α-amylase mutant and use thereof

Abstract

Belonging to the technical fields of genetic engineering and enzyme engineering, disclosed are an α-amylase mutant and a use thereof. Compared with a parent α-amylase having an amino acid sequence shown in SEQ ID NO: 1, the α-amylase mutant has disulfide bond site pair mutations, the disulfide bond site pairs having the following characteristics: i. not being within 5 Å of an active site of the α-amylase mutant; ii. the difference in amino acid position numbering being greater than 10; iii. the distance between the SG atoms of the two cysteines forming the disulfide bond is within 5 Å; iv. the Chi3 angle satisfies 60°<Chi3<120° or -60°>Chi3>-120°. The provided α-amylase mutant exhibits significantly improved thermostability and / or acid resistance, with most α-amylase mutants possessing both excellent acid resistance and excellent thermostability, and higher hydrolytic activity toward starch, better meeting the needs of deep-processing industries using starch as a raw material, such as sugar production, brewing, alcohol and organic acids.

Description

Alpha-amylase mutants and use thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to alpha-amylase mutants and use thereof. BACKGROUND

[0002] Enzyme molecule modification can be achieved by protein rational design or directed evolution technology. By modifying natural enzymes, new enzymes with higher stability, higher activity, higher selectivity and higher tolerance to extreme environments can be provided for industrial production. Rational design is one of the important methods in protein engineering. Based on the clear structure, function and molecular mechanism of protein-related properties, the changes of specific amino acid sites in protein molecules are first designed theoretically, thereby obtaining some mutants with special properties. The difficulty of this research lies in how to find effective modification points.

[0003] At present, the number of protein sequences stored in the three databases of Genbank, EMBL and DDBJ is growing at a geometric rate, while the growth rate of the number of protein three-dimensional structure information recorded in the PDB (Protein Data Bank) database is far behind. Therefore, it is extremely difficult to obtain protein structure by experimental methods, and thus using homology modeling technology to obtain the three-dimensional structure of protein has become a common bioinformatics method for modern biologists. At present, commonly used protein homology modeling programs include Swiss-Model, CPHmodel, SDSC1, 3D-jigsaw, InsightII, sybyl, COMPOSER, Modeller, AlphaFold2, etc.

[0004] After determining the three-dimensional structure of the protein, the increasingly mature molecular simulation technology provides strong support for solving the association between the three-dimensional structure and the function of the protein. Molecular simulation technology can be used to simulate various dynamic behaviors of molecules, glassy molecular structure, characteristics of molecular motion, folding and unfolding of proteins, etc. Common molecular force fields include Amber (Assisted Model Building with Energy Refinement) and Charmm (Chemistry at HARvard Molecular Mechanics).

[0005] Amber is used for computational simulation of biological macromolecules such as proteins, nucleic acids, and sugars. AMBER provides two parts of content, one is a set of molecular mechanics force fields for simulating biological molecules, and the other is a program for molecular simulation, including source code and demonstration.

[0006] CHARMM is a widely recognized and applied molecular simulation program for the simulation of biological macromolecules, including molecular dynamics, energy minimization, and Monte Carlo simulations. The CHARMM force field used by the program can provide users with empirical energy calculations for a variety of small molecules, macromolecules (including proteins, nucleic acids, and sugars), such as thermodynamic free energy, folding free energy.

[0007] Alpha-amylase (alpha-1, 4-glucan-4-glucanohydrolase, E.C. 3.2.1.1) constitutes a group of enzymes that catalyze the hydrolysis of starch, as well as other linear and branched 1, 4-glycosidic oligo- and polysaccharides. Alpha-amylases can be applied in the initial stage of starch processing (liquefaction), wet corn milling, alcohol production, as cleaning agents in detergent matrices, for starch desizing, for the baking industry and the beverage industry, for drilling processes in oil mines, for deinking processes in recycled paper, and for animal feed.

[0008] Although the currently available alpha-amylases have achieved some success in the above application fields, in recent years, with the changes in the process conditions of the starch raw material processing industry, the alcohol industry, the enzyme preparation industry is required to continuously update and improve the types of enzymes to meet the industrial needs. For example, in the process of the starch saccharification industry, starch is generally first liquefied using amylase, and then saccharifying enzyme is added for saccharification to produce glucose.

[0009] However, the currently available amylases are suitable for a pH range of 6.0-6.5, with an optimum pH of 6.0, and are inactivated below a pH of 5.0, while the optimum pH for the saccharification step is about 4.5. Moreover, the current sugar production process widely uses jet liquefaction, with a jet temperature generally at 105-108°C, and a 5-6 min residence time in the intermediate high temperature holding tank. Therefore, after completing the step of starch liquefaction, the pH and temperature need to be repeatedly adjusted before adding saccharifying enzyme, causing complexity in production and environmental problems.

[0010] In traditional white wine production, due to incomplete solid-state fermentation, there is generally more than 10% residual starch in the distiller's grains, and the pH in the distiller's grains is very low. When the distiller's grains are returned to the pit for fermentation, the acidic pH environment is not suitable for the action of alpha-amylase.

[0011] In summary, the current alpha-amylase is still difficult to meet the requirements of the starch raw material processing industry for high temperature resistance, acid resistance, and high activity, and cannot be well adapted to the application of the deep processing industry of starch raw materials such as sugar production, brewing, alcohol production, and organic acid production. Therefore, it is of great significance to use genetic engineering to modify alpha-amylase and develop alpha-amylase that can further improve the resistance to high temperature and / or acid, for the deep processing industry of starch raw materials.

[0012] SUMMARY

[0013] The present application aims to solve at least one of the above technical problems in the prior art. To this end, the present application proposes an alpha-amylase mutant and its application.

[0014] The present application aims to provide an alpha-amylase mutant with high temperature resistance and / or acid resistance. The present application uses a computer-aided method to predict the disulfide bond site generated in the structure of the amylase according to the spatial structure of the parent alpha-amylase and the functional requirements, and finally analyzes the influence of the mutation of the amino acid site capable of forming a disulfide bond on the formation of hydrogen bonds and salt bridges inside the enzyme molecule to determine the site for introducing a disulfide bond in the structure of the amylase protein. The alpha-amylase mutant provided by the present application has good high temperature resistance and / or acid resistance, and can better meet the needs of the deep processing industry of starch as raw material such as sugar making, brewing, alcohol, and organic acid.

[0015] The present application provides an alpha-amylase mutant.

[0016] Specifically, the alpha-amylase mutant has alpha-amylase activity, and compared with the parent alpha-amylase having the amino acid sequence shown in SEQ ID NO: 1, the alpha-amylase mutant has a disulfide bond site pair mutation, and the disulfide bond site pair has the following characteristics:

[0017] i. not in the active site of the alpha-amylase mutant in the range;

[0018] ii. the difference in amino acid site number is greater than 10;

[0019] iii. the distance between the two cysteine SG atoms forming the disulfide bond is within ;

[0020] iv. the Chi3 angle is 60°<Chi3<120° or -60°>Chi3>-120°.

[0021] The alpha-amylase mutant has improved properties compared with the parent alpha-amylase having the amino acid sequence shown in SEQ ID NO: 1, and the improved properties include increased pH stability and / or increased thermal stability.

[0022] In some embodiments of the present application, the pH stability includes obvious acid resistance under storage at pH 4.0 for 2h.

[0023] In some embodiments of the present application, the increased thermal stability includes increased stability at 70-99℃, and / or, after dilution by 10 times with a buffer solution at pH 4.5, enhanced stability after heat treatment at 95℃.

[0024] In some embodiments of the present application, the Chi3 angle of the pair of disulfide bond sites is 65° < Chi3 < 120° or the Chi3 angle is -65° > Chi3 > -120°.

[0025] In some embodiments of the present application, the alpha-amylase mutant has a mutation of the pair of disulfide bond sites as shown in any one of (1) to (43) as compared to a parent alpha-amylase having an amino acid sequence as shown in SEQ ID NO: 1:

[0026] (1) Q97C, D227C; (2) P346C, K381C; (3) A2C, E416C; (4) E120C, S131C; (5) N127C, G192C; (6) D184C, K238C; (7) P243C, T281C; (8) Y266C, N291C; (9) V325C, A348C; (10) W183C, D195C; (11) G66C, T76C; (12) Y99C, G228C; (13) Q356C, Q397C; (14) T140C, A200C; (15) M204C, F241C; (16) V221C, T253C; (17) D18C, S53C; (18) Y60C, G109C; (19) Y394C, V414C; (20) W183C, N193C; (21) S297C, D341C; (22) D286C, L313C; (23) G301C, D428C; (24) A110C, W139C; (25) E412C, G439C; (26) V116C, A138C; (27) A27C, A90C; (28) G108C, Y199C; (29) S433C, K469C; (30) E120C, R174C; (31) P384C, T451C; (32) T410C, M436C; (33) G20C, G79C; (34) G408C, L425C; (35) F12C, P44C; (36) L22C, Y78C; (37) L289C, T312C; (38) T410C, A423C; (39) D286C, T323C; (40) L450C, V479C; (41) V116C, W158C; (42) M314C, E357C; (43) Q10C, W42C.

[0027] The present application also provides a nucleic acid molecule.

[0028] In particular, a nucleic acid molecule encoding the above alpha-amylase mutant.

[0029] The application also provides a recombinant expression vector.

[0030] In particular, the recombinant expression vector comprises the nucleic acid molecule.

[0031] In some embodiments of the application, the carrier of the recombinant expression vector is a plasmid; preferably, the plasmid comprises a pBE-S plasmid.

[0032] The application also provides a recombinant bacterium.

[0033] In particular, the recombinant bacterium comprises the nucleic acid molecule or the recombinant expression vector.

[0034] In some embodiments of the application, the recombinant bacterium is selected from an Escherichia coli cell or a Bacillus cell.

[0035] The application also provides an enzyme-containing composition.

[0036] In particular, the enzyme-containing composition comprises the alpha-amylase mutant.

[0037] The application also provides the use of the alpha-amylase mutant.

[0038] In particular, the alpha-amylase mutant is used in the production of sugar syrup and / or fermentation products, such as sugar production, brewing, or other deep processing of starch as raw material.

[0039] In some embodiments of the application, the process for producing sugar syrup and / or fermentation products comprises the following steps: (a) liquefying starch-containing material in the presence of the alpha-amylase mutant; (b) saccharifying the liquefied material; and (c) fermenting with a fermenting organism.

[0040] Compared with the prior art, the application has the following beneficial effects:

[0041] Compared with the parent alpha-amylase, the alpha-amylase mutant provided by the application has significantly improved heat resistance and / or acid resistance, most of the alpha-amylase mutants have excellent acid resistance and heat resistance, and have higher catalytic activity for starch hydrolysis, which can better meet the needs of the deep processing industry of starch as raw material, such as sugar production, brewing, alcohol, organic acid, etc. BRIEF DESCRIPTION OF DRAWINGS

[0042] FIG. 1 is a schematic diagram of the Chi3 angle in the embodiments of the application;

[0043] FIG. 2 is a sequence homology comparison diagram of the parent alpha-amylase and PDB ID: 4UZU;

[0044] FIG. 3 is a graph of the heat resistance test results of the alpha-amylase mutant;

[0045] Figure 4 is a graph of the results of acid resistance testing of the alpha-amylase mutants;

[0046] Figure 5 is a graph of the results of thermal stability testing of the alpha-amylase mutants under acidic conditions;

[0047] Figure 6 is a graph of the results of liquefaction experiments of corn starch;

[0048] Figure 7 is a graph of the results of liquefaction experiments of corn powder for alcohol. DETAILED DESCRIPTION

[0049] In order to make the skilled person more clearly understand the technical solutions described in the present application, the following examples are given for illustration. It should be noted that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0050] The biological materials, reagents or devices used in the following examples, if not specifically stated, can be obtained from conventional commercial channels, or can be obtained by known methods. The molecular biology experimental methods not specifically described in the following examples are carried out according to the specific methods listed in the book "Molecular Cloning Laboratory Guide" (third edition) by J. Sambrook, or according to the instructions of the reagent kit and product.

[0051] Definitions

[0052] Alpha-amylase: The term "alpha-amylase" means a 1,4-alpha-D-glucan glucanohydrolase (EC. 3.2.1.1) that catalyzes the hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides. Alpha-amylase activity can be determined using methods known in the art, such as the iodine test method in "Alpha-amylase preparation GB / T 24401-2009".

[0053] According to the present application, a variant that shows an improved property under at least one test condition is considered to have an improved property compared to the parent alpha-amylase. For the purposes of the present application, in some embodiments of the present application, to meet the requirements of the deep processing industry of starch raw materials such as sugar, brewing, alcohol, organic acid, etc., the improved property is increased pH stability, for example, increased acid environment temperature resistance. In some embodiments of the present application, the improved property is increased thermal stability, for example, increased high temperature stability. In some embodiments of the present application, the improved property is both increased thermal stability and increased pH stability, for example, both increased acid environment temperature resistance and increased high temperature stability.

[0054] Some of the alpha-amylase mutants of the present application have increased stability at 70-99°C, are significantly acid tolerant when stored at pH 4.0 for 2h, and also have enhanced stability after heat treatment at 95°C in 10-fold diluted buffer at pH 4.5, compared to the parent alpha-amylase. Some of the alpha-amylase mutants of the present application have increased stability at 70-99°C, compared to the parent alpha-amylase. Some of the alpha-amylase mutants of the present application are significantly acid tolerant when stored at pH 4.0 for 2h, compared to the parent alpha-amylase. Some of the alpha-amylase mutants of the present application also have enhanced stability after heat treatment at 95°C in 10-fold diluted buffer at pH 4.5, compared to the parent alpha-amylase. It is understood that the alpha-amylase mutants of the present application have at least one advantage of heat tolerance or acid tolerance, compared to the parent alpha-amylase.

[0055] Parent or parent alpha-amylase: The term "parent" or "parent alpha-amylase" means an alpha-amylase having the amino acid sequence as set forth in SEQ ID NO: 1.

[0056] Variant, mutant: The term "variant" "mutant" means a polypeptide having alpha-amylase activity comprising a mutation (i.e., a substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to the parent alpha-amylase set forth in SEQ ID NO: 1. A substitution means the replacement of the amino acid occupying a position with a different amino acid; a deletion means the removal of the amino acid occupying a position; and an insertion means the addition of an amino acid after the amino acid occupying a position, with the proviso that the amino acid occupying the position is adjacent and immediately followed by the amino acid. The mutants of the present application have at least 20%, e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the alpha-amylase activity of the mature polypeptide of SEQ ID NO: 1.

[0057] Coding sequence: The term "coding sequence" or "coding region" means a polynucleotide sequence, which specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by the open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG, and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence can be a sequence of genomic DNA, cDNA, synthetic polynucleotide, and / or recombinant polynucleotide.

[0058] Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polypeptide. Control sequences are operabiy linked to polynucleotides encoding polypeptides and can be native or foreign to those polynucleotides, and are native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator sequence. The control sequences can provide multiple links for the introduction of specific restriction sites used in the ligation of polynucleotides encoding polypeptides.

[0059] Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modifications, translation, post-translational modifications, and secretion. Expression can be measured, for example, to detect increased expression, by techniques known in the art, measuring levels of mRNA and / or translated polypeptide.

[0060] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operabiy linked to control sequences providing for its expression.

[0061] Fermentable medium: The term "fermentable medium" or "fermentation medium" refers to a medium comprising one or more (e.g., two, several) sugars, such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides, wherein the medium is capable of being partially converted (fermented) by a host cell into a desired product, such as ethanol. In some cases, the fermentation medium is derived from a natural source, such as sugar cane, starch, or cellulose; and can be pre-treated for enzymatic hydrolysis (saccharification) from such sources. The term fermentation medium is understood herein to refer to a medium prior to the addition of a fermenting organism, for example, a medium resulting from a saccharification process, and a medium used in a simultaneous saccharification and fermentation process (SSF).

[0062] For the purposes described herein, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., Trends Genet. 2000, 16, 276-277) (preferably version 3.0.0 or later), with optional parameters set to gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the Needle program is used as the percent identity, and is calculated as the number of identical matched amino acid residues divided by the length of the reference sequence, multiplied by 100 (using the -nobrief option).

[0063] (identical residues x 100) / (length of the reference sequence - total number of gaps in the alignment).

[0064] Variant nomenclature: For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid residues in other alpha-amylases, the amino acid sequence of the alpha-amylase mutant is aligned with the polypeptide disclosed in SEQ ID NO: 1, and the amino acid position numbering corresponding to any of the amino acid residues in the mature polypeptide disclosed in SEQ ID NO: 1 is determined using the Needleman-Wunsch algorithm as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later).

[0065] In describing the mutants, accepted IUPAC single letter or three letter amino acid abbreviations are used for ease of reference.

[0066] Substitution: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. For example, a substitution of glutamine for cysteine at position 97 is designated "Gln97Cys" or "Q97C".

[0067] Amino acid site number difference: According to the present application, "amino acid site number difference" refers to the difference between two amino acid site number values, used to express the distance between two amino acids in the sequence, that is, to express how many amino acids exist between the two amino acids. In the embodiments of the present application, the amino acid site number difference of the disulfide bond site pair is greater than 10, which means that the amino acids between the disulfide bond site pair are more than 10 amino acids apart. For example, the disulfide bond site pair Q97C, D227C, the amino acids between the disulfide bond site pair are 130 amino acids apart.

[0068] Cysteine SG atom: that is, the gamma sulfur atom of cysteine. The mercapto groups of two cysteine residues in a peptide chain undergo an oxidation reaction, so that a covalent bond is formed between the gamma sulfur atoms, that is, a disulfide bond. In some embodiments of the present application, the distance between the two cysteine SG atoms forming the disulfide bond is within 2.5 A; in some embodiments of the present application, the distance between the two cysteine SG atoms forming the disulfide bond is within 2.0 A; in some embodiments of the present application, the distance between the two cysteine SG atoms forming the disulfide bond is within 1.5 A; in some embodiments of the present application, the distance between the two cysteine SG atoms forming the disulfide bond is within 1.0 A; in some embodiments of the present application, the distance between the two cysteine SG atoms forming the disulfide bond is within 0.5 A.

[0069] Chi3 angle: abbreviation "χ3", "χ" is the 22nd letter in the Greek alphabet. Chi3 angle refers to the dihedral angle defined by the twist of Cis-Si-Sj-Cjs, as shown in Figure 1. In the embodiments of the present application, the Chi3 angle of the disulfide bond site pair is 60° < Chi3 < 120° or -60° > Chi3 > -120°. In some embodiments of the present application, the Chi3 angle of the disulfide bond site pair is 65° < Chi3 < 120° or the Chi3 angle is -65° < Chi3 < -120°.

[0070] Example 1: Rational design of amylases

[0071] 1.1 De novo modeling

[0072] ​​​​​​The parent alpha-amylase gene sequence (SEQ ID NO: 1) of the present application has the lowest proportion of essential cysteines forming disulfide bond pairs, only one, with potential for disulfide bond design. Sequence searching and homology alignment using the BLAST server in the GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank / ) showed that Alpha-amylase (PDB ID: 4UZU) also derived from Geobacillus stearothermophilus has 98.34% homology with the amylase sequence of the present application. The parent alpha-amylase (SEQ ID NO: 1) of the present application belongs to CAZY family GH13, which is one of the most in-depth studied in all glycoside hydrolase families. Mechanically, alpha-amylase hydrolyzes alpha-1, 4-glycosidic bonds in starch through a covalent glycosyl enzyme intermediate. This mechanism is very clear and requires a catalytic nucleophile, an acid / base, and an "auxiliary" residue to complete. As shown in Figure 2, the parent alpha-amylase (SEQ ID NO: 1) of the present application has 98.34% sequence homology with PDB ID: 4UZU, and the key catalytically active site can be determined as the catalytic triad consisting of Asp232, Glu262, and Asp329. A major feature of the design of disulfide bonds in the present application is that the disulfide bond site pair is not in the active site of the alpha-amylase mutant range, thereby avoiding affecting the substrate catalytic activity of the alpha-amylase.

[0073] AlphaFold2 is a deep neural network learning model based on the Transformer architecture, providing end-to-end protein structure prediction with very high modeling accuracy; based on the AlphaFold2 computing service built on this platform, the protein sequence is input to predict the three-dimensional structure of the enzyme. After running, five structure files are generated, of which ranked_0 is the highest scoring structure model with a pLDDT value of 98.55, and the difference in RMSD from the resolved structure 4UZU is 0.715. Therefore, the most reliable structure model is selected for further analysis.

[0074] 1.2 Structure conformation rationality evaluation

[0075] ​The structural model obtained by the foregoing step is evaluated for protein structure conformation rationality using the SAVES v6.0 (https: / / saves.mbi.ucla.edu / ) scoring program, and is presented in the form of a Ramachandran plot. In general, the proportion of amino acid residues falling within the allowed region and the maximum allowed region is higher than 90% of the entire protein, and it can be considered that the conformation of the model conforms to the rules of stereochemistry. A model with more than 90% of residues in the most favorable region is considered to be of high quality, and a reliable model should have more than 80% of residues. The results of the evaluation are as follows: the overall structure quality score of the model predicted by AlphaFold2 is 95.7717, the Ramachandran plot shows that 87.0% of the residues in the predicted model are in the most favorable region, and the results show that the model predicted by AlphaFold2 has very high precision and comprehensive and intuitive reasonable conformation. Therefore, the model predicted by AlphaFold2 is selected for subsequent experiments.

[0076] 1.3 Disulfide bond design

[0077] To explore a reasonable and effective disulfide bond pair and improve the thermostability of amylase, the method uses a deep learning algorithm model to screen disulfide bond pairs under certain conditions: a) the disulfide bond site pair is not within the 5A range of the active region; b) the amino acid sequence difference of the disulfide bond site pair is greater than 10; c) the distance between the SG atoms of the cysteine of the disulfide bond site pair after mutation is within 5A; d) the Chi3 angle of the disulfide bond site pair after mutation is between 80<Chi3<120 or -80>Chi3>-120. The final summary of the calculation results is shown in Table 1. A total of 43 pairs of disulfide bond candidate mutation libraries are screened under the above conditions, which can be verified by experiments;

[0078] Table 1

[0079] Example 2, construction and expression of amylase and its variants

[0080] 2.1 Materials and reagents

[0081] 2.1.1 Strains and vectors:

[0082] The expression strain containing the amylase amyS gene and variants, Escherichia coli TOP10, Bacillus sp. WB600, vector pBE-S, antibiotics, kanamycin, ampicillin were purchased from Shanghai Generay Biotech Co., Ltd.

[0083] 2.1.2 Enzymes and kits:

[0084] SuperFidelity 2x Master Mix PCR polymerase, restriction enzymes were purchased from NEB company, plasmid extraction kit, purification kit were purchased from Shanghai Sangon Biotech Co., Ltd.

[0085] 2.1.3 Culture medium:

[0086] The culture medium of E. coli was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). The LB+Amp medium was LB medium added with ampicillin at a final concentration of 100 μg / mL; the culture medium of Bacillus was TB medium (11.8 g / L tryptone, 23.6 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4), and the TB+Kan medium was TB medium added with kanamycin at a final concentration of 20 μg / mL.

[0087] 2.1.4 Chemical reagents:

[0088] Soluble starch was purchased from Huzhou Zhanwang Pharmaceutical Co., Ltd., and other reagents were purchased from Guangzhou Chemical Reagent Factory.

[0089] 2.2 Amylase activity determination method

[0090] The amylase enzyme activity was determined by the iodine method in the national standard “α-amylase preparation GB / T 24401-2009”. The α-amylase preparation can randomly cut the α-1, 4 glucoside bond in the starch molecular chain into short chain dextrin, a small amount of maltose and glucose, and the blue-purple characteristic reaction of starch to iodine gradually disappears, showing brown red. The color disappearance speed is related to the enzyme activity, and the enzyme activity can be calculated by the absorbance after reaction.

[0091] 1 g of solid enzyme powder or 1 mL of liquid enzyme, under the condition of 70°C and pH 6.0, 1 min to liquefy 1 mg of soluble starch, which is 1 enzyme activity unit, expressed as “U / g or U / mL”.

[0092] The parent sequence and its species were disclosed, and the subsequent site number was based on the parent sequence. The α-amylase (amyS) gene of the mutant Geobacillus stearothermophilus (Genebank, AGK25234.1) was disclosed, and the amino acid sequence was shown in SEQ ID NO. 1.

[0093] 2.2 Expression of amylase and its variants

[0094] 2.2.1 Synthesis of high-temperature amylase amyS gene and construction of vector

[0095] Mutated high-temperature alpha-amylase (amyS) gene of Geobacillus stearothermophilus (Genebank, AGK25234.1), the amino acid sequence of which corresponds to alpha-amylase is shown in SEQ ID NO. 1 (denoted as parent alpha-amylase).

[0096] NdeI and SalI enzyme cutting sites were introduced at the 5' end and 3' end of the high-temperature alpha-amylase amyS gene respectively, and were connected to the pUC57-amp vector. The pUC57-amyS was inoculated into LBA culture medium and cultured overnight. The plasmid was extracted, cut with NdeI and SalI, and the target gene fragment was recovered by gel cutting and connected to the expression vector pBE-S to obtain the expression vector pBE-amyS.

[0097] 2.2.2 Mutation introduction of disulfide bond

[0098] The first cysteine was introduced into the amylase amyS gene using the pBE-amyS as the template by PCR method. After DpnI (DpnI endonuclease) digestion, the target fragment was recovered by gel cutting, and the decomposed product was transformed into E. coli TOP10 competent cells by chemical transformation heat shock method. The recombinant transformants were verified by bacterial liquid PCR, and the plasmid of the correct transformants was extracted for sequencing to determine the corresponding mutant, thereby obtaining the expression vector pBE-amyS-M1-1. The second cysteine was introduced into the amylase amyS gene using the pBE-amyS-M1-1 as the template by PCR method. After DpnI (DpnI endonuclease) digestion, the target fragment was recovered by gel cutting, and the decomposed product was transformed into E. coli TOP10 competent cells by chemical transformation heat shock method. The recombinant transformants were verified by bacterial liquid PCR, and the plasmid of the correct transformants was extracted for sequencing to determine the corresponding mutant, thereby obtaining the expression vector pBE-amyS-M1 containing a pair of cysteine mutations. The chemical transformation method was used to transform into WB600 chemical transformation competent cells to obtain Bacillus recombinant transformants.

[0099] 2.2.3 Shake flask fermentation of mutant strains containing disulfide bond

[0100] The recombinant transformants obtained in step 2 were picked one by one with toothpicks into 250 mL shake flasks, 40 mL of TB culture medium was added to each shake flask, and the culture was incubated at 37°C, 220 rpm, 85% humidity for about 48 h. The supernatant was obtained by centrifugation, and the parent alpha-amylase gene and the mutant were purified by affinity chromatography purification method to prepare enzyme solution of each alpha-amylase mutant.

[0101] Product effect test

[0102] 1. Detection of optimal reaction pH of α-amylase mutants

[0103] The enzyme activity of α-amylase was measured at 70°C under the conditions of pH 4.0, pH 4.5, pH 6.0 and pH 7.0, respectively. The results are shown in Table 2.

[0104] The enzyme activity of the parent α-amylase measured under the condition of pH 6.0 was used as a control to calculate the relative enzyme activity of each mutant enzyme under different pH conditions.

[0105] The relative enzyme activity refers to the ratio of the activity of an enzyme under certain conditions to the activity of the enzyme under the optimal reaction conditions. The calculation formula of the relative enzyme activity is as follows: relative enzyme activity = (activity of the enzyme being measured / activity of the enzyme under the optimal reaction conditions) x 100%.

[0106] Table 2

[0107] As shown in Table 2, the optimal reaction pH of the α-amylase mutants is 6, and most of the α-amylase mutants also exhibit high enzyme activity at pH 4 and pH 5.

[0108] 2. Detection of optimal reaction temperature of α-amylase mutants

[0109] The enzyme activity of the parent α-amylase and the α-amylase mutants was measured at 60°C, 70°C, 80°C and 90°C under the condition of pH 6.0, respectively. The results are shown in Table 3.

[0110] The enzyme activity of the parent α-amylase measured at 70°C was used as a control to calculate the relative enzyme activity of each enzyme under different temperature conditions.

[0111] The relative enzyme activity refers to the ratio of the activity of an enzyme under certain conditions to the activity of the enzyme under the optimal reaction conditions. The calculation formula of the relative enzyme activity is as follows: relative enzyme activity = (activity of the enzyme being measured / activity of the enzyme under the optimal reaction conditions) x 100%.

[0112] Table 3

[0113] As shown in Table 3, the optimal reaction temperature of the α-amylase mutants is 70°C, and the mutants also exhibit high enzyme activity at 80°C and 90°C.

[0114] 3. Heat resistance of α-amylase mutants

[0115] The purified parent alpha-amylase and alpha-amylase mutant enzyme solution was heat treated at 99°C for 30 minutes, then cooled on ice, and then enzyme activity was detected at 70°C, pH 6.0. The enzyme activity retention rate was calculated, and the calculation formula was as follows: enzyme activity retention rate = (enzyme activity after heat storage / enzyme activity before heat storage) x 100%. The heat resistance test results are shown in Table 4 and Figure 3. In Figure 3, the parent alpha-amylase is denoted as parent, and alpha-amylase mutants 1-43 are denoted as 1-43 (hereinafter the same).

[0116] Table 4

[0117] As can be seen from Table 4 and Figure 3, the enzyme activity retention rates of alpha-amylase mutants 1-7, alpha-amylase mutant 9, alpha-amylase mutant 12, alpha-amylase mutants 14-22, alpha-amylase mutants 26-31, alpha-amylase mutants 33-36, alpha-amylase mutant 38, alpha-amylase mutants 41-43 were significantly higher than that of the parent alpha-amylase after being treated at 99°C for 30 minutes, and the heat resistance was significantly improved.

[0118] 4. Acid resistance of alpha-amylase mutants

[0119] The purified parent alpha-amylase and alpha-amylase mutant enzyme solution was stored at pH 4.0 for 2h, and then enzyme activity was detected at 70°C, pH 6.0. The enzyme activity retention rate was calculated, and the calculation formula was as follows: enzyme activity retention rate = (enzyme activity after heat storage / enzyme activity before heat storage) x 100%. The acid resistance test results are shown in Table 5 and Figure 4.

[0120] Table 5

[0121] As can be seen from Table 5 and Figure 4, the enzyme activity retention rates of alpha-amylase mutants 1-4, alpha-amylase mutant 8, alpha-amylase mutant 10, alpha-amylase mutants 12-19, alpha-amylase mutants 21-26, alpha-amylase mutants 28-42 were significantly higher than that of the parent alpha-amylase after being stored at pH 4.0 for 2h, and the acid resistance was significantly improved.

[0122] 5. Heat stability detection of alpha-amylase mutants under acidic conditions

[0123] The purified parent alpha-amylase and alpha-amylase mutant enzyme solution was diluted 10 times with a buffer at pH 4.5, and after heat treatment at 95°C for 2 minutes, the enzyme activity was detected at 70°C and pH 6.0. The enzyme activity retention rate was calculated according to the following formula: enzyme activity retention rate = (enzyme activity after heat storage / enzyme activity before heat storage) x 100%. The results of the heat stability test are shown in Table 6 and Figure 5.

[0124] Table 6

[0125] As can be seen from Table 6 and Figure 5, the enzyme activity retention rates of alpha-amylase mutants 1-9, alpha-amylase mutants 11-13, alpha-amylase mutants 15-31, alpha-amylase mutants 33-38, and alpha-amylase mutants 41-43 were still higher than that of the parent alpha-amylase after dilution 10 times with a buffer at pH 4.5 and heat treatment at 95°C for 2 minutes, and the heat stability of the alpha-amylases under acidic conditions was significantly improved.

[0126] 6. Liquefaction experiment of corn starch

[0127] A 34% corn starch solution was prepared, and the pH was adjusted to 4.5 with dilute sulfuric acid. 500 g of the corn starch solution was added to a liquefaction tank, and 15 U / g of the parent alpha-amylase and each alpha-amylase mutant enzyme solution was added, respectively. The reaction process was set as follows: 50°C for 1 min, 70°C for 5 min, and 95°C for 70 min. After the reaction was completed, the reducing sugar content of each experimental group was detected, and the results are shown in Table 7 and Figure 6.

[0128] Table 7

[0129] As can be seen from Table 7 and Figure 6, the use of the enzyme solution of alpha-amylase mutants 1-9, alpha-amylase mutants 11-13, alpha-amylase mutants 15-23, alpha-amylase mutants 25-30, alpha-amylase mutants 33-35, alpha-amylase mutants 37-38, and alpha-amylase mutants 41-43 for the experiment of corn starch liquefaction had a better liquefaction effect than the parent alpha-amylase, and the reducing sugar content after liquefaction was higher than that of the parent alpha-amylase.

[0130] 7. Liquefaction experiment of corn powder for alcohol

[0131] The corn syrup with dry substance concentration of 25% (DS=25%) was configured, and the pH was adjusted to 4.8 by dilute sulfuric acid. 500 g of the corn syrup was added into a liquefaction tank, and 15 U / g of the parent alpha-amylase and each alpha-amylase mutant enzyme solution was added. The reaction process was set as: 60℃ for 30 min-88℃ for 120 min-30℃ for 30 min. After the reaction was completed, the reducing sugar content was detected, and the results are shown in Table 8 and FIG. 7.

[0132] Table 8

[0133] As shown in Table 8 and FIG. 7, the corn syrup was liquefied by using the enzyme solution of the alpha-amylase mutants 1-12, the alpha-amylase mutants 14-16, the alpha-amylase mutants 19-30, the alpha-amylase mutants 33-35, the alpha-amylase mutants 38-39, and the alpha-amylase mutants 41-43, and the liquefaction effect was better than that of the parent alpha-amylase, and the reducing sugar content after liquefaction was higher than that of the parent alpha-amylase.

[0134] In summary, compared with the parent alpha-amylase, the alpha-amylase mutants provided in the embodiments of the present application have at least one advantage of heat resistance or acid resistance, and most of the alpha-amylase mutants have excellent acid resistance and heat resistance, and have higher catalytic activity of starch hydrolysis, which can better meet the needs of the deep processing industry of starch as raw material such as sugar making, brewing, alcohol, organic acid, etc.

[0135] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as the limitation of the patent scope of the present application. It should be noted that for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the patent protection scope of the present application should be subject to the appended claims.

Claims

1. An alpha-amylase mutant, characterized in that, The alpha-amylase mutant has a disulfide bond site pair mutation compared to a parent alpha-amylase having an amino acid sequence as set forth in SEQ ID NO: 1, the disulfide bond site pair has the following characteristics: i. is not in the active site of the alpha-amylase mutant in the range of 0°<Chi3<120° or -60°>Chi3>-120°. ii. the difference of amino acid site number is greater than 10; iii. the distance between the two cysteine SG atoms forming the disulfide bond is in the range of 2.0 to 2.5 A in the range of 0°<Chi3<120° or -60°>Chi3>-120°. iv. the Chi3 angle is 60°<Chi3<120° or -60°>Chi3>-120°.

2. The alpha-amylase mutant according to claim 1, characterized in that, The Chi3 angle of the disulfide bond site pair is 65°<Chi3<120° or the Chi3 angle is -65°>Chi3>-120°.

3. The alpha-amylase mutant according to claim 1, having improved properties compared to the parent alpha-amylase of the amino acid sequence shown in SEQ ID NO: 1, characterized in that, The improved properties include increased pH stability and / or increased thermal stability.

4. The alpha-amylase mutant according to claim 3, characterized in that, The pH stability includes acid resistance at pH 4.0 for 2h.

5. The alpha-amylase mutant according to claim 3, characterized in that, The increased thermal stability includes increased stability at 70-99℃, and / or, enhanced stability after heat treatment at 95℃ after 10-fold dilution with a buffer at pH 4.

5.

6. The alpha-amylase mutant according to any one of claims 1 to 3, characterized in that, The alpha-amylase mutant has a disulfide bond site pair mutation as set forth in any one of (1)-(43) compared to a parent alpha-amylase having an amino acid sequence as set forth in SEQ ID NO: 1: (1) Q97C, D227C; (2) P346C, K381C; (3) A2C, E416C; (4) E120C, S131C; (5) N127C, G192C; (6) D184C, K238C; (7) P243C, T281C; (8) Y266C, N291C; (9) V325C, A348C; (10) W183C, D195C; (11) G66C, T76C; (12) Y99C, G228C; (13) Q356C, Q397C; (14) T140C, A200C; (15) M204C, F241C; (16) V221C, T253C; (17) D18C, S53C; (18) Y60C, G109C; (19) Y394C, V414C; (20) W183C, N193C; (21) S297C, D341C; (22) D286C, L313C; (23) G301C, D428C; (24) A110C, W139C; (25) E412C, G439C; (26) V116C, A138C; (27) A27C, A90C; (28) G108C, Y199C; (29) S433C, K469C; (30) E120C, R174C; (31) P384C, T451C; (32) T410C, M436C; (33) G20C, G79C; (34) G408C, L425C; (35) F12C, P44C; (36) L22C, Y78C; (37) L289C, T312C; (38) T410C, A423C; (39) D286C, T323C; (40) L450C, V479C; (41) V116C, W158C; (42) M314C, E357C; (43) Q10C, W42C.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the alpha-amylase mutant of any one of claims 1 to 6.

8. A recombinant expression vector, characterized in that, comprising the nucleic acid molecule of claim 7.

9. The recombinant expression vector of claim 8, wherein, The vector of the recombinant expression vector is a plasmid; preferably, the plasmid comprises a pBE-S plasmid.

10. A recombinant bacterium, characterized in that, comprising the nucleic acid molecule of claim 7 or the recombinant expression vector of claim 8.

11. The recombinant bacteria of claim 10, wherein The recombinant bacteria are selected from the group consisting of E. coli and Bacillus.

12. An enzyme-containing composition comprising, The enzyme-containing composition comprises the alpha-amylase mutant of any one of claims 1 to 6.

13. Use of the alpha-amylase mutant of any one of claims 1 to 6 in the production of sugar syrup and / or alcohol.

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