Α-amylase mutant, and preparation method therefor and use thereof

Abstract

Provided in the present invention are an α-amylase mutant having improved enzyme activity, thermal stability and / or low pH stability, a polynucleotide encoding the α-amylase mutant, a recombinant expression vector containing the polynucleotide, a host cell expressing the α-amylase mutant, a composition containing the α-amylase mutant, and a preparation method therefor and the use thereof.

Description

Alpha-amylase mutants, methods for making and using the same

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application No. CN202410968595.3, filed on July 18, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of protein engineering, in particular to alpha-amylase mutants, methods for making and using the same. BACKGROUND

[0004] Alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, E.C. 3.2.1.1) is capable of hydrolyzing alpha-1,4-glucosidic bonds in starch molecules, thereby hydrolyzing starch into dextrin, oligosaccharide and monosaccharide. Alpha-amylase is one of the earliest, most widely used and largest enzyme preparation products in industry, accounting for about 25% of the global enzyme preparation market. Alpha-amylase can be used in the initial stage of starch processing (liquefaction) in food processing, as a cleaning agent in detergent matrix in household care, for paper desizing in the textile industry, and for making drug carriers and drug precursors in the pharmaceutical field.

[0005] In the starch sugar, citric acid, alcohol and other industries, starch is first liquefied by amylase, and then saccharified by saccharifying enzyme to produce glucose for subsequent production. However, amylase is easily inactivated above 100℃ or below pH 5.0, while the jet liquefaction temperature in the starch liquefaction process can reach above 110℃, and the optimal pH value of the subsequent saccharification step is about 4.5, so the starch sugar industry needs alpha-amylase that is resistant to high temperature and acid. Alpha-amylase BLA from Bacillus licheniformis (SEQ ID NO: 1) has been widely concerned due to its relatively excellent high-temperature and acid resistance. Novozymes disclosed in international patent application WO2002 / 010355 a mutant LE399 of hybrid alpha-amylase composed of N-terminal 1-37 amino acid residues from alpha-amylase from Bacillus amyloliquefaciens and C-terminal 40-483 amino acid residues from alpha-amylase from Bacillus licheniformis, which has improved stability under high temperature and low pH conditions compared to wild-type BLA. However, there is still a need for more new alpha-amylases with improved properties, i.e., alpha-amylases with higher stability under high temperature and low pH conditions. SUMMARY

[0006] The present invention aims to provide alpha-amylase mutants having improved enzyme activity, thermal stability and / or low pH stability, in particular having improved stability at high temperature, low pH conditions compared to LE399 (SEQ ID NO: 2), the amino acid positions described herein corresponding to the amino acid positions of SEQ ID NO: 1.

[0007] In one aspect, the present invention provides an alpha-amylase mutant comprising the following amino acid alterations relative to the amino acid sequence set forth in SEQ ID NO: 2: V3(AAPF), D114W, L134R, N172R, S187D, N188P, H247Y, and Q360C, and an amino acid substitution forming a non-native disulfide bond comprising a pair of amino acid substitutions selected from the group consisting of: N126C and G191C, A29C and R93C, and Q26C and S89C; wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1; and wherein the alpha-amylase mutant has amylase activity.

[0008] In some embodiments, the alpha-amylase mutant further comprises the amino acid substitutions H68W, F201Y, D416V, and R437W relative to the amino acid sequence set forth in SEQ ID NO: 2.

[0009] In some embodiments, the alpha-amylase mutant further comprises an amino acid substitution at position 205, which is H205D or H205Y, relative to the amino acid sequence set forth in SEQ ID NO: 2.

[0010] In some embodiments, the alpha-amylase mutant further comprises one or more amino acid substitutions selected from S148N, Y156H, K213T, N265G, H31S, and S320A, relative to the amino acid sequence set forth in SEQ ID NO: 2.

[0011] In some embodiments, the alpha-amylase mutant has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0012] In some embodiments, the alpha-amylase mutant comprises any one of the following combinations of amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 2:

[0013] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + K213T + H247Y + Q360C + D416V + R437W + Q26C + S89C;

[0014] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + K213T + H247Y + Q360C + D416V + R437W + A29C + R93C;

[0015] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + H205D + H31S + N265G;

[0016] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + Y156H + S148N + N265G;

[0017] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + K213T + H247Y + Q360C + D416V + R437W + N126C + G191C + Y156H + S148N + H205D + H31S;

[0018] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H247Y + Q360C + D416V + R437W + N126C + G191C + S320A + S148N + H205D;

[0019] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + S320A + Y156H + H31S;

[0020] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + K213T + H247Y + Q360C + D416V + R437W + N126C + G191C + S320A + Y156H + H205D + N265G;

[0021] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + N265G + H31S; and

[0022] V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + S148N + H31S + N265G.

[0023] In further aspects, the present application provides polynucleotides encoding the above-described alpha-amylase mutants, recombinant expression vectors comprising the polynucleotides, host cells comprising the above-described alpha-amylase mutants or the recombinant expression vectors. In some embodiments, the host cell is a bacterium. In some embodiments, the bacterium is from the genus Bacillus. In some embodiments, the host cell is B. subtilis or B. licheniformis.

[0024] In further aspects, the present application provides methods of making the above-described alpha-amylase mutants, comprising the steps of: (a) cultivating the host cell of the present application under conditions suitable for expression of the alpha-amylase mutant; and (b) recovering the expressed alpha-amylase mutant. The present application also provides alpha-amylase mutants obtained by the above-described methods.

[0025] In further aspects, the present application provides compositions comprising the above-described alpha-amylase mutants and one or more additional enzymes. In some embodiments, the one or more additional enzymes are selected from the group consisting of alpha-amylases, beta-amylases, cellulases, glucoamylases, hemicellulases, isoamylases, isomerases, lipases, phytases, proteases, and pullulanases.

[0026] In further aspects, the present application provides uses of the above-described alpha-amylase mutants and the above-described compositions for liquefying starch-containing material, for washing, for desizing textiles, and for producing bakery products. DETAILED DESCRIPTION

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0028] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict between the present specification (including that of definitions) and that of a reference, the present specification controls. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0029] The terms "about" and "approximately" when used with a numerical value, generally mean that the value and the entire value of the variable are within the measurement or experimental error (e.g., 95% confidence interval of the mean) or within a wider range of the specified value (e.g., ±5% or ±10%).

[0030] The terms "comprising" or variations such as "comprise" or "comprises" or "including" or variations such as "includes" or "include" or "containing" or variations such as "contains" or "contain", are to be construed as incorporating by reference all such steps or elements, but not to exclude any other steps or elements. "Consisting essentially of" means excluding any additional step or component of any kind. "Consisting of means excluding any additional step or component of any kind. The term "comprising" also includes the term "consisting of" and "consisting essentially of".

[0031] When referring to a numerical range, the specific values of the upper and lower limits of the range are to be construed as having been specifically and individually stated. Also, any intermediate range described within the numerical range is to be construed as having been specifically excluded from the numerical range. Further, any intermediate ranges, sub-ranges, and all individual values described in the numerical range can be excluded from the numerical range.

[0032] The term "and / or" should be understood to mean either of the two elements or a combination of the two elements.

[0033] The term "mutant" refers to a polypeptide having one or more insertions, deletions, and / or substitutions of amino acids relative to a parent polypeptide. A substitution refers to replacing an amino acid occupying a position with a different amino acid; a deletion refers to removing an amino acid occupying a position; and an insertion refers to adding one or several (e.g., 1-5) amino acids adjacent to an amino acid occupying a position. The mutant retains at least one activity of the parent polypeptide, but can vary in the level of activity, e.g., the mutant can be unchanged or improved in at least one activity or property relative to the parent polypeptide.

[0034] The term "non-native disulfide bond" refers to a disulfide bond that is not present in native alpha-amylase or alpha-amylase mutant LE399, which is introduced by protein engineering, e.g., by changing one or more amino acids to cysteine residues.

[0035] The term "parent" refers to a polypeptide that is changed to produce a mutant, which can be a naturally occurring (wild-type) polypeptide or a mutant thereof. In some embodiments, the parent polypeptide is mutant LE399 of a hybrid alpha-amylase consisting of N-terminal 1-37 amino acid residues from alpha-amylase from B. amyloliquefaciens and C-terminal 40-483 amino acid residues from alpha-amylase from B. licheniformis, which has an amino acid sequence shown in SEQ ID NO: 2, and the activity or property thereof can include basic alpha-amylase activity, and can further include thermal stability and / or pH stability, etc.

[0036] The term "alpha-amylase activity" refers to an activity of hydrolyzing alpha-1,4- glycosidic bonds in a starch molecule to generate dextrin, oligosaccharide, and monosaccharide.

[0037] The term "stability" refers to a property of an alpha-amylase or a mutant thereof that maintains a certain amount of enzyme activity under certain environmental conditions during storage, use, or treatment, etc. For example, "thermal stability" or "stability at high temperature" refers to an alpha-amylase or a mutant thereof that maintains a certain amount of enzyme activity after a period of time at a certain temperature or at a higher temperature; "low pH stability" refers to an alpha-amylase or a mutant thereof that maintains a certain amount of enzyme activity after a period of time at a lower pH. In a comparison involving, for example, stability, "increased stability" refers to an alpha-amylase or a mutant thereof that has a higher residual enzyme activity under a certain environment (e.g., after a period of time at a high temperature and / or at a low pH) compared to other alpha-amylase mutants and / or parent alpha-amylases and / or wild-type alpha-amylases.

[0038] The term "residual enzyme activity" refers to a percentage of enzyme activity of an alpha-amylase or a mutant thereof under a certain environment (e.g., after a period of time at a certain temperature and / or at a certain pH, preferably at a high temperature and / or at a low pH) relative to the enzyme activity of the enzyme without the corresponding treatment, wherein the enzyme activity before and after the treatment is determined by the same method.

[0039] The term "wild-type" enzyme refers to an enzyme expressed by a naturally occurring organism or cell (e.g., a bacterium or a fungus). The "naturally occurring" refers to not being mutagenized or genetically manipulated by humans.

[0040] The term "recombinant" when used with reference to a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, a recombinant cell expresses genes that it does not normally express, or expresses genes in an amount different from that which it normally expresses.

[0041] The term "heterologous" means that the nucleic acid or polypeptide is introduced into a cell in an artificial manner and / or is not naturally found in the cell in which it is present.

[0042] The term "expression" in the context of the present application includes any step involved in the production of an alpha-amylase mutant of the application, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0043] The term "expression vector" is defined herein as a linear or circular DNA molecule comprising a polynucleotide encoding a protein, such as an alpha-amylase mutant of the application, and which is operably linked to additional nucleotides (e.g., control sequences) that provide for its expression.

[0044] The term "host cell" includes a cell that is transformed, transfected, transduced or the like with a nucleic acid construct or expression vector comprising a polynucleotide encoding an alpha-amylase mutant and progeny of the same that is not identical to the parent cell due to mutations that occur during replication and expresses an alpha-amylase mutant of the application from the cell.

[0045] The term "control sequence" refers to nucleic acid sequences necessary for expression of a polynucleotide encoding an alpha-amylase mutant of the application. Each control sequence can be heterologous (i.e., foreign) to the polynucleotide encoding the alpha-amylase mutant or homologous (i.e., native) to the polynucleotide encoding the alpha-amylase mutant. These control sequences include, but are not limited to, a leader, a polyadenylation sequence, a pre-pro peptide sequence, a promoter, a signal peptide sequence and a transcription terminator. In some embodiments, the control sequences include at least a promoter and transcriptional and translational stop signals.

[0046] The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. A mature polypeptide may, for example, be free of a signal peptide after N-terminal processing.

[0047] The term "signal peptide" refers to a peptide that is operably linked in-frame to the amino terminus of a mature polypeptide and directs secretion of the encoded polypeptide.

[0048] The term "sequence identity" is used to describe the relatedness of two amino acid sequences or two nucleotide sequences. "Sequence identity" means the percentage of nucleic acid base or amino acid residue positions in the two sequences that are identical (i.e., 100% homologous) when aligned for maximum sequence comparison over a comparison window. The two sequences can be aligned for optimal comparison by adding or deleting a number of positions to or from one sequence. The percentage sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the results by 100 to yield the percentage of sequence identity. Sequence identity between two amino acid sequences can be determined using available local alignment tools (e.g., BLAST) or global alignment tools (e.g., using the Needleman-Wunsch algorithm).

[0049] "Position corresponding to" is the position in the queried amino acid sequence that corresponds to a particular position in the reference amino acid sequence when the queried amino acid sequence is optimally aligned with the reference amino acid sequence. In the present invention, the positions of the amino acids in the described alpha-amylase mutants are determined based on the amino acid sequence shown in reference amino acid sequence SEQ ID NO: 1.

[0050] Mutants of the present invention are denoted by the amino acids in conventional one-letter or three-letter designation. As is well known to those skilled in the art, the amino acid one-letter designations are as follows: A for alanine; C for cysteine; D for aspartic acid; E for glutamic acid; F for phenylalanine; G for glycine; H for histidine; I for isoleucine; K for lysine; L for leucine; M for methionine; N for asparagine; P for proline; Q for glutamine; R for arginine; S for serine; T for threonine; V for valine; W for tryptophan; and Y for tyrosine. In this document, the following nomenclature is used: original amino acid, position, substituted amino acid. For example, an amino acid change can be denoted in the form "H68W", where 68 refers to the substitution at the 68th amino acid, the H in front of 68 is the amino acid occupying that position before the substitution, and the W after 68 is the amino acid occupying that position after the substitution. When an amino acid change is denoted in the form "V3(AAPF)", it means that the amino acid at that position before the change is V, and after the change is AAPF. Multiple mutations are connected by a plus sign (+), for example "H68W+D114W" represents a substitution of histidine (H) with tryptophan (W) at position 68 and a substitution of aspartic acid (D) with tryptophan (W) at position 114.

[0051] Unless otherwise indicated, herein nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation.

[0052] The present application relates to alpha-amylase mutants having alpha-amylase activity, thermostability, and / or low pH stability, particularly stability at high temperature and low pH. In some embodiments, the alpha-amylase mutants of the present application have increased alpha-amylase activity, increased thermostability, and / or increased low pH stability compared to the parent alpha-amylase.

[0053] In some embodiments, the alpha-amylase mutants of the present application can exhibit significantly increased thermostability and / or low pH stability compared to the parent alpha-amylase, which makes them particularly useful in industrial production and can be widely applied in the fields of food, home care, textile, feed or medicine, specifically including but not limited to starch liquefaction, saccharification, fermentation, brewing, baking, textile desizing, textile washing, and increase of digestibility in animal feed.

[0054] In some embodiments, the alpha-amylase mutants of the present application comprise amino acid changes relative to the parent alpha-amylase, which will be described in detail below. In some embodiments, the parent alpha-amylase can be mutant LE399 of BLA, such as the alpha-amylase shown in SEQ ID NO: 2. In some embodiments, the parent alpha-amylase is a mature polypeptide (e.g., the mature polypeptide of LE399) which does not comprise a signal peptide. In some embodiments, the parent alpha-amylase comprises a signal peptide (e.g., LE399 comprising a signal peptide, such as a polypeptide with a signal peptide added to the N-terminus of SEQ ID NO: 2).

[0055] In some embodiments, the alpha-amylase mutants of the present application comprise the following amino acid changes relative to the amino acid sequence shown in SEQ ID NO: 2: V3(AAPF), D114W, L134R, N172R, S187D, N188P, H247Y, and Q360C. In some embodiments, the alpha-amylase mutants can introduce a non-native disulfide bond through amino acid substitution. In some embodiments, the alpha-amylase mutants can further comprise one or more pairs of amino acid substitutions selected from the group consisting of: N126C and G191C, A29C and R93C, and Q26C and S89C. In some embodiments, the non-native disulfide bond is formed through one or more pairs of amino acid substitutions selected from the group consisting of: N126C and G191C, A29C and R93C, and Q26C and S89C, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1, and the alpha-amylase mutant has amylase activity.

[0056] In some embodiments, the alpha-amylase mutant further comprises the amino acid substitutions H68W, F201Y, D416V, and R437W relative to the amino acid sequence set forth in SEQ ID NO: 2.

[0057] In some embodiments, the alpha-amylase mutant further comprises an amino acid substitution at position 205, which is H205D or H205Y, relative to the amino acid sequence set forth in SEQ ID NO: 2.

[0058] Without being bound by theory, the introduction of a non-native disulfide bond in the alpha-amylase mutant of the present application can further improve the alpha-amylase activity, thermal stability, and / or low-pH stability of the alpha-amylase mutant. In some embodiments, the disulfide bond is formed by the amino acid substitutions N126C and G191C. In some embodiments, the disulfide bond is formed by the amino acid substitutions A29C and R93C. In some embodiments, the disulfide bond is formed by the amino acid substitutions Q26C and S89C.

[0059] In some embodiments, the alpha-amylase mutant further comprises the amino acid substitution S148N, or no amino acid change at position 148, relative to the amino acid sequence set forth in SEQ ID NO: 2, based on the above amino acid changes. In some embodiments, the alpha-amylase mutant further comprises the amino acid substitution Y156H, or no amino acid change at position 156, relative to the amino acid sequence set forth in SEQ ID NO: 2, based on the above amino acid changes. In some embodiments, the alpha-amylase mutant further comprises the amino acid substitution K213T, or no amino acid change at position 213, relative to the amino acid sequence set forth in SEQ ID NO: 2, based on the above amino acid changes. In some embodiments, the alpha-amylase mutant further comprises the amino acid substitution N265G, or no amino acid change at position 265, relative to the amino acid sequence set forth in SEQ ID NO: 2, based on the above amino acid changes. In some embodiments, the alpha-amylase mutant further comprises the amino acid substitution H31S, or no amino acid change at position 31, relative to the amino acid sequence set forth in SEQ ID NO: 2, based on the above amino acid changes. In some embodiments, the alpha-amylase mutant further comprises the amino acid substitution S320A, or no amino acid change at position 320, relative to the amino acid sequence set forth in SEQ ID NO: 2, based on the above amino acid changes.

[0060] It is understood that in the present application, when referring to an alpha-amylase mutant comprising an amino acid change at certain positions, it does not exclude amino acid changes at other positions, for any one of the not mentioned positions the amino acid can be changed or not changed relative to SEQ ID NO: 2. In some embodiments, the alpha-amylase mutant comprises only the listed amino acid changes relative to SEQ ID NO: 2, and no amino acid changes at any of the not listed positions.

[0061] In some embodiments, the alpha-amylase mutant of the present application has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, for example at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity. In some embodiments, the alpha-amylase mutant of the present application is sequence aligned to the amino acid sequence set forth in SEQ ID NO: 2 over the amino acid sequence of the mature polypeptide (e.g. the polypeptide sequence without or free of the signal peptide).

[0062] In some embodiments, the alpha-amylase mutant of the present application is a mature polypeptide which does not comprise a signal peptide and / or a leader sequence. In some embodiments, the alpha-amylase mutant of the present application comprises a signal peptide and / or a leader sequence. In some embodiments, the signal peptide can have the amino acid sequence as set forth in SEQ ID NO: 5.

[0063] The alpha-amylase mutant of the present application has alpha-amylase activity as well as stability at high temperature and low pH. In some embodiments, the alpha-amylase mutant of the present application has increased alpha-amylase enzyme activity and / or increased stability at high temperature and low pH compared to the parent alpha-amylase or other alpha-amylase mutants. By "high temperature" is meant at least about 80°C, at least about 90°C, or at least about 100°C, for example 80°C-120°C, preferably 95°C-115°C, more preferably 100°C-110°C. By "low pH" is meant a pH value of 4.0-5.0, preferably about 4.5.

[0064] In some embodiments, the alpha-amylase activity of the alpha-amylase mutants of the present application is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% higher, for example, but not limited to, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% higher, compared to the parent alpha-amylase or other alpha-amylase mutants. The enzyme activity can be detected, for example, by the iodine-starch colorimetric method, for example, by reacting the alpha-amylase with soluble starch, followed by the addition of iodine solution, and determining the enzyme activity by detecting the absorbance at a specific wavelength (e.g., 660 nm) and comparing with a control. In some embodiments, the enzyme activity assay is performed at 70°C, pH 6.0. In some embodiments, the enzyme activity assay is performed at 100°C, pH 4.5.

[0065] In some embodiments, the alpha-amylase mutants of the present application have higher residual enzyme activity at high temperature conditions, particularly at 80°C-120°C (e.g., about 100°C), compared to the parent alpha-amylase or wild-type alpha-amylase or other alpha-amylase mutants. In some embodiments, the alpha-amylase mutants of the present application have higher residual enzyme activity at 100°C, compared to the parent alpha-amylase or wild-type alpha-amylase or other alpha-amylase mutants. In some embodiments, the alpha-amylase mutants of the present application have higher residual enzyme activity after being kept at 100°C for 30 minutes, compared to the parent alpha-amylase or wild-type alpha-amylase or other alpha-amylase mutants. In some embodiments, the alpha-amylase mutants of the present application have residual enzyme activity of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, after being kept at 100°C for 30 minutes. In some embodiments, the enzyme activity assay of the mutants without corresponding treatment is performed at 70°C.

[0066] In some embodiments, the alpha-amylase mutants of the application have a higher residual enzyme activity at low pH conditions, in particular at a pH of 4.0-5.0 (e.g. about 4.5), than the parent alpha-amylase or wild-type alpha-amylase or other alpha-amylase mutants. In some embodiments, the alpha-amylase mutants of the application have a higher residual enzyme activity at pH 4.5 than the parent alpha-amylase or wild-type alpha-amylase or other alpha-amylase mutants. In some embodiments, the alpha-amylase mutants of the application have a residual enzyme activity of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%, for example but not limited to, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%, after 30 minutes at pH 4.5. In some embodiments, the enzyme activity assay of the mutants without the corresponding treatment is performed at conditions of pH 6.0.

[0067] In some embodiments, the alpha-amylase mutants of the application have a higher residual enzyme activity at high temperature and low pH conditions, in particular at 80°C-120°C, pH of 4.0-5.0 (e.g. 100°C, pH 4.5), than the parent alpha-amylase or wild-type alpha-amylase or other alpha-amylase mutants. In some embodiments, the alpha-amylase mutants of the application have a higher residual enzyme activity at 100°C, pH 4.5 than the parent alpha-amylase or wild-type alpha-amylase or other alpha-amylase mutants. In some embodiments, the alpha-amylase mutants of the application have a higher residual enzyme activity after 30 minutes at 100°C, pH 4.5 than the parent alpha-amylase or wild-type alpha-amylase or other alpha-amylase mutants. In some embodiments, the alpha-amylase mutants of the application have a residual enzyme activity of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%, for example but not limited to, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%, after 30 minutes at 100°C, pH 4.5. In some embodiments, the enzyme activity assay of the mutants without the corresponding treatment is performed at conditions of 70°C, pH 6.0.

[0068] The present application also relates to methods of improving the enzymatic activity, thermostability, and / or low pH stability of an alpha-amylase, comprising making the above-mentioned mutations to a parent alpha-amylase (e.g., the Bacillus licheniformis alpha-amylase mutant LE399). The above-mentioned mutations can be made using any method known in the art, such as site-directed mutagenesis, homologous recombination, etc.

[0069] The present application also relates to polynucleotides encoding the alpha-amylase mutants of the present application, as well as recombinant expression vectors comprising the polynucleotides for expressing the alpha-amylase mutants. The polynucleotides can further comprise a coding sequence for a signal peptide to allow the mutants to be secreted extracellularly upon expression.

[0070] The expression vectors comprise polynucleotides encoding the alpha-amylase mutants of the present application operably linked to one or more control sequences that direct the expression of the coding sequence for the alpha-amylase mutants in a suitable host cell. Control sequences include, but are not limited to, promoters, terminators, leader sequences, polyadenylation sequences, signal peptide sequences, etc. A constitutive promoter or an inducible promoter can be used. Control sequences suitable for different host cells are well known to those skilled in the art. In some embodiments, the promoter can be the amyE gene promoter of Bacillus subtilis, the terminator can be the terminator of the Bacillus amyloliquefaciens alpha-amylase gene amyQ, and the signal peptide sequence can be the sequence encoding the signal peptide of the Bacillus licheniformis amylase gene amyL. In some embodiments, the promoter sequence can be the nucleotide sequence set forth as SEQ ID NO: 3. In some embodiments, the terminator sequence can be the nucleotide sequence set forth as SEQ ID NO: 4.

[0071] The expression vector can be an autonomously replicating vector, i.e., a vector that is capable of replicating independently of the chromosomal DNA of the host cell, such as plasmids or artificial chromosomes, or can be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome. The expression vector can comprise one or more selectable markers that permit easy selection of transformed, transfected, or transduced cells, for example, those that confer resistance to antibiotics, heavy metals, etc. The expression vector can also comprise an origin of replication to allow the expression vector to be capable of autonomous replication in the host cell. The selection of these elements is routine in the art, and methods for constructing recombinant expression vectors utilizing these elements are well known in the art, for example, see J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition. In some embodiments, the polynucleotide encoding the alpha-amylase mutant of the present application is inserted into the plasmid vector pUC57 to make an expression vector for expressing the alpha-amylase mutant.

[0072] The present application also relates to recombinant host cells for expressing the alpha-amylase mutants of the present application. The host cell can be a prokaryotic cell or a eukaryotic cell, for example, it can be a bacterial cell, a fungal cell, a plant cell, an insect cell, or an animal cell (e.g., a mammalian cell). In some embodiments, the host cell can be a fungus, for example, a fungus from the genus Aspergillus, for example, Aspergillus niger, Aspergillus oryzae, etc. In some embodiments, the host cell can be a bacterium, for example, a bacterium from the genus Bacillus, for example, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, etc. In some embodiments, Bacillus subtilis is used as the host cell. Methods for introducing a polynucleotide into a host cell are well known to those skilled in the art, for example, by chemical transformation (e.g., CaCl2treatment), electroporation, gene gun, etc.

[0073] The present application also relates to a method of preparing the above-mentioned alpha-amylase mutants, comprising: (a) culturing the host cell of the present application under conditions suitable for expression of the alpha-amylase mutant; and (b) recovering the alpha-amylase mutant. In some embodiments, the alpha-amylase mutant expression in the host cell can be constitutive expression or inducible expression, depending on the control sequence (e.g. promoter) used to direct the expression of the alpha-amylase mutant. When the alpha-amylase mutant expression in the host cell is inducible expression, step (a) can further comprise inducing the expression of the alpha-amylase mutant, the inducer used depending on the control sequence (e.g. promoter) used to direct the expression of the alpha-amylase mutant.

[0074] In some embodiments, the method of preparing the alpha-amylase mutant comprises: (a) inserting the polynucleotide encoding the alpha-amylase mutant of the present application into plasmid vector pUC57 to prepare a recombinant expression vector for expressing the alpha-amylase mutant; (b) transforming the recombinant expression vector prepared in step (a) into Bacillus subtilis to obtain a host cell expressing the alpha-amylase mutant; (c) culturing the host cell to express the alpha-amylase mutant; (d) recovering the alpha-amylase mutant.

[0075] The conditions suitable for expression of the alpha-amylase mutant are well known to those skilled in the art and can be selected according to the kind of host cell. For example, the host cell can be cultured using a nutrient medium containing a carbon source, a nitrogen source and / or inorganic salts, and the growth conditions such as temperature and / or nutrient supply can be controlled. In some embodiments, the host cell is cultured at 37°C. The host cell can be cultured in a shake flask or subjected to fermentation culture in a laboratory or industrial fermenter, for example, continuous fermentation, batch fermentation, fed-batch fermentation or solid-state fermentation culture. In some embodiments, the host cell is cultured by shake flask fermentation, preferably at 220 rpm.

[0076] The alpha-amylase mutant can be secreted into the culture medium, which can then be recovered from the culture medium. The alpha-amylase mutant can be recovered using methods known in the art. For example, the alpha-amylase mutant can be recovered from the nutrient medium by various conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation and / or precipitation. The alpha-amylase mutant can also not be secreted, i.e. expressed intracellularly and retained intracellularly, in which case the alpha-amylase mutant can be recovered after lysing the cells. In some embodiments, the alpha-amylase mutant is recovered by collecting the fermentation broth.

[0077] The enzyme activity of the produced alpha-amylase (including the alpha-amylase mutants described herein) can be detected using a variety of methods known in the art. For example, the enzyme activity of the alpha-amylase can be determined by detecting the activity of the alpha-amylase in catalyzing the hydrolysis of starch. The enzyme activity assay method can be, for example, an iodine-starch colorimetric method, e.g., reacting the alpha-amylase with soluble starch, followed by adding iodine solution, and determining the enzyme activity by detecting the absorbance at a specific wavelength (e.g., 660 nm) and comparing with a control. In some embodiments, the alpha-amylase activity is detected at 70°C, pH 6.0.

[0078] The alpha-amylase mutants of the present application have higher enzyme activity, good thermal stability, and stability at low pH, and can be widely used in various fields, such as food, home care, textile, feed, or pharmaceutical fields. The use of alpha-amylases in these fields is known, and the alpha-amylases of the present application will achieve better results than the parent alpha-amylase or wild-type alpha-amylase or other alpha-amylase mutants in these fields due to their good performance.

[0079] As known to those skilled in the art, in industrial production, starch liquefaction is usually carried out at high temperature (e.g., 80-120°C) and low pH (e.g., pH 4.0-5.0), and thus the alpha-amylases provided herein having higher stability at high temperature and low pH will be particularly useful in liquefying starch-containing materials. They can maintain higher enzyme activity (i.e., the activity of hydrolyzing alpha-1,4-glycosidic bonds in starch molecules to generate dextrin, oligosaccharides, and monosaccharides) at high temperature and / or low pH, thereby improving the efficiency of starch liquefaction, optimizing the effect of starch liquefaction, and reducing the amount of enzyme added while achieving comparable results to other alpha-amylases.

[0080] In the fields including but not limited to the above (e.g., starch liquefaction), the alpha-amylase mutants of the present application can be used together with additional enzymes in the form of a composition, which can comprise one or more of the alpha-amylase mutants of the present application and one or more additional enzymes. In some embodiments, the additional enzyme is one or more selected from the group consisting of alpha-amylases, beta-amylases, cellulases, glucoamylases, hemicellulases, isoamylases, isomerases, lipases, phytases, proteases, and pullulanases. In some embodiments, the alpha-amylase in the additional enzyme is different from the alpha-amylase mutants of the present application included in the composition, e.g., having a different sequence, a different source, and / or a different functional activity.

[0081] In some embodiments, the alpha-amylase mutants or compositions of the present application can be used for liquefying starch or starch-containing materials, and thus can be used for laundry, textile desizing, production of baked products. For example, in the food industry, the alpha-amylase can be used in the fields of starch processing, alcohol brewing and bread baking, and specifically can be used for starch liquefaction, saccharification, production of high-maltose syrup, production of high-glucose syrup, etc. For example, in the household care field, the alpha-amylase can be used for removing starch-containing dirt and stains in dishwashing and laundry methods. For example, in the textile field, the alpha-amylase can be used for degradation of starch slurry, improving desizing efficiency. For example, in the feed field, the alpha-amylase can be used as a feed additive to increase digestibility and improve the nutritional value of feed. For example, in the medical field, the alpha-amylase can be used for the preparation of drug carriers, sustained-release agents, etc.

[0082] The present application is further described by the following examples, which should not be construed as limiting the present application. The reagents used in the following examples are commercially available unless otherwise specified.

[0083] Example 1: Construction of LE399 and its mutant strains

[0084] 1) The amino acid sequence of LE399 is shown in SEQ ID NO: 2. The promoter of LE399 gene uses the promoter of the alpha-amylase gene amyE of Bacillus subtilis (SEQ ID NO: 3), the terminator uses the terminator of the alpha-amylase gene amyQ of Bacillus amyloliquefaciens (SEQ ID NO: 4), and the signal peptide uses the signal peptide of the amylase gene amyL of Bacillus licheniformis (SEQ ID NO: 5). The complete expression frame of LE399 containing the above-mentioned amyE promoter, amyL signal peptide, LE399 gene and amyQ terminator is synthesized by Kingsriver Biotech Co., Ltd. according to the codon bias of Bacillus subtilis, and is named “LE399 expression frame fragment”. The LE399 Bacillus subtilis integration plasmid pUC57-LE399-Cm-amyE is constructed by the following method:

[0085] The 532 bp homologous arm upstream of amyE (fragment 1) was amplified using the genomic DNA of Bacillus subtilis ATCC6051a as template and using amyE-5'-F and amyE-5'-R as primers; the 592 bp homologous arm downstream of amyE (fragment 2) was amplified using the genomic DNA of Bacillus subtilis ATCC6051a as template and using amyE-3'-F and amyE-3'-R as primers. The 1264 bp Cm expression cassette was amplified using the genomic DNA of Bacillus licheniformis ATCC14580 as template and using Cm-F and Cm-R as primers. The three fragments and the "LE399 expression cassette fragment" were amplified using overlap PCR to obtain the complete knock-in fragment amyE upstream homologous arm-LE399 complete expression cassette-Cm-amyE downstream homologous arm. The knock-in fragment and the pUC57 plasmid were linearized using EcoRI / Hind III and then ligated using T4 DNA ligase to construct the integration plasmid pUC57-LE399-Cm-amyE. The sequence of the integration plasmid is shown in SEQ ID NO: 6, and the sequences of the primers used are shown in Table 1. The underlined parts represent the enzyme digestion sites.

[0086] Table 1. Primer names and sequences

[0087] 2) The pUC57-LE399-Cm-amyE integration plasmid obtained by the above construction was transformed into the competent cells of Bacillus subtilis ATCC6051a by the conventional Bacillus subtilis chemical transformation method. The cells were cultured at 37°C and 200 rpm for 2 h, then 100 μL of the cell solution was diluted 100-fold and spread on LB solid medium containing 5 ng / μL chloramphenicol (formula: 1% peptone, 0.5% yeast extract, 1% NaCl, 1.5% agar). After overnight culture at 37°C, random clones were selected for colony PCR and sequencing verification to obtain the Bacillus subtilis LE399 expression strain.

[0088] Referring to the construction methods of the LE399 Bacillus subtilis integration plasmid and expression strain described in 1) and 2) above, the Bacillus subtilis integration plasmids and expression strains of the mutants (named X1-X17) in Table 2 were constructed. The difference is that the LE399 gene is replaced by the gene of each mutant shown in Table 2.

[0089] Table 2. Mutants

[0090] Example 2: Shake flask fermentation

[0091] The Bacillus subtilis expression strains of LE399 and its mutants obtained in Example 1 were subjected to shake flask fermentation. The experimental method was as follows:

[0092] Respectively, an appropriate amount of bacteria was inoculated into a 250ml flask containing 50ml LB liquid seed medium (formula: 1% peptone, 0.5% yeast extract, 1% NaCl), 37℃, 220rpm incubated overnight, then inoculated into 50ml AKP fermentation medium (formula: 10% glucose, 6% bean cake powder, 1% anhydrous disodium hydrogen phosphate) containing 50ml 250ml flask, 37℃, 220rpm incubated for 48h, then the fermentation broth was collected, and an appropriate amount of fermentation broth was used for detection of amylase activity.

[0093] Example 3: Enzyme activity detection of amylase

[0094] Definition of amylase activity unit: 1mL of liquid enzyme at 70℃, pH 6.0, 1min liquefies 1mg of soluble starch, which is 1 enzyme activity unit, expressed in U / mL.

[0095] The enzyme activity detection method is as follows: 20ml 20g / L soluble starch solution is mixed with 5ml pH 6.0 phosphate buffer, preheated at 70℃ for 8min, 1.0ml diluted enzyme solution is added, and the reaction is accurately carried out for 5min, then 1ml reaction solution is taken, 0.5ml 0.1mol / L hydrochloric acid solution and 5ml dilute iodine solution are added into a test tube, shaken well, and 0.5ml 0.1mol / L hydrochloric acid solution and 5ml dilute iodine solution are used as blank, the absorbance is measured at 660nm wavelength, the concentration of the test enzyme solution is obtained according to the absorbance and alpha-amylase enzyme concentration control table in appendix B of GB 1886.174-2016 "National food safety standard food additives food industry enzyme preparations", and the enzyme activity of the test sample is obtained according to the calculation formula X=C*N*16.67 (wherein X represents enzyme activity, C is the concentration of the test enzyme sample, N is the dilution multiple of the sample, and 16.67 is the conversion coefficient calculated according to the definition of enzyme activity).

[0096] Relative enzyme activity refers to the enzyme activity of each mutant relative to LE399, that is, the enzyme activity of LE399 is set as 100%, and the ratio (expressed as a percentage) of the enzyme activity of each mutant relative to the enzyme activity of LE399.

[0097] pH 4.5 heat-resistant residual enzyme activity refers to the percentage of enzyme activity after incubation of enzyme solution at pH 4.5, 100℃ for 30min relative to the enzyme activity before incubation. The enzyme activity detection method before and after incubation is the same as the above enzyme activity detection method. The experimental results are shown in Table 3.

[0098] Table 3 Relative enzyme activity and pH 4.5 heat-resistant residual enzyme activity of LE399 and its mutants

[0099] The amino acid sequence involved in the application is as follows:

[0100] SEQ ID NO: 1 (B. licheniformis alpha-amylase BLA amino acid sequence)

[0101] SEQ ID NO: 2 (LE399 amino acid sequence)

[0102] SEQ ID NO: 3 (B. subtilis alpha-amylase gene amyE promoter)

[0103] SEQ ID NO: 4 (B. amyloliquefaciens alpha-amylase gene amyQ terminator sequence)

[0104] SEQ ID NO: 5 (B. licheniformis amylase gene amyL signal peptide amino acid sequence)

[0105] SEQ ID NO: 6 (integration plasmid pUC57-LE399-Cm-amyE sequence)

[0106] The embodiments of the present application are not limited to the above-described examples, and various changes and improvements can be made in form and details by those skilled in the art without departing from the spirit and scope of the present application, and such are considered to fall within the scope of the present application.

Claims

1. An alpha-amylase mutant comprising the following amino acid alterations relative to the amino acid sequence set forth in SEQ ID NO: 2: V3(AAPF), D114W, L134R, N172R, S187D, N188P, H247Y, and Q360C, and an amino acid substitution forming a non-native disulfide bond comprising a pair of amino acid substitutions selected from the group consisting of: N126C and G191C, A29C and R93C, and Q26C and S89C; wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1; and wherein the alpha-amylase mutant has amylase activity.

2. The alpha-amylase mutant of claim 1, further comprising the amino acid substitutions H68W, F201Y, D416V, and R437W relative to the amino acid sequence set forth in SEQ ID NO:

2.

3. The alpha-amylase mutant of claim 2, further comprising an amino acid substitution at position 205, which is H205D or H205Y, relative to the amino acid sequence set forth in SEQ ID NO:

2.

4. The alpha-amylase mutant of claim 3, further comprising one or more amino acid substitutions selected from S148N, Y156H, K213T, N265G, H31S, and S320A relative to the amino acid sequence set forth in SEQ ID NO:

2.

5. The alpha-amylase mutant of any one of claims 1-4, having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

2.

6. The alpha-amylase mutant of any one of claims 1-4, comprising any one of the following combinations of amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 2: V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + K213T + H247Y + Q360C + D416V + R437W + Q26C + S89C; V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + K213T + H247Y + Q360C + D416V + R437W + A29C + R93C; V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H247Y + Q360C + D416V + R437W + N126C + G191C + H205D + H31S + N265G; ​ ​ ​ ​ V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + Y156H + S148N + N265G; V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + S320A + Y156H + H31S; V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + S320A + S148N + H205D; V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + S320A + Y156H + H31S; V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + S320A + Y156H + H205D + N265G; V3(AAPF) + H68W + D114W + L134R + N172R + S187D + N188P + F201Y + H205Y + H247Y + Q360C + D416V + R437W + N126C + G191C + S320A + Y156H + H205D + N265G; 7. A polynucleotide encoding the alpha-amylase mutant of any one of claims 1-6.

8. A recombinant expression vector comprising the polynucleotide of claim 7.

9. A host cell comprising the polynucleotide of claim 7 or the recombinant expression vector of claim 8.

10. The host cell of claim 9, which is a bacterium.

11. The host cell of claim 10, wherein the bacterium is from the genus Bacillus.

12. The host cell of any one of claims 9-11, which is B. subtilis or B. licheniformis.

13. A method of making the alpha-amylase mutant of any one of claims 1-6, comprising the steps of: ​ (a) cultivating the host cell of any of claims 9-11 under conditions suitable for expression of the alpha-amylase mutant; (b) recovering the expressed alpha-amylase mutant.

14. An alpha-amylase mutant obtained by the method of claim 13.

15. A composition comprising the alpha-amylase mutant of any of claims 1-6 and one or more additional enzymes.

16. The composition of claim 15, wherein the one or more additional enzymes are selected from the group consisting of alpha-amylases, beta-amylases, cellulases, glucoamylases, hemicellulases, isoamylases, isomerases, lipases, phytases, proteases, and pullulanases.

17. Use of the alpha-amylase mutant of any of claims 1-6 or the composition of any of claims 15-16 for liquefying starch-containing material.

18. Use of the alpha-amylase mutant of any of claims 1-6 or the composition of any of claims 15-16 for washing.

19. Use of the alpha-amylase mutant of any of claims 1-6 or the composition of any of claims 15-16 for desizing of textiles.

20. Use of the alpha-amylase mutant of any of claims 1-6 or the composition of any of claims 15-16 for producing a baked product.

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