Glycerol phosphate oxidase mutant and its preparation method and application

Abstract

The present application relates to a glycerol phosphate oxidase mutant and a preparation method and application thereof. Compared with SEQ ID NO.1, the amino acid sequence of the glycerol phosphate oxidase mutant comprises one or more of the following amino acid substitution sites: 16th, 74th, 118th, 132nd, 137th, 150th, 244th, 265th, 280th, 304th, 330th, 357th, 362nd, 367th, 368th, 383rd, 387th, 395th, 396th, 402nd, 403rd, 442nd, 463rd, 493rd, 516th, 517th, 564th, 575th, 581st, 589th and 590th. The glycerol phosphate oxidase mutant has the advantages of high expression level and high enzyme activity.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a glycerol phosphate oxidase mutant and a preparation method and application thereof. Background Art

[0002] Triglyceride (TG) is an important component of lipids. Elevated triglyceride in serum is common in familial lipid metabolism disorders, nephrotic syndrome, diabetes, hypothyroidism, etc. The determination of triglyceride is helpful for understanding blood lipid metabolism, liver function (liver is one of the organs that synthesize triglyceride) and the diagnosis of diseases caused by atherosclerosis. The glycerol phosphate oxidase method for detecting triglycerides is simple, fast and accurate, and can be automatically batch operated on a biochemical analyzer. It is a routine method for determining triglycerides in clinical laboratories. Glycerol phosphate oxidase (G3PO, EC1.1.3.21) is the key enzyme for enzymatic detection of TG. The glycerol phosphate oxidase used in in vitro diagnostic reagents mainly comes from streptococci, coccocci, yeast and Escherichia coli. Its preparation method is mainly through the fermentation of wild bacteria or the expression of glycerol phosphate oxidase from different species in Escherichia coli to obtain recombinant enzymes. Due to the complex strain culture conditions and cumbersome production processes, the existing production methods have problems such as low enzyme yield and inability to produce on a large scale, which hinders the application of glycerol phosphate oxidase. Summary of the invention

[0003] Based on this, it is necessary for the present application to provide a glycerol phosphate oxidase mutant with significantly improved expression level and its preparation method and application.

[0004] The specific technical solutions are as follows:

[0005] A glycerol phosphate oxidase mutant, which, compared with the glycerol phosphate oxidase shown in SEQ ID NO.1, comprises an amino acid substitution at one or more of the following positions: position 16, position 74, position 118, position 132, position 137, position 150, position 244, position 265, position 280, position 304, position 330, position 357, position 362, position 367, position 368, position 383, position 387, position 395, position 396, position 402, position 403, position 442, position 463, position 493, position 516, position 517, position 564, position 575, position 581, position 589 and position 590.

[0006] In one embodiment, the glycerol phosphate oxidase mutant comprises one or more of the following amino acid substitutions:

[0007] Glutamine at position 16 was replaced by glutamic acid, phenylalanine at position 74 was replaced by glycine, phenylalanine at position 118 was replaced by glutamic acid, asparagine at position 132 was replaced by glycine, lysine at position 137 was replaced by proline, glutamic acid at position 150 was replaced by lysine, phenylalanine at position 244 was replaced by lysine, lysine at position 265 was replaced by serine, glutamine at position 280 was replaced by glycine, glutamine at position 304 was replaced by glutamic acid, glutamic acid at position 330 was replaced by threonine, tyrosine at position 357 was replaced by glutamic acid, asparagine at position 362 was replaced by lysine, aspartic acid at position 367 was replaced by glutamic acid, lysine at position 368 was replaced by glutamic acid, asparagine at position 383 was replaced by glycine. substituted by glutamine or lysine, substituted by glutamine at position 387, substituted by glutamine or lysine at position 395, substituted by lysine at position 396, substituted by asparagine or lysine at position 402, substituted by lysine at position 403, substituted by asparagine at position 442, substituted by lysine at position 463, substituted by threonine at position 493, substituted by serine at position 516, substituted by arginine at position 517, substituted by arginine at position 564, substituted by lysine at position 575, substituted by aspartic acid at position 581, substituted by proline at position 589, substituted by alanine at position 590, and substituted by lysine at position 591.

[0008] The nucleic acid molecule comprises a nucleotide fragment encoding the glycerol phosphate oxidase mutant.

[0009] The recombinant expression vector comprises the nucleic acid molecule.

[0010] The recombinant host cell comprises the nucleic acid molecule or the recombinant expression vector.

[0011] The preparation method of the glycerol phosphate oxidase mutant comprises the following steps:

[0012] The recombinant host cell is cultured, and the glycerol phosphate oxidase mutant is isolated from the obtained culture product.

[0013] The application of the glycerol phosphate oxidase mutant in the preparation of a triglyceride detection reagent.

[0014] Compared with the traditional technology, this application has the following beneficial effects:

[0015] The present application performs amino acid substitution on the sequence of wild-type glycerol phosphate oxidase and constructs a recombinant expression vector to express a glycerol phosphate oxidase mutant. The mutant has high enzyme activity and the expression level of the enzyme is significantly improved, which reduces the cost of using glycerol phosphate oxidase, greatly improves the availability of glycerol phosphate oxidase, and promotes the development of triglyceride detection reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the detection result of the expression amount of the glycerol phosphate oxidase mutant, wherein M represents the protein molecular weight standard, 1 represents the wild-type glycerol phosphate oxidase induced in a small amount, 2 represents the glycerol phosphate oxidase mutant TST1 induced in a small amount, 3 represents the glycerol phosphate oxidase mutant TST2 induced in a small amount, and 4 represents the glycerol phosphate oxidase mutant TST3 induced in a small amount;

[0017] Figure 2 The purity identification results of SDS-PAGE, where M represents the protein molecular weight standard; 1 represents the bacterial protein after induction; 2 represents the supernatant protein after bacterial crushing and centrifugation; 3 represents the protein not bound to the purification column; and 4 represents the mutant glycerol phosphate oxidase after purification. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0020] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0021] One embodiment of the present application provides a glycerophosphate oxidase mutant with significantly improved expression and high enzyme activity. Compared with the glycerophosphate oxidase shown in SEQ ID NO.1, the glycerophosphate oxidase mutant comprises amino acid substitutions at one or more of the following sites:

[0022] 16th, 74th, 118th, 132nd, 137th, 150th, 244th, 265th, 280th, 304th, 330th, 357th, 362nd, 367th, 368th, 383rd, 387th, 395th, 396th, 402nd, 403rd, 442nd, 463rd, 493rd, 516th, 517th, 564th, 575th, 581st, 589th and 590th.

[0023] The amino acid sequence of the wild-type glycerol phosphate oxidase gene is shown in SEQ ID NO. 1, which is derived from the Streptococcus sp. strain.

[0024] In a specific example, the glycerol phosphate oxidase mutant comprises one or more of the following amino acid substitutions:

[0025] Glutamine at position 16 is replaced by glutamic acid, phenylalanine at position 74 is replaced by glycine, phenylalanine at position 118 is replaced by glutamic acid, asparagine at position 132 is replaced by glycine, lysine at position 137 is replaced by proline, glutamic acid at position 150 is replaced by lysine, phenylalanine at position 244 is replaced by lysine or tyrosine, lysine at position 265 is replaced by serine, glutamine at position 280 is replaced by glycine, glutamine at position 304 is replaced by glutamic acid, glutamic acid at position 330 is replaced by threonine, tyrosine at position 357 is replaced by glutamic acid, asparagine at position 362 is replaced by lysine, aspartic acid at position 367 is replaced by glutamic acid, lysine at position 368 is replaced by glutamic acid, asparagine at position 383 is replaced by substituted with glycine, arginine at position 387 is substituted with glutamine or lysine, asparagine at position 395 is substituted with lysine, histidine at position 396 is substituted with asparagine or lysine, aspartic acid at position 402 is substituted with lysine, glutamic acid at position 403 is substituted with asparagine, arginine at position 442 is substituted with lysine, lysine at position 463 is substituted with threonine, glutamic acid at position 493 is substituted with serine, lysine at position 516 is substituted with arginine, glutamic acid at position 517 is substituted with arginine, glutamic acid at position 564 is substituted with lysine, glutamic acid at position 575 is substituted with aspartic acid, glutamic acid at position 581 is substituted with proline, lysine at position 589 is substituted with alanine and arginine at position 590 is substituted with lysine.

[0026] In one specific example, the amino acid substitutions include substitution of glutamine at position 16 with glutamic acid, substitution of phenylalanine at position 74 with glycine, substitution of phenylalanine at position 118 with glutamic acid, substitution of asparagine at position 132 with glycine, substitution of lysine at position 137 with proline, substitution of glutamic acid at position 150 with lysine, substitution of phenylalanine at position 244 with lysine, substitution of lysine at position 265 with serine, substitution of glutamic acid at position 330 with threonine, substitution of aspartic acid at position 367 with glutamic acid, substitution of lysine at position 368 with glutamic acid, substitution of asparagine at position 383 with glycine, substitution of arginine at position 387 with glutamine. substituted with asparagine or lysine, substituted with lysine for asparagine at position 395, substituted with asparagine or lysine for histidine at position 396, substituted with lysine for asparagine at position 402, substituted with asparagine for glutamic acid at position 403, substituted with asparagine for arginine at position 442, substituted with lysine for lysine at position 463, substituted with threonine for glutamic acid at position 493, substituted with serine for lysine at position 516, substituted with arginine for glutamic acid at position 517, substituted with arginine for glutamic acid at position 564, substituted with lysine for glutamic acid at position 575, substituted with aspartic acid for lysine at position 589, substituted with alanine for lysine at position 590, and substituted with arginine at position 591.

[0027] In one specific example, the amino acid substitutions include substitution of glutamine at position 16 with glutamic acid, substitution of phenylalanine at position 74 with glycine, substitution of phenylalanine at position 118 with glutamic acid, substitution of asparagine at position 132 with glycine, substitution of glutamic acid at position 150 with lysine, substitution of phenylalanine at position 244 with lysine, substitution of glutamine at position 280 with glycine, substitution of glutamine at position 304 with glutamic acid, substitution of asparagine at position 362 with lysine, substitution of aspartic acid at position 367 with glutamic acid, substitution of lysine at position 368 with glutamic acid, substitution of asparagine at position 383 with glycine, and substitution of glutamic acid at position 384 with lysine. The arginine at position 7 is substituted by glutamine or lysine, the asparagine at position 395 is substituted by lysine, the histidine at position 396 is substituted by asparagine or lysine, the aspartic acid at position 402 is substituted by lysine, the arginine at position 442 is substituted by lysine, the lysine at position 463 is substituted by threonine, the glutamic acid at position 493 is substituted by serine, the lysine at position 516 is substituted by arginine, the glutamic acid at position 517 is substituted by arginine, the glutamic acid at position 564 is substituted by lysine, the glutamic acid at position 575 is substituted by aspartic acid, the lysine at position 589 is substituted by alanine and the arginine at position 590 is substituted by lysine.

[0028] In one specific example, the amino acid substitutions include substitution of glutamine at position 16 with glutamic acid, substitution of phenylalanine at position 74 with glycine, substitution of phenylalanine at position 118 with glutamic acid, substitution of asparagine at position 132 with glycine, substitution of lysine at position 137 with proline, substitution of glutamic acid at position 150 with lysine, substitution of phenylalanine at position 244 with lysine, substitution of lysine at position 265 with serine, substitution of glutamine at position 280 with glycine, substitution of glutamine at position 304 with glutamic acid, substitution of glutamic acid at position 330 with threonine, substitution of tyrosine at position 357 with glutamic acid, substitution of asparagine at position 362 with lysine, substitution of aspartic acid at position 367 with glutamic acid, substitution of lysine at position 368 with glutamic acid, and substitution of threonine at position 383 with glutamic acid. The asparagine at position 3 is substituted by glycine, the arginine at position 387 is substituted by glutamine or lysine, the asparagine at position 395 is substituted by lysine, the histidine at position 396 is substituted by asparagine or lysine, the aspartic acid at position 402 is substituted by lysine, the glutamic acid at position 403 is substituted by asparagine, the arginine at position 442 is substituted by lysine, the lysine at position 463 is substituted by threonine, the glutamic acid at position 493 is substituted by serine, the lysine at position 516 is substituted by arginine, the glutamic acid at position 517 is substituted by arginine, the glutamic acid at position 564 is substituted by lysine, the glutamic acid at position 575 is substituted by aspartic acid, the glutamic acid at position 581 is substituted by proline, the lysine at position 589 is substituted by alanine, and the arginine at position 590 is substituted by lysine.

[0029] In a specific example, the amino acid fragment of the glycerol phosphate oxidase mutant is a sequence shown in any one of SEQ ID NOs. 2-4.

[0030] One embodiment of the present application further provides a nucleic acid molecule comprising a nucleotide fragment encoding the glycerol phosphate oxidase mutant. Any nucleotide fragment capable of encoding and expressing the glycerol phosphate oxidase mutant is protected by the present application. Optionally, the nucleotide fragment is a sequence shown in any one of SEQ ID NOs. 5 to 7.

[0031] One embodiment of the present application also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecule.

[0032] Those skilled in the art should know that, in addition to the above nucleotide fragments, the vector expressing the glycerol phosphate oxidase mutant provided by the present application may also include nucleotide sequences such as promoters, enhancers, and non-coding regions to achieve the purpose of improving the expression amount, expression efficiency, product activity, and other aspects of the gene. In addition, the vector may also include a nucleotide sequence of a tag for the purpose of conveniently detecting the expression of the product.

[0033] The recombinant expression vector provided in the present application can be a recombinant recombinant vector obtained by connecting any existing vector in the prior art to the above-mentioned nucleic acid molecule. In a specific example, the vector can be selected from at least one of a plasmid, a virus and a bacteriophage. Optionally, the plasmid includes the pET series.

[0034] One embodiment of the present application also provides a recombinant host cell, comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector.

[0035] The host cell used to express the glycerol phosphate oxidase mutant of the present application can be any host commonly used in the art, and is not particularly limited. The host cell includes a prokaryotic cell and / or a eukaryotic cell, optionally, a prokaryotic cell such as Escherichia coli such as BL21 (DE3) strain, Bacillus subtilis, etc.; optionally, a eukaryotic cell such as a yeast cell, a plant cell, an insect cell, and an animal cell such as a mammalian cell (e.g., a mouse cell, a human cell, etc.).

[0036] One embodiment of the present application further provides a method for preparing the above-mentioned glycerol phosphate oxidase mutant, which can significantly increase the expression level of the enzyme, and specifically comprises the following steps a to b:

[0037] Step a, constructing a recombinant expression vector containing the coding gene of the above glycerol phosphate oxidase mutant.

[0038] In a specific example, the construction of the recombinant expression vector includes the following steps:

[0039] A nucleic acid fragment for synthesizing a glycerophosphate oxidase mutant, as shown in any one of SEQ ID NOs. 2 to 4; and

[0040] The above nucleic acid fragments were respectively inserted into expression vectors.

[0041] Wherein, the expression vector is selected from pET series plasmids; optionally, the pET series plasmid is pET28 plasmid.

[0042] Step b, introducing the above recombinant expression vector into host cells, culturing the host cells, expressing the protein, and obtaining a glycerol phosphate oxidase mutant.

[0043] The method for introducing the expression vector into the host is not particularly limited as long as it can introduce the recombinant expression vector into the host, and the introduction method includes heat shock method, electroporation method, calcium ion method, spheroplast method and the like.

[0044] The glycerol phosphate oxidase mutant provided in the present application has high enzyme activity and can be used to measure triglycerides.

[0045] Among them, the principle of glycerol phosphate oxidase to determine triglycerides:

[0046] Triglycerides in serum can be hydrolyzed into glycerol and free fatty acids by lipoproteinase. Glycerol generates 3-phosphoglycerol under the action of ATP and glycerol kinase, and then generates dihydroxyacetone phosphate and H2O2 by glycerol phosphate oxidase. H2O2, 4-APP and TOOS generate red quinone compounds under the action of peroxidase. The degree of color development is proportional to the concentration of triglycerides.

[0047] The principle of detecting glycerol phosphate oxidase in the present application is: glycerol phosphate oxidase catalyzes disodium glycerol phosphate to generate hydrogen peroxide (H2O2), and the generated hydrogen peroxide and 4-aminoantipyrine react under the catalysis of horseradish peroxidase to generate colored quinoneimine (quinoneimine dye), and a spectrophotometer is used to measure the change in the absorbance of the reaction solution at 555nm to determine the change in the concentration of the colored quinoneimine, thereby calculating the GPO enzyme activity.

[0048] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0049] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, the deviations caused by instrument test accuracy or operation accuracy are allowed.

[0050] 1. The wild-type amino acid sequence of glycerophosphate oxidase derived from Streptococcus sp. strain was obtained from databases such as NCBI as shown in SEQ ID NO. 1. By artificially designing the sequence, at least one of the following amino acids was substituted in the wild-type amino acid sequence: glutamine (Q) at position 16 was substituted with glutamic acid (E), phenylalanine (F) at position 74 was substituted with glycine (G), phenylalanine (F) at position 118 was substituted with glutamic acid (E), asparagine (N) at position 132 was substituted with glycine (G), lysine (K) at position 137 was substituted with proline (P), glutamic acid (E) at position 150 was substituted with lysine (K), phenylalanine (F) at position 244 was substituted with lysine (K), Lysine (K) at position 65 was substituted with serine (S), glutamine (Q) at position 280 was substituted with glycine (G), glutamine (Q) at position 304 was substituted with glutamic acid (E), glutamic acid (E) at position 330 was substituted with threonine (T), tyrosine (Y) at position 357 was substituted with glutamic acid (E), asparagine (N) at position 362 was substituted with lysine (K), aspartic acid (D) at position 367 was substituted with glutamic acid (E), lysine (K) at position 368 was substituted with glutamic acid (E), asparagine (N) at position 383 was substituted with glycine (G) , the arginine (R) at position 387 is substituted with glutamine (Q) or lysine (K), the asparagine (N) at position 395 is substituted with lysine (K), the histidine (H) at position 396 is substituted with asparagine (N) or lysine (K), the aspartic acid (D) at position 402 is substituted with lysine (K), the glutamic acid (E) at position 403 is substituted with asparagine (N), the arginine (R) at position 442 is substituted with lysine (K), the lysine (K) at position 463 is substituted with threonine (T), the glutamic acid (E) at position 493 is substituted with serine (S), the Lysine (K) at position 516 was substituted with arginine (R), glutamic acid (E) at position 517 was substituted with arginine (R), glutamic acid (E) at position 564 was substituted with lysine (K), glutamic acid (E) at position 575 was substituted with aspartic acid (D), glutamic acid (E) at position 581 was substituted with proline (P), lysine (K) at position 589 was substituted with alanine (A), and arginine (R) at position 590 was substituted with lysine (K), and glycerol phosphate oxidase mutants TST1, TST2, and TST3 with increased enzyme expression were obtained, and the amino acid sequences were SEQ ID NO.2 (TST1), SEQ ID NO.3 (TST2), and SEQ ID NO.4 (TST3), respectively. The plasmid synthesis was entrusted to Nanjing Qingke Biotechnology Co., Ltd., and the synthetic gene contained NdeI and XhoI restriction sites, and the amino terminus of the recombinant GPO was fused with a His tag.Finally, the recombinant plasmid pET-28a-GPO was transformed into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli expressing the glycerol phosphate oxidase mutant.

[0051] The amino acid sequence of SEQ ID NO.1 is as follows: MFSNKTRQDSIQKMQ Q EELDLLIIGGGITGAGVAVQAAASGIKTGLIEMQDFAEGTSSRSTKLVHGGIRYLKT F DVEVVADTVGERAVVQGIAPHIPKPDPMLLPIYEDEGATTFNM F SVKVAMDLYDKLA N VTGT K YENYTLTPEEVL E REPFLKEGLKGAGVYLDFRNNDARLVIDNIKKAAEDGAYLVSKMKAVGFLYEGDQIVGVKARDLLTDEVIEIKAKLVINTSGPWVDKVRNLN F TRPVSPKMRPTKGIHLVVDA K KLPVPQPTYFDTGKQDGRMVFAIPRENKTYFGTTDTDYQGDFTDPKVTQEDVDYLLDVINHRYP E ANITLADIEASWAGLRPLLIGNSGSDYNGGDNGSIS DK SFNKVVDTVSEYKE N KVS R AEEVEDVL NH LENSR DE KAPSTISRGSSLEREPDGLLTLSGGKITDYRKMAEGAL R LIRQLLKEEYGIETKEIDSK K YQISGGNFDPTKLEETVTELAKEGVAAGL E EEDATYIADFYGTNARRIFELA KE MAPYPGLSLAESARLRYGLEEEMVLAPGDYLIRRTNHLLFERDQLD E IKQPVIDAIAEYFGWTEEEKAQQT K RLEALIAESDLRELKGEK.

[0052] The amino acid sequence of SEQ ID NO.2 is as follows:

[0053] MFSNKTRQDSIQKMQ E EELDLLIIGGGITGAGVAVQAAASGIKTGLIEMQDFAEGTSSRSTKLVHGGIRYLKT G DVEVVADTVGERAVVQGIAPHIPKPDPMLLPIYEDEGATTFNM E SVKVAMDLYDKLA G VTGT P YENYTLTPEEVL K REPFLKKEGLKGAGVYLDFRNNDARLVIDNIKKAAEDGAYLVSKMKAVGFLYEGDQIVGVKARDLLTDEVIEIKAKLVINTSGPWVDKVRNLN Y TRPVSPKMRPTKGIHLVVDA S KLPVPQPTYFDTGKQDGRMVFAIPRENKTYFGTTDTDYQGDFTDPKVTQEDVDYLLDVINHRYP T ANITLADIEASWAGLRPLLIGNSGSDYNGGDNGSIS EE SFNKVVDTVSEYKE G KVS Q AEVEDVL KN LENSR KN KAPSTISRGSSLEREPDGLLTLSGGKITDYRKMAEGAL K LIRQLLKEEYGIETKEIDSK T YQISGGNFDPTKLEETVTELAKEGVAAGL S EEDATYIADFYGTNARRIFELA RR MAPYPGLSLAESARLRYGLEEEMVLAPGDYLIRRTNHLLFERDQLD K IKQPVIDAIA D YFGWTEEEKAQQTAKLEALIAESDLRELKGEK。

[0054] The amino acid sequence of SEQ ID NO.3 is as follows:

[0055] MFSNKTRQDSIQKMQ EEELDLLIIGGGITGAGVAVQAAASGIKTGLIEMQDFAEGTSSRSTKLVHGGIRYLKT G DVEVVADTVGERAVVQGIAPHIPKPDPMLLPIYEDEGATTFNM E SVKVAMDLYDKLA G VTGTKYENYTLTPEEVL K REPFLKKEGLKGAGVYLDFRNNDARLVIDNIKKAAEDGAYLVSKMKAVGFLYEGDQIVGVKARDLLTDEVIEIKAKLVINTSGPWVDKVRNLN K TRPVSPKMRPTKGIHLVVDAKKLPVPQPTYFDTGK G DGRMVFAIPRENKTYFGTTDTDY E GDFTDPKVTQEDVDYLLDVINHRYPEANITLADIEASWAGLRPLLIGNSGSDYNGGD K GSIS EE SFNKVVDTVSEYKE G KVS K AEVEDVL KK LENSR K EKAPSTISRGSSLEREPDGLLTLSGGKITDYRKMAEGAL K LIRQLLKEEYGIETKEIDSK T YQISGGNFDPTKLEETVTELAKEGVAAGL S EEDATYIADFYGTNARRIFELA RR MAPYPGLSLAESARLRYGLEEEMVLAPGDYLIRRTNHLLFERDQLD K IKQPVIDAIA D YFGWTEEEKAQQT AK LEALIAESDLRELKGEK。

[0056] The amino acid sequence of SEQ ID NO.4 is as follows:

[0057] MFSNKTRQDSIQKMQ E EELDLLIIGGGITGAGVAVQAAASGIKTGLIEMQDFAEGTSSRSTKLVHGGIRYLKT GDVEVVADTVGERAVVQGIAPHIPKPDPMLLPIYEDEGATTFNM E SVKVAMDLYDKLA G VTGT P YENYTLTPEEVL K REPFLKKEGLKGAGVYLDFRNNDARLVIDNIKKAAEDGAYLVSKMKAVGFLYEGDQIVGVKARDLLTDEVIEIKAKLVINTSGPWVDKVRNLN K TRPVSPKMRPTKGIHLVVDA S KLPVPQPTYFDTGK G DGRMVFAIPRENKTYFGTTDTDY E GDFTDPKVTQEDVDYLLDVINHRYP T ANITLADIEASWAGLRPLLIGNSGSD E NGGD K GSIS EE SFNKVVDTVSEYKE G KVS Q AEVEDVL KN LENSR KN KAPSTISRGSSLEREPDGLLTLSGGKITDYRKMAEGAL K LIRQLLKEEYGIETKEIDSK T YQISGGNFDPTKLEETVTELAKEGVAAGL S EEDATYIADFYGTNARRIFELA RR MAPYPGLSLAESARLRYGLEEEMVLAPGDYLIRRTNHLLFERDQLD K IKQPVIDAIA D YFGWT P EEKAQQT AK LEALIAESDLRELKGEK。

[0058] The nucleotide sequence of TST1 is shown in SEQ ID NO.5:

[0059]

[0060] The nucleotide sequence of TST2 is shown in SEQ ID NO.6:

[0061] ATGTTCTCTAACAAAACCCGTCAGGACTCTATCCAGAAAATGCAGGAAGAAGAACTGGACCTGCTGATCATCGGTGGTGGTATCACCGGTGCTGGTGTT

[0062] GCTGTTCAGGCTGCTGCTTCTGGTATCAAAACCGGTCTGATCGAAATGCAG

[0063] GACTTCGCTGAAGGTACCTCTTCTCGTTCTACCAAACTGGTTCACGGTGGT

[0064] ATCCGTTACCTGAAAACCGGTGACGTTGAAGTTGTTGCTGACACCGTTGGT

[0065] GAACGTGCTGTTGTTCAGGGTATCGCTCCGCACATCCCGAAACCGGACCC

[0066] GATGCTGCTGCCGATCTACGAAGACGAAGGTGCTACCACCTTCAACATGG

[0067] AATCTGTTAAAGTTGCTATGGACCTGTACGACAAACTGGCTGGTGTTACCG

[0068] GTACCAAATACGAAAACTACACCCTGACCCCGGAAGAAGTTCTGAAACGT

[0069] GAACCGTTCCTGAAAAAAGAAGGTCTGAAAGGTGCTGGTGTTTACCTGGA

[0070] CTTCCGTAACAACGACGCTCGTCTGGTTATCGACAACATCAAAAAAGCTGC

[0071] TGAAGACGGTGCTTACCTGGTTTCTAAAATGAAAGCTGTTGGTTTCCTGTA

[0072] CGAAGGTGACCAGATCGTTGGTGTTAAAGCTCGTGACCTGCTGACCGACG

[0073] AAGTTATCGAAATCAAAGCTAAACTGGTTATCAACACCTCTGGTCCGTGGG

[0074] TTGACAAAGTTCGTAACCTGAACAAAACCCGTCCGGTTTCTCCGAAAATG

[0075] CGTCCGACCAAAGGTATCCACCTGGTTGTTGACGCTAAAAAACTGCCGGT

[0076] TCCGCAGCCGACCTACTTCGACACCGGTAAAGGTGACGGTCGTATGGTTTT

[0077] CGCTATCCCGCGTGAAAACAAAACCTACTTCGGTACCACCGACACCGACT

[0078] ACGAAGGTGACTTCACCGACCCGAAAGTTACCCAGGAAGACGTTGACTAC

[0079] CTGCTGGACGTTATCAACCACCGTTACCCGGAAGCTAACATCACCCTGGCT

[0080] GACATCGAAGCTTCTTGGGCTGGTCTGCGTCCGCTGCTGATCGGTAACTCT

[0081] GGTTCTGACTACAACGGTGGTGACAAAGGTTCTATCTCTGAAGAATCTTTC

[0082] AACAAAGTTGTTGACACCGTTTCTGAATACAAAGAAGGTAAAGTTTCTAA

[0083] AGCTGAAGTTGAAGACGTTCTGAAAAAACTGGAAAACTCTCGTAAAGAA

[0084] AAAGCTCCGTCTACCATCTCTCGTGGTTCTTCTCTGGAACGTGAACCGGAC

[0085] GGTCTGCTGACCCTGTCTGGTGGTAAAATCACCGACTACCGTAAAATGGCT

[0086] GAAGGTGCTCTGAAACTGATCCGTCAGCTGCTGAAAGAAGAATACGGTAT

[0087] CGAAACCAAAGAAATCGACTCTAAAACCTACCAGATCTCTGGTGGTAACT

[0088] TCGACCCGACCAAACTGGAAGAAACCGTTACCGAACTGGCTAAAGAAGG

[0089] TGTTGCTGCTGGTCTGTCTGAAGAAGACGCTACCTACATCGCTGACTTCTA

[0090] CGGTACCAACGCTCGTCGTATCTTCGAACTGGCTCGTCGTATGGCTCCGTA

[0091] CCCGGGTCTGTCTCTGGCTGAATCTGCTCGTCTGCGTTACGGTCTGGAAGA

[0092] AGAAATGGTTCTGGCTCCGGGTGACTACCTGATCCGTCGTACCAACCACCT

[0093] GCTGTTCGAACGTGACCAGCTGGACAAAATCAAACAGCCGGTTATCGACG

[0094] CTATCGCTGACTACTTCGGTTGGACCGAAGAAGAAAAAGCTCAGCAGACC

[0095] GCTAAACTGGAAGCTCTGATCGCTGAATCTGACCTGCGTGAACTGAAAGGTGAAAAATAA。

[0096] The nucleotide sequence of TST3 is shown in SEQ ID NO.7:

[0097] ATGTTCTCTAACAAAACCCGTCAGGACTCTATCCAGAAAATGCAGGAAGAAGAACTGGACCTGCTGATCATCGGTGGTGGTATCACCGGTGCTGGTGTTGCTGTTCAGGCTGCTGCTTCTGGTATCAAAACCGGTCTGATCGAAATGCAGGACTTCGCTGAAGGTACCTCTTCTCGTTCTACCAAACTGGTTCACGGTGGTATCCGTTACCTGAAAACCGGTGACGTTGAAGTTGTTGCTGACACCGTTGGTGAACGTGCTGTTGTTCAGGGTATCGCTCCGCACATCCCGAAACCGGACCCGATGCTGCTGCCGATCTACGAAGACGAAGGTGCTACCACCTTCAACATGGAATCTGTTAAAGTTGCTATGGACCTGTACGACAAACTGGCTGGTGTTACCGGTACCCCGTACGAAAACTACACCCTGACCCCGGAAGAAGTTCTGAAACGTGAACCGTTCCTGAAAAAAGAAGGTCTGAAAGGTGCTGGTGTTTACCTGGA

[0098] CTTCCGTAACAACGACGCTCGTCTGGTTATCGACAACATCAAAAAAGCTGC

[0099] TGAAGACGGTGCTTACCTGGTTTCTAAAATGAAAGCTGTTGGTTTCCTGTA

[0100] CGAAGGTGACCAGATCGTTGGTGTTAAAGCTCGTGACCTGCTGACCGACG

[0101] AAGTTATCGAAATCAAAGCTAAACTGGTTATCAACACCTCTGGTCCGTGGG

[0102] TTGACAAAGTTCGTAACCTGAACAAAACCCGTCCGGTTTCTCCGAAAATG

[0103] CGTCCGACCAAAGGTATCCACCTGGTTGTTGACGCTTCTAAACTGCCGGTT

[0104] CCGCAGCCGACCTACTTCGACACCGGTAAAGGTGACGGTCGTATGGTTTTC

[0105] GCTATCCCGCGTGAAAACAAAACCTACTTCGGTACCACCGACACCGACTA

[0106] CGAAGGTGACTTCACCGACCCGAAAGTTACCCAGGAAGACGTTGACTACC

[0107] TGCTGGACGTTATCAACCACCGTTACCCGACCGCTAACATCACCCTGGCTG

[0108] ACATCGAAGCTTCTTGGGCTGGTCTGCGTCCGCTGCTGATCGGTAACTCTG

[0109] GTTCTGACGAAAACGGTGGTGACAAAGGTTCTATCTCTGAAGAATCTTTCA

[0110] ACAAAGTTGTTGACACCGTTTCTGAATACAAAGAAGGTAAAGTTTCTCAG

[0111] GCTGAAGTTGAAGACGTTCTGAAAAACCTGGAAAACTCTCGTAAAAACA

[0112] AAGCTCCGTCTACCATCTCTCGTGGTTCTTCTCTGGAACGTGAACCGGACG

[0113] GTCTGCTGACCCTGTCTGGTGGTAAAATCACCGACTACCGTAAAATGGCTG

[0114] AAGGTGCTCTGAAACTGATCCGTCAGCTGCTGAAAGAAGAATACGGTATC

[0115] GAAACCAAAGAAATCGACTCTAAAACCTACCAGATCTCTGGTGGTAACTT

[0116] CGACCCGACCAAACTGGAAGAAACCGTTACCGAACTGGCTAAAGAAGGT

[0117] GTTGCTGCTGGTCTGTCTGAAGAAGACGCTACCTACATCGCTGACTTCTAC

[0118] GGTACCAACGCTCGTCGTATCTTCGAACTGGCTCGTCGTATGGCTCCGTAC

[0119] CCGGGTCTGTCTCTGGCTGAATCTGCTCGTCTGCGTTACGGGTCTGGAAGAA

[0120] GAAATGGTTCTGGCTCCGGGTGACTACCTGATCCGTCGTACCAACCACCTG

[0121] CTGTTCGAACGTGACCAGCTGGACAAAATCAAACAGCCGGTTATCGACGC

[0122] TATCGCTGACTACTTCGGTTGGACCCCGGAAGAAAAAGCTCAGCAGACCG

[0123] CTAAACTGGAAGCTCTGATCGCTGAATCTGACCTGCGTGAACTGAAAGGTGAAAAATAA.

[0124] 2. Streak three recombinant E. coli containing pET-28a-GPO plasmids on a solid LB plate containing 50 μg / mL kanamycin, and culture at 37°C for 12-16 hours to obtain a single clone. Pick a single clone of recombinant E. coli on the plate and culture it in 5 ml liquid LB medium at 37°C overnight. Inoculate the overnight cultured bacterial solution into 5 ml liquid LB medium at a 1% inoculum, culture at 37°C until the biomass OD600 reaches 0.6-0.8, then add the inducer isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM and continue to culture at 16°C for 16-20 hours, and collect the bacteria by centrifugation at 4°C.

[0125] The collected bacteria were resuspended in Lysis buffer (50mM Tris buffer pH8.0, 100mM NaCl), and ultrasonically disrupted (power 450W, ultrasonic 5s, interval 10s) for 30min, and then centrifuged at 15000rpm4℃ for 15min to collect the supernatant, which is the crude enzyme solution. The expression level of glycerol phosphate oxidase was measured. The wild-type glycerol phosphate oxidase expression strain was used as a control, and the construction method of the wild-type glycerol phosphate oxidase expression strain was consistent with step 1 in Example 1. By comparing the expression levels of the wild-type and three mutant single colonies, the results are as follows Figure 1 As shown, the expression level of the glycerol phosphate oxidase mutant was significantly higher than that of the wild-type enzyme, and a recombinant Escherichia coli strain with high expression of the glycerol phosphate oxidase mutant was obtained.

[0126] Example 2 Purification of glycerol phosphate oxidase mutants

[0127] The selected glycerol phosphate oxidase mutant recombinant strain and wild-type strain were cultured in 5L LB medium at 37°C until OD600 was 0.6-0.8, IPTG inducer was added to a final concentration of 0.5mM, and cultured at 16°C for 16-20h. The cells were collected by centrifugation at 6000rpm, suspended in 200ml Lysis buffer (50mM Tris buffer pH8.0, 100mM NaCl), broken by high-pressure homogenizer, centrifuged at 14000rpm for 40 minutes, and the precipitate was removed. Purification was performed using HisCap6FF pre-packed columns from Tiandirenhe Company: 5 column volumes of 50 mM Tris buffer (pH 8.0) were used to balance the purification column, sample was loaded at a flow rate of 2 ml / min, and after loading, 50 mM Tris buffer (pH 8.0) was used for washing, and finally 50 mM Tris buffer (pH 8.0) containing 250 mM imidazole was used for elution. The protein components were collected and dialyzed to remove excess imidazole, and purity was identified using SDS-PAGE. The results are shown below. Figure 2 As shown, the molecular weight of the obtained glycerol phosphate oxidase is about 72 kDa.

[0128] Example 3 Enzyme activity detection of glycerol phosphate oxidase mutants

[0129] The enzyme activity detection of the glycerol phosphate oxidase mutant includes the following steps:

[0130] (1) Preparation of reaction solution: 0.1 M DL-α-glycerophosphate, 0.125% Triton X-100, 50 mM Tris-HCl (Ph8.0) buffer, 75 U / ml peroxidase, 7.5 mM TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt), 7.5 mM 4-AAP (4-aminoantipyrine).

[0131] (2) Enzyme diluent: 20 mM Tris-HCl (Ph8.0) buffer containing 0.2% BSA.

[0132] (3) Sample preparation: Dilute the sample to 0.15-0.35 U / ml with enzyme diluent.

[0133] (4) Detection steps for detecting the enzyme activity of glycerol phosphate oxidase mutants:

[0134] 1) Heat the reaction solution in a 37°C metal bath for 2 min;

[0135] 2) Add 1 mL of reaction solution into a 1 mL cuvette;

[0136] 3) Add 0.02 mL of diluted enzyme solution and mix with the reaction solution;

[0137] 4) The spectrophotometer monitors the absorbance change (△As) within 1 min at a wavelength of 555 nm;

[0138] 5) Replace the diluted enzyme solution with enzyme diluent, and follow the above steps to measure the blank absorbance change (△Ab), △A = △As-△Ab

[0139] 6) The calculation formula of the enzyme activity of the glycerol phosphate oxidase mutant is:

[0140]

[0141] Weight activity(U / mg)=Volume activity×1 / C

[0142] In the formula,

[0143] 1.02: total volume of reaction solution (mL);

[0144] 0.02: volume of enzyme solution after dilution (mL);

[0145] 1.0: optical path length;

[0146] df: dilution factor;

[0147] 1 / 2: 1 mol of hydrogen peroxide generates 1 / 2 mol of quinone dye substances;

[0148] C: enzyme concentration (mg / mL);

[0149] 39.2: Under standard reaction conditions, the molar absorption coefficient of the chromophore at 555 nm (cm 2 / μmol).

[0150] The enzyme activity of glycerol phosphate oxidase was quantified according to the absorbance change amplitude per minute. Each unit of enzyme activity was defined as the amount of enzyme required to produce one molecule of hydrogen peroxide per minute. The enzyme activity determination results are shown in Table 1. The enzyme activity of the purified glycerol phosphate oxidase mutant TST2 was slightly higher than that of the wild-type glycerol phosphate oxidase.

[0151] Table 1

[0152] sample Specific enzyme activity (U / mg) Wild-type glycerol phosphate oxidase 24.1 Glycerol phosphate oxidase mutant TST2 27.5

[0153] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0154] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A glycerol phosphate oxidase mutant, characterized in that: Compared with the glycerol phosphate oxidase shown in SEQ ID NO.1, the glycerol phosphate oxidase mutant has the following amino acid substitutions: Glutamine at position 16 was replaced by glutamic acid, phenylalanine at position 74 was replaced by glycine, phenylalanine at position 118 was replaced by glutamic acid, asparagine at position 132 was replaced by glycine, glutamic acid at position 150 was replaced by lysine, phenylalanine at position 244 was replaced by lysine, glutamine at position 280 was replaced by glycine, glutamine at position 304 was replaced by glutamic acid, asparagine at position 362 was replaced by lysine, aspartic acid at position 367 was replaced by glutamic acid, lysine at position 368 was replaced by glutamic acid, asparagine at position 383 was replaced by glycine, and asparagine at position 387 was replaced by Arginine was substituted with lysine, asparagine at position 395 was substituted with lysine, histidine at position 396 was substituted with lysine, aspartic acid at position 402 was substituted with lysine, arginine at position 442 was substituted with lysine, lysine at position 463 was substituted with threonine, glutamic acid at position 493 was substituted with serine, lysine at position 516 was substituted with arginine, glutamic acid at position 517 was substituted with arginine, glutamic acid at position 564 was substituted with lysine, glutamic acid at position 575 was substituted with aspartic acid, lysine at position 589 was substituted with alanine and arginine at position 590 was substituted with lysine.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide fragment encoding the glycerol phosphate oxidase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 2.

4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector includes one or more of a plasmid and a virus.

5. A recombinant host cell, characterized in that The recombinant host cell comprises the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3 or 4.

6. The recombinant host cell according to claim 5, characterized in that The host cells include prokaryotic cells.

7. The recombinant host cell according to claim 6, characterized in that The prokaryotic cells include Escherichia coli.

8. The method for preparing the glycerol phosphate oxidase mutant according to claim 1, characterized in that: The preparation method comprises the following steps: Cultivate the recombinant host cell according to any one of claims 5 to 7, and isolate the glycerol phosphate oxidase mutant from the obtained culture product.

9. Use of the glycerol phosphate oxidase mutant according to claim 1 in preparing a triglyceride detection reagent.

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