferably from 2.0 to 2.5.

[0074] Coding sequence: As used herein, the term "coding sequence" means a polynucleotide sequence that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence can be a DNA, cDNA, RNA, synthetic, or recombinant nucleotide sequence.

[0075] Control sequences: The term "control sequences" as used herein refers to nucleic acid sequences necessary for expression of a polynucleotide encoding a variant of the present application. Each control sequence can be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the variant or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, pre-pro peptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. The control sequences can be provided with linkers for easy attachment to the polynucleotide encoding the variant. The term "control sequences" is intended to include, at a minimum, components whose presence is necessary for expression of a polynucleotide encoding a variant of the present application. In some embodiments, the control sequences include components that, when the components are present in the proper position relative to the polynucleotide encoding the variant, allow for expression of the polynucleotide.

[0076] Operably linked: The term "operably linked" means that the control sequences are placed at appropriate positions in relation to the coding sequence of a polynucleotide such that the control sequences direct expression of the coding sequence.

[0077] Expression: The term "expression" in the context of the present application includes any step involved in the production of a beta-mannanase of the present application, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0078] Expression vector: The term "expression vector" is defined herein as a linear or circular DNA molecule comprising a polynucleotide encoding a protein, such as a beta-mannanase of the present application, and said polynucleotide is operably linked to additional nucleotides that provide for its expression.

[0079] Host cell: The term "host cell" as used herein includes a cell that is transformed, transfected, transduced or the like with a nucleic acid construct or expression vector comprising a polynucleotide encoding a beta-mannanase and from which a beta-mannanase can be expressed according to the methods of the present application. Preferably, the recombinant host is modified to express and secrete a beta-mannanase of the present application as its main activity or one of its main activities. This can be done by deleting the coding for the main endogenous secreted enzymes and by integrating the heterologous gene to a locus with high expression and production levels.

[0080] Unless otherwise defined, all scientific and technical terms used herein have the meanings that are commonly understood by one of ordinary skill in the art. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0081] The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein" and "peptide" and are used interchangeably. The conventional one-letter code or three-letter code for amino acid residues is used, with the amino acid sequence presented in the standard amino to carboxy terminal orientation (i.e., N→C).

[0082] For substitutions of amino acids, the following nomenclature is used: original amino acid, position, substituted amino acid. For example, substitution of lysine ("L-Lys" or "K") at position 316 with proline ("L-Pro" or "P") is designated "Lys316Pro" or "K316P".

[0083] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". A particular sequence has at least a certain percentage of amino acid residues identical with a specified reference sequence when aligned using the CLUSTALW algorithm with preset parameters. The preset parameters for the CLUSTALW algorithm are: gap count as non-identical residues compared to the reference sequence. Gaps occurring at either end are included. For example, a variant 500 amino acid residue polypeptide which lacks the C-terminal five amino acid residues has a percentage sequence identity of 99% (495 / 500 identical residues x 100) relative to the parent polypeptide. Such a variant is encompassed by the language "a variant having at least 99% sequence identity with the parent".

[0084] As used herein, the term "about" means ±10%. The terms "comprising", "including", "having" mean "including but not limited to". As used herein, the term "about" means up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in certain cases up to or below 20% deviation from the referenced value, the deviation range including integer values and, if applicable, non-integer values, constituting a continuous range. DETAILED DESCRIPTION

[0085] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein like or similar elements are designated by like or similar reference numerals throughout the several views. The embodiments described below are exemplary in nature, and are intended to be illustrative of the present application rather than to be limiting of the present application.

[0086] The following disclosure provides many different embodiments, or examples, for implementing different aspects of the present application. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present application, the particular methods and materials described herein are illustrative only. Accordingly, it is to be understood that the present application is not limited to the particular methodology, protocols, and reagents described, as such may vary. Except as otherwise indicated, the embodiments of the application are presented by way of example only and are not intended to limit the scope of the application. 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 the application pertains. In case of conflict between any document incorporated by reference and the disclosure contained in the specification, the specification will control.

[0087] Unless otherwise indicated, the molecular biology experiments described in the following examples were performed using standard techniques known in the art, as described in Sambrook, Molecular Cloning: A Laboratory Manual (3rd Ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), or as described in the manufacturer's manuals.

[0088] The application will now be described in greater detail by way of illustrative examples, which are not intended to limit the scope of the application. In particular, it is noted that the reagents used in the examples are commercially available unless otherwise specified.

[0089] Experimental materials and reagents:

[0090] 1. Strains and vectors: The expression host Pichia pastoris X-33 (Invitrogen), the expression plasmid vector pPICZαA (Invitrogen), E. coli strain Top 10 and the strain containing the β-mannanase gene and the expression plasmid were preserved in the laboratory.

[0091] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from Fermentas Company, ligase was purchased from Promaga Company, DNA polymerase was purchased from Beijing Zongshi Jin Bio. Plasmid extraction kit and purification kit were purchased from Shanghai Shenguo Company. Antibiotic Zeocin was purchased from Invitrogen Company, and others were domestic analytical reagents (all can be purchased from ordinary biochemical reagent companies).

[0092] 3. Culture medium:

[0093] LB solid medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 1% agar powder, pH 7.0.

[0094] LB-A resistant medium is LB medium with a final concentration of 100 μg / mL ampicillin. LB-Z resistant medium is LB medium with a final concentration of 25 μg / mL Zeocin.

[0095] YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

[0096] YPD-Z resistance medium is YPD medium with final concentration of 100 ug / mL Zeocin.

[0097] Induction medium BMGY: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (v / v).

[0098] Induction medium BMMY: 0.5% methanol instead of glycerol, the rest of the components are the same as BMGY.

[0099] Fermentation basic salt medium: diammonium phosphate 5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate heptahydrate 1.5%, potassium sulfate 1.95%, calcium sulfate 0.1%, antifoam 0.03%, after sterilization, add 4.35 mL PTM1 per liter.

[0100] PTM1 (trace salt solution): copper sulfate 0.6%, potassium iodide 0.018%, manganese sulfate monohydrate 0.3%, sodium molybdate dihydrate 0.02%, boric acid 0.002%, cobalt chloride hexahydrate 0.05%, zinc chloride 2%, ferric sulfate heptahydrate 6.5%, concentrated sulfuric acid 0.5%, biotin 0.02%.

[0101] 4. Enzyme activity determination of β-mannanase

[0102] The activity of β-mannanase was detected according to the method of GB / T 36861-2018.

[0103] Determination of standard curve:

[0104] 1) Accurately weigh 0.1000 g of D-mannose dried (105°C) to constant weight in a centrifuge tube, accurately weigh 10 g of pH 5.50.1M acetic acid-sodium acetate buffer, dissolve thoroughly, this solution is 10 g / L of D-mannose stock solution.

[0105] 2) Dilute the above D-mannose stock solution 10 times (for example: 1 mL of 10 g / L mannose stock solution is added to 9 mL of acetic acid-sodium acetate buffer), which is 1 g / L of mannose solution. In this way, the mannose solution is diluted into standard solutions of different concentrations of 0 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL and 700 μg / mL.

[0106] 3) Take 8 test tubes numbered 0-7, put them in the test tube rack, and add 1 mL of the above standard solution of different concentrations and 1 mL of pH 5.5 0.1M acetic acid-sodium acetate buffer to 0-7 test tubes, respectively.

[0107] 4) Add 2.5 mL of DNS color developing solution with the dispenser, mix well. Put 8 test tubes into the boiling water bath and time accurately for 5 min. Take out, ice bath for 2 min, and add 8 mL of distilled water with the bottle opening dispenser.

[0108] 5) 0 is blank control, measure the absorbance value at 540 nm. Draw the standard curve with D-mannose concentration as Y axis and absorbance value A as X axis, and obtain the linear regression equation.

[0109] Determination of sample enzyme activity:

[0110] 1) According to the sample quantity, prepare the corresponding clean test tubes on the metal test tube rack, weigh the sample, and do three parallel samples. Each weighed sample needs a blank control, and the shake flask sample does not need a parallel sample. Do one blank control for each batch.

[0111] 2) Take 1.0 ml of 0.6% mannose substrate in the test tube with the external pipette, and preheat the test tube in the 37°C water bath for 10 min. Accurately time and add 1.0 ml of diluted enzyme solution, shake by hand for 3 times with an interval of 15 s, and add the second sample in the same way. Accurately react for 30 min, add 2.5 ml of DNS solution to terminate the reaction, and use the vortex instrument to vortex for 3 s after all the addition is completed.

[0112] 3) Boil in boiling water for 5 min, immediately ice bath for 2 min, and add 8 ml of distilled water after taking out and vortexing. The blank control is to add 1.0 ml of diluted enzyme solution, first add 2.5 ml of DNS, then add 1.0 ml of 0.6% mannose, and the rest of the operation is the same as the sample.

[0113] 4) Centrifuge to take the supernatant, use UV5200 or UV5800 spectrophotometer for colorimetry, colorimetric wavelength is 540 nm, first adjust zero with the blank control, then measure the sample absorbance value and record. Calculate the enzyme activity according to the D-mannose standard curve.

[0114] Enzyme activity (u / ml) = (OD*K+b)*n / 180.2 / 30*1000

[0115] Where: n - enzyme dilution factor OD - sample absorbance value detected

[0116] b - intercept of the standard curve K - slope of the standard curve

[0117] 30 - reaction time, min 180.2 - molecular weight of mannose, g / mol

[0118] Enzyme activity definition: the amount of enzyme required to release 1 umol of reducing sugar per minute from a solution of mannan with a concentration of 3 mg / ml at pH 5.5 and a temperature of 37°C for exactly 30 min is one unit of enzyme activity.

[0119] Specific enzyme activity is defined as the number of units of enzyme activity per 1 mg of protein and is expressed as U / mg.

[0120] Example 1 Construction of expression plasmids of parent β-mannanase and its mutants

[0121] The parent β-mannanase is β-mannanase ManA (UniProtKB / Swiss-Prot: A2QKT4.1) derived from Aspergillus niger CBS 513.88, and its amino acid sequence is shown as SEQ ID NO: 1. The present inventors designed mutant sequences in combination with molecular dynamics simulation and structure-based protein sequence generation model to obtain β-mannanase mutants ManA-mutl, ManA-mut2, ManA-mut3, ManA-mut4, ManA-mut5, ManA-mut6, ManA-mut7, ManA-mut8 and ManA-mut9, and the mutant mutation sites are summarized in Table 1.

[0122] Table 1 Summary of mutant mutation sites

[0123] The codon optimization was performed on the amino acid sequences of ManA and its mutants by using the Pichia pastoris codon table. The genes encoding β-mannanase and its mutants were synthesized by using the whole gene synthesis method, and the gene synthesis was completed by Nanjing Kingsriver Biotechnology Co., Ltd. The synthesized genes have EcoRI and XbaI enzyme cutting sites at both ends. The synthesized genes were cut by EcoRI and XbaI, the target gene fragments were recovered by gel recovery, the products were purified and recovered, and were connected to the expression vector pPICZαA. The ligation products were transformed into E. coli Top10 competent cells by using the chemical transformation heat shock method, and were inoculated on LB-Z resistant plates and cultured at 37°C. After the transformants appeared on the plates, single colonies were picked and inoculated in LB-Z resistant test tubes and cultured at 37°C. The recombinant transformants were verified by bacterial liquid PCR, and the plasmids of the correct transformants were extracted for sequencing, obtaining the expression vectors pPICZαA-ManA, pPICZαA ManA-mut1, pPICZαA-ManA-mut2, pPICZαA ManA-mut3, pPICZαA-ManA-mut4, pPICZαA-ManA-mut5, pPICZαA-ManA-mut6, pPICZαA-ManA-mut7, pPICZαA-ManA-mut8 and pPICZαA-ManA-mut9.

[0124] Example 2 Construction of expression strains of parent β-mannanase and its variants

[0125] The expression vectors pPICZαA-ManA, pPICZαA-ManA-mut1 and pPICZαA-ManA-mut2, pPICZαA-ManA-mut3, pPICZαA-ManA-mut4, pPICZαA-ManA-mut5, pPICZαA-ManA-mut6, pPICZαA-ManA-mut7, pPICZαA-ManA-mut8 and pPICZαA-ManA-mut9 with correct sequencing in Example 1 were linearized by using the restriction endonuclease PmeI, the linear plasmid fragments were recovered and purified, and were transformed into Pichia pastoris X33 competent cells by using the electroporation method. YPD-Z resistant medium was used for screening, and yeast recombinant transformants were obtained.

[0126] Preparation of Pichia pastoris X-33 competent cells: 1) inoculate Pichia pastoris X-33 single colony into 5 ml YPD medium, and cultivate at 30°C, 250 rpm / min for 24 h; 2) take 1 ml of the culture to inoculate into a 500 ml flask containing 100 ml YPD medium, and cultivate for 12-16 h, until OD600=0.8-1.2, then transfer the bacterial solution into a 50 ml pre-cooled centrifuge tube, centrifuge at 4°C, 5000 g for 5 min, and discard the supernatant; 3) resuspend the bacterial cells with 25 ml of pre-cooled sterile water, centrifuge at 4°C, 5000 g for 5 min, discard the supernatant, and repeat this step 2-3 times; 4) resuspend the bacterial cells with 5 ml of pre-cooled 1 mol / L sorbitol per tube, centrifuge at 4°C, 5000 g for 5 min, discard the supernatant, and repeat this step 2-3 times; 5) finally resuspend with 200 μL of sorbitol, and aliquot into 1.5 ml centrifuge tubes, 80 μL per tube.

[0127] Transformation screening: 1) take one competent cell, add 10 μL of linearized and concentrated DNA, mix gently, and transfer into a pre-cooled electrode cup with a size of 0.2 cm, and ice-bath for 5 min; 2) perform electroporation under the parameters of 1.5 Kv, 25 μF, and 200 Ω, after the completion of electroporation, quickly add 1 ml of pre-cooled 1 mol / L sorbitol, then transfer the mixture into a 1.5 ml centrifuge tube, and recover at 30°C for 1 h; 3) take 200 μL and spread on YPD-Z solid medium containing 100 μg / ml Zeocin, and cultivate the plate at 30°C for 3 days, observe the positive transformants, use PCR to identify positive single colonies, and send the PCR positive colonies for sequencing verification.

[0128] Example 3 Expression and purification of parent β-mannanase and its variants

[0129] The recombinant transformants were inoculated into 50 ml of BMGY medium, respectively, and cultured at 30°C with shaking at 220 rpm for 18 h. The bacterial cells were obtained by centrifugation, and an appropriate amount of the bacterial cells was transferred into 50 ml of BMMY medium to make the bacterial cell concentration reach OD600=1. The bacterial cells were further cultured at 30°C with shaking at 250 rpm, and 1% of the culture was added with methanol every 24 h. After 5 days of induction and expression, the culture was centrifuged to obtain the supernatant. The crude enzyme solution was salted out with 70% ammonium sulfate, and then purified by Sephadex G-75 gel filtration chromatography and DEAE-Sepharose Fast Flow ion exchange chromatography. After purification, the purified sample was detected by SDS-PAGE to be a single band, and the results are shown in Figure 1. The purified sample was subjected to β-mannanase activity determination, and the relative specific activity was calculated. As shown in Table 2, the enzyme activity of the parent β-mannanase ManA was set as 100%, and the ratio of the enzyme activity of each mutant to the enzyme activity of the parent β-mannanase ManA was the relative specific activity. As shown in Table 2, compared with the parent β-mannanase ManA, the relative specific activities of the mutants ManA-mut1 to ManA-mut9 were significantly improved, with the highest improvement of up to 234%.

[0130] Table 2 Comparison of specific activities of the parent β-mannanase ManA and the mutants

[0131] Example 4 Detection of heat resistance of the parent β-mannanase and its variants

[0132] The purified sample was diluted to about 100 U / mL with a sodium acetate buffer at pH 5.5, and then treated at different temperatures for 3 min, respectively. The residual enzyme activity was determined, and the relative enzyme activity was calculated with the enzyme activity of the untreated sample as 100%. The results are shown in Table 3. As shown in Table 3, compared with the parent β-mannanase ManA, the thermal stabilities of the mutants ManA-mut1 to ManA-mut9 were significantly improved, and the residual enzyme activity was greater than 45% after 5 min of water bath at 90°C, while the parent had only about 23%.

[0133] Table 3 Comparison of heat resistance of the parent β-mannanase ManA and the mutants

[0134] Example 5 Detection of acid resistance of the parent β-mannanase ManA and the mutants

[0135] The acid resistance of the parent β-mannanase ManA and the mutants was tested in a sodium phosphate dibasic-citric acid buffer at pH 2.0 and 2.5 at a temperature of 37°C. The residual enzyme activity of the parent β-mannanase ManA and the mutants was detected after 4 hours of treatment at pH 2.0 and 2.5, respectively, and the untreated sample was used as a control. The residual rate of enzyme activity under acidic conditions was calculated, and the results are shown in Table 4. The results show that the mutants ManA-mutl to ManA-mut9 have higher acid resistance and can better adapt to the acidic environment of the gastrointestinal tract of animals.

[0136] Table 4 Comparison of acid resistance of the parent β-mannanase ManA and the mutants

[0137] Example 6 Fermentation and enzymatic hydrolysis of palm kernel meal using the parent β-mannanase ManA and the mutants

[0138] The palm kernel meal was mixed with the corresponding amount of enzyme preparation and microbial agent. The enzyme preparation included 500 U / g of raw material of mannanase, 200 U / g of raw material of cellulase, 200 U / g of raw material of α-galactosidase, 200 U / g of raw material of pectinase, 100 U / g of raw material of alkaline protease, and 100 U / g of raw material of neutral protease, which were all products produced by the company. The microbial agent included 15 g / ton of raw material of Lactobacillus plantarum (100 billion / g), 30 g / ton of raw material of Bacillus subtilis (100 billion / g), and 30 g / ton of raw material of Saccharomyces cerevisiae (200 billion / g). The corresponding amount of water at 35-38°C was added and mixed uniformly, and then the mixture was placed in an incubator and fermented at 37°C for 72 hours. The reducing sugar content of the hydrolyzate was detected, and the results are shown in Table 5. The results show that the reducing sugar content of the palm kernel meal hydrolyzed by the mutants ManA-mutl to ManA-mut9 was significantly higher than that of the parent β-mannanase ManA, indicating that the performance of the mutants in hydrolyzing palm kernel meal was significantly better than that of the parent β-mannanase ManA.

[0139] Table 5 Detection of reducing sugar content of palm kernel meal hydrolyzed by the parent β-mannanase ManA and the mutants

[0140] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. Furthermore, a person skilled in the art can combine and integrate different embodiments or examples described in the present specification.

[0141] Although the embodiments of the present application have been shown and described above, it is to be understood that the above-described embodiments are exemplary, and are not to be construed as limiting the present application, and that those skilled in the art can make changes, modifications, substitutions and variations of the above-described embodiments within the scope of the present application.

[0142] Sequence information:

[0143] SEQ ID NO: 1 (ManA)

[0144] SEQ ID NO: 2: (ManA-mutl)

[0145] SEQ ID NO: 3: (ManA-mut2)

[0146] SEQ ID NO: 4: (ManA-mut3)

[0147] SEQ ID NO: 5: (ManA-mut4)

[0148] SEQ ID NO: 6: (ManA-mut5)

[0149] SEQ ID NO: 7: (ManA-mut6)

[0150] SEQ ID NO: 8: (ManA-mut7)

[0151] SEQ ID NO: 9: (ManA-mut8)

[0152] SEQ ID NO: 10: (ManA-mut9)

Claims

1. A beta-mannanase mutant, the amino acid sequence of which has at least 90% sequence identity to a parent beta-mannanase set forth in SEQ ID NO: 1 and comprises the following combination of amino acid mutations: K316P, D319N, and G321H, the positions being numbered with reference to SEQ ID NO: 1, wherein the variant has beta-1,4-D-mannosidic bond hydrolytic activity.

2. The beta-mannanase mutant of claim 1, further comprising at least one, two, three, four, or more of the amino acid mutations in S24A, S155K, S173G, G192S, A195P, G256Q, T298E, G301A, or N322F.

3. The beta-mannanase mutant of claim 2, comprising the following combination of amino acid mutations: 1) K316P, D319N, G321H, and N322F; 2) S173G, G192S, A195P, T298E, K316P, D319N, G321H, and N322F; 3) S155K, A195P, K316P, D319N, G321H, and N322F; 4) G192S, A195P, K316P, D319N, G321H, and N322F; 5) S173G, A195P, T298E, K316P, D319N, G321H, and N322F; 6) S173G, T298E, G301A, K316P, D319N, G321H, and N322F; 7) S24A, S173G, G256Q, G192S, A195P, K316P, D319N, G321H, and N322F; 8) S155K, G192S, A195P, G301A, K316P, D319N, G321H, and N322F; 9) G192S, A195P, T298E, G301A, K316P, D319N, G321H, and N322F.

4. The beta-mannanase mutant of any one of claims 1-3, the amino acid sequence of which has at least 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.4%, 97.5%, 98%, 98.5%, or 99% sequence identity to a parent beta-mannanase set forth in SEQ ID NO:

1.

5. The beta-mannanase mutant of any one of claims 1-4, the amino acid sequence of which corresponds to SEQ ID NO: 2-10, respectively.

6. An isolated polynucleotide selected from: a) a polynucleotide or complementary DNA encoding the beta-mannanase mutant of any one of claims 1 to 5; b) a polynucleotide comprising a nucleotide sequence that is degenerate to the nucleotide sequence of the polynucleotide of a), or the complementary strand of said polynucleotide.

7. A vector comprising the isolated polynucleotide of claim 6.

8. A host cell comprising the isolated polynucleotide of claim 6 or the vector of claim 7.

9. The host cell of claim 8, which is a filamentous fungus or a yeast.

10. The host cell of claim 9, which is Aspergillus niger, Aspergillus oryzae, Trichoderma reesei or Pichia pastoris.

11. A method of producing the beta-mannanase mutant of any one of claims 1 to 5, said method comprising the steps of transforming a host cell with an expression vector encoding said polypeptide variant, and culturing said host cell under conditions that allow expression of said mutant, and optionally recovering and purifying said mutant.

12. An enzyme preparation comprising one or more beta-mannanase mutants of any one of claims 1 to 5.

13. The enzyme preparation of claim 12, further comprising at least one enzyme selected from the group consisting of cellulases, amylases, lipases, proteases, hemicellulases, ligninases, pectinases, xylanases, beta-glucanases, alpha-galactosidases and glucose oxidases.

14. Use of the beta-mannanase mutant of any one of claims 1 to 5 or the enzyme preparation of any one of claims 12-13 in the field of daily chemicals, food, chemical industry, medicine, agriculture or feed.

15. The use of claim 14, which comprises use in detergents, feed, yeast processing, papermaking, biomass utilization and petroleum industry.

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