Native starch saccharifying enzyme mutant, encoding gene and application thereof

Abstract

The application relates to the technical field of genetic engineering, and discloses a raw starch saccharifying enzyme mutant, a coding gene thereof and application. The raw starch saccharifying enzyme mutant provided by the application has a lower optimum temperature of reaction, the degradation efficiency of the enzyme on a raw corn starch reaction substrate is also improved, the catalytic activity of the raw starch saccharifying enzyme mutant is improved, and the raw starch saccharifying enzyme mutant has important application value in the fields of reducing sugar production and ethanol production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a raw starch saccharifying enzyme mutant, its encoding gene, and its applications. Background Technology

[0002] In recent years, raw amylases have received increasing attention due to their ability to directly degrade raw starch granules. Among them, raw starch saccharifying enzyme (RSGA) degrades raw starch granules to produce only glucose at temperatures below starch gelatinization temperature, eliminating the need for high-temperature starch gelatinization in traditional starch sugar production processes, simplifying the process. At the same time, the application of raw starch saccharifying enzymes at low temperatures relative to gelatinization temperature reduces energy consumption and improves production efficiency, thus showing broad development prospects.

[0003] In recent years, the discovery and research of starch-saccharifying enzymes have been gradually carried out. New enzyme genes with the function of degrading raw starch have been discovered in nature from different sources, such as *Aspergillus niger*, *Penicillium*, and *Aspergillus pumilus*. Through expression and structural analysis of related genes, we have gained a basic understanding of the specificity and structure of starch degradation. However, a starch-saccharifying enzyme that can efficiently catalyze the degradation of raw corn starch under relatively low temperature conditions has not yet been obtained.

[0004] Starch saccharifying enzymes derived from *Aspergillus fumigatus* have been identified in previous studies as having the function of degrading raw corn starch. Improving the enzyme's ability to degrade raw corn starch at lower temperatures is currently an important research direction. Therefore, there is an urgent need to further improve the catalytic activity of starch saccharifying enzymes and lower their optimal reaction temperature in order to obtain starch saccharifying enzymes with enzymatic properties more suitable for industrial applications. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a raw starch saccharifying enzyme mutant, its encoding gene, and its applications. This raw starch saccharifying enzyme mutant has high catalytic activity and a low optimal temperature, enabling it to exhibit good degradation effects on raw starch, especially raw corn starch, at lower temperatures.

[0006] To achieve the above objectives, the present invention provides a raw starch saccharifying enzyme mutant, wherein the raw starch saccharifying enzyme mutant comprises the following amino acid sequence:

[0007] (a) An amino acid sequence as shown in SEQ ID NO: 2; or

[0008] (b) A derivative protein obtained by substituting, deleting or adding at least one amino acid of the amino acid sequence shown in SEQ ID NO: 2, with an enzyme activity not less than 95% of that of (a).

[0009] A second aspect of the present invention provides a gene capable of encoding a raw starch saccharifying enzyme mutant as described above.

[0010] A third aspect of the present invention provides a recombinant vector containing the gene as described above.

[0011] A fourth aspect of the present invention provides a recombinant strain containing the gene as described above or the recombinant vector as described above.

[0012] The fifth aspect of the present invention provides a method for preparing a raw starch saccharifying enzyme mutant, the method comprising culturing the recombinant strain as described above and separating and purifying the culture product to obtain the raw starch saccharifying enzyme mutant.

[0013] The sixth aspect of the present invention provides the application of the raw starch saccharifying enzyme mutant as described above, the encoding gene as described above, the recombinant vector as described above, and the recombinant strain as described above in the degradation of raw starch.

[0014] The seventh aspect of the present invention provides the use of the previously described starch saccharifying enzyme mutant, the previously described encoding gene, the previously described recombinant vector, and the previously described recombinant strain in the preparation of reducing sugars.

[0015] Compared with wild-type raw starch saccharifying enzyme, the optimal reaction temperature of the raw starch saccharifying enzyme mutant provided by this invention is reduced from 70℃ to 60℃, and the degradation efficiency of the reaction substrate raw corn starch is increased by 20%. The catalytic activity of the raw starch saccharifying enzyme mutant is improved, which has important application value in the fields of reducing sugar production and ethanol production. Attached Figure Description

[0016] Figure 1 This is a comparison chart of the optimal reaction temperatures of the raw starch saccharifying enzyme mutants G101T / Q113T and pRSGA in Example 3. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] The first aspect of this invention provides a raw starch saccharifying enzyme mutant, the raw starch saccharifying enzyme mutant comprising the following amino acid sequence:

[0019] (a) An amino acid sequence as shown in SEQ ID NO: 2; or

[0020] (b) A derivative protein obtained by substituting, deleting or adding at least one amino acid of the amino acid sequence shown in SEQ ID NO: 2, with an enzyme activity not less than 95% of that of (a).

[0021] SEQ ID NO: 2

[0022] APQLSARATGSLDSWLGTETTVALNGILANIGADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNLTLQKVITEYVNSTAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRPQRDGPALRATALIDFGNWLIDN GYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCYMQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPRALANHKVYTDSFRSVYAINSGIPQG AAVSAGRYPEDVYYNGNPWFLTTLAAAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSTFTDIINAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFLTANMRRNGVVPAPWGAASANSVPSSCSM GSATGTYSTATATSWPSTLTSGSPGSTTTVGTTTSTTSGTATETACATPTAVAVTFNEIATTTYGENVYIVGSISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAACGVSTATENDTWR

[0023] In some embodiments of the present invention, the enzyme activity of the raw starch saccharifying enzyme mutant containing amino acid sequence (b) can be 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.8%, 99.9%, or 100% of the enzyme activity of the raw starch saccharifying enzyme mutant containing amino acid sequence (a), or it can be any intermediate value between any two of the above values.

[0024] Enzyme activity, also known as enzyme activity, is characterized by enzyme activity units. In this invention, unless otherwise specified, an enzyme activity unit is defined as the amount of enzyme required to hydrolyze a 20 g / L raw starch aqueous solution to produce 1 μmol of reducing sugar (calculated as glucose) in 1 hour under reaction conditions of pH 4.6 and 40°C.

[0025] In this invention, unless otherwise specified, "RSGA" and "wild-type raw starch saccharifying enzyme" refer to wild-type raw starch saccharifying enzyme derived from the microorganism *Aspergillus fumigatus*, and "pRSGA" refers to the wild-type raw starch saccharifying enzyme derived from *Aspergillus fumigatus* expressed in *Pichia pastoris*. The amino acid sequence of the wild-type raw starch saccharifying enzyme derived from *Aspergillus fumigatus* has 612 amino acids (SEQ ID NO: 10), and its encoding gene is 1836 nucleotides in length (SEQ ID NO: 11).

[0026] SEQ ID NO: 10:

[0027] APQLSARATGSLDSWLGTETTVALNGILANIGADGAYAKSAKPGIIIASPSTSEPDYYYTWTRDAALVTKVLVDLFRNGNLGLQKVITEYVNSQAYLQTVSNPSGGLASGGLAEPKYNVDMTAFTGAWGRPQRDGPALRATALIDFGNWLIDN GYSSYAVNNIWPIVRNDLSYVSQYWSQSGFDLWEEVNSMSFFTVAVQHRALVEGSTFAKRVGASCSWCDSQAPQILCYMQSFWTGSYINANTGGGRSGKDANTVLASIHTFDPEAGCDDTTFQPCSPALANHKVYTDSFRSVYAINSGIPQG AAVSAGRYPEDVYYNGNPWFLTTLAAAEQLYDAIYQWKKIGSISITSTSLAFFKDIYSSAAVGTYASSTSTFTDIINAVKTYADGYVSIVQAHAMNNGSLSEQFDKSSGLSLSARDLTWSYAAFLTANMRRNGVVPAPWGAASANSVPSSCSMGSATGTYSTATATSWPSTLTSGSPGSTTTVGTTTSTTSGTATETACATPTAVAVTFNEIATTTYGENVYIVGSISELGNWDTSKAVALSASKYTSSNNLWYVSVTLPAGTTFEYKYIRKESDGSIVWESDPNRSYTVPAACGVSTATENDTWR

[0028] SEQ ID NO: 11

[0029]

[0030] In this invention, the amino acid sequence shown in SEQ ID NO: 2 is obtained by mutating the 101st amino acid, glycine (Gly), to threonine (Thr) and the 113th amino acid, glutamine (Gln), to threonine (Thr) in the amino acid sequence of wild-type raw starch saccharifying enzyme (SEQ ID NO: 10) derived from Aspergillus fumigatus.

[0031] The 20 amino acid residues that make up proteins can be divided into four categories according to the polarity of their side chains: 1. Nonpolar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro); 2. Polar, uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), aspartic acid (Asn), glutamine (Gln), and tyrosine (Tyr); 3. Positively charged amino acids: arginine (Arg), lysine (Lys), and histidine (His); 4. Negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "Biochemistry" (Second Edition), Volume 1, Shen Tong and Wang Jingyan, pp. 82-83, Higher Education Press, December 1990). If a substitution of an amino acid residue belonging to the same category occurs in a protein, such as Arg replacing Lys or Leu replacing Ile, the function of that residue in the protein's structural domain (e.g., providing a positive charge or forming a hydrophobic pocket structure) remains unchanged, and the protein's three-dimensional structure is not affected; therefore, the protein can still perform its function. This substitution of an amino acid residue belonging to the same category can occur at any amino acid residue position in the aforementioned amylase mutant.

[0032] As previously stated, the starch saccharifying enzyme mutant provided by this invention can be further modified or mutated to obtain derived proteins. The term "derived protein" in this invention refers to a protein that differs from the starch saccharifying enzyme mutant having the aforementioned amino acid sequence, may also differ in the form of modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. The induced variants can be obtained through various techniques, such as random mutations generated by radiation or mutagens, or through techniques such as site-directed mutagenesis or other known molecular biology techniques. The "derived protein" also includes analogs containing residues of naturally occurring L-type amino acids (such as D-type amino acids), and analogs containing non-naturally occurring or synthetic amino acids (such as β-amino acids, γ-amino acids, etc.).

[0033] Modifications (typically without altering the primary structure, i.e., without changing the amino acid sequence) include chemically derived forms of proteins, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those occurring during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to glycosylating enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Furthermore, modifications can also be applied to proteins that improve their resistance to proteolysis or optimize their solubility.

[0034] According to a preferred embodiment of the present invention, the raw starch saccharifying enzyme mutant further contains an amino acid tag that is easy to separate and purify.

[0035] Any amino acid tag commonly used in protein purification in the art can be applied to this invention, as long as it does not affect enzyme activity. Preferably, the amino acid tag is at least one of a multihistidine tag, a GST tag, and a MBP maltose-binding protein tag. The GST tag utilizes the principle of GST fusion protein covalently binding to immobilized glutathione (GSH) via a disulfide bond, followed by GSH exchange elution for purification. This invention does not impose any particular limitation on the specific sequence of the amino acid tag; for example, the amino acid tag may include the following amino acid sequences:

[0036] Multihistidine tag: HHHHHH (SEQ ID NO: 7)

[0037] GST Label:

[0038] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAY SKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK(SEQ ID NO:8)

[0039] MBP Maltose-Binding Protein Tag:

[0040] MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQS GLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYE NGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT(SEQ ID NO:9)

[0041] In a preferred embodiment, to further improve the purification effect, the tag is a multihistidine tag.

[0042] A second aspect of the present invention provides a gene capable of encoding a raw starch saccharifying enzyme mutant as described above.

[0043] In this invention, there are no particular limitations on the specific sequence of the coding gene, as long as it can encode the aforementioned starch saccharifying enzyme mutant. Those skilled in the art can design and construct the relevant coding gene based on the amino acid sequence of the starch saccharifying enzyme mutant and codon tables and gene construction techniques known in the art.

[0044] In some embodiments of the present invention, the nucleotide sequence of the gene encoding the aforementioned raw starch saccharifying enzyme mutant is shown in SEQ ID NO: 1.

[0045] SEQ ID NO: 1

[0046] GCCCCGCAGCTGAGCGCACGTGCAACTGGTTCTTTAGACAGTTGGCTGGGTACCGAAACCACCGTGGCACTGAACGGTATTCTGGCAAACATCGGCGCCGATGGTGCATACGCCAAGAGCGCAAAACCGGGCATCATCATTGCAAGCCCGAGCACCAGCGAGCCGGATTACTACTATACTTGGACACGCGATGCCGCATTAGTTACCAAGGTGCTGGTTGATTTATTCCGCAATGGCAATCTG ACT CTGCAGAAAGTGATCACCGAGTATGTGAACAGC ACA

[0047] The underlined parts represent mutation sites.

[0048] In some embodiments of the present invention, the coding gene may also contain a coding gene for a tag that facilitates protein purification (e.g., a coding gene for an amino acid tag as described above).

[0049] As is well known in the art, of the 20 different amino acids that make up proteins, except for Met (ATG) or Trp (TGG), which are encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D, page 950). That is, due to the degeneracy of the genetic codon, most amino acids are determined by more than one codon. The substitution of the third nucleotide in a triplet codon often does not change the amino acid composition; therefore, genes encoding the same protein can have different nucleotide sequences. Based on known codon tables, those skilled in the art can deduce the nucleotide sequences of genes encoding these proteins from the amino acid sequences disclosed in this invention, and from the amino acid sequences of starch-amylase mutants obtained from these sequences that exhibit unchanged or substantially unchanged activity. These nucleotide sequences can be obtained through biological methods (such as PCR or mutagenesis) or chemical synthesis methods; therefore, all such nucleotide sequences should be included within the scope of this invention. Conversely, using the DNA sequence disclosed herein, methods known in the art, such as those of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989), can be employed to modify the nucleic acid sequence provided in this invention to obtain an amino acid sequence consistent with the alkane hydroxylase activity described in this invention. Therefore, in this invention, the coding gene can also be codon optimized according to the characteristics of the expression vector or the needs of in vitro expression to improve the expression efficiency or expression level in the in vitro expression vector. This invention does not impose any particular limitation on the specific codon liberalization method, as long as the optimized coding gene can express a protein having the aforementioned amino acid sequence (a) or (b).

[0050] A third aspect of the present invention provides a recombinant vector containing the gene as described above.

[0051] In this invention, the recombinant vector can be any existing vector commonly used for in vitro protein expression, and the aforementioned coding gene is introduced into it using genetic engineering techniques. This invention does not impose any particular limitation on the specific vector used; those skilled in the art can select one according to actual needs and requirements (e.g., based on the required expression efficiency, expression level, and the requirements of the selected expression vector for the recombinant vector). Preferably, to further improve the expression level and extracellular expression of the recombinant protein, the vector is pPIC9K.

[0052] A fourth aspect of the present invention provides a recombinant strain containing the gene as described above or the recombinant vector as described above.

[0053] In this invention, any existing expression system in the art can be used to construct the recombinant strain, as long as it can express the aforementioned amylase mutant. In some embodiments of this invention, when the recombinant strain contains the recombinant vector as described above, the recombinant vector can be transformed, transduced, or transfected into the host cell (strain) using conventional methods in the art, such as chemical transformation by calcium chloride method or electroporation transformation, preferably electroporation transformation; wherein, the method and conditions of electroporation transformation are conventional operations in the art and will not be described in detail here. The host cell can be a prokaryotic cell or a eukaryotic cell, as long as it can serve as the host cell for the recombinant vector. Preferably, the host cell is at least one of Pichia pastoris, Escherichia coli, and Bacillus subtilis. Preferably, in order to achieve high-density fermentation and production of recombinant protein, the host cell is Pichia pastoris, and more preferably Pichia pastoris GS115.

[0054] The fifth aspect of the present invention provides a method for preparing a raw starch saccharifying enzyme mutant, the method comprising culturing the recombinant strain as described above and separating and purifying the culture product to obtain the raw starch saccharifying enzyme mutant.

[0055] The present invention does not impose any particular restrictions on the specific culture methods and conditions of the recombinant strains. Those skilled in the art can adjust the culture conditions according to actual conditions and needs (such as the type of strain selected and the culture requirements, the requirements for expression efficiency and expression level, etc.).

[0056] In some embodiments of the present invention, the culture includes inducing protein expression.

[0057] In some preferred embodiments of the present invention, when the recombinant strain is Pichia pastoris, the culture includes: inoculating Pichia pastoris into YPD medium for a first culture at 28-30°C for 16-18 hours to obtain a first fermentation broth; centrifuging the first fermentation broth to collect the bacterial cells, then resuspending the bacterial cells in BMMY medium to induce protein expression, and performing a second fermentation at 28-30°C for 120-130 hours to obtain a second fermentation broth. The component content and preparation method of the YPD and BMMY mediums can be conventionally selected in the art and will not be elaborated further here.

[0058] In this invention, any existing methods and conditions for separating and purifying culture products can be used to separate and purify the raw starch saccharifying enzyme mutant, as long as the separation and purification conditions have no or negligible impact on the enzyme activity of the raw starch saccharifying enzyme mutant. For example, nickel affinity chromatography can be used to separate and purify the raw starch saccharifying enzyme mutant.

[0059] In some preferred embodiments of the present invention, the method for preparing the raw starch saccharifying enzyme mutant includes: (1) determining the mutation site based on the structure of wild-type raw starch saccharifying enzyme and designing mutation primers for site-directed mutagenesis; (2) using the vector pPIC9K-pRSGA carrying the gene encoding wild-type raw starch saccharifying enzyme as a template to construct the pPIC9K-G101T / Q113T mutant plasmid by site-directed mutagenesis, wherein the mutant plasmid contains the nucleotide sequence shown in SEQ ID NO: 1; (3) transforming the pPIC9K-G101T / Q113T mutant plasmid into Pichia pastoris GS115 competent cells by electroporation, and selecting the verified positive clones for fermentation culture; (4) after fermentation, collecting the supernatant of the fermentation broth by centrifugation, and purifying the raw starch saccharifying enzyme mutant by nickel column chromatography. The preparation method of the vector pPIC9K-pRSGA is as follows: the wild-type raw starch saccharifying enzyme gene (nucleotide sequence SEQ ID NO: 11) from Aspergillus fumigatus is linked into the pPICK9K vector by a one-step cloning method, and after transformation into Escherichia coli JM109, single colonies are selected for sequencing verification to obtain the plasmid.

[0060] The sixth aspect of the present invention provides the application of the raw starch saccharifying enzyme mutant as described above, the encoding gene as described above, the recombinant vector as described above, and the recombinant strain as described above in the degradation of raw starch.

[0061] In some embodiments of the present invention, the raw starch is raw corn starch.

[0062] The seventh aspect of the present invention provides the use of the previously described starch saccharifying enzyme mutant, the previously described encoding gene, the previously described recombinant vector, and the previously described recombinant strain in the preparation of reducing sugars.

[0063] The present invention will be described in detail below through embodiments.

[0064] Unless otherwise specified, the experimental methods and equipment described in the following embodiments are conventional methods and equipment. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.

[0065] In the following embodiments, unless otherwise specified, all sequencing and primer construction were outsourced to Shanghai Sangon Biotech Co., Ltd.

[0066] LB solid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar powder 2 g / L.

[0067] LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0068] YPD medium: yeast extract 10g / L, peptone 20g / L, glucose 20g / L.

[0069] BMMY medium: yeast extract 10 g / L, peptone 20 g / L, methanol 1 (v / v)%, YNB 13.4 g / L, biotin 4 × 10⁻⁶ -5 g / L, 100mM potassium phosphate buffer (pH 6.0). Methanol should be added immediately before use.

[0070] The pPICK9K-pRSGA plasmid was obtained by ligating the wild-type raw amylase gene (nucleotide sequence SEQ ID NO: 11, synthesized by Suzhou Genewiz Biotechnology Co., Ltd.) from Aspergillus fumigatus into the pPICK9K vector in a one-step cloning process. After transforming Escherichia coli JM109, single colonies were selected and sequenced for verification.

[0071] The pPICK9K plasmid was purchased from Invitrogen.

[0072] Example 1

[0073] This embodiment illustrates the construction of a vector containing the gene sequence of a raw starch saccharifying enzyme mutant and the construction of a recombinant strain.

[0074] (1) The pPIC9K-G101T / Q113T mutant plasmid was constructed using the PCR site-directed mutagenesis method.

[0075] Using pPIC9K-pRSGA plasmid as a template, and with forward mutation primer 1 (SEQ ID NO: 3) and reverse mutation primer 1 (SEQ ID NO: 4) as primers, the site-directed mutation G101T was introduced by PCR1 amplification. After sequencing verification, the plasmid (named pPIC9K-G101T plasmid) was successfully constructed. The sequences of forward and reverse mutation primers 1 are shown in Table 1, with the underlined mutation sites. The composition of the PCR1 reaction system is shown in Table 2. The PCR1 amplification conditions were: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 5 min 40 s, for 25 cycles, and finally incubation at 72℃ for 5 min.

[0076] Table 1

[0077] Forward mutation primer 1 (SEQ ID NO: 3) <![CDATA[5’-CGCAATGGCAATCTG ACT CTGCAGAAAGTG-3’]]> Reverse mutation primer 1 (SEQ ID NO: 4) 5'-CAGATTGCCATTGCGGAATAAATCAACCAGC-3' Forward mutation primer 2 (SEQ ID NO: 5) <![CDATA[5’-CCGAGTATGTGAACAGC ACA GCTTATCTG-3’]]> Reverse mutation primer 2 (SEQ ID NO: 6) 5'-TGTTCACATACTCGGTGATCACTTTCTGCA-3'

[0078] Table 2

[0079]

[0080]

[0081] Using pPIC9K-G101T plasmid as a template, and with forward mutation primer 2 (SEQ ID NO: 5) and reverse mutation primer 2 (SEQ ID NO: 6) as primers, the site-directed mutation Q113T was introduced by PCR2 amplification. Sequencing verification results showed that no other mutations appeared besides the two required mutation sites. Therefore, the mutant plasmid (named pPIC9K-G101T / Q113T mutant plasmid) was successfully constructed. The sequences of forward and reverse mutation primers 2 are shown in Table 1, with underlined mutation sites. The composition of the PCR2 reaction system is shown in Table 3. The PCR2 amplification conditions were: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 5 min 40 s, for 25 cycles, and finally incubation at 72℃ for 5 min.

[0082] The mutant plasmid (named pPIC9K-G101T / Q113T mutant plasmid) contains the nucleotide sequence shown in SEQ ID NO: 1, which encodes a raw starch saccharifying enzyme mutant (named G101T / Q113T, amino acid sequence shown in SEQ ID NO: 2).

[0083] Table 3

[0084]

[0085] (2) Construction of recombinant strains

[0086] After confirming the correct band size by agarose gel electrophoresis, the PCR amplification products obtained through the above steps were digested with the restriction endonuclease DpnI and then recovered by column extraction (using a Thermo Fisher Scientific kit). The products were then transformed into *E. coli* JM109 competent cells via electroporation. After overnight culture in LB solid medium containing 50 μg / mL ampicillin, single clones were selected and transferred to the appropriate liquid LB medium. Plasmids were then extracted and sequenced for verification. The correctly sequenced mutant plasmid pPIC9K-G101T / Q113T was linearized by restriction endonuclease SalI and recovered (using a Thermo Fisher Scientific column recovery kit). This plasmid was then transformed into *Pichia pastoris* GS115 competent cells via electroporation, and single clones were selected for verification. The correctly verified single clones were the recombinant strain.

[0087] The digestion system and conditions for the restriction endonuclease DpnI are shown in Table 4, and the digestion system and conditions for the restriction endonuclease SalI are shown in Table 5.

[0088] Table 4

[0089]

[0090] Table 5

[0091]

[0092] Example 2

[0093] This example illustrates the expression and purification method of a raw starch saccharifying enzyme mutant.

[0094] The recombinant strain obtained in Example 1 was inoculated into a 250 mL shake flask containing 40 mL of YPD medium and cultured at 30 °C for 24 h to obtain the first fermentation broth. The fermentation broth was collected and centrifuged. The bacterial cells were washed 2-3 times with sterile physiological saline and resuspended in 50 mL of BMMY medium. Fermentation was continued at 30 °C for 120 h to obtain the second fermentation broth. The second fermentation broth was centrifuged at 4 °C and 5000 rpm for 15 min to obtain the fermentation supernatant for purification.

[0095] Because the mutated gene encoding amylosaccharidase carries a histidine tag, nickel affinity chromatography was used to separate and purify the target protein. The specific procedure is as follows: The fermentation supernatant was filtered through a 0.22 μm aqueous membrane to remove impurities, yielding the supernatant. This supernatant was loaded onto a nickel affinity chromatography column (Histrap 5 mL FF) pre-equilibrated with buffer A (20 mM KH2PO4-K2HPO4, pH 7.4). After reequilibration, the protein was eluted with different concentrations of buffer B (20 mM KH2PO4-K2HPO4, pH 7.4, 500 mM imidazole). The eluent from each absorption peak was collected, and enzyme activity was measured (using soluble starch as a substrate). The collected eluent from the absorption peaks was then subjected to electrophoresis. After electrophoresis verification, the purified enzyme solution of the amylosaccharidase mutant G101T / Q113T (i.e., the target protein) was obtained.

[0096] The method for determining enzyme activity (using soluble starch as a substrate) is as follows: the substrate is a 20 g / L soluble starch aqueous solution; the reaction system consists of 400 μL substrate, 400 μL acetate-sodium acetate buffer (pH 4.6), and 200 μL purified enzyme solution. The reaction is carried out at 40℃ for 10 min. Immediately after reaction, 1 mL of DNS is added to terminate the reaction. The reducing sugar (calculated as glucose) produced in the system is determined using the DNS method. The control is the inactivated enzyme solution. The reducing sugar produced in the enzyme activity reaction system is calculated based on the glucose standard curve.

[0097] In this embodiment, the enzyme activity unit of raw starch saccharifying enzyme based on soluble starch is defined as follows: the amount of enzyme required to hydrolyze a 20 g / L soluble starch aqueous solution to produce 1 μmol of reducing sugar in 1 min under reaction conditions of pH 4.6 and 40℃ is defined as one enzyme activity unit (U).

[0098] Example 3

[0099] This example illustrates the effect of temperature on the enzyme activity of pRSGA and the starch saccharifying enzyme mutants G101T / Q113T using soluble starch as a substrate.

[0100] The method for obtaining pRSGA is as follows: pPICK9K-pRSGA plasmid is transformed into competent cells of Pichia pastoris GS115 by electroporation, and single clones are selected for verification. The verified single clones are then expressed and purified for pRSGA according to the method in Example 2. After electrophoresis verification, the purified enzyme solution of pRSGA is obtained.

[0101] The purified enzyme solution of pRSGA and the purified enzyme solution of the raw starch saccharifying enzyme mutant G101T / Q113T prepared in Example 2 were tested according to the following method: The purified enzyme solution was reacted for 10 min at specific reaction conditions (30℃, 40℃, 50℃, 60℃, 70℃, 80℃), and the enzyme activity of raw starch saccharifying enzyme (using soluble starch as substrate) was determined. The relative values ​​(relative enzyme activity) at other temperatures were calculated with the highest enzyme activity as 100%.

[0102] The method for determining the enzyme activity of raw starch saccharifying enzyme using soluble starch as a substrate is as follows: the substrate is a 20 g / L soluble starch aqueous solution, and the reaction system is: 400 μL substrate, 400 μL acetate-sodium acetate buffer at pH 4.6, and 200 μL purified enzyme solution. After reacting at the reaction temperature (30℃, 40℃, 50℃, 60℃, 70℃, 80℃) for 10 min, 1 mL DNS is immediately added to terminate the reaction. The reducing sugar (calculated as glucose) produced in the system is determined by the DNS method. The control is the inactivated enzyme solution. The reducing sugar produced in the enzyme activity reaction system is calculated based on the glucose standard curve.

[0103] In this embodiment, the enzyme activity unit of raw starch saccharifying enzyme under different reaction conditions is defined as follows: the amount of enzyme required to hydrolyze 20 g / L soluble starch aqueous solution to produce 1 μmol of reducing sugar in 1 min under specific reaction conditions (30℃, 40℃, 50℃, 60℃, 70℃, 80℃) at pH 4.6 is defined as one enzyme activity unit (U).

[0104] The results are as follows Figure 1 As shown. (Through) Figure 1 It can be seen that, compared with pRSGA, the optimal reaction temperature of the raw starch saccharifying enzyme mutant G101T / Q113T is reduced from 70℃ to 60℃, which is suitable for the future requirement of degrading raw starch at a temperature lower than the gelatinization temperature, and has important application value.

[0105] Example 4

[0106] This embodiment illustrates the determination of kinetic parameters of pRSGA and the raw starch saccharifying enzyme mutant G101T / Q113T using raw corn starch as a substrate.

[0107] The method for obtaining pRSGA is as follows: pPICK9K-pRSGA plasmid is transformed into competent cells of Pichia pastoris GS115 by electroporation, and single clones are selected for verification. The verified single clones are then expressed and purified for pRSGA according to the method in Example 2. After electrophoresis verification, the purified enzyme solution of pRSGA is obtained.

[0108] The kinetic parameters of pRGSA and the raw starch saccharifying enzyme mutant were obtained according to the following method:

[0109] (1) Using raw corn starch suspensions of 0 g / L, 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, 15 g / L, 17.5 g / L and 20 g / L as substrates, the purified enzyme solution was reacted at 40℃ for 60 min, and the enzyme activity of raw starch saccharifying enzyme (based on raw corn starch suspension) was determined.

[0110] (2) The enzyme activities measured under different concentrations of raw starch suspension were plotted using the Michaelis-Menten equation with double reciprocals to obtain the regression to the Xianning equation and calculate the relevant K. m and k cat value.

[0111] The method for determining the enzyme activity of raw starch saccharifying enzyme (using raw corn starch as substrate) is as follows: Raw corn starch suspensions at specific concentrations (0 g / L, 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, 15 g / L, 17.5 g / L, and 20 g / L) are used as substrates (prepared with pH 4.6 acetic acid-sodium acetate buffer). The reaction system consists of 20 mL substrate and 0.5 mL enzyme solution. The reaction is carried out at 40℃ for 60 min, and 0.5 mL of 0.1 mol / L NaOH is added immediately to terminate the reaction. The control is the inactivated enzyme solution. After the reaction, the reaction solution is centrifuged at 8000 r / min for 5 min, and the reducing sugar concentration (calculated as glucose) of the supernatant is determined using the DNS method, thereby calculating the corresponding raw starch saccharifying enzyme activity.

[0112] In this embodiment, the enzyme activity of raw starch saccharifying enzyme at different substrate concentrations is defined as follows: under reaction conditions of pH 4.6 and 40℃, the amount of enzyme required to hydrolyze a raw corn starch suspension of a specific concentration (0 g / L, 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, 15 g / L, 17.5 g / L, 20 g / L) to produce 1 μmol of reducing sugar in 1 hour is defined as one enzyme activity unit (U).

[0113] Enzyme reaction kinetic parameter k cat / K m The results are shown in Table 6. The results show that the k values ​​of the raw starch saccharifying enzyme mutant G101T / Q113T are... cat / K m The increased value makes it more suitable for catalyzing the reaction of raw corn starch.

[0114] Table 6. Kinetic parameters of enzyme-catalyzed reactions

[0115]

[0116] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention. SEQUENCE LISTING <110> Jilin COFCO Biochemical Co., Ltd. Jiangnan University <120> Starch saccharifying enzyme mutants, their encoding genes, and applications <130> I74963JLZL <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 1836 <212> DNA <213> Artificial Sequence <220> <223> This sequence is artificially synthesized <400> 1 gccccgcagc tgagcgcacg tgcaactggt tctttagaca gttggctggg taccgaaacc 60 accgtggcac tgaacggtat tctggcaaac atcggcgccg atggtgcata cgccaagagc 120 gcaaaaccgg gcatcatcat tgcaagcccg agcaccagcg agccggatta ctactatact 180 tggacacgcg atgccgcatt agttaccaag gtgctggttg atttattccg caatggcaat 240 ctgactctgc agaaagtgat caccgagtat gtgaacagca cagctttct gcagaccgtg 300 agcaatccga gcggtggttt agcaagcggt ggtttagcag aaccgaaata caatgtggat 360 atgacagcat tcaccggcgc atggggtcgt ccgcagcgtg atggtccggc cttacgtgcc 420 accgctttaa ttgactttgg taattggctg atcgacaacg gctatagcag ctacgccgtt 480 aataacatct ggccgatcgt tcgtaacgat ctgagctacg tgagccagta ctggagtcag 540 agcggcttcg atttatggga agaggtgaac agcatgagct tcttcaccgt ggccgtgcag 600 catcgtgcac tggtggaagg cagcacattt gccaaacgcg ttggtgccag ttgcagctgg 660 tgtgatagcc aagctccgca gattttatgc tatatgcaga gtttttggac cggcagctat 720 atcaacgcca ataccggtgg tggtcgtagc ggtaaagatg caaacaccgt gctggcaagc 780 atccatacct tcgatcccga agctggttgt gacgatacca cctttcagcc gtgtagcccg 840 cgtgctttag ccaatcataa agtgtacacc gacagcttcc gtagcgtgta tgcaatcaac 900 agtggtatcc cgcaaggtgc agccgtgagt gctggtcgct atccggaaga cgtgtactac 960 aacggcaatc cgtggtttct gaccacactg gcagcagccg agcaactgta tgatgccatt 1020 tatcagtgga agaaaattgg cagtatcagcattaccogca ccagcttagc atttttcaaa 1080 gatatctaca gtagcgccgc agtgggtacc tatgccagca gtaccagtac atttacagat 1140 attatcaacg ccgtgaagac ctatgcagac ggctacgtta gcattgtgca agctcacgcc 1200 atgaacaacg gctctttaag tgaacagttc gacaaaagca gcggtttaag tctgagtgca 1260 cgcgatttaa cttggagtta tgccgccttt ttaaccgcaa atatgcgtcg caatggtgtt 1320 gtgcccgctc cttggggtgc cgcaagcgca aatagcgtgc cgagcagttg cagcatgggt 1380 agcgccaccg gtacctacag cacagccaca gcaaccagtt ggccgagcac tttaaccagt 1440 ggtagccccg gtagcaccac aaccgttggc accacaacaa gcaccaccag tggcaccgcc 1500 accgaaaccg catgtgccac cccgacagcc gtggccgtta cctttaatga gattgccacc 1560 accacctatg gcgaaaacgt gtacatcgtg ggcagcatca gcgaactggg taattgggat 1620 accagcaagg cagtggcttt aagtgccagt aaatatacaa gcagtaataa tctgtggtat 1680 gtgagtgtga ctttaccggc tggtaccacc ttcgagtaca agtacattcg caaagagagc 1740 gacggcagca tcgtgtggga aagcgacccg aaccgtagtt acacagttcc cgctgcatgt 1800 ggcgttagca ccgcaaccga gaacgacact tggcgt 1836 <210> 2 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> This sequence is artificially synthesized <400> 2 Ala Pro Gln Leu Ser Ala Arg Ala Thr Gly Ser Leu Asp Ser Trp Leu 1 5 10 15 Gly Thr Glu Thr Thr Val Ala Leu Asn Gly Ile Leu Ala Asn Ile Gly 20 25 30 Ala Asp Gly Ala Tyr Ala Lys Ser Ala Lys Pro Gly Ile Ile Ile Ala 35 40 45 Ser Pro Ser Thr Ser Glu Pro Asp Tyr Tyr Tyr Thr Trp Thr Arg Asp 50 55 60 Ala Ala Leu Val Thr Lys Val Leu Val Asp Leu Phe Arg Asn Gly Asn 65 70 75 80 Leu Thr Leu Gln Lys Val Ile Thr Glu Tyr Val Asn Ser Thr Ala Tyr 85 90 95 Leu Gln Thr Val Ser Asn Pro Ser Gly Gly Leu Ala Ser Gly Gly Leu 100 105 110 Ala Glu Pro Lys Tyr Asn Val Asp Met Thr Ala Phe Thr Gly Ala Trp 115 120 125 Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Leu Ile 130 135 140 Asp Phe Gly Asn Trp Leu Ile Asp Asn Gly Tyr Ser Ser Tyr Ala Val 145 150 155 160 Asn Asn Ile Trp Pro Ile Val Arg Asn Asp Leu Ser Tyr Val Ser Gln 165 170 175 Tyr Trp Ser Gln Ser Gly Phe Asp Leu Trp Glu Glu Val Asn Ser Met 180 185 190 Ser Phe Phe Thr Val Ala Val Gln His Arg Ala Leu Val Glu Gly Ser 195 200 205 Thr Phe Ala Lys Arg Val Gly Ala Ser Cys Ser Trp Cys Asp Ser Gln 210 215 220 Ala Pro Gln Ile Leu Cys Tyr Met Gln Ser Phe Trp Thr Gly Ser Tyr 225 230 235 240 Ile Asn Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp Ala Asn Thr 245 250 255 Val Leu Ala Ser Ile His Thr Phe Asp Pro Glu Ala Gly Cys Asp Asp 260 265 270 Thr Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Val 275 280 285 Tyr Thr Asp Ser Phe Arg Ser Val Tyr Ala Ile Asn Ser Gly Ile Pro 290 295 300 Gln Gly Ala Ala Val Ser Ala Gly Arg Tyr Pro Glu Asp Val Tyr Tyr 305 310 315 320 Asn Gly Asn Pro Trp Phe Leu Thr Thr Leu Ala Ala Ala Glu Gln Leu 325 330 335 Tyr Asp Ala Ile Tyr Gln Trp Lys Lys Ile Gly Ser Ile Ser Ile Thr 340 345 350 Ser Thr Ser Leu Ala Phe Phe Lys Asp Ile Tyr Ser Ser Ala Ala Val 355 360 365 Gly Thr Tyr Ala Ser Ser Thr Ser Thr Phe Thr Asp Ile Ile Asn Ala 370 375 380 Val Lys Thr Tyr Ala Asp Gly Tyr Val Ser Ile Val Gln Ala His Ala 385 390 395 400 Met Asn Asn Gly Ser Leu Ser Glu Gln Phe Asp Lys Ser Ser Gly Leu 405 410 415 Ser Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Phe Leu Thr 420 425 430 Ala Asn Met Arg Arg Asn Gly Val Val Pro Ala Pro Trp Gly Ala Ala 435 440 445 Ser Ala Asn Ser Val Pro Ser Ser Cys Ser Met Gly Ser Ala Thr Gly 450 455 460 Thr Tyr Ser Thr Ala Thr Ala Thr Ser Trp Pro Ser Thr Leu Thr Ser 465 470 475 480 Gly Ser Pro Gly Ser Thr Thr Thr Val Gly Thr Thr Thr Ser Thr Thr 485 490 495 Ser Gly Thr Ala Thr Glu Thr Ala Cys Ala Thr Pro Thr Ala Val Ala 500 505 510 Val Thr Phe Asn Glu Ile Ala Thr Thr Thr Tyr Gly Glu Asn Val Tyr 515 520 525 Ile Val Gly Ser Ile Ser Glu Leu Gly Asn Trp Asp Thr Ser Lys Ala 530 535 540 Val Ala Leu Ser Ala Ser Lys Tyr Thr Ser Ser Asn Asn Leu Trp Tyr 545 550 555 560 Val Ser Val Thr Leu Pro Ala Gly Thr Thr Phe Glu Tyr Lys Tyr Ile 565 570 575 Arg Lys Glu Ser Asp Gly Ser Ile Val Trp Glu Ser Asp Pro Asn Arg 580 585 590 Ser Tyr Thr Val Pro Ala Ala Cys Gly Val Ser Thr Ala Thr Glu Asn 595 600 605 Asp Thr Trp Arg 610 <210> 3 <211> 30 <212> DNA <213> Artificial sequence <220> <223> This sequence is artificially synthesized. <400> 3 cgcaatggca atctgactct gcagaaagtg 30 <210> 4 <211> 31 <212> DNA <213> Artificial sequence <220> <223> This sequence is artificially synthesized. <400> 4 cagattgcca ttgcggaata aatcaaccag c 31 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> This sequence is artificially synthesized. <400> 5 ccgagtatgt gaacagcaca gcttatctg 29 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> This sequence is artificially synthesized. <400> 6 tgttcacata ctcggtgatc actttctgca 30 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> This sequence is artificially synthesized. <400> 7 His His His His His His 1 5 <210> 8 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> This sequence is artificially synthesized. <400> 8 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Three Gln Ser Met With Arg Tyr With Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Val Leu Asp With Arg Tyr Gly Val Ser Arg With Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe was synthesized by Arg and Glu synthesized by Pro Gln and Asp synthesized by Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys 210 215 <210> 9 <211> 392 <212> PRT <213> Artificial Sequence <220> <223> This sequence is artificially synthesized. <400> 9 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Val Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys 340 345 350 Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360 365 Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp 370 375 380 Glu Ala Leu Lys Asp Ala Gln Thr 385 390 <210> 10 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> This sequence is artificially synthesized. <400> 10 Ala Pro Gln Leu Ser Ala Arg Ala Thr Gly Ser Leu Asp Ser Trp Leu 1 5 10 15 Gly Thr Glu Thr Thr Val Ala Leu Asn Gly Ile Leu Ala Asn Ile Gly 20 25 30 Ala Asp Gly Ala Tyr Ala Lys Ser Ala Lys Pro Gly Ile Ile Ile Ala 35 40 45 Ser Pro Ser Thr Ser Glu Pro Asp Tyr Tyr Tyr Thr Trp Thr Arg Asp 50 55 60 Ala Ala Leu Val Thr Lys Val Leu Val Asp Leu Phe Arg Asn Gly Asn 65 70 75 80 Leu Gly Leu Gln Lys Val Ile Thr Glu Tyr Val Asn Ser Gln Ala Tyr 85 90 95 Leu Gln Thr Val Ser Asn Pro Ser Gly Gly Leu Ala Ser Gly Gly Leu 100 105 110 Ala Glu Pro Lys Tyr Asn Val Asp Met Thr Ala Phe Thr Gly Ala Trp 115 120 125 Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Leu Ile 130 135 140 Asp Phe Gly Asn Trp Leu Ile Asp Asn Gly Tyr Ser Ser Tyr Ala Val 145 150 155 160 Asn Asn Ile Trp Pro Ile Val Arg Asn Asp Leu Ser Tyr Val Ser Gln 165 170 175 Tyr Trp Ser Gln Ser Gly Phe Asp Leu Trp Glu Glu Val Asn Ser Met 180 185 190 Ser Phe Phe Thr Val Ala Val Gln His Arg Ala Leu Val Glu Gly Ser 195 200 205 Thr Phe Ala Lys Arg Val Gly Ala Ser Cys Ser Trp Cys Asp Ser Gln 210 215 220 Ala Pro Gln Ile Leu Cys Tyr Met Gln Ser Phe Trp Thr Gly Ser Tyr 225 230 235 240 Ile Asn Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp Ala Asn Thr 245 250 255 Val Leu Ala Ser Ile His Thr Phe Asp Pro Glu Ala Gly Cys Asp Asp 260 265 270 Thr Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Val 275 280 285 Tyr Thr Asp Ser Phe Arg Ser Val Tyr Ala Ile Asn Ser Gly Ile Pro 290 295 300 Gln Gly Ala Ala Val Ser Ala Gly Arg Tyr Pro Glu Asp Val Tyr Tyr 305 310 315 320 Asn Gly Asn Pro Trp Phe Leu Thr Thr Leu Ala Ala Ala Glu Gln Leu 325 330 335 Tyr Asp Ala Ile Tyr Gln Trp Lys Lys Ile Gly Ser Ile Ser Ile Thr 340 345 350 Ser Thr Ser Leu Ala Phe Phe Lys Asp Ile Tyr Ser Ser Ala Ala Val 355 360 365 Gly Thr Tyr Ala Ser Ser Thr Ser Thr Phe Thr Asp Ile Ile Asn Ala 370 375 380 Val Lys Thr Tyr Ala Asp Gly Tyr Val Ser Ile Val Gln Ala His Ala 385 390 395 400 Met Asn Asn Gly Ser Leu Ser Glu Gln Phe Asp Lys Ser Ser Gly Leu 405 410 415 Ser Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Phe Leu Thr 420 425 430 Ala Asn Met Arg Arg Asn Gly Val Val Pro Ala Pro Trp Gly Ala Ala 435 440 445 Ser Ala Asn Ser Val Pro Ser Ser Cys Ser Met Gly Ser Ala Thr Gly 450 455 460 Thr Tyr Ser Thr Ala Thr Ala Thr Ser Trp Pro Ser Thr Leu Thr Ser 465 470 475 480 Gly Ser Pro Gly Ser Thr Thr Thr Val Gly Thr Thr Thr Ser Thr Thr 485 490 495 Ser Gly Thr Ala Thr Glu Thr Ala Cys Ala Thr Pro Thr Ala Val Ala 500 505 510 Val Thr Phe Asn Glu Ile Ala Thr Thr Thr Tyr Gly Glu Asn Val Tyr 515 520 525 Ile Val Gly Ser Ile Ser Glu Leu Gly Asn Trp Asp Thr Ser Lys Ala 530 535 540 Val Ala Leu Ser Ala Ser Lys Tyr Thr Ser Ser Asn Asn Leu Trp Tyr 545 550 555 560 Val Ser Val Thr Leu Pro Ala Gly Thr Thr Phe Glu Tyr Lys Tyr Ile 565 570 575 Arg Lys Glu Ser Asp Gly Ser Ile Val Trp Glu Ser Asp Pro Asn Arg 580 585 590 Ser Tyr Thr Val Pro Ala Ala Cys Gly Val Ser Thr Ala Thr Glu Asn 595 600 605 Asp Thr Trp Arg 610 <210> 11 <211> 1836 <212> DNA <213> Artificial Sequence <220> <223> This sequence is artificially synthesized. <400> 11 gccccgcagc tgagcgcacg tgcaactggt tctttagaca gttggctggg taccgaaacc 60 accgtggcac tgaacggtat tctggcaaac atcggcgccg atggtgcata cgccaagagc 120 gcaaaaccgg gcatcatcat tgcaagcccg agcaccagcg agccggatta ctactatact 180 tggacacgcg atgccgcatt agttaccaag gtgctggttg atttattccg caatggcaat 240 ctgggtctgc agaaagtgat caccgagtat gtgaacagcc aagcttatct gcagaccgtg 300 agcaatccga gcggtggttt agcaagcggt ggtttagcag aaccgaaata caatgtggat 360 atgacagcat tcaccggcgc atggggtcgt ccgcagcgtg atggtccggc cttacgtgcc 420 accgctttaa ttgactttgg taattggctg atcgacaacg gctatagcag ctacgccgtt 480 aataacatct ggccgatcgt tcgtaacgat ctgagctacg tgagccagta ctggagtcag 540 agcggcttcg atttatggga agaggtgaac agcatgagct tcttcaccgt ggccgtgcag 600 catcgtgcac tggtggaagg cagcacattt gccaaacgcg ttggtgccag ttgcagctgg 660 tgtgatagcc aagctccgca gattttatgc tatatgcaga gtttttggac cggcagctat 720 atcaacgcca ataccggtgg tggtcgtagc ggtaaagatg caaacaccgt gctggcaagc 780 atccatacct tcgatcccga agctggttgt gacgatacca cctttcagcc gtgtagcccg 840 cgtgctttag ccaatcataa agtgtacacc gacagcttcc gtagcgtgta tgcaatcaac 900 agtggtatcc cgcaaggtgc agccgtgagt gctggtcgct atccggaaga cgtgtactac 960 aacggcaatc cgtggtttct gaccacactg gcagcagccg agcaactgta tgatgccatt 1020 tatcagtgga agaaaattgg cagtatcagc attaccagca ccagcttagc atttttcaaa 1080 gatatctaca gtagcgccgc agtgggtacc tatgccagca gtaccagtac atttacagat 1140 attatcaacg ccgtgaagac ctatgcagac ggctacgtta gcattgtgca agctcacgcc 1200 atgaacaacg gctctttaag tgaacagttc gacaaaagca gcggtttaag tctgagtgca 1260 cgcgatttaa cttggagtta tgccgccttt ttaaccgcaa atatgcgtcg caatggtgtt 1320 gtgcccgctc cttggggtgc cgcaagcgca aatagcgtgc cgagcagttg cagcatgggt 1380 agcgccaccg gtacctacag cacagccaca gcaaccagtt ggccgagcac tttaaccagt 1440 ggtagccccg gtagcaccac aaccgttggc accacaacaa gcaccaccag tggcaccgcc 1500 accgaaaccg catgtgccac cccgacagcc gtggccgtta cctttaatga gattgccacc 1560 accacctatg gcgaaaacgt gtacatcgtg ggcagcatca gcgaactggg taattgggat 1620 accagcaagg cagtggcttt aagtgccagt aaatatacaa gcagtaataa tctgtggtat 1680 gtgagtgtga ctttaccggc tggtaccacc ttcgagtaca agtacattcg caaagagagc 1740 gacggcagca tcgtgtggga aagcgacccg aaccgtagtt acacagttcc cgctgcatgt 1800 ggcgttagca ccgcaaccga gaacgacact tggcgt 1836

Claims

1. A mutant of raw starch saccharifying enzyme, characterized in that, The amino acid sequence of the raw starch saccharifying enzyme mutant is shown in SEQ ID NO:

2.

2. A gene capable of encoding the raw starch saccharifying enzyme mutant of claim 1.

3. The gene according to claim 2, wherein, The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

4. A recombinant vector, characterized in that, The recombinant vector contains the gene as described in claim 2 or 3.

5. A recombinant bacterial strain, characterized in that, The recombinant strain contains the gene described in claim 2 or 3 or the recombinant vector described in claim 4.

6. The recombinant strain according to claim 5, wherein, The recombinant strain is Pichia pastoris (Pichia pastoris). Pichia pastoris ), Escherichia coli ( Escherichia coli Bacillus subtilis ( Bacillus subtilis At least one of the following.

7. A method for preparing a raw starch saccharifying enzyme mutant, characterized in that, The method includes culturing the recombinant strain according to claim 5 or 6 and separating and purifying the culture product to obtain the raw starch saccharifying enzyme mutant.

8. The application of the raw starch saccharifying enzyme mutant of claim 1, the gene of claim 2 or 3, the recombinant vector of claim 4, and the recombinant strain of claim 5 or 6 in the degradation of raw starch.

9. The application according to claim 8, wherein, The raw starch is raw corn starch.

10. The use of the raw starch saccharifying enzyme mutant of claim 1, the gene of claim 2 or 3, the recombinant vector of claim 4, and the recombinant strain of claim 5 or 6 in the preparation of glucose.

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