α-Glucosidase Mutants, Kits and Their Applications

Abstract

The present invention provides an α-glucosidase mutant, a kit and its application. The α-glucosidase mutant includes: (a) a protein mutated based on the α-glucosidase shown in SEQ ID NO: 1, and the mutation includes mutations occurring at one or more sites of V298, E283 or R338; (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having α-glucosidase activity. It can solve the problem of low specific enzyme activity of α-glucosidase in the prior art and is applicable to the field of α-glucosidase modification.

Description

Technical Field

[0001] The present invention relates to the field of α-glucosidase modification, and in particular, to an α-glucosidase mutant, a kit and their applications. Background Art

[0002] α-Glucosidase is a glycoside hydrolase, which is an essential enzyme in the sugar metabolism pathway of organisms. It directly participates in the metabolism pathways of starch and glycogen. Its main function is to hydrolyze glucosidic bonds and release glucose as a product. It can cleave the α-1,4 glycosidic bond at the non-reducing end of oligosaccharide substrates to release glucose, or transfer the released glucose residue to another saccharide substrate to form an α-1,6 glycosidic bond. In the field of in vitro diagnosis, α-glucosidase is mainly used for the detection of α-amylase. However, the specific enzyme activity of the existing α-glucosidase on the market is still relatively low, and there are problems such as a large addition amount and high price. Therefore, it is urgent to screen out α-glucosidase with high specific enzyme activity suitable for α-amylase kits. Summary of the Invention

[0003] The main object of the present invention is to provide an α-glucosidase mutant, a kit and their applications to solve the problem of low specific enzyme activity of α-glucosidase in the prior art.

[0004] To achieve the above object, according to the first aspect of the present invention, there is provided an α-glucosidase mutant, which includes: (a) a protein mutated based on the α-glucosidase shown in SEQ ID NO: 1, and the mutation includes mutations at one or more sites of V298, E283 or R338; (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having α-glucosidase activity.

[0005] Further, in (a), the types of amino acids substituted at the V298 site include V298F, V298Y or V298H, the type of amino acid substituted at the E283 site includes E283R, and the type of amino acid substituted at the R338 site includes R338Y; wherein, the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid.

[0006] Further, the mutation includes any one of the following amino acid mutation combinations: V298F+E283R, V298Y+E283R, V298H+E283R or V298H+R338Y; wherein, V298F+E283R is the protein shown in SEQ ID NO: 2, V298Y+E283R is the protein shown in SEQ ID NO: 3; V298H+E283R is the protein shown in SEQ ID NO: 4; V298H+R338Y is the protein shown in SEQ ID NO: 5; or the α-glucosidase mutant includes a protein having more than 70% homology with the protein shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and having α-glucosidase activity.

[0007] Further, the α-glucosidase mutant includes a protein having more than 80%, preferably more than 85%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in (a) and having α-glucosidase activity.

[0008] To achieve the above object, according to the second aspect of the present invention, there is provided a DNA molecule encoding any one of the above α-glucosidase mutants.

[0009] To achieve the above object, according to the third aspect of the present invention, there is provided a recombinant plasmid ligated with the above DNA molecule.

[0010] To achieve the above object, according to the fourth aspect of the present invention, there is provided a host cell containing the above DNA molecule or the above recombinant plasmid.

[0011] Further, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli; preferably, Escherichia coli includes JM109 or Rosetta strain.

[0012] To achieve the above object, according to the fifth aspect of the present invention, there is provided a kit including α-glucosidase, and the α-glucosidase includes any one of the above α-glucosidase mutants.

[0013] Further, the above kit is used for detecting α-amylase and further includes at least one of the following: a substrate, a buffer or an ionic additive; the substrate can be further hydrolyzed by α-glucosidase after being hydrolyzed by α-amylase to produce p-nitrophenol; preferably, the substrate includes p-nitrophenyl-D-maltoheptaose; preferably, the buffer includes Tris buffer; preferably, the ionic additive includes sodium chloride and / or calcium chloride; preferably, α-glucosidase and the substrate are separately provided in the kit.

[0014] By applying the technical solution of the present invention, site-directed mutagenesis is carried out on the V298, E283 or R338 site of wild-type α-glucosidase to obtain an α-glucosidase mutant. This mutant has a higher specific enzyme activity than wild-type α-glucosidase and can be applied to the detection of α-amylase. Specific Embodiments

[0015] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0016] Term Explanation:

[0017] Specific enzyme activity: The specific activity of an enzyme refers to the number of enzyme activity units per milligram of protein.

[0018] As mentioned in the background art, the wild-type α-glucosidase extracted in the prior art has poor specific enzyme activity. When applied to the detection of α-amylase, a large amount of α-glucosidase needs to be added, resulting in high cost and insensitive reaction, and inaccurate final detection results. Therefore, in this application, the inventors attempt to develop an α-glucosidase mutant, a kit and its application to improve the specific enzyme activity of α-glucosidase. On this basis, the applicant proposes a series of protection schemes for this application.

[0019] In the first typical embodiment of this application, an α-glucosidase mutant is provided. The mutant includes: (a) a protein mutated based on the α-glucosidase shown in SEQ ID NO: 1, and the mutation includes mutations at one or more of the sites V298, E283 or R338; (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having α-glucosidase activity.

[0020] SEQ ID NO: 1:

[0021] MKKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKIILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHLFDVKQPDLNWENSEVRQALYDMINWWLDKGIDGFRIDAISHIKKKPGLPDLPNPKGLKYVPSFAAHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFHMIFQFEHLGLWKRKADGSIDVRRLKRTLTKWQKGLENRGWNALFLENHDLPRSVSTWGNDREYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVQFSDIRDYRDVAALRLYELERANGRTHEEVMKIIWKTGRDNSRTPMQWSDAPNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGAYDLLLENHPSIYAYTRTLGRDRALIIVNVSDRPSLYRYDGFRLQSSDLALSNYPVRPHKNATRFKLKPYEARVYIWKE。

[0022] In a preferred embodiment, in the above (a), the types of amino acids substituted at the V298 site include V298F, V298Y or V298H, the types of amino acids substituted at the E283 site include E283R, and the types of amino acids substituted at the R338 site include R338Y; wherein, the letter before the number represents the original amino acid, and the letter after the number represents the mutant amino acid.

[0023] In a preferred embodiment, the α-glucosidase mutation comprises any one of the following amino acid mutation combinations: V298F + E283R, V298Y + E283R, V298H + E283R or V298H + R283Y; wherein, V298F + E283R is the protein shown in SEQ ID NO: 2, V298Y + E283R is the protein shown in SEQ ID NO: 3; V298H + E283R is the protein shown in SEQ ID NO: 4; V298H + R338Y is the protein shown in SEQ ID NO: 5; the α-glucosidase mutant comprises a protein having more than 70% homology with the proteins shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and having α-glucosidase activity.

[0024] SEQ ID NO: 2:

[0025] MKKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKIILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHLFDVKQPDLNWENSEVRQALYDMINWWLDKGIDGFRIDAISHIKKKPGLPDLPNPKGLKYVPSFAAHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFHMIFQFRHLGLWKRKADGSIDFRRLKRTLTKWQKGLENRGWNALFLENHDLPRSVSTWGNDREYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVQFSDIRDYRDVAALRLYELERANGRTHEEVMKIIWKTGRDNSRTPMQWSDAPNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGAYDLLLENHPSIYAYTRTLGRDRALIIVNVSDRPSLYRYDGFRLQSSDLALSNYPVRPHKNATRFKLKPYEARVYIWKE.

[0026] SEQ ID NO: 3:

[0027] MKKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKIILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHLFDVKQPDLNWENSEVRQALYDMINWWLDKGIDGFRIDAISHIKKKPGLPDLPNPKGLKYVPSFAAHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFHMIFQFRHLGLWKRKADGSIDYRRLKRTLTKWQKGLENRGWNALFLENHDLPRSVSTWGNDREYWAESAKALGALYFFMQGTPFIY QGQEIGMTNVQFSDIRDYRDVAALRLYELERANGRTHEEVMKIIWKTGRDNSRTPMQWSDAPNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGAYDLLLENHPSIYAYTRTLGRDRALIIVNVSDRPSLYRYDGFRLQSSDLALSNYPVRPHKNATRFKLKPYEARVYIWKE。

[0028] SEQ ID NO: 4:

[0029] MKKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKIILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHLFDVKQPDLNWENSEVRQALYDMINWWLDKGIDGFRIDAISHIKKKPGLPDLPNPKGLKYVPSFAAHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFHMIFQFRHLGLWKRKADGSIDHRRLKRTLTKWQKGLENRGWNALFLENHDLPRSVSTWGNDREYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVQFSDIRDYRDVAALRLYELERANGRTHEEVMKIIWKTGRDNSRTPMQWSDAPNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGAYDLLLENHPSIYAYTRTLGRDRALIIVNVSDRPSLYRYDGFRLQSSDLALSNYPVRPHKNATRFKLKPYEARVYIWKE。

[0030] SEQ ID NO: 5:

[0031] MKKTWWKEGVAYQIYPRSFMDANGDGIGDLRGIIEKLDYLVELGVDIVWICPIYRSPNADNGYDISDYYAIMDEFGTMDDFDELLAQAHRRGLKIILDLVINHTSDEHPWFIESRSSRDNPKRDWYIWRDGKDGREPNNWESIFGGSAWQYDERTGQYYLHLFDVKQPDLNWENSEVRQALYDMINWWLDKGIDGFRIDAISHIKKKPGLPDLPNPKGLKYVPSFAAHMNQPGIMEYLRELKEQTFARYDIMTVGEANGVTVDEAEQWVGEENGVFHMIFQFEHLGLWKRKADGSIDHRRLKRTLTKWQKGLENRGWNALFLENHDLPRSVSTWGNDYEYWAESAKALGALYFFMQGTPFIYQGQEIGMTNVQFSDIRDYRDVAALRLYELERANGRTHEEVMKIIWKTGRDNSRTPMQWSDAPNAGFTTGTPWIKVNENYRTINVEAERRDPNSVWSFYRQMIQLRKANELFVYGAYDLLLENHPSIYAYTRTLGRDRALIIVNVSDRPSLYRYDGFRLQSSDLALSNYPVRPHKNATRFKLKPYEARVYIWKE。

[0032] In a preferred embodiment, the α-glucosidase mutant comprises a protein having a homology of more than 80%, preferably more than 85%, more preferably more than 95%, and further preferably more than 99% with the amino acid sequence defined in (a) and having α-glucosidase activity.

[0033] The above amino acid mutations were all tested and explored in the examples of this application. Compared with the parent having the amino acid sequence shown in SEQ ID NO: 1, they all have α-glucosidase activity. The above mutation sites are all mutations around the amino acid active site. Such mutations can improve the activity of the protein. For mutations far from the active site, the impact on enhancing the activity of the protein is relatively small. Therefore, proteins having a homology of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% or more with the above amino acid sequence and having the same ability to improve specific enzyme activity can be obtained.

[0034] The homology in this specification refers to the "identity" between amino acid sequences, that is, the total ratio of amino acid residues of the same type in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool), FASTA, etc.

[0035] The proteins mentioned above that have a homology of 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, and even 99.9% or more) with the protein provided by sequence (a) and have the same function, their active sites, active pockets, active mechanisms, protein structures, etc. are probably the same as those of the protein provided by sequence (a), and they are homologous proteins obtained by amino acid mutation.

[0036] The acquisition of the above-mentioned identical sequences can be achieved through amino acid substitution, replacement, etc. Generally, for the rules of substitution, replacement, etc., the effects are similar after the mutual substitution of amino acids with similar properties. For the convenience of description, the abbreviations of amino acid residues are listed as follows first: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0037] For the substitution or replacement of amino acids, for example, in the above-mentioned homologous proteins, conservative amino acid substitutions may occur. "Conservative amino acid substitutions" include but are not limited to:

[0038] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0039] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0040] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0041] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0042] Those skilled in the art can also perform conservative substitutions on amino acids according to amino acid substitution rules well-known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0043] When site-directed mutagenesis of the α-glucosidase mutant is carried out in this application, two rounds of PCR amplification are performed. The first round of PCR in this application is "mutation PCR" or "mutant primer PCR", that is, specific primers are used to guide the generation of mutants. These specific primers usually contain changes at the mutation sites to ensure the correct selection of mutants as much as possible and can generate enough template DNA containing the correct mutants for the second round of PCR. The second round of PCR in this application is "amplification PCR" or "primer amplification PCR", that is, the DNA fragments generated in the first round of PCR, and the forward and reverse primers of the target fragment are used to amplify the full-length DNA fragment of α-glucosidase containing the mutation site, so that all DNA strands of α-glucosidase can be extended. Therefore, by performing two rounds of PCR, the stability and accuracy of the α-glucosidase mutant in this application can be ensured.

[0044] In the second typical embodiment of this application, a DNA molecule is provided, and this DNA molecule encodes any one of the above-mentioned α-glucosidase mutants.

[0045] The above DNA can encode the above-mentioned α-glucosidase mutant and can be ligated to a recombinant plasmid to form circular DNA. Both the above DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc. to obtain the above-mentioned α-glucosidase mutant.

[0046] In the third typical embodiment of this application, a recombinant plasmid is provided, and the above-mentioned recombinant plasmid is ligated with the above-mentioned DNA molecule.

[0047] In the fourth typical embodiment of this application, a host cell is provided, and this host cell contains the above-mentioned DNA molecule or the above-mentioned recombinant plasmid.

[0048] In a preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli; preferably, Escherichia coli JM109 includes the Rosstta strain. Preferably, the above-mentioned host cell is not a plant cell or an animal cell.

[0049] Using the above host cell, it is possible to replicate the recombinant plasmid in the host cell, and also to transcribe and translate the DNA molecule carried on the recombinant plasmid to obtain a large amount of α-glucosidase mutants. Using existing techniques, the host cell can be disrupted, and the protein can be purified after disruption or by other means to obtain α-glucosidase mutants. The host cell is a host cell not derived from plants or animals.

[0050] The Escherichia coli expression system is a prokaryotic expression system that can express a variety of foreign genes in Escherichia coli cells, including genes of fungi, plants, bacteria, and viruses.

[0051] The advantages of the Escherichia coli expression system include:

[0052] (1) Compared with other expression systems, Escherichia coli grows rapidly and has a shorter expression cycle;

[0053] (2) Escherichia coli cells are easy to grow, easy to scale up culture, have high yields, and are beneficial to large-scale expression of recombinant proteins;

[0054] (3) The expression conditions of Escherichia coli are simple, the cost is low, and it is easy to scale up production.

[0055] The Rosstta strain used in this application belongs to one of the host cells of the Escherichia coli expression system. Rosstta is derived from the Origami series of host bacteria. This series of strains contains the original TrxB and Gor mutant genes of the Origami strain, as well as rare codons of eukaryotic cells in the Rosetta series of strains. This characteristic can improve the correct folding rate of disulfide bonds in the cells, enabling efficient protein expression; when it is applied to the expression, preparation, and purification of proteins, it has the advantages of economy, rapidity, high yield, higher safety, and wide application.

[0056] In the fifth typical embodiment of this application, a kit is provided. The kit includes α-glucosidase, and the α-glucosidase includes any one of the above α-glucosidase mutants.

[0057] Preferably, the above kit is used to detect the α-amylase activity in a sample.

[0058] In a preferred embodiment, the above kit is used to detect α-amylase, and the kit further includes at least one of the following: a substrate, a buffer, a serum protein, or an ionic additive; wherein, the substrate can be further hydrolyzed by α-glucosidase to produce p-nitrophenol after being hydrolyzed by α-amylase. Preferably, the substrate includes p-nitrophenyl-D-maltoheptaose; preferably, the buffer includes Tris buffer; preferably, the ionic additive includes sodium chloride and / or calcium chloride; preferably, the serum protein includes bovine serum albumin; preferably, the α-glucosidase and the substrate are separately provided in the kit.

[0059] The detection principle of the α-amylase assay kit of the present application is as follows: when using p-nitrophenyl-D-maltoheptaose (PNPG7) as the substrate, PNPG7 is hydrolyzed by α-amylase in the sample to generate the intermediate product PNPG3, and then α-glucosidase is used as the auxiliary enzyme to hydrolyze the intermediate product to release p-nitrophenol, thereby causing an increase in the absorbance at 405 nm. This change is proportional to the activity of α-amylase in the sample. Therefore, the activity of α-amylase in the sample can be calculated by the rate of increase in the absorbance value at 405 nm.

[0060] When using any of the above α-glucosidase mutants to determine the activity of α-amylase, the deviation value is less than 3% compared with the wild-type α-glucosidase, which is beneficial to improving the sensitivity and accuracy of the α-amylase activity determination.

[0061] The beneficial effects of the present application will be further explained in detail below with specific examples.

[0062] Example 1

[0063] The wild-type α-glucosidase was expressed by Nanjing Genscript Biotech Corporation according to the sequence shown in SEQ ID NO: 1 (NCBI GeneBank: BAA12704.1).

[0064] 1. Site-directed mutagenesis of α-glucosidase

[0065] Mutation sites: V298F + E283R.

[0066] The forward and reverse mutation primers containing the mutation sites were designed and synthesized, and the primer sequences are shown in Table 1.

[0067] Table 1

[0068]

[0069] The intermediate DNA fragments amplified in this example and the primers for amplifying each fragment are shown in Table 2.

[0070] Table 2

[0071] Intermediate DNA fragment (numbers represent positions) Forward primer Reverse primer 1-E283R Primer-1F (BamHI) Primer-2R (E283R) E283R-V298F Primer-2F (E283R) Primer-3R (V298F) V298F-555 Primer-3F (V298F) Primer-4R (XhoI)

[0072] The first round of PCR: using the target gene as the template and paired with specific primers, the intermediate DNA fragment required for the mutant was amplified, and the reaction system and reaction conditions are shown in Table 3 and Table 4.

[0073] Table 3

[0074]

[0075] Table 4

[0076]

[0077] After the PCR reaction, the PCR product was subjected to 1% agarose gel electrophoresis. The gel block containing the target fragment was cut out and the target fragment was recovered using a gel extraction kit for later use.

[0078] Second-round PCR: Using the middle DNA fragment as a template, paired with upstream and downstream specific primers containing BamHI and XhoI restriction sites, the mutant DNA fragment was amplified. Each fragment and the template primers are shown in Table 5.

[0079] Table 5

[0080]

[0081] The reaction system and reaction conditions for the second-round PCR are shown in Table 6 and Table 7.

[0082] Table 6

[0083]

[0084] Table 7

[0085]

[0086] After the PCR reaction, the PCR product was subjected to 1% agarose gel electrophoresis. The gel block containing the target fragment was cut out and the target fragment was recovered using a gel extraction kit for later use.

[0087] 2. Construction of mutant recombinant bacteria

[0088] The PCR product after amplification and the vector plasmid pET28a (+) Were digested with BamHI and XhoI. The digestion reaction conditions were: 37°C, 3 h. The specific reaction system is shown in Table 8 and Table 9.

[0089] Table 8

[0090] Component Volume PCR recovered product 300 ng BamHI enzyme 0.5 μL XhoI enzyme 0.5 μL 10×Tango buffer 10 μL Sterile water Make up to 50 μL Total volume 50 μL

[0091] Table 9

[0092] Component Volume Vector 1 μg BamHI enzyme 0.5 μL XhoI enzyme 0.5 μL 10×Tango buffer 10 μL Sterile water Make up to 50 μL Total volume 50 μL

[0093] After the digestion products were recovered using a gel extraction kit, they were ligated. The ligation reaction conditions were: 22°C, 1 h. The specific reaction system is shown in Table 10.

[0094] Table 10

[0095] Component Volume Vector 4.5 μL Fragment 6.5 μL 10×T4 buffer 1.5 μL T4 DNA ligase buffer 0.5 μL Sterile water Make up to 15 μL Total volume 15 μL

[0096] After the ligation reaction, the ligation product was transformed into competent JM109 cells, and the bacterial solution was screened for positive clones by PCR.

[0097] The positive clone strains screened out were cultured on a large scale. After extracting the target plasmid, it was transformed into Rosetta strains. The specific steps are as follows:

[0098] (1) Take out the Rosetta strain containing Rosetta competent cells from the -80 °C refrigerator, quickly insert the centrifuge tube containing the strain into ice. After the bacterial mass melts, add the target plasmid and gently mix by flicking the bottom of the centrifuge tube with your finger. Let the centrifuge tube containing the Rosetta strain and the target plasmid stand in ice for 30 min;

[0099] (2) Heat shock at 42 °C for 90 s, quickly put the centrifuge tube back on ice and let it stand for 3 min;

[0100] (3) Spread all the bacterial solution in the centrifuge tube onto a solid LB medium containing the corresponding antibiotic;

[0101] (4) Invert the plate and incubate it overnight in a 37 °C incubator. Identify and screen the successfully positive cloned Rosetta positive recombinant bacteria by PCR.

[0102] 3. Induction expression

[0103] (1) Bacterial strain culture: Inoculate the screened Rosetta positive recombinant bacteria into 10 mL of liquid LB medium containing the corresponding antibiotic, and culture overnight at 37 °C and 200 rpm.

[0104] (2) Large-scale culture: Inoculate the overnight cultured bacterial solution into liquid LB medium containing the corresponding antibiotic at a ratio of 1:100, and culture at 37 °C and 230 rpm for 3 - 5 h until the OD600 reaches 1.2.

[0105] (3) Induction: Pre-cool the bacterial solution in a 4 °C refrigerator for about 30 min, add IPTG to a final concentration of 0.5 mM, and induce culture at 25 °C and 180 rpm for 16 h;

[0106] (4) Bacterial cell collection: Centrifuge at 8000 rpm for 10 min, discard the supernatant, and freeze the bacterial cell pellet in a -80 °C refrigerator for later use.

[0107] 4. Purification of the target protein

[0108] The expression plasmid pET28a contains a His tag. The His-tag can have special interactions with various metal ions. Nickel chloride in the Ni column can bind to the fusion protein with His-Tag and can also bind to imidazole. When the tagged protein binds to the nickel ion medium in the pores on the surface of the chromatography column, imidazole at different concentrations passes through the chromatography column, and the tagged protein coordinated with nickel, impurity proteins, etc. will be eluted respectively, thus obtaining the target protein with high purity. The recombinant mutant and wild-type α-glucosidase proteins were purified through the Ni column according to the conventional purification process, and then the protein concentration was measured using the Coomassie Brilliant Blue method.

[0109] 5. Performance Verification

[0110] (1) Determination of the specific enzyme activity of wild-type recombinant α-glucosidase and α-glucosidase mutant

[0111] The detection instrument is the self-produced biochemical analyzer BC1200 of Shenzhen New Industries Biomedical Co., Ltd.

[0112] The reagent p-nitrophenyl-α-D-glucopyranoside involved was purchased from Sigma.

[0113] The detection principle is that p-nitrophenyl-α-D-glucopyranoside (PNPG) generates p-nitrophenol (PNP) and α-D-glucose under the action of α-glucosidase. Near the wavelength of 400 nm, the rising rate of the absorbance of p-nitrophenol is proportional to the activity of α-glucosidase. One enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of p-nitrophenol in 1 min. The specific enzyme activity (U / mg) = enzyme activity (U / mL) / enzyme protein concentration (mg / mL) × 100%.

[0114] The detection steps are as follows:

[0115] 1) Drawing of the PNP standard curve

[0116] Weigh 0.0139 g of PNP and dissolve it with PBS (pH = 7.0) to prepare a stock solution of 10 μmol / mL. Dilute the stock solution into several gradients such as 0.05 μmol / mL, 0.1 μmol / mL... 1 μmol / mL, and then measure the absorbance at OD400 nm to make a PNP-absorbance value curve (three parallels for each gradient).

[0117] 2) Determination of the specific enzyme activity

[0118] Take 0.5 mL of the crude enzyme solution diluted 100-fold, add 0.5 mL of PBS buffer, incubate in a 30 °C water bath for 10 min, then add 2 mL of preheated 10 mM PBS buffer, and immediately add 2 mL of 1 mol / L Na2CO3 to stop the reaction after reacting at 30 °C for 20 min. Measure the absorbance value at OD400nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0119] The specific enzyme activity results are shown in Table 17.

[0120] (2) Thermal stability of wild type and mutant 4

[0121] The thermal stability of the enzyme was determined by storing the enzyme in a buffer of 50 mM Hepes, 0.5 M NaCl, pH 7.0, and incubating at 4 °C and 37 °C for 11 days. The residual enzyme activity was calculated with the enzyme activity incubated at 4 °C as 100%.

[0122] The thermal stability results are shown in Table 18.

[0123] Example 2

[0124] In this example, the mutation sites for site-directed mutagenesis of α-glucosidase are: V298Y + E283R.

[0125] Design and synthesize forward and reverse mutation primers containing the mutation sites. The primer sequences are shown in Table 11.

[0126] Table 11

[0127]

[0128] The intermediate DNA fragments amplified in this example and the primers for amplifying each fragment are shown in Table 12.

[0129] Table 12

[0130] Intermediate DNA fragment (numbers represent positions) Forward primer Reverse primer 1-E283R Primer-1F (BamHI) Primer-2R (E283R) E283R-V298Y Primer-2F (E283R) Primer-5R (V298Y) V298Y-555 Primer-5F (V298Y) Primer-4R (XhoI)

[0131] The remaining steps in this example are the same as those in Example 1. The results of the specific enzyme activity measurement are shown in Table 17, and the thermal stability test results are shown in Table 18.

[0132] Example 3

[0133] In this example, the mutation sites for site-directed mutagenesis of α-glucosidase are: V298H + E283R.

[0134] Design and synthesize forward and reverse mutation primers containing the mutation sites. The primer sequences are shown in Table 13.

[0135] Table 13

[0136]

[0137] The intermediate DNA fragments amplified in this example and the primers for amplifying each fragment are shown in Table 14.

[0138] Table 14

[0139] Intermediate DNA fragment (numbers represent positions) Forward primer Reverse primer 1-E283R Primer-1F (BamHI) Primer-2R (E283R) E283R-V298H Primer-2F (E283R) Primer-6R (V298H) V298H-555 Primer-6F (V298H) Primer-4R (XhoI)

[0140] The remaining steps in this example are the same as those in Example 1. The results of specific enzyme activity measurement are shown in Table 17, and the results of thermal stability detection are shown in Table 18.

[0141] Example 4

[0142] The mutation sites of site-directed mutagenesis of α-glucosidase in this example: V298H + R338Y.

[0143] Design and synthesize forward and reverse mutation primers containing the mutation sites. The primer sequences are shown in Table 15.

[0144] Table 15

[0145]

[0146] The intermediate DNA fragments amplified in this example and the primers for amplifying each fragment are shown in Table 16.

[0147] Table 16

[0148] Intermediate DNA fragment (numbers represent positions) Forward primer Reverse primer 1-V298H Primer-1F (BamHI) Primer-6R (V298H) V298H-R338Y Primer-6F (V298H) Primer-7R (R338Y) R338Y-555 Primer-7F (R338Y) Primer-4R (XhoI)

[0149] The remaining steps in this example are the same as those in Example 1. The results of specific enzyme activity measurement are shown in Table 17, and the results of thermal stability detection are shown in Table 18.

[0150] Table 17

[0151] Number First measured value Second measured value Third measured value Average value Wild type 30.2 34.5 32.2 32.3 Example 1 65.1 65.5 65.3 65.3 Example 2 97.4 98.2 97.1 97.57 Example 3 75.2 73.4 76.3 75.0 Example 4 155.6 150.1 161.3 155.7

[0152] Compared with the wild-type α-glucosidase, the specific enzyme activities of the α-glucosidase mutants in Examples 1, 2, 3, and 4 have all increased significantly. Among them, the increase in specific enzyme activity of the α-glucosidase mutant in Example 4 is the largest, about 5 times that of the wild-type α-glucosidase, and the α-glucosidase mutants in Examples 1, 2, and 3 are about 2 - 3 times that of the wild-type α-glucosidase.

[0153] Table 18

[0154] Number Residual enzyme activity ratio Wild type 96.7％ Example 1 68％ Example 2 67.8％ Example 3 85.7％ Example 4 95.9％

[0155] Both the wild-type α-glucosidase and the α-glucosidase mutants of Examples 1, 2, 3, and 4 exhibited good thermal stability and could meet the requirements of practical use. Among them, the residual enzyme activity of the α-glucosidase in Example 4 was above 95%, and the residual enzyme activities of the α-glucosidase mutants in Examples 1, 2, and 3 were above 65%.

[0156] Application of the mutant of Example 5 in an α-amylase kit

[0157] Fix the following reaction system and compare the applications of the wild-type α-glucosidase and the α-glucosidase mutants of Examples 1-4 in α-amylase detection.

[0158] R1: 50 mmol / L Tris buffer, 50 mmol / L sodium chloride, α-glucosidase;

[0159] R2: 0.9 mmol / L p-nitrophenyl-D-maltoheptaose, 50 mmol / L Tris buffer, and 5 mmol / L calcium chloride;

[0160] The specific detection steps are as follows:

[0161] Add 5 μL of the sample and 200 μL of reagent R1, mix well, incubate at 37 °C for 3 min, then add 50 μL of reagent R2, mix well, after a 1-min delay at 37 °C, read the absorbance value, read once every 1 min, read twice in total, and calculate the average change rate of absorbance per minute ΔA / min.

[0162] Using 16 mg of the wild-type glycosidase as a control, add 5.5 mg of the mutant in Example 1, 3.7 mg of the mutant in Example 2, 2.3 mg of the mutant in Example 3, and 2.3 mg of the mutant in Example 4 to detect three samples with known target values of 91, 150, and 305. The results are shown in Table 19.

[0163] Table 19

[0164]

[0165] As shown in the results in the table, the α-glucosidase mutants in Examples 1, 2, 3, and 4 all exhibited good detection accuracy, with a deviation from the sample target value of less than 3%.

[0166] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: By performing site-directed mutagenesis on the V298 site of the α-glucosidase protein with E283 or R338, namely V298F+E283R, V298Y+E283R, V298H+E283R or V298H+R283Y, an α-glucosidase with a specific enzyme activity increased by at least 2 times and having thermal stability is obtained. When it is applied to an α-amylase kit, compared with the wild-type α-glucosidase, the α-glucosidase mutant disclosed in this application has higher sensitivity in detecting α-amylase, meeting the detection requirements, and the deviation from the sample target value is less than 3%, improving the accuracy of the detection results.

[0167] The foregoing is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An α-glucosidase mutant, characterized in that the mutation in the α-glucosidase mutant is selected from any one of the following amino acid mutation combinations: V298F + E283R, V298Y + E283R, V298H + E283R or V298H + R338Y; wherein, V298F + E283R is the protein shown in SEQ ID NO: 2, V298Y + E283R is the protein shown in SEQ ID NO: 3; V298H + E283R is the protein shown in SEQ ID NO: 4; V298H + R338Y is the protein shown in SEQ ID NO:

5.

2. A DNA molecule, characterized in that, The DNA molecule encodes the α-glucosidase mutant according to claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid is ligated with the DNA molecule according to claim 2.

4. A host cell, characterized in that, The host cell contains the DNA molecule according to claim 2 or the recombinant plasmid according to claim 3, and the host cell is a non-animal or plant variety.

5. The host cell according to claim 4, wherein The host cell is a prokaryotic cell or a eukaryotic cell.

6. The host cell according to claim 5, wherein The prokaryotic cell is Escherichia coli.

7. The host cell according to claim 6, wherein The Escherichia coli is JM109 or Rosetta strain.

8. A kit, characterized in that, The kit includes α-glucosidase, and the α-glucosidase is the α-glucosidase mutant according to claim 1.

9. The kit according to claim 8, wherein The kit is used for detecting α-amylase and further includes at least one of the following: a substrate, a buffer or an ionic additive; The substrate can be further hydrolyzed by the α-glucosidase to produce p-nitrophenol after being hydrolyzed by α-amylase.

10. The kit according to claim 9, characterized in that, The substrate is p-nitrophenyl-D-maltoheptaose.

11. The kit according to claim 9, wherein The buffer is Tris buffer.

12. The kit according to claim 9, wherein The ionic additive is sodium chloride and / or calcium chloride.

13. The kit according to claim 9 or 10, characterized in that, The α-glucosidase and the substrate are separately provided in the kit.

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