Method for preparing an immobilized enzyme or an immobilized protein and the immobilized enzyme or the immobilized protein

Abstract

Disclosed are a method for preparing an immobilized enzyme or an immobilized protein and the immobilized enzyme or the immobilized protein. The method comprises: covalently binding SpyCatcher on a carrier; constructing a target enzyme or a target protein fused with SpyTag at an N terminal or a C terminal; and contacting the carrier covalently bound with SpyCatcher with the target enzyme or the target protein fused with SpyTag. The immobilized enzyme or the immobilized protein comprises a carrier, SpyCatcher covalently bound with the carrier, and a target enzyme or a target protein fused with SpyTag at an N terminal or a C terminal, which is covalently bound with the carrier through the SpyTag and the SpyCatcher. The present application also relates to a carrier covalently bound with SpyCatcher and the use thereof for selectively immobilizing a target enzyme or a target protein fused with SpyTag at an N terminal or a C terminal from a protein mixture.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing an immobilized enzyme or an immobilized protein, and in particular to a method for preparing an immobilized enzyme or an immobilized protein by purifying a target enzyme or a target protein fused with SpyTag from a protein mixture and immobilizing the target enzyme or the target protein onto a carrier to which SpyCatcher is bound, and an immobilized enzyme or an immobilized protein obtained by the method. The present application also relates to a carrier covalently bound with SpyCatcher and the use thereof for selectively immobilizing a target enzyme or a target protein fused with SpyTag at the N-terminus or the C-terminus from a protein mixture. TECHNICAL BACKGROUND

[0002] Enzymes (proteins) have the advantages of mild reaction conditions, high selectivity, high specificity, high conversion rate, etc. as biological catalysts, and thus have good prospects for industrial applications. However, free enzymes are far from meeting the requirements of industrial applications due to their properties, and thus have many limitations in industrial applications. In addition, target enzymes are often produced together with other impurities during preparation, and these impurities often need to be removed by purification methods, and additional purification steps will inevitably increase the cost. On the other hand, in order to meet the needs of large-scale use in industrial applications and improve the separation efficiency of enzymes and reaction solutions, immobilized enzymes are often used in industrial applications.

[0003] Generally, the purification of enzymes and the immobilization of enzymes are two very important links in the industrial applications of enzymes. In order to reduce process steps, improve efficiency, and reduce cost, attempts have been made to complete the purification and immobilization of enzymes in one step, for example, in Gaberc-Porekar V, Menart V, Gaberc-Porekar V, et al. Perspectives of immobilized-metal affinity chromatography [J]. J biochem biophys methods. 2001, 49: 335-360, it is reported that immobilized metal affinity chromatography method is used, but in this method, oxidizing agent and reducing agent are needed, the reaction conditions are not mild enough for enzymes, and at the same time, metal ion leaching occurs in this method, resulting in unstable binding of enzymes to the carrier. SUMMARY

[0004] In order to solve the above problems, the present invention provides a method for preparing an immobilized enzyme or immobilized protein that is simple, mild in conditions, and stable in binding between the enzyme and the carrier. The method separates the target enzyme or target protein from the foreign protein in one step and binds the target enzyme or target protein to the carrier to obtain the immobilized enzyme or immobilized protein. The obtained immobilized enzyme or immobilized protein has high stability, maintains high enzymatic activity and can meet the needs of industrial applications. In addition, the present invention also provides a carrier covalently bound to SpyCatcher, and relates to the use of the carrier covalently bound to SpyCatcher in selectively immobilizing the target enzyme or target protein fused with SpyTag at the N-terminus or C-terminus from a protein mixture.

[0005] The above problems are solved by the following technical solutions.

[0006] 1. A method for preparing an immobilized enzyme or immobilized protein, the method comprising:

[0007] - Covalently attaching SpyCatcher to the carrier;

[0008] -Construct a target enzyme or target protein with SpyTag fused to the N-terminus or C-terminus;

[0009] - The carrier covalently bound with SpyCatcher and the target enzyme or target protein fused with SpyTag are brought into contact with each other.

[0010] 2. The method of item 1, wherein the SpyCatcher has a gene coding sequence of SEQ ID No. 1, or a gene coding sequence of SEQ ID No. 3, or a gene coding sequence of SEQ ID No. 6, or a gene coding sequence of SEQ ID No. 7.

[0011] 3. The method of item 1 or 2, wherein the SpyTag has a gene coding sequence of SEQ ID No. 2.

[0012] 4. The method according to any one of items 1 to 3, wherein the carrier is an epoxy carrier or an aldehyde agarose carrier.

[0013] 5. The method according to any one of items 1 to 4, wherein the target enzyme is L-phenylserine aldolase (LPA) or leucine dehydrogenase (LDH), and the target protein is green fluorescent protein (GFP). Preferably, the L-phenylserine aldolase has a gene coding sequence of SEQ ID No. 8, and the leucine dehydrogenase has a gene coding sequence of SEQ ID No. 9.

[0014] 6. An immobilized enzyme or immobilized protein obtained by the method of any one of items 1-5.

[0015] 7. An immobilized enzyme or immobilized protein comprising a carrier, a SpyCatcher covalently bound to the carrier, and a target enzyme or target protein fused with a SpyTag at the N-terminus or C-terminus, wherein the target enzyme or target protein is covalently bound to the carrier through the SpyTag and the SpyCatcher.

[0016] 8. The immobilized enzyme or immobilized protein of item 7, wherein the SpyCatcher has a gene coding sequence of SEQ ID No. 1, or a gene coding sequence of SEQ ID No. 3, or a gene coding sequence of SEQ ID No. 6, or a gene coding sequence of SEQ ID No. 7.

[0017] 9. The immobilized enzyme or immobilized protein of item 7 or 8, wherein the SpyTag has a gene coding sequence of SEQ ID No. 2.

[0018] 10. The immobilized enzyme or immobilized protein of any one of items 7-9, wherein the carrier is an epoxy-based carrier or a glyoxal agarose carrier.

[0019] 11. The immobilized enzyme or immobilized protein of any one of items 7-10, wherein the target enzyme is L-phenylserine aldolase or leucine dehydrogenase, and the target protein is green fluorescent protein, preferably, the L-phenylserine aldolase has a gene coding sequence of SEQ ID No. 8, and the leucine dehydrogenase has a gene coding sequence of SEQ ID No. 9.

[0020] 12. A carrier covalently bound with a SpyCatcher.

[0021] 13. The carrier of item 12, wherein the SpyCatcher has a gene coding sequence of SEQ ID No. 1, or a gene coding sequence of SEQ ID No. 3, or a gene coding sequence of SEQ ID No. 6, or a gene coding sequence of SEQ ID No. 7.

[0022] 14. The carrier of item 12 or 13, wherein the carrier is an epoxy-based carrier or a glyoxal agarose carrier.

[0023] 15. Use of a carrier covalently bound with a SpyCatcher for selectively immobilizing a target enzyme or target protein fused with a SpyTag at the N-terminus or C-terminus from a protein mixture.

[0024] 16. The use of item 15, wherein the SpyCatcher has the gene coding sequence of SEQ ID No. 1, or has the gene coding sequence of SEQ ID No. 3, or has the gene coding sequence of SEQ ID No. 6, or has the gene coding sequence of SEQ ID No. 7.

[0025] 17. The use of item 15 or 16, wherein the SpyTag has the gene coding sequence of SEQ ID No. 2.

[0026] 18. The use of any one of items 15-17, wherein the carrier is an epoxy-based carrier or a glyoxal agarose carrier.

[0027] 19. The use of any one of items 15-18, wherein the target enzyme is L-phenylserine aldolase or leucine dehydrogenase, and the target protein is green fluorescent protein, preferably, the L-phenylserine aldolase has the gene coding sequence of SEQ ID No. 8, and the leucine dehydrogenase has the gene coding sequence of SEQ ID No. 9.

[0028] The method for preparing the immobilized enzyme or the immobilized protein of the present application has the advantage that the target enzyme or the target protein can be separated from the protein mixture and immobilized in one step, with a high immobilization rate, and the obtained immobilized enzyme maintains high enzyme activity and good thermal stability, and the obtained immobilized protein has good thermal stability. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The process for preparing the immobilized enzyme or the immobilized protein with the glyoxal agarose as the carrier is schematically represented. DETAILED DESCRIPTION

[0030] Unless otherwise defined, all publications, patents, patent applications and other references mentioned in this specification are incorporated herein by reference in their entirety.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification, including definitions, controls.

[0032] Unless otherwise stated, all percentages, parts, ratios, etc. are by weight.

[0033] When a quantity, concentration, or other value or parameter is expressed in a range, preferred range, or a range "between" two values, it is understood that the intervening range of values or intervening ranges of values, between any stated value or intervening value in the stated range, is encompassed even if that intervening value or intervening range is not expressly stated. The number of values recited in the range is not intended to limit the scope of the range but merely to describe more specifically the range. Unless otherwise stated, the numerical values recited in this application are intended to be approximations.

[0034] In this document, the terms "formed from" or "consisting of" are synonymous with "comprising." As used herein, the terms "comprise," "comprises," "comprising," "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion.

[0035] Also, the use of "a" or "an" to describe certain elements or components is not intended to be construed as excluding pluralities or as requiring solely a single one of the elements or components. Thus, "a" or "an" shall be read to include one or at least one, and the singular number of elements or components shall not exclude the plural unless expressly stated.

[0036] Unless otherwise indicated, the materials, methods, and examples of the application are illustrative only and not limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the application, suitable methods and materials are described in the following.

[0037] When the term "about" is used to describe the endpoints of a value or range, it is understood that the endpoints are intended to be included in the range. In the present application, each recited numerical value should be considered to be modified by "about."

[0038] In one aspect of the application, there is provided a method of preparing an immobilized enzyme or an immobilized protein, the method comprising:

[0039] - covalently binding SpyCatcher to a support;

[0040] - constructing a target enzyme or a target protein fused with SpyTag at the N-terminus or the C-terminus;

[0041] - contacting the support covalently bound with SpyCatcher with the target enzyme or the target protein fused with SpyTag.

[0042] In the present application, SpyCatcher and SpyTag have the meanings conventionally used in the art. SpyTag is a polypeptide segment and SpyCatcher is a protein corresponding thereto, and they can spontaneously form an isopeptide bond to covalently bind (Zakeri BF, J.O.; Celik, E.; Chittock, E.C.; Schwarz-Linek, U.; Moy, V.T.; Howarth, M. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 2012; 2012, 109(12),: E690-E7.).

[0043] Preferably, in the present application, SpyCatcher has the genetic coding sequence of SEQ ID No. 1, or has the genetic coding sequence of SEQ ID No. 3, or has the genetic coding sequence of SEQ ID No. 6, or has the genetic coding sequence of SEQ ID No. 7.

[0044] In the present application, in order to distinguish from the description, the SpyCatcher having the genetic coding sequence of SEQ ID No. 1 is called oSpyCatcher, the SpyCatcher having the genetic coding sequence of SEQ ID No. 6 is called aSpyCatcher, the SpyCatcher having the genetic coding sequence of SEQ ID No. 7 is called cSpyCatcher, and the SpyCatcher having the genetic coding sequence of SEQ ID No. 3 is called mSpyCatcher. It should be understood that the above-mentioned oSpyCatcher, aSpyCatcher, cSpyCatcher and mSpyCatcher are all encompassed in the scope of the term "SpyCatcher".

[0045] The SpyCatcher having the gene coding sequence of SEQ ID No. 3, SEQ ID No. 6 or SEQ ID No. 7 is a mutant of the SpyCatcher having the gene coding sequence of SEQ ID No. 1. Among them, the SpyCatcher having the gene coding sequence of SEQ ID No. 6 is a mutant obtained by adding multiple lysine peptide chains (LysGlyLysGlyLysGly) to the C-terminus of the SpyCatcher having the gene coding sequence of SEQ ID No. 1; the SpyCatcher having the gene coding sequence of SEQ ID No. 7 is a mutant obtained by mutating the lysines at positions 28, 37 and 64 to arginines in the SpyCatcher having the gene coding sequence of SEQ ID No. 1; and the SpyCatcher having the gene coding sequence of SEQ ID No. 3 is a mutant obtained by combining the above two mutation methods in the SpyCatcher having the gene coding sequence of SEQ ID No. 1.

[0046] In the method of the present application, the method of covalently binding the SpyCatcher to the carrier is a conventional enzyme (protein) immobilization method in the art. The immobilization method herein is performed by binding the amino group in the SpyCatcher to the group on the carrier capable of forming a covalent bond with the amino group.

[0047] In the present application, the carriers having the aSpyCatcher, cSpyCatcher and mSpyCatcher bound thereto can further improve the immobilization rate and stability of the target enzyme or target protein, as compared to the carrier having the oSpyCatcher bound thereto. Without wishing to be bound by theory, the inventors believe that adding the peptide chain containing multiple lysines (LysGlyLysGlyLysGly) to the C-terminus of the oSpyCatcher (aSpyCatcher) can enable the oSpyCatcher to be directionally immobilized to the carrier and more firmly bound, and mutating the lysines at positions 28, 37 and 64 to arginines in the oSpyCatcher (cSpyCatcher) can enable the immobilization direction of the SpyCatcher on the carrier to be more conducive to the subsequent binding of the SpyTag, and thus the mSpyCatcher combining the two mutation methods has both advantages.

[0048] Generally, when the carrier is an agarose carrier, the agarose carrier is first pretreated with propylene glycol and sodium periodate to have an aldehyde group, and then combined by forming a covalent bond between the amino group in SpyCatcher and the aldehyde group on the carrier. When the carrier is an epoxy carrier, generally no pretreatment of the carrier is needed, and the combination is directly by forming a covalent bond between the amino group in SpyCatcher and the epoxy group on the carrier. When other conventional carriers in the art are used, SpyCatcher is covalently bound to the carrier by a treatment or combination method known in the art.

[0049] Preferably, the SpyTag in the present application has the gene coding sequence of SEQ ID No. 2.

[0050] In the present application, SpyTag can be fused to the N-terminus or C-terminus of the target enzyme or target protein. The SpyTag peptide segment can be fused to the N-terminus or C-terminus of the target enzyme or target protein by conventional methods in the art, for example, the Megawhop method to obtain a gene encoding a fusion protein.

[0051] In the present application, the carrier covalently bound with SpyCatcher is contacted with the target enzyme or target protein fused with SpyTag, and the contacting conditions are not particularly limited, for example, the contacting can be carried out at room temperature and pressure, for example, at 4-37°C (for example, 20-35°C), for example, for 8-20 hours, for example, 10-16 hours, and the pH is also not particularly limited, as long as it is suitable for the presence of the protein without denaturation, for example, pH 5-10, pH 5.5-9.

[0052] In another aspect of the present application, it relates to an immobilized enzyme or immobilized protein obtained by the method according to the present application.

[0053] In yet another aspect of the present application, it relates to an immobilized enzyme or immobilized protein,

[0054] - a carrier;

[0055] - SpyCatcher covalently bound to the carrier; and

[0056] - a target enzyme or target protein fused with SpyTag at the N-terminus or C-terminus,

[0057] wherein the target enzyme or target protein is immobilized on the carrier by covalent binding of the SpyTag to SpyCatcher.

[0058] The foregoing description and definitions in the method for preparing an immobilized enzyme or immobilized protein also apply to the immobilized enzyme or immobilized protein of the present aspect.

[0059] Another aspect of the present application relates to a carrier covalently bound with SpyCatcher. The content of covalently binding SpyCatcher to a carrier described in the above method for preparing immobilized enzymes or immobilized proteins is also applicable to this aspect.

[0060] Another aspect of the present application relates to the use of a carrier covalently bound to SpyCatcher for selectively immobilizing a target enzyme or target protein fused with SpyTag at the N-terminus or C-terminus from a protein mixture. The aforementioned description and definition of the method for preparing immobilized enzymes or immobilized proteins also apply to the use in this aspect.

[0061] Example

[0062] The present invention is described in more detail below through examples, but it should be understood that the scope of the present invention is not limited to the examples.

[0063] Assay and calculation methods used in the examples:

[0064] 1 ) Thermal stability of enzymes

[0065] The free enzyme or immobilized enzyme was incubated at 60°C and samples were taken at regular intervals to determine the residual activity.

[0066] 2) Immobilization rate of green fluorescent protein (GFP)

[0067] The fluorescence intensity of free GFP and immobilized GFP in 100 mM phosphate buffer (PB) at 28°C and pH 8.5 was measured using a multifunctional microplate reader at excitation and emission wavelengths of 488 nm and 508 nm, respectively.

[0068] The calculation method for the SpyTag-GFP immobilization rate is:

[0069] SpyTag-GFP immobilization rate = (SpyTag-GFP fluorescence in the supernatant at the zero time of immobilization - residual SpyTag-GFP fluorescence in the supernatant at the end of immobilization) / SpyTag-GFP fluorescence in the supernatant at the zero time of immobilization × 100%

[0070] 3) L-phenylserine aldolase (LPA) activity and immobilization rate

[0071] LPA enzyme activity is defined as the amount of enzyme required to catalyze the conversion of L-phenylserine to 1 μmol of benzaldehyde per minute at 30°C, pH 8.5, and an L-phenylserine concentration of 10 mmol / L. One activity unit (U) was defined as the amount of enzyme required to catalyze the conversion of L-phenylserine to 1 μmol of benzaldehyde per minute.

[0072] The concentration of generated benzaldehyde was measured at 290 nm using an EnSpire 2300 multifunctional microplate reader at 30°C to calculate the enzyme activity.

[0073] The calculation method of SpyTag-LPA immobilization rate is:

[0074] SpyTag-LPA immobilization rate = (SpyTag-LPA enzyme activity in supernatant at zero time of immobilization - SpyTag-LPA enzyme activity in supernatant at the end of immobilization) / SpyTag-LPA enzyme activity in supernatant at zero time of immobilization x 100%

[0075] 4) Enzyme activity and immobilization rate of leucine dehydrogenase activity (LDH)

[0076] LDH enzyme activity definition: Prepare a substrate solution containing 4.5 mM 2-oxobutyric acid and 0.204 mM NADH with 900 mM NH3-NH4Cl buffer at pH = 9.5, detect enzyme activity at 30°C with EnSpire2300 multifunctional enzyme labeler, and the amount of enzyme required to catalyze the consumption of 1 μmol NADH per minute is defined as 1 U.

[0077] Determine the change value and time of NADH concentration with EnSpire2300 multifunctional enzyme labeler at 30°C at ultraviolet 340 nm to calculate enzyme activity.

[0078] The calculation method of SpyTag-LDH immobilization rate is:

[0079] SpyTag-LDH immobilization rate = (SpyTag-LDH enzyme activity in supernatant at zero time of immobilization - SpyTag-LDH enzyme activity in supernatant at the end of immobilization) / SpyTag-LDH enzyme activity in supernatant at zero time of immobilization x 100%

[0080] Example A: Preparation of SpyCatcher conjugated carriers

[0081] Construction of SpyCatcher genetically engineered expression bacteria and cultivation

[0082] Using the method of Megawhop (Sanchis J, Layla Fernández, Carballeira J D, et al. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates [J]. Applied Microbiology and Biotechnology, 2008, 81(2): 387-397.), pET28a SpyTag-GFP-SpyCatcher with the gene coding sequence of SEQ ID No. 5 was used as a template to construct pET28a-oSpyCatcher; the primers with the DNA sequences of SEQ ID No. 14 and SEQ ID No. 15 were used to obtain the first round of PCR products by PCR, and then the first round of PCR products were used as long primers for the second round of whole plasmid amplification to obtain the second round of PCR products; the second round of PCR products obtained by Megawhop were subjected to Dpnl enzyme digestion, and then the Dpnl enzyme digestion was transformed into E. coli BL21 (DE3) competent cells, which were verified by DNA sequencing of the plasmid to obtain the genetically engineered strain E. coli BL21 (DE3) / pET28a-oSpyCatcher.

[0083] Using pET28a-oSpyCatcher as a template, the primers with the DNA sequences of SEQ ID No. 16 and SEQ ID No. 17 were used to construct a mutant with the gene coding sequence of SEQ ID No. 6 (aSpyCatcher, gene coding sequence SEQ ID No. 6) with a poly-lysine peptide chain (LysGlyLysGlyLysGly) at the C-terminus by the method of Megawhop, which was verified by sequencing, and finally the genetically engineered strain E. coli BL21 (DE3) / pET28a-aSpyCatcher was obtained.

[0084] Similarly, with pET28a-oSpyCatcher as a template, primers of SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20 and SEQ ID No. 21 DNA sequences were used to construct a mutant in which three lysines (28th, 37th and 64th) were mutated to arginines (cSpyCatcher, gene coding sequence SEQ ID No. 7) by the Megawhop method. Finally, the genetic engineering strain E. coli BL21(DE3) / pET28a-cSpyCatcher was obtained.

[0085] Finally, the two mutation methods were combined to construct the mutant (mSpyCatcher). With cSpyCatcher as a template, primers of SEQ ID No. 16 and SEQ ID No. 17 DNA sequences were used to construct a mutant (mSpyCatcher, gene coding sequence SEQ ID No. 3) by the Megawhop method using the primers used to construct aSpyCatcher. The genetic engineering strain obtained was E. coli BL21(DE3) / pET28a-mSpyCatcher.

[0086] The genetic engineering E. coli BL21(DE3) / pET28a-oSpyCatcher, BL21(DE3) / pET28a-mSpyCatcher, BL21(DE3) / pET28a-aSpyCatcher and BL21(DE3) / pET28a-cSpyCatcher were cultured in LB medium at 37°C and 200 r / min for 12 h, inoculated into lactose medium at 1% inoculation amount, and cultured at 28°C and 200 r / min for 24 h. The bacterial cells were harvested by centrifugation at 4000 rpm for 10 min. The compositions of the LB medium and the lactose medium were as follows:

[0087] LB medium: 1% NaCl, 1% peptone, 0.5% yeast powder and 50 μg / mL kanamycin.

[0088] Lactose self-induction medium: peptone 10 g / L, yeast powder 5 g / L, Na2HPO4 8.95 g / L, KH2PO4 3.4 g / L, NH4Cl 2.67 g / L, Na2SO4 0.7 g / L, MgSO4 0.24 g / L, glycerol 5 g / L, glucose 0.5 g / L, lactose 2 g / L and 50 μg / mL kanamycin.

[0089] Purification of SpyCatcher

[0090] The harvested bacteria were resuspended in 0.1 M phosphate buffer (pH 8.5) and sonicated. The conditions of sonication were: 5 seconds of sonication, 5 seconds of interval, 99 cycles, 200 W of sonication power, and twice of sonication. The supernatant was obtained by centrifugation at 20000 rpm for 5 min at 4°C.

[0091] The various SpyCatcher protein solutions were loaded onto Ni-NTA resin columns, and the pH value was adjusted to pH 8.5. The columns were washed with 30 mM imidazole buffer to remove the impurities, and then eluted with 200 mM imidazole buffer to obtain the various SpyCatcher purified solutions, namely oSpyCatcher purified solution, aSpyCatcher purified solution, cSpyCatcher purified solution, and mSpyCatcher purified solution. The solutions were stored at 4°C for later use.

[0092] i) Immobilization on a glyoxal agarose carrier

[0093] The washed 6% cross-linked agarose was weighed at 5.6 g, and 4 mL of a mixture of 1 M NaOH and 0.5 M NaBH4 was slowly added in an ice bath, followed by the addition of 1.6 mL of glycidol (final concentration 2 M). The reaction was carried out at 25°C for 15 h in a 200 rpm shaker. The stirred agarose was washed and dried. 5 g of the agarose was weighed and added to 43 mL of deionized water, and then 0.23 g of sodium periodate (final concentration 20 mM) was added. The reaction was carried out at 25°C for 2 h in a 100 rpm shaker. The support was washed with a large amount of deionized water (500 mL) and filtered.

[0094] 1 g of the support was weighed and added to different amounts of the above-mentioned SpyCatcher purified solutions and 0.2 M sodium bicarbonate buffer, and the pH of the system was adjusted to 10. The reaction was carried out at 25°C for 3 h in a 100 rpm shaker, and then a certain amount of sodium borohydride was added to make the final concentration 1 mg / ml. The reaction was carried out at room temperature for 30 min with gentle stirring, and then washed with 25 mM phosphate buffer (pH 8.0) and dried. oSpyCatcher-agarose support, aSpyCatcher-agarose support, cSpyCatcher-agarose support, and mSpyCatcher-agarose support were obtained, respectively. The washed and dried supports were stored for later use.

[0095] ii) Immobilization on an epoxy carrier

[0096] Take 1 g of the carrier, add different amounts of the above-mentioned SpyCatcher purified liquid and 1.25M phosphate buffer, and adjust the pH of the system to 8. 25℃, 200rpm shaking bed for 24h, respectively, to obtain oSpyCatcher-epoxy carrier, aSpyCatcher-epoxy carrier, cSpyCatcher-epoxy carrier and mSpyCatcher-epoxy carrier. After washing and drying, they are ready for use.

[0097] Example B: Preparation of SpyTag fused enzymes or proteins

[0098] Using the method of Megawhop, the pET28a-SpyTag-GFP with SEQ ID No. 4 was constructed using the aforementioned pET28a SpyTag-GFP-SpyCatcher as a template, using the primer of SEQ ID No. 12 and SEQ ID No. 13 DNA sequence, and the first round of PCR product was obtained by PCR, and then the first round of PCR product was used as a long primer for the second round of whole plasmid amplification. The second round of PCR product obtained by Megawhop was subjected to Dpnl enzyme digestion, and then the Dpnl enzyme digestion was transformed into E. coli BL21 (DE3) competent cells, and the genetic engineering strain E. coli BL21 (DE3) / pET28a-SpyTag-GFP was obtained by sequencing verification. The method of GoldenGate (Engler, C., & Marillonnet, S. (2014). Golden gate cloning. In DNA cloning and assembly methods (pp. 119-131). Humana Press, Totowa, NJ.) was used to replace the GFP in pET-SpyTag-GFP with L-phenylalanine aldolase (LPA) with SEQ ID No. 8 gene coding sequence and leucine dehydrogenase (LDH) with SEQ ID No. 9 gene coding sequence.

[0099] Firstly, the plasmid backbone (pET-SpyTag plasmid backbone) containing BsaI enzyme cutting site and the target fragment (LPA and LDH) need to be constructed. When constructing the plasmid backbone, the primers with SEQ ID No. 22 and SEQ ID No. 23 are used, and when constructing the target fragment (LPA and LDH), the primers with SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27 are used. The recombinant plasmids pET-SpyTag-LPA (the gene coding sequence of SpyTag-LPA is SEQ ID No. 10) and pET-SpyTag-LDH (the gene coding sequence of SpyTag-LDH is SEQ ID No. 11) are obtained by enzyme cutting and ligation method through Golden Gate method.

[0100] The genetically engineered E. coli BL21 (DE3) / pET28a-SpyTag-GFP, BL21 (DE3) / pET28a-SpyTag-LPA or BL21 (DE3) / pET-SpyTag-LDH were cultured in LB medium at 37℃, 200r / min for 12h, inoculated into lactose medium with 1% inoculation amount, and cultured at 28℃, 200r / min for 24h. The bacterial cells were harvested by centrifugation at 4000rpm for 10min. Among them, the components of LB medium and lactose medium are as follows:

[0101] LB medium: 1% NaCl, 1% peptone, 0.5% yeast powder and 50μg / mL kanamycin.

[0102] Lactose self-induction medium: peptone 10g / L, yeast powder 5g / L, Na2HPO4 8.95g / L, KH2PO4 3.4g / L, NH4Cl 2.67g / L, Na2SO4 0.7g / L, MgSO4 0.24g / L, glycerol 5g / L, glucose 0.5g / L, lactose 2g / L, 50μg / mL kanamycin.

[0103] The harvested bacterial cells were resuspended with 0.1M phosphate buffer (pH8.5) and the bacterial cells were broken by ultrasonic wave. The ultrasonic conditions were: ultrasonic for 5 seconds, interval for 5 seconds, 99 cycles, ultrasonic power 200W, and the bacterial cells were broken twice. The supernatant was the heterologous protein solution containing the target protein (SpyTag-GFP, SpyTag-LPA or SpyTag-LDH) fused with SpyTag after centrifugation at 20000rpm for 5min at 4℃.

[0104] Example C1 : Immobilized GFP: oSpyCatcher-agarose carrier + SpyTag-GFP

[0105] The oSpyCatcher-Agarose carrier was prepared as in Example A, using 1 mg of oSpyCatcher for immobilization per gram of carrier.

[0106] The prepared oSpyCatcher-Agarose carrier was mixed with SpyTag-GFP prepared as in Example B in a centrifuge tube, 5 mg of SpyTag-GFP was added per gram of oSpyCatcher-Agarose carrier, and was placed at 28°C, with pH adjusted to 5.5 using HC1.

[0107] The supernatant was sampled at zero time of mixing, and the supernatant was sampled after 20 h of mixing, and the fluorescence value of the supernatant was measured as before. According to the aforementioned formula, the immobilization rate of SpyTag-GFP on the oSpyCatcher-Agarose carrier was 40.78% after 20 h.

[0108] Example C2: Immobilized GFP: mSpyCatcher-agarose carrier + SpyTag-GFP

[0109] The mSpyCatcher-Agarose carrier was prepared as in Example A, using 1 mg of mSpyCatcher for immobilization per gram of carrier.

[0110] The prepared mSpyCatcher-Agarose carrier was mixed with SpyTag-GFP prepared as in Example B in a centrifuge tube, 5 mg of SpyTag-GFP was added per gram of mSpyCatcher-Agarose carrier, and was placed at 28°C, with pH adjusted to 5.5 using HC1.

[0111] The supernatant was sampled at zero time of mixing, and the supernatant was sampled after 20 h of mixing, and the fluorescence value of the supernatant was measured as before. According to the aforementioned formula, the immobilization rate of SpyTag-GFP on the mSpyCatcher-Agarose carrier was 55.56% after 20 h.

[0112] Example C3: Immobilized GFP: oSpyCatcher-agarose carrier + SpyTag-GFP

[0113] The oSpyCatcher-Agarose carrier was prepared as in Example A, using 5 mg of oSpyCatcher for immobilization per gram of carrier.

[0114] The prepared oSpyCatcher-Agarose carrier was mixed with SpyTag-GFP prepared as in Example B in a centrifuge tube, 5 mg of SpyTag-GFP was added per gram of oSpyCatcher-Agarose carrier, and was placed at 30°C, with pH adjusted to 6.5 using HC1.

[0115] The supernatant was sampled at the zero time of mixing, and the supernatant was sampled after 16h of mixing, and the fluorescence value of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-GFP on the oSpyCatcher-agarose carrier was 71.21% after 16h.

[0116] Example C4: Immobilized GFP: mSpyCatcher-agarose carrier + SpyTag-GFP

[0117] The mSpyCatcher-agarose carrier was prepared as in Example A, and 5mg of mSpyCatcher was used for immobilization per gram of the carrier.

[0118] The prepared mSpyCatcher-agarose carrier was mixed with SpyTag-GFP prepared in Example B above in a centrifuge tube, 5mg of SpyTag-GFP was added per gram of the mSpyCatcher-agarose carrier, and it was placed at 30°C, and the pH was adjusted to 6.5 with HC1.

[0119] The supernatant was sampled at the zero time of mixing, and the supernatant was sampled after 16h of mixing, and the fluorescence value of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-GFP on the mSpyCatcher-agarose carrier was 82.90% after 16h.

[0120] Example C5: Immobilized LPA: oSpyCatcher-agarose carrier + SpyTag-LPA

[0121] The oSpyCatcher-agarose carrier was prepared as in Example A, and 5mg of oSpyCatcher was used for immobilization per gram of the carrier.

[0122] The prepared oSpyCatcher-agarose carrier was mixed with SpyTag-LPA prepared in Example B above in a centrifuge tube, 5mg of SpyTag-LPA was added per gram of the oSpyCatcher-agarose carrier, and it was placed at 25°C, and the pH was adjusted to 7.5 with HC1.

[0123] The supernatant was sampled at the zero time of mixing, and the supernatant was sampled after 22h of mixing, and the enzyme activity of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-LPA on the oSpyCatcher-agarose carrier was 59% after 22h.

[0124] Thermal stability:

[0125] The thermal stability of the free LPA enzyme and the immobilized SpyTag-LPA in this example was measured according to the aforementioned measurement method, respectively.

[0126] The free LPA enzyme activity at time 0 was 9.50 U / mL, and after 100 min, the free LPA enzyme activity was 2.67 U / mL, and the residual enzyme activity ratio was 28.13%.

[0127] The immobilized LPA enzyme activity at time 0 was 26.96 U / g, and after 100 min, the immobilized LPA enzyme activity was 16.17 U / g, and the residual enzyme activity ratio was 59.96%.

[0128] Example C6: Immobilized LPA: mSpyCatcher-agarose carrier + SpyTag-LPA

[0129] The mSpyCatcher-agarose carrier was prepared as in Example A, and 5 mg of mSpyCatcher was used for immobilization per gram of carrier.

[0130] The prepared mSpyCatcher-agarose carrier was mixed with the SpyTag-LPA prepared in Example B above in a centrifuge tube, 5 mg of SpyTag-LPA was added per gram of mSpyCatcher-agarose carrier, and it was placed at 25°C, and the pH was adjusted to 7.5 with HC1.

[0131] The supernatant was sampled at the mixing time 0, and the supernatant was sampled after 22 h of mixing, and the enzyme activity of the supernatant was measured as described above. According to the calculation according to the above formula, the immobilization rate of SpyTag-LPA on the mSpyCatcher-agarose carrier was 73% after 22 h.

[0132] Thermal stability:

[0133] According to the above-mentioned determination method, the thermal stability of the free LPA enzyme and the immobilized SpyTag-LPA in this example was determined, respectively.

[0134] The free enzyme LPA enzyme activity at time 0 was 9.50 U / mL, and after 100 min, the free LPA enzyme activity was 2.67 U / mL, and the residual enzyme activity ratio was 28.13%.

[0135] The immobilized LPA enzyme activity at time 0 was 34.07 U / g, and after 100 min, the immobilized LPA enzyme activity was 22.84 U / g, and the residual enzyme activity ratio was 67.05%.

[0136] Example C7: Immobilized LDH: oSpyCatcher-agarose carrier + SpyTag-LDH

[0137] The oSpyCatcher-agarose carrier was prepared as in Example A, and 5 mg of oSpyCatcher was used for immobilization per gram of carrier.

[0138] The prepared oSpyCatcher-agarose carrier was mixed with the SpyTag-LDH prepared in Example B in a centrifuge tube, 5 mg of SpyTag-LDH was added per gram of oSpyCatcher-agarose carrier, and was placed at 20°C, pH = 8.5.

[0139] The supernatant was sampled at zero time of mixing, and the supernatant was sampled after 15 h of mixing, and the enzyme activity of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-LDH on the oSpyCatcher-agarose carrier was 54% after 15 h.

[0140] Thermal stability:

[0141] The thermal stability of free LDH enzyme and the immobilized SpyTag-LDH in this example was measured according to the aforementioned determination method, respectively.

[0142] The enzyme activity of free LDH enzyme at 0 time was 12.72 U / mL, and the enzyme activity of free LDH enzyme after 100 min was 2.83 U / mL, and the residual enzyme activity ratio was 22.24%.

[0143] The enzyme activity of immobilized LDH enzyme at 0 time was 15.92 U / g, and the enzyme activity of immobilized LDH enzyme after 100 min was 14.68 U / g, and the residual enzyme activity ratio was 92.19%.

[0144] Example C8: Immobilized LDH: mSpyCatcher-agarose carrier + SpyTag-LDH

[0145] The mSpyCatcher-agarose carrier was prepared as in Example A, and 5 mg of mSpyCatcher was used for immobilization per gram of carrier.

[0146] The prepared mSpyCatcher-agarose carrier was mixed with the SpyTag-LDH prepared in Example B in a centrifuge tube, 5 mg of SpyTag-LDH was added per gram of mSpyCatcher-agarose carrier, and was placed at 20°C, pH = 8.5.

[0147] The supernatant was sampled at zero time of mixing, and the supernatant was sampled after 15 h of mixing, and the enzyme activity of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-LDH on the mSpyCatcher-agarose carrier was 85% after 15 h.

[0148] Thermal stability:

[0149] The thermal stability of free LDH enzyme and the immobilized SpyTag-LDH in this example was measured according to the aforementioned determination method, respectively.

[0150] The free LDH enzyme activity at time 0 was 12.72 U / mL, and after 100 min, the free LDH enzyme activity was 2.83 U / mL, and the residual enzyme activity ratio was 22.24%.

[0151] The immobilized LDH enzyme activity at time 0 was 23.46 U / g, and after 100 min, the immobilized LDH enzyme activity was 22.29 U / g, and the residual enzyme activity ratio was 95.02%.

[0152] Example C9: Immobilized GFP: aSpyCatcher-agarose carrier + SpyTag-GFP

[0153] The cSpyCatcher-agarose carrier was prepared as in Example A, using 5 mg of cSpyCatcher for immobilization per gram of carrier.

[0154] The prepared cSpyCatcher-agarose carrier was mixed with the SpyTag-GFP prepared in Example B above in a centrifuge tube, 5 mg of SpyTag-GFP was added per gram of cSpyCatcher-agarose carrier, and was placed at 28°C, and the pH was adjusted to 7.5 with HC1.

[0155] The supernatant was sampled at zero time of mixing, and the supernatant was sampled after 20 h of mixing, and the fluorescence value of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-GFP on the cSpyCatcher-agarose carrier was 77.30% after 20 h.

[0156] Example C10: Immobilized GFP: cSpyCatcher-agarose carrier + SpyTag-GFP

[0157] The cSpyCatcher-agarose carrier was prepared as in Example A, using 5 mg of cSpyCatcher for immobilization per gram of carrier.

[0158] The prepared cSpyCatcher-agarose carrier was mixed with the SpyTag-GFP prepared in Example B above in a centrifuge tube, 5 mg of SpyTag-GFP was added per gram of cSpyCatcher-agarose carrier, and was placed at 28°C, and the pH was adjusted to 7.5 with HC1.

[0159] The supernatant was sampled at zero time of mixing, and the supernatant was sampled after 20 h of mixing, and the fluorescence value of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-GFP on the cSpyCatcher-agarose carrier was 77.30% after 20 h.

[0160] Example C1 1 : Immobilized GFP: oSpyCatcher-epoxy carrier + SpyTag-GFP

[0161] An oSpyCatcher-epoxy support was prepared as in Example A, using 20 mg oSpyCatcher per gram of support for immobilization.

[0162] The prepared oSpyCatcher-epoxy vector and the SpyTag-GFP prepared in Example B were mixed in a centrifuge tube, with 5 mg of SpyTag-GFP added per gram of aSpyCatcher-epoxy vector. The mixture was placed at 28° C. and the pH was adjusted to 7.5 with HCl.

[0163] The supernatant was sampled at time zero and 14 h after mixing, and the fluorescence value of the supernatant was measured as described above. According to the above formula, the immobilization rate of SpyTag-GFP on the oSpyCatcher-epoxy support was 50.0% after 14 h.

[0164] Example C12: Immobilized GFP: mSpyCatcher-epoxy carrier + SpyTag-GFP

[0165] The mSpyCatcher-epoxy carrier was prepared as in Example A, and 20 mg of mSpyCatcher was used per gram of carrier for immobilization.

[0166] The prepared mSpyCatcher-epoxy vector and the SpyTag-GFP prepared in Example B were mixed in a centrifuge tube, with 5 mg of SpyTag-GFP added per gram of mSpyCatcher-epoxy vector. The mixture was placed at 28° C. and the pH was adjusted to 7.5 with HCl.

[0167] The supernatant was sampled at time zero and 14 h after mixing, and the fluorescence of the supernatant was measured as described above. According to the above formula, the immobilization rate of SpyTag-GFP on the mSpyCatcher-epoxy support was 72.0% after 14 h.

[0168] Comparative Example 1 : Blank glyoxal agarose carrier + SpyTag-GFP

[0169] A blank acetaldehyde agarose carrier was prepared as in Example A, that is, 1 g of activated acetaldehyde agarose carrier was weighed, deionized water and 0.2 M sodium bicarbonate buffer were added, and the pH of the system was adjusted to 10. The mixture was shaken at 25° C. and 100 rpm for 3 h to obtain a blank acetaldehyde agarose carrier.

[0170] The prepared blank acetaldehyde agarose carrier was mixed with the SpyTag-GFP prepared in Example B above in a centrifuge tube, 5 mg of SpyTag-GFP was added per gram of blank acetaldehyde agarose carrier, and the mixture was placed at 28° C. and the pH was adjusted to 8.0 with HCl.

[0171] The supernatant was sampled at the zeroth hour of mixing, and the supernatant was sampled after 20 h of mixing, and the fluorescence value of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-GFP on the blank acetaldehyde agarose carrier was about 0% after 20 h. At the same time, under the detection of a fluorescence microscope, the blank acetalaldehyde agarose carrier particles after the immobilization treatment had no fluorescence.

[0172] Comparative Example 2: Blank glyoxal agarose carrier + SpyTag-LPA

[0173] The blank acetaldehyde agarose carrier was prepared as in Comparative Example 1.

[0174] The prepared blank acetaldehyde agarose carrier was mixed with SpyTag-LPA prepared in Example B above in a centrifuge tube, 5 mg of SpyTag-LPA was added per gram of blank acetaldehyde agarose carrier, and the system was placed at 25°C, and the pH was adjusted to 7.5 with HC1.

[0175] The supernatant was sampled at the zeroth hour of mixing, and the supernatant was sampled after 22 h of mixing, and the enzyme activity of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-LPA on the blank acetaldehyde agarose carrier was about 0% after 22 h. At the same time, the blank acetaldehyde agarose carrier particles after the immobilization treatment had no detectable enzyme activity.

[0176] Comparative Example 3: Blank glyoxal agarose carrier + SpyTag-LDH

[0177] The blank acetaldehyde agarose carrier was prepared as in Comparative Example 1.

[0178] The prepared blank acetaldehyde agarose carrier was mixed with SpyTag-LDH prepared in Example B above in a centrifuge tube, 5 mg of SpyTag-LDH was added per gram of blank acetaldehyde agarose carrier, and the system was placed at 30°C, and the pH was adjusted to 8.5.

[0179] The supernatant was sampled at the zeroth hour of mixing, and the supernatant was sampled after 15 h of mixing, and the enzyme activity of the supernatant was measured as described previously. According to the aforementioned formula, the immobilization rate of SpyTag-LDH on the blank acetaldehyde agarose carrier was about 0% after 15 h. At the same time, the blank acetaldehyde agarose carrier particles after the immobilization treatment had no detectable enzyme activity.

[0180] Comparative Example 4: Blank epoxy carrier + SpyTag-GFP

[0181] The blank epoxy carrier was prepared as in Example A, that is, 1 g of epoxy carrier was weighed, deionized water and 1.25 M phosphate buffer were added, and the pH of the system was adjusted to 8-9. The system was placed in a 25°C, 200 rpm shaker for 24 h to obtain the blank epoxy carrier.

[0182] The prepared blank epoxy carrier was mixed with the SpyTag-GFP prepared in Example B above in a centrifuge tube, with 5 mg of SpyTag-GFP added per gram of blank epoxy carrier, and the mixture was placed at 28° C. and pH=8.5.

[0183] The supernatant was sampled at time zero and 20 hours after mixing, and fluorescence was measured as described above. According to the aforementioned formula, the immobilization efficiency of SpyTag-GFP on the blank epoxy-based support was approximately 0% after 20 hours. Furthermore, fluorescence microscopy revealed no fluorescence on the immobilized blank epoxy-based support particles.

[0184] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included within the scope of the present invention. SEQUENCE LISTING <110> University of Science and Technology Beijing <120> Method for preparing immobilized enzyme or immobilized protein and immobilized enzyme or immobilized protein <130> I2019TC3529CB <160> 27 <210> 1 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> oSpyCatcher gene coding sequence <400> 1 gggagtggtg gcagcggagg cgccatggtt gataccttat caggtttatc aagtgagcaa 60 ggtcagtccg gtgatatgac aattgaagaa gatagtgcta cccatattaa attctcaaaa 120 cgtgatgagg acggcaaaga gttagctggt gcaactatgg agttgcgtga ttcatctggt 180 aaaactatta gtacatggat ttcagatgga caagtgaaag atttctacct gtatccagga 240 aaatatacat ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc 300 tttacagtta atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct 360 catatttaat ga 372 <210> 2 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Gene coding sequence for SpyTag <400> 2 atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60 ggt 63 <210> 3 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> Gene coding sequence for mSpyCatcher <400> 3 gggagtggtg gcagcggagg cgccatggtt gataccttat caggtttatc aagtgagcaa 60 ggtcagtccg gtgatatgac aattgaagaa gatagtgcta cccatattcg cttctcaaaa 120 cgtgatgagg acggccgaga gttagctggt gcaactatgg agttgcgtga ttcatctggt 180 aaaactatta gtacatggat ttcagatgga caagtgcgcg atttctacct gtatccagga 240 aaatatacat ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc 300 tttacagtta atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct 360 catatttaat gaaagggcaa aggcaaaggc 390 <210> 4 <211> 777 <212> DNA <213> Artificial Sequence <220> <223> SpyTag-GFP的基因编码序列 <400> 4 atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60 ggtatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat 120 ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac 180 ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca 240 cttgtcacta ctttcggtta tggtgttcaa tgctttgcga gatacccaga tcatatgaaa 300 cagcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt 360 ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt 420 gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac 480 aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat 540 ggaatcaaag ttaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca 600 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 660 tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc 720 cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaa 777 <210> 5 <211> 1149 <212> DNA <213> Artificial Sequence <220> <223> Gene coding sequence for SpyTag-GFP-SpyCatcher <400> 5 atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60 ggtatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat 120 ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac 180 ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca 240 cttgtcacta ctttcggtta tggtgttcaa tgctttgcga gatacccaga tcatatgaaa 300 cagcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt 360 ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt 420 gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac 480 aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat 540 ggaatcaaag ttaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca 600 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 660 tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc 720 cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaaggg 780 agtggtggca gcggaggcgc catggttgat accttatcag gtttatcaag tgagcaaggt 840 cagtccggtg atatgacaat tgaagaagat agtgctaccc atattaaatt ctcaaaacgt 900 GAGAAGAAGA AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA AGAAG 48 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 48 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 48 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 48 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 48 <210> 6 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> Gene coding sequence for aSpyCatcher <400> 6 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 48 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 48 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 48 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 48 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 48 tttacagtta atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct 360 catatttaat gaaagggcaa aggcaaaggc 390 <210> 7 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Gene coding sequence of cSpyCatcher <400> 7 gggagtggtg gcagcggagg cgccatggtt gataccttat caggtttatc aagtgagcaa 60 ggtcagtccg gtgatatgac aattgaagaa gatagtgcta cccatattcg cttctcaaaa 120 cgtgatgagg acggccgaga gttagctggt gcaactatgg agttgcgtga ttcatctggt 180 aaaactatta gtacatggat ttcagatgga caagtgcgcg atttctacct gtatccagga 240 aaatatacat ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc 300 tttacagtta atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct 360 catatttaat ga 372 <210> 8 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Gene coding sequence of LPA <400> 8 atgaatggtg aaacatcacg tccaccagct ctaggttttt catcagataa tattgctggt 60 gcttcaccag aagttgctca agctctagtt aaacattctt ctggtcaggc tggtccgtac 120 ggtactgacg aactgaccgc tcaggttaaa cgtaaattct gcgaaatctt cgaacgtgac 180 gttgaagttt tcctggttcc gaccggtact gctgctaacg ctctgtgcct gtctgctatg 240 accccgccgt ggggtaacat ctactgccac ccggcttctc acatcaacaa cgacgaatgc 300 ggtgctccgg aatttttctc taacggtgct aaactgatga ccgttgacgg tccggctgct 360 aaactggaca tcgttcgtct gcgtgaacgt acccgtgaaa aagttggtga cgttcacacc 420 acccagccgg cttgcgtttc tatcacccag gctaccgaag ttggttctat ctacaccctg 480 gacgaaatcg aagctatcgg tgacgtttgc aaatcttctt ctctgggtct gcacatggac 540 ggttctcgtt tcgctaacgc tctggtttct ctgggttgct ctccggctga aatgacctgg 600 aaagctggtg ttgacgctct gtctttcggt gctaccaaaa acggtgttct ggctgctgaa 660 gctatcgttc tgttcaacac ctctctggct accgaaatgt cttaccgtcg taaacgtgct 720 ggtcacctgt cttctaaaat gcgtttcctg tctgctcaga tcgacgctta cctgaccgac 780 gacctgtggc tgcgtaacgc tcgtaaagct aacgctgctg ctcagcgtct ggctcagggt 840 ctggaaggtc tgggtggtgt tgaagttctg ggtggtactg aagctaacat cctgttctgc 900 cgtctggact ctgctatgat cgacgctctg ctgaaagctg gtttcggttt ctaccacgac 960 cgttggggtc cgaacgttgt tcgtttcgtt acctctttcg ctaccaccgc tgaagacgtt 1020 gaccacctac taaatcaagt acgactagct gctgatcgta ctcaggaacg ataa 1074 <210> 9 <211> 1101 <212> DNA <213> Artificial Sequence <220> <223> Gene coding sequence of LDH <400> 9 atgaccctgg aaatcttcga atacctggaa aaatacgact acgaacaggt tgttttctgc 60 caggacaaag aatctggtct gaaagctatc atcgctatcc acgacaccac cctgggtccg 120 gctctgggtg gtacccgtat gtggacctac gactctgaag aagctgctat cgaagacgct 180 ctgcgtctgg ctaaaggtat gacctacaaa aacgctgctg ctggtctgaa cctgggtggt 240 gctaaaaccg ttatcatcgg tgacccgcgt aaagacaaat ctgaagctat gttccgtgct 300 ctgggtcgtt acatccaggg tctgaacggt cgttacatca ccgctgaaga cgttggtacc 360 accgttgacg acatggacat catccacgaa gaaaccgact tcgttaccgg tatctctccg 420 tctttcggtt cttctggtaa cccgtctccg gttaccgcgt atggtgtata ccggggtatg 480 aaagctgctg ctaaggaggc gttcggtacc gacaatctgg aaggtaaagt tatcgctgtt 540 cagggtgttg gtaacgttgc ttaccacctg tgcaaacacc tgcacgctga aggtgctaaa 600 ctgatcgtta ccgacatcaa caaagaagct gttcagcgtg ctgttgaaga attcggtgct 660 tctgctgttg aaccgaacga aatctacggt gttgaatgcg acatctacgc tccgtgcgct 720 ctgggtgcta ccgttaacga cgaaaccatc ccgcagctga aagctaaagt tatcgctggt 780 tctgctaaca accagctgaa agaagaccgt cacggtgaca tcatccacga aatgggtatc 840 gtttacgctc cggactacgt tatcaacgct ggtggtgtta tcaacgttgc tgacgaactg 900 tacggttaca accgtgaacg tgctctgaaa cgtgttgaat ctatctacga caccatcgct 960 aaagttatcg aaatctctaa acgtgacggt atcgctacct acgttgctgc tgaccgtctg 1020 gctgaagaac gtatcgcttc tctgaaaaac tctcgttcta cctacctgcg taacggtcat 1080 gatattattt ctcgtcgtta a 1101 <210> 10 <211> 1137 <212> DNA <213> Artificial Sequence <220> <223> Gene coding sequence of SpyTag-LPA <400> 10 atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60 ggtatgaatg gtgaaacatc acgtccacca gctctaggtt tttcatcaga taatattgct 120 ggtgcttcac cagaagttgc tcaagctcta gttaaacatt cttctggtca ggctggtccg 180 tacggtactg acgaactgac cgctcaggtt aaacgtaaat tctgcgaaat cttcgaacgt 240 gacgttgaag ttttcctggt tccgaccggt actgctgcta acgctctgtg cctgtctgct 300 atgaccccgc cgtggggtaa catctactgc cacccggctt ctcacatcaa caacgacgaa 360 tgcggtgctc cggaattttt ctctaacggt gctaaactga tgaccgttga cggtccggct 420 gctaaactgg acatcgttcg tctgcgtgaa cgtacccgtg aaaaagttgg tgacgttcac 480 accacccagc cggcttgcgt ttctatcacc caggctaccg aagttggttc tatctacacc 540 ctggacgaaa tcgaagctat cggtgacgtt tgcaaatctt cttctctggg tctgcacatg 600 gacggttctc gtttcgctaa cgctctggtt tctctgggtt gctctccggc tgaaatgacc 660 tggaaagctg gtgttgacgc tctgtctttc ggtgctacca aaaacggtgt tctggctgct 720 gaagctatcg ttctgttcaa cacctctctg gctaccgaaa tgtcttaccg tcgtaaacgt 780 gctggtcacc tgtcttctaa aatgcgtttc ctgtctgctc agatcgacgc ttacctgacc 840 gacgacctgt ggctgcgtaa cgctcgtaaa gctaacgctg ctgctcagcg tctggctcag 900 ggtctggaag gtctgggtgg tgttgaagtt ctgggtggta ctgaagctaa catcctgttc 960 tgccgtctgg actctgctat gatcgacgct ctgctgaaag ctggtttcgg tttctaccac 1020 gaccgttggg gtccgaacgt tgttcgtttc gttacctctt tcgctaccac cgctgaagac 1080 gttgaccacc tactaaatca agtacgacta gctgctgatc gtactcagga acgataa 1137 <210> 11 <211> 1164 <212> DNA <213> Artificial Sequence <220> <223> Gene coding sequence of SpyTag-LDH <400> 11 atgggagccc acatcgtgat ggtggacgcc tacaagccga cgaagggttc agggggttcc 60 ggtatgaccc tggaaatctt cgaatacctg gaaaaatacg actacgaaca ggttgttttc 120 tgccaggaca aagaatctgg tctgaaagct atcatcgcta tccacgacac caccctgggt 180 ccggctctgg gtggtacccg tatgtggacc tacgactctg aagaagctgc tatcgaagac 240 gctctgcgtc tggctaaagg tatgacctac aaaaacgctg ctgctggtct gaacctgggt 300 ggtgctaaaa ccgttatcat cggtgacccg cgtaaagaca aatctgaagc tatgttccgt 360 gctctgggtc gttacatcca gggtctgaac ggtcgttaca tcaccgctga agacgttggt 420 accaccgttg acgacatgga catcatccac gaagaaaccg acttcgttac cggtatctct 480 ccgtctttcg gttcttctgg taacccgtct ccggttaccg cgtatggtgt ataccggggt 540 atgaaagctg ctgctaagga ggcgttcggt accgacaatc tggaaggtaa agttatcgct 600 gttcagggtg ttggtaacgt tgcttaccac ctgtgcaaac acctgcacgc tgaaggtgct 660 aaactgatcg ttaccgacat caacaaagaa gctgttcagc gtgctgttga agaattcggt 720 gcttctgctg ttgaaccgaa cgaaatctac ggtgttgaat gcgacatcta cgctccgtgc 780 gctctgggtg ctaccgttaa cgacgaaacc atcccgcagc tgaaagctaa agttatcgct 840 ggttctgcta acaaccagct gaaagaagac cgtcacggtg acatcatcca cgaaatgggt 900 atcgtttacg ctccggacta cgttatcaac gctggtggtg ttatcaacgt tgctgacgaa 960 ctgtacggtt acaaccgtga acgtgctctg aaacgtgttg aatctatcta cgacaccatc 1020 gctaaagtta tcgaaatctc taaacgtgac ggtatcgcta cctacgttgc tgctgaccgt 1080 ctggctgaag aacgtatcgc ttctctgaaa aactctcgtt ctacctacct gcgtaacggt 1140 catgatatta tttctcgtcg ttaa 1164 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> SpyTag-GFP upper primer <400> 12 gaactataca aatgaaagct tgcggccgca ct 32 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> SpyTag-GFP downstream primer <400> 13 ttcatttgta tagttcatcc atgccatgtg t 31 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> oSpyCatcher upstream primer <400> 14 gcagccatgg gagtggtggc agcggagg 28 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> oSpyCatcher downstream primer <400> 15 ccactcccat ggctgccgcg cggcacca 28 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> aSpyCatcher upstream primer <400> 16 aagggcaaag gcaaaggcaa gcttgcggcc gcact 35 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> aSpyCatcher downstream primer <400> 17 gcctttgcct ttgccctttc attaaatatg agcgtc 36 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> cSpyCatcher (28 and 37) upstream primer <400> 18 caaaacgtga tgaggacggc cgagagttag ctggtgcaac 40 <210> 19 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> cSpyCatcher (28 and 37) downstream primer <400> 19 tcctcatcac gttttgagaa gcgaatatgg gtagcactat ctt 43 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> cSpyCatcher (64) upstream primer <400> 20 gacaagtgcg cgatttctac ctgtatccag 30 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> cSpyCatcher (64 bit) downstream primer <400> 21 gtagaaatcg cgcacttgtc catctgaaat 30 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> pET28a-SpyTag plasmid backbone upstream primer <400> 22 aaaaggtctc agcttctaac aaagcccgaa agg 33 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pET28a-SpyTag plasmid backbone downstream primer <400> 23 ttttggtctc taccggaacc ccctgaa 27 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LPA upstream primer <400> 24 ttttggtctc tcggtatgaa tggtgaaaca 30 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LPA downstream primer <400> 25 ttttggtctc tgcttttatc gttcctgagt 30 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LDH upper primer <400> 26 ttttggtctc tcggtatgac cctggaaatc 30 <210> 27 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> LDH lower primer <400> 27 ttttggtctc tgcttttaac gacgagaaat a 31

Claims

1. A method for preparing an immobilized enzyme or immobilized protein, the method comprising: - Covalently binding SpyCatcher to a carrier, wherein the gene coding sequence of SpyCatcher is shown in SEQ ID No. 3, or SEQ ID No. 6, or SEQ ID No. 7; the carrier is an epoxy carrier or an aldehyde agarose carrier; - Constructing a target enzyme or target protein with SpyTag fused to the N-terminus or C-terminus, wherein the gene coding sequence of the SpyTag is shown in SEQ ID No. 2; - The carrier covalently bound with SpyCatcher and the target enzyme or target protein fused with SpyTag are brought into contact with each other. 2 . The method according to claim 1 , wherein the target enzyme is L-phenylserine aldolase or leucine dehydrogenase, and the target protein is green fluorescent protein (GFP).

3. The method according to claim 2, wherein the gene coding sequence of the L-phenylserine aldolase is shown in SEQ ID No. 8, and the gene coding sequence of the leucine dehydrogenase is shown in SEQ ID No.

9.

4. An immobilized enzyme or immobilized protein comprising: - a carrier, wherein the carrier is an epoxy carrier or an aldehyde agarose carrier; - a SpyCatcher covalently bound to the carrier, wherein the gene coding sequence of the SpyCatcher is shown as SEQ ID No. 3, or as SEQ ID No. 6, or as SEQ ID No. 7; and - a target enzyme or target protein having SpyTag fused to the N-terminus or C-terminus; the gene coding sequence of the SpyTag is shown in SEQ ID No. 2; in, The target enzyme or target protein is immobilized on the carrier through covalent binding of the SpyTag and the SpyCatcher. 5 . The immobilized enzyme or immobilized protein according to claim 4 , wherein the target enzyme is L-phenylserine aldolase or leucine dehydrogenase, and the target protein is green fluorescent protein (GFP). 6 . The immobilized enzyme or immobilized protein according to claim 5 , wherein the gene coding sequence of the L-phenylserine aldolase is shown in SEQ ID No. 8, and the gene coding sequence of the leucine dehydrogenase is shown in SEQ ID No.

9.

7. A carrier covalently bound to SpyCatcher, wherein: The gene coding sequence of the SpyCatcher is shown as SEQ ID No. 3, or as SEQ ID No. 6, or as SEQ ID No. 7; the vector is an epoxy vector or an acetaldehyde agarose vector.

8. Use of a carrier covalently bonded with SpyCatcher for selectively immobilizing a target enzyme or target protein fused with SpyTag at the N-terminus or C-terminus from a protein mixture, wherein: The gene coding sequence of the SpyCatcher is shown in SEQ ID No.3, or SEQ ID No.6, or SEQ ID No.7; the vector is an epoxy vector or an acetaldehyde agarose vector; the gene coding sequence of the SpyTag is shown in SEQ ID No.

2. 9 . The method according to claim 8 , wherein the target enzyme is L-phenylserine aldolase or leucine dehydrogenase, and the target protein is green fluorescent protein (GFP).

10. The use according to claim 9, wherein the gene coding sequence of the L-phenylserine aldolase is shown as SEQ ID No. 8, and the gene coding sequence of the leucine dehydrogenase is shown as SEQ ID No. 9.

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