CN112679701B - Immobilized lysine endopeptidase and preparation method and application thereof - Google Patents
Immobilized lysine endopeptidase and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of bioengineering, in particular to immobilized lysine endopeptidase as well as a preparation method and application thereof, wherein the preparation method comprises the steps of firstly, reacting an epoxy group of an epoxy carrier with an amino group of D-glutamic acid-1-tert-butyl ester, modifying the D-glutamic acid-1-tert-butyl ester on the epoxy carrier, and then activating the D-glutamic acid-1-tert-butyl ester by using an amino acid carboxyl activating agent to obtain an activated modified epoxy carrier modified by the D-glutamic acid-1-tert-butyl ester; incubating the modified epoxy carrier with Lys-C to obtain immobilized Lys-C; insulin analogs were then prepared using immobilized Lys-C. The method of the invention innovatively uses the covalent bonding method to immobilize the Lys-C, has high immobilization efficiency, is easy to recover the immobilized Lys-C, can be repeatedly used, and retains the enzyme activity of more than 70 percent of the original activity after being repeatedly used for 10 times.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to immobilized lysine endopeptidase and a preparation method and application thereof.
Background
Lysyl endopeptidase (EC 3.4.21.50) is a serine protease, also known as Lys-C endonuclease, originally discovered and isolated from Agrobacterium by Masaki et al [ Biochim Biophys acta.1981; 660(1):44-50. Protein Enzymes: spring and Cysteine peptases, 126-137. lysyl endopeptidase prophase is reported to be mainly obtained by a method of wild strain Expression, but the Expression level is too low to meet the industrial requirements when Lys-C is expressed by wild strains, for example, the natural Expression level of Lys-C of lysobacter origin is lower than 60U/L fermentation broth, and the natural Expression level of Lys-C of achromobacter origin is only 32U/L fermentation broth [ FEMS Microbiology Letters,213(1), (13) -20. Journal of Biological Chemistry,273(27), (273), (16792-16797. Protein Expression and Purification,118, 31-38. ]; in order to overcome the defect of low expression level of wild strains, the recombinant lysyl endopeptidase is prepared by adopting a gene recombination method, but the yield of Lys-C is not obviously improved, so the cost is high. Lys-C endonuclease has a broad pH tolerance and a stronger surfactant tolerance, can specifically recognize and cleave lysine residues in peptide chains, has a higher activity and specificity compared to trypsin [ CN109486800B ], and is an important tool enzyme widely used in fields such as proteomics [ Rapid Communications in Mass Spectrometry,27(14), 1669-1672 ], and biopharmaceuticals, in particular in the preparation of Insulin [ CN102816785A, Biotechnology and Bioengineering,37(7), 693-695. ChemBiochem,9(18), 2989-2996 ], such as Insulin terrestris (Insulin Destemir), Insulin degluidec (Insulin degreec) and Insulin Aspart [ Insulin on Biological in (799), 808).
As the reaction of Lys-C is generally carried out in aqueous solution, substrates, products and enzyme are difficult to separate from the solution after the reaction is finished, and the repeated use of the Lys-C is not facilitated, but the problems can be solved by immobilization, US4634671 reports a method for immobilizing Lys-C enzyme based on a cross-linking method, but the immobilization method is difficult to control the reaction conditions well, the immobilization time is up to 72 hours, the immobilized enzyme yield is low (14.7-48%), and methods such as gel filtration, ultrafiltration and the like are adopted for recovering the immobilized enzyme, so that the operation is relatively complicated, and the industrial application is not facilitated.
The covalent binding method has been developed as one of the most active methods for the enzyme-carrier binding, with good stability and reusability [ Applied Science and conversion Technology,2017,26(6): 157-. Epoxy groups in the epoxy carrier can be covalently bonded with functional groups (amino, thiol and phenols) on enzyme molecules, so that the enzyme molecules are immobilized on the surface of the carrier, and although the covalent immobilization improves the stability of the enzyme, the enzyme activity in the affinity reaction is reduced, and the reproducibility is poor; in addition, during the immobilization process, the conformation of the enzyme may be altered, resulting in the enzyme becoming inactive.
Since the epoxy group can react with the N-terminal amino group of the protein and the side chain groups of lysine, cysteine, histidine and the like to cause multi-site immobilization between the enzyme and the carrier, so that the activity of the enzyme is influenced, and the activity of immobilized Lys-C obtained by immobilizing Lys-C by using the traditional epoxy immobilization method is extremely low, the epoxy carrier needs to be modified, a modified epoxy carrier is developed and Lys-C is immobilized, and a method for preparing insulin by using the immobilized Lys-C is urgently needed.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide an immobilized lysine endopeptidase, and a preparation method and use thereof, which are used to solve the problems of the prior art.
To achieve the above and other related objects, the present invention provides a method for preparing a modified epoxy carrier, comprising the steps of:
1) mixing D-glutamic acid-1-tert-butyl ester with an epoxy carrier to obtain an epoxy carrier modified by the D-glutamic acid-1-tert-butyl ester;
2) mixing the D-glutamic acid-1-tert-butyl ester modified epoxy carrier obtained in the step 1) with an amino acid carboxyl activating agent to obtain an activated D-glutamic acid-1-tert-butyl ester modified epoxy carrier, namely the modified epoxy carrier.
The invention also provides a modified epoxy carrier prepared by the method.
The invention also provides a preparation method of the immobilized Lys-C, which comprises the step of mixing the Lys-C with the modified epoxy carrier to obtain the immobilized Lys-C.
The invention also provides immobilized Lys-C, which is prepared by the method.
Use of said immobilized Lys-C in the preparation of an insulin analogue intermediate or an insulin analogue.
The invention also provides a preparation method of the insulin analogue intermediate, which comprises the steps of mixing insulin precursor protein with the immobilized Lys-C for reaction, and respectively recovering the immobilized Lys-C and enzyme digestion products after the reaction is finished, wherein the enzyme digestion products are the insulin analogue intermediate.
The present invention also provides a method for preparing an insulin analogue, the method comprising the steps of: the insulin analogue intermediate is subjected to a transpeptidation reaction.
As described above, the immobilized lysine endopeptidase, the preparation method and the use thereof of the present invention have the following beneficial effects:
(1) the method innovatively uses a covalent bonding method to immobilize Lys-C, and has high immobilization efficiency and high enzyme activity recovery rate.
(2) The immobilized Lys-C of the method is easy to recover and can be reused for 10 times, and the enzyme activity is kept to be more than 70 percent of the original activity.
(3) The method has simple operation and low cost, and is beneficial to industrial application.
Drawings
FIG. 1 shows a schematic diagram of modified and immobilized Lys-C of an epoxy carrier of the present invention, wherein A is a schematic diagram of L-Glu-OtBu modified epoxy carrier; b is a schematic diagram of activating L-Glu-OtBu; c is a schematic diagram of modified epoxy carrier immobilized Lys-C.
FIG. 2 shows the sequence diagram of human insulin lacking threonine B30 (D30 protein).
FIG. 3 shows the sequence diagram of insulin aspart (M30 protein) lacking threonine at position B30.
Figure 4 shows a schematic sequence of human insulin precursor.
FIG. 5 shows a schematic sequence of insulin aspart precursor.
FIG. 6 shows a proteolytic cleavage assay of human insulin precursor according to the invention.
FIG. 7 is a molecular weight measurement chart of B30-deficient threonine human insulin (D30) according to the present invention.
FIG. 8 is a diagram showing the cleavage reaction assay of insulin aspart precursor protein of the present invention.
FIG. 9 shows a diagram of the transpeptidation reaction assay for the M30 protein of the present invention.
FIG. 10 is a graph showing a molecular weight measurement of threonine aspartate according to the present invention.
FIG. 11 reaction scheme of deprotection group of dry powder of insulin aspart threonine ester.
Detailed Description
The invention provides a preparation method of a modified epoxy carrier, which comprises the following steps:
1) mixing D-glutamic acid-1-tert-butyl ester with an epoxy carrier to obtain an epoxy carrier modified by the D-glutamic acid-1-tert-butyl ester;
2) mixing the D-glutamic acid-1-tert-butyl ester modified epoxy carrier obtained in the step 1) with an amino acid carboxyl activating agent to obtain the activated D-glutamic acid-1-tert-butyl ester modified epoxy carrier.
The epoxy carrier is a carrier with epoxy functional groups on the surface. The mass molar ratio of the epoxy carrier to the D-glutamic acid-1-tert-butyl ester is 1 g: 0.05-0.1M. In one embodiment, the epoxy carrier is selected from epoxy immobilized enzyme carriers.
In one embodiment, step 1) is carried out in an organic solvent. The organic solvent is one or more of DMF, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, dioxane and N-methylpyrrolidone.
In one embodiment, the concentration of D-glutamic acid-1-tert-butyl ester in the step 1) is 0.05-0.1 mol/L.
In one embodiment, the reaction temperature in step 1) is 32 to 40 ℃.
In one embodiment, the reaction time of step 1) is 10 to 14 hours.
In one embodiment, the product of step 1) is also washed and blocked after the reaction of D-glutamic acid-1-tert-butyl ester (i.e., L-Glu-OtBu) with the epoxy carrier is completed.
In one embodiment, the washing solution is selected from organic solvents. The organic solvent is one or more of DMF, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, dioxane and N-methylpyrrolidone.
The purpose of the blocking is to finish the reaction of the epoxy group which does not react with the D-glutamic acid-1-tert-butyl ester, so as to avoid the epoxy group participating in the next reaction and influencing the reaction efficiency. In one embodiment, the blocking buffer is selected from glycine or ethanolamine. The mass-to-volume ratio of the product to the blocking buffer was 1 g: 3-12 mL.
In one embodiment, the epoxy carrier is LX-1000EP (New science and technology materials, Inc. of Xian blue), and the reaction scheme of step 1) is shown in FIG. 1A.
In one embodiment, the mass molar ratio of the D-glutamic acid-1-tert-butyl ester modified epoxy support to the amino acid carboxyl activating agent in step 2) is 1 g: 0.05-0.1M.
In one embodiment, the step 2) is carried out in an organic solvent, and the concentration of the amino acid carboxyl activator is 0.05-0.1 mol/L.
In one embodiment, the reaction conditions in step 2) are 22-28 ℃.
In one embodiment, the reaction time of step 2) is 6-10 h.
In one embodiment, the step 2) reaction is followed by washing with an organic solvent.
In one embodiment, the reaction scheme for step 2) is as shown in fig. 1B.
The amino acid carboxyl activating agent is selected from one or more of O- (N-succinimidyl) -NNNN-tetramethyltetrafluoroborate urea (TSTU), diisopropylcarbodiimide (DIPCDI), N-Dicycloethylcarbodiimide (DCC), N-hydroxy phenylpropanetriazole (HOBt), pentafluorophenol ester (PfP ester) and hydroxy diimidazole (CDI).
The modified epoxy carrier obtained by the reaction is an epoxy carrier modified by succinimidyl activated D-glutamic acid-1-tert-butyl ester (LX-1000 EP-D-Glu-OtBu-TSTU).
The invention also provides a modified epoxy carrier obtained by the method.
The invention also provides a preparation method of the immobilized Lys-C, which comprises the step of mixing the Lys-C with the modified epoxy carrier to obtain the immobilized Lys-C.
In one embodiment, the mass ratio of the modified epoxy carrier to Lys-C is 1 g: 0.001 to 0.1 g.
Specifically, the preparation method is carried out in a fixed buffer solution, and the concentration of Lys-C is 0.1-100 mg/mL. In one embodiment, the fixation buffer comprises 1, 4 butanediol, DMSO, and water.
In one embodiment, the reaction temperature is 20 to 30 ℃.
In one embodiment, the pH of the reaction system is 8.5 to 9.5.
In one embodiment, the reaction time is 20 to 40 min.
After the reaction is finished, the product is collected by filtration and washed by washing buffer, and finally immobilized Lys-C is obtained.
In one embodiment, the reaction scheme is as shown in figure 1C.
The preparation method uses a covalent bonding method to immobilize Lys-C, the immobilization efficiency is high, and the enzyme activity recovery rate can reach 70%.
The invention also provides immobilized Lys-C obtained by the method.
The immobilized Lys-C is easy to recover and can be reused for 10 times, and the enzyme activity is kept to be more than 70% of the original activity.
The invention also provides the use of said immobilized Lys-C in the preparation of insulin analogues.
The insulin analogue refers to a substance which can simulate the secretion of normal insulin and is similar to the insulin in structure.
The invention provides a preparation method of an insulin analogue intermediate, which comprises the steps of mixing insulin precursor protein with immobilized Lys-C for reaction, and respectively recovering the immobilized Lys-C and a restriction enzyme product after the reaction is finished, wherein the restriction enzyme product is the insulin analogue intermediate.
Current systems for the industrial expression of recombinant insulin include saccharomyces cerevisiae and pichia pastoris, because the complete human proinsulin cannot be expressed in the yeast system, and secretory expression of single-chain insulin precursors can be significantly improved by removing threonine at position B30 from the molecule encoding proinsulin cDNA and replacing the C peptide with a short C peptide. On this basis, by designing a lysine site for cleavage by lysyl endopeptidase at the C-terminus of the leader peptide and the C-terminus of the short C-peptide, proinsulin can be cleaved enzymatically by lysyl endopeptidase.
Different insulin analogue intermediates were prepared, using different insulin precursor proteins. For example, a human insulin precursor protein and an insulin aspart precursor protein which are respectively used for preparing a B30-deleted threonine human insulin analogue (D30 protein, the sequence of which is shown in figure 2) and B30-deleted threonine insulin aspart (M30 protein, the sequence of which is shown in figure 3) are prepared. The leader peptide and C peptide have different sequences and sequences according to different manufacturers, but they have a common part, and the sequences of human insulin precursor protein and insulin aspart precursor protein are schematically shown in FIGS. 4 and 5, respectively, and the leader peptide and C peptide have no uniform sequence and thus have no specific amino acids.
The insulin analogue intermediates include D30 protein and M30 protein. Different insulins can be obtained by continuing the reaction of the insulin analogue intermediate. Performing transpeptidation reaction on the D30 protein to obtain recombinant human insulin; insulin detemir is obtained by connecting a 14C fatty acid side chain to the lysine residue at the position 29 of the B chain of the D30 protein, and insulin degludec is obtained by connecting a 16C fatty acid side chain to the lysine residue at the position 29 of the B chain of the D30 protein through a glutamic acid linker; the insulin aspart can be obtained by performing transpeptidation reaction on the M30 protein.
In one embodiment, the mass ratio of insulin precursor protein to immobilized Lys-C is 50: 1 to 3.
In one embodiment, the reaction temperature is 25 to 37 ℃.
In one embodiment, the reaction pH is 8.5 to 10.5.
In one embodiment, the reaction time is 8 to 16 hours.
In one embodiment, the reaction is performed in a buffer. The buffer solution is Tris solution, sodium carbonate-sodium bicarbonate buffer solution (20mM), borax-sodium hydroxide buffer solution (50mM) and disodium hydrogen phosphate-sodium hydroxide buffer solution (50 mM).
In one embodiment, insulin precursor protein is first dissolved in a buffer, preheated to the reaction temperature, adjusted to the reaction pH, and then immobilized Lys-C is added. After the reaction is finished, the reaction product is subjected to conventional operations such as purification, precipitation, washing filtration, drying and the like.
The present invention also provides a method for preparing an insulin analogue, the method comprising the steps of: the insulin analogue intermediate is subjected to a transpeptidation reaction.
The transpeptidation reaction refers to a reaction which is an enzymatic synthesis, i.e., a reaction in which peptide bond synthesis is performed by a reverse reaction of protease.
In one embodiment, the method further comprises subjecting the product obtained from the transpeptidation reaction to a deprotection group reaction.
For example, transpeptidation reaction is carried out on M30 protein to obtain insulin aspart threonine ester, deprotection group reaction is further carried out on the insulin aspart threonine ester, and the insulin aspart can be obtained after the reaction is finished.
Specifically, the preparation method of insulin aspart comprises the following steps:
1) mixing the insulin analogue intermediate M30 protein with threonine ester and enzyme to obtain insulin aspart threonine ester;
2) carrying out deprotection group reaction on the threonine ester of the insulin aspart to obtain the insulin aspart.
In one embodiment, the mass ratio of M30 protein to threonine ester in step 1) is 1: 7 to 13.
In one embodiment, the reaction temperature in step 1) is 25 to 37 ℃.
In one embodiment, the reaction pH in step 1) is 8.0-10.0.
In one embodiment, the reaction time in step 1) is 16-24 hours.
In one embodiment, the reaction of step 1) is performed in a mixed solution of a buffer and an organic solvent.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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 invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
EXAMPLE 1 preparation of modified epoxy Carrier
Modification: weighing 1g of epoxy carrier LX-1000EP (New science and technology materials Co., Ltd., Xian blue), adding 0.1M of D-glutamic acid-1-tert-butyl ester solution dissolved in DMF, stirring and reacting at 37 ℃ for 12h, after the reaction is finished, centrifugally collecting the epoxy carrier, washing the epoxy carrier with DMF, then adding 12 times of sealing buffer solution (3M glycine, pH 8.5) by volume, stirring and reacting at 37 ℃ for 12h, and after the sealing is finished, centrifugally removing the sealing solution to obtain the D-glutamic acid-1-tert-butyl ester modified epoxy carrier;
activation: adding 0.1M O- (N-succinimidyl) -NNNN-tetramethyltetrafluoroborate urea solution dissolved in tetrahydrofuran into the D-glutamic acid-1-tert-butyl ester modified epoxy carrier obtained in the step I, stirring and reacting for 8h at 25 ℃, after the reaction is finished, centrifugally collecting the carrier, and washing with tetrahydrofuran to obtain a succinimidyl activated D-glutamic acid-1-tert-butyl ester modified epoxy carrier (LX-1000 EP-D-Glu-OtBu-TSTU);
example 2 immobilized lysine endopeptidase (Lys-C)
50mg of the modified epoxy vector (LX-1000EP-D-Glu-OtBu-TSTU) was weighed, 0.8U of Lys-C was added to a fixation buffer (1, 4-butanediol: DMSO: water: 1: 2), and the reaction was stirred at 25 ℃ for 30 min; at the same time, 50mg of unmodified epoxy vector (LX-1000EP) was weighed, 0.8U Lys-C was added to 1M phosphate buffer (pH 7.0), and the reaction was stirred at 20 ℃ for 24h [ Biomacromolecules 2003,4,772-777], as a control.
After completion of the reaction, the supernatant was removed by centrifugation, and the carrier was washed 5 times with 5-fold volume of PBS buffer (0.1M, pH 7.5) to obtain immobilized Lys-C. And detecting the enzyme activity of Lys-C supernatant before and after immobilization, and calculating the immobilization amount and immobilization efficiency of Lys-C. The correlation calculation formula is as follows:
Lys-C fixed quantity (U) is total enzyme activity before fixation (U) -total enzyme activity after fixation (U) (1)
Example 3 Lys-C Activity assay
Lys-C enzyme activity determination principle: Lys-C catalyzes the hydrolysis of na-benzoyl-DL-lysine-4-nitrophenylamine hydrobromide to p-nitroaniline with a maximum absorbance at 405 nm.
Detection of free Lys-C enzyme activity: 2.6mL of 0.2mol/L AMP (2-amino-2-methyl-1, 3-propanediol) buffer (pH9.5) was mixed with 0.3mL of 2.5mmol/L substrate (N.alpha. -benzoyl-DL-lysine-4-nitrophenylamine hydrobromide) solution, and after incubation at 30 ℃ for 5 minutes, 0.1mL of enzyme solution (in 2mmol/L Tris-HCl buffer, pH 8.0) was added, mixed immediately, incubated at 30 ℃ for 25 minutes, followed by 1mL of stop solution (a mixed solution of 55mL of water and 45mL of acetic acid) and the absorbance at 405nm was measured. Enzyme solution was not added to the blank control. One Lys-C activity unit (U) is defined as the amount of enzyme required to produce 1. mu. mol of p-nitroanilide per minute at 30 ℃ pH 9.5.
Wherein, Delta A/min represents the variation value of absorbance per minute, namely the slope; molar extinction coefficient of S-p-nitroaniline (9.62 cm)2/. mu.mol); d-optical path (cell optical path is about 1 cm); vt-represents the total volume of the reaction solution (4 mL); vs-represents the volume of the sample enzyme solution (0.1 mL); x-represents the dilution factor of the enzyme solution of the sample.
Detection of immobilized Lys-C enzyme activity: 2.6mL of 0.2mol/L AMP (2-amino-2-methyl-1, 3-propanediol) buffer (pH9.5) was mixed with 0.3mL of 2.5mmol/L substrate (N.alpha. -benzoyl-DL-lysine-4-nitrophenylamine hydrobromide) solution, and after incubation at 30 ℃ for 5 minutes, 5mg of immobilized Lys-C was added, immediately mixed, incubated at 30 ℃ for 25 minutes, followed by addition of 1mL of stop buffer (a mixed solution of 55mL of water and 45mL of acetic acid), and the supernatant was taken to measure its absorbance at 405 nm. Wherein the blank control group was not added with immobilized Lys-C.
Wherein, Delta A/min represents the variation value of absorbance per minute, namely the slope; molar extinction coefficient of S-p-nitroaniline (9.62 cm)2/. mu.mol); d-optical path (cell optical path is about 1 cm); vt-represents the total volume of the reaction solution (4 mL); m-represents the weighed amount of the immobilized enzyme (0.005 g).
The immobilization results are shown in Table 1, and the immobilization efficiency and the enzyme activity recovery rate of the modified epoxy carrier for Lys-C are more than 80%, and are respectively 97% and 85%; and the traditional epoxy immobilization method has the immobilization efficiency of 82% for Lys-C, and the recovery rate of enzyme activity is only 31%.
TABLE 1 results of Lys-C immobilization by epoxy Carrier
EXAMPLE 4 immobilization of Lys-C under different conditions
Fixation of Lys-C at different temperatures: 50mg of the modified epoxy carrier (LX-1000EP-D-Glu-OtBu-TSTU) was weighed, 0.8U Lys-C was added thereto, and the mixture was stirred at 20 ℃ and 25 ℃ and 30 ℃ for 30 minutes to react, and the immobilization results are shown in Table 2.
Fixation of Lys-C at different pH: 50mg of each modified epoxy carrier (LX-1000EP-D-Glu-OtBu-TSTU) was weighed, 0.8U Lys-C was added, the pH of each of the immobilization buffers (1, 4-butanediol: DMSO: water ═ 1: 1: 2) was adjusted to 8.0, 8.5, 9.0, 9.5, and 10.0, and the reaction was stirred at 25 ℃ for 30 minutes, and the immobilization results are shown in Table 3.
Immobilization of epoxy vehicle to Lys-C in different ratios: 50mg of the modified epoxy carrier (LX-1000EP-D-Glu-OtBu-TSTU) was weighed out, and 0.2U, 0.4U, 0.8U and 1.6U of Lys-C were added thereto, followed by reaction with stirring at 25 ℃ for 30 min. The immobilization results are shown in Table 4.
After completion of the reaction, the supernatant was removed by centrifugation, and the carrier was washed 5 times with 5-fold volume of PBS buffer (0.1M, pH 7.5) to obtain immobilized Lys-C. And detecting the enzyme activity of Lys-C supernatant before and after immobilization, and calculating the immobilization amount, immobilization efficiency and enzyme activity recovery rate of Lys-C. The correlation calculation methods are shown in equations (1), (2) and (5), respectively.
TABLE 2 results of immobilization of Lys-C at different temperatures for modified epoxy carriers
The results of the immobilization of Lys-C by the modified epoxy carrier at different temperatures are shown in Table 2, and from 20 ℃ to 30 ℃, the immobilization efficiency and the enzyme activity recovery rate of the modified epoxy carrier for Lys-C are increased, respectively increased from 74% to 95% and 65% to 86%, wherein the efficiency is not obviously increased from 25 ℃ to 30 ℃.
TABLE 3 results of immobilization of Lys-C at different pH with modified epoxy vehicle
The results of the immobilization of the epoxy carrier on Lys-C under different pH values are shown in Table 3, from pH 8.0-10.0, the immobilization efficiency of the modified epoxy carrier on Lys-C is increased to 97% from 72%, but the enzyme activity recovery rate difference is large, and from pH 8.5-9.5, the enzyme activity recovery rate is between 81-89%; when the pH value is 8.0 and 10.0, the recovery rate of the enzyme activity is only 55 percent and 63 percent respectively.
TABLE 4 immobilization results of Lys-C of different masses
As shown in Table 4, the results of immobilization of Lys-C with different masses showed that the immobilization efficiency and the recovery rate of enzyme activity were gradually decreased with the increase of the amount of Lys-C introduced.
Example 5 immobilized Lys-C repeat use
Mixing 2.6mL of 0.2mol/L AMP (2-amino-2-methyl-1, 3-propanediol) buffer solution (pH9.5) with 0.3mL of 2.5mmol/L substrate (N alpha-benzoyl-DL-lysine-4-nitrophenylamine hydrobromide) solution, incubating at 30 ℃ for 5 minutes, adding 5mg of immobilized Lys-C, immediately mixing uniformly, incubating at 30 ℃ for 25 minutes, adding 1mL of stop solution (mixed solution of 55mL of water and 45mL of acetic acid), filtering and recovering the immobilized Lys-C, taking supernatant to measure the absorbance at 405nm, and calculating the enzyme activity of the immobilized Lys-C as original enzyme activity data; adding the recovered immobilized Lys-C into an AMP (2.6mL of 0.2mol/L) and substrate (0.3mL of 2.5mmol/L) mixed solution preheated for 5 minutes at 30 ℃, immediately mixing uniformly, incubating for 25 minutes at 30 ℃, then adding 1mL of stop solution (55mL of water and 45mL of acetic acid mixed solution), filtering and recovering the immobilized Lys-C, simultaneously taking the supernatant, measuring the absorbance of the supernatant at 405nm, and calculating the enzyme activity of the immobilized Lys-C to be used as the enzyme activity data of a second reaction; by analogy, the recovered immobilized Lys-C was reacted 10 times to calculate the enzyme activity of immobilized Lys-C for each time (blank control without immobilized Lys-C). The results are shown in Table 5.
TABLE 5 immobilized Lys-C repeat use
As shown in Table 5, the activity of the immobilized Lys-C remained more than 70% of the original activity after repeating the reaction 10 times.
Example 6 application of immobilized Lys-C to preparation of human insulin analog lacking B30 threonine
The current systems for the industrial expression of recombinant human insulin include saccharomyces cerevisiae and pichia pastoris, because the complete human proinsulin cannot be expressed in the yeast system, and the secretory expression of single-chain insulin precursors can be significantly improved by removing threonine at position B30 in the molecule encoding proinsulin cDNA and replacing C peptide with short C peptide. On this basis, a lysine site for cleavage by lysyl endopeptidase was designed at the C-terminus of the leader peptide and the C-terminus of the short C-peptide, and thus proinsulin was digested with lysyl endopeptidase to obtain B30 threonine-deleted human insulin (hereinafter referred to as D30 protein). The D30 protein is transpeptidated to obtain recombinant human insulin. The D30 protein has a 14C fatty acid side chain connected to the lysine residue at position 29 of the B chain to obtain insulin detemir. Deglutated insulin can be obtained by attaching a 16C fatty acid side chain to the lysine residue at position 29 of the B chain of D30 protein via a glutamic acid linker. In this example, the enzyme digestion reaction of insulin precursor protein was performed using immobilized lysyl endopeptidase to prepare D30 protein.
Human insulin precursor protein was prepared according to the reference (liuhai peak. technical study of conversion of recombinant insulin precursor to human insulin and insulin detemir [ D ]. university of east china, 2014). Dissolving 2000mg of human insulin precursor protein freeze-dried powder in 100mL of 50mM Tris solution, preheating to 30 ℃, adjusting the pH to 9.0 by using a sodium hydroxide solution, adding 0.12g of immobilized Lys-C, stirring at 30 ℃ for reacting for 8 hours, filtering the reaction solution, and recovering the immobilized Lys-C.
The cleavage efficiency was determined by means of a C18 column (diameter 4.6mm, length 200mm, particle size 5 μm). Mobile phase A: 0.05% trifluoroacetic acid-water, mobile phase B: 0.05% trifluoroacetic acid-acetonitrile; elution gradient: 20% B-35B, 30min, flow rate 1 mL/min. The reaction solution (20. mu.L) was poured into a chromatograph, and the column temperature was 30 ℃ and the wavelength was 214nm, and a chromatogram was recorded, as shown in FIG. 6. The enzyme digestion efficiency is calculated to be 83.19% (by adopting an area normalization method, the enzyme digestion efficiency is calculated according to the proportion of the peak area of the B30 threonine human insulin lacking in the enzyme digestion product to the total area). Compared with free Lys-C enzyme digestion, the enzyme digestion efficiency has no obvious difference.
And (3) carrying out reverse-phase chromatography purification on the enzyme digestion reaction liquid, collecting D30 fraction, precipitating, washing, filtering and drying to obtain a product with the molecular weight consistent with the theoretical molecular weight (5706.48Da) of D30.
Example 7 application of immobilized Lys-C to preparation of insulin aspart
The preparation process of the insulin aspart comprises the enzyme digestion of an insulin aspart precursor, namely, the specific site is cut by tool enzyme to obtain the insulin aspart (hereinafter referred to as M30 protein) lacking B30 threonine; performing transpeptidation reaction on M30 protein by using tool enzyme to obtain insulin aspart threonine ester, and performing deprotection reaction on the insulin aspart to obtain insulin aspart; this example used immobilized lysyl endopeptidase to perform the cleavage reaction of insulin aspart precursor and transpeptidation reaction of M30 protein.
Insulin aspart precursors were prepared according to the reference (preparation and purification of li-epi-short C-peptide insulin aspart [ D ] university of chongqing, 2017.). Dissolving 2000mg of insulin aspart precursor freeze-dried powder in 100mL of 100mM Tris solution, preheating to 37 ℃, adjusting the pH to 9.5 by using 1M sodium hydroxide solution, adding 0.12g of immobilized Lys-C, stirring at 37 ℃ for reacting for 16h, filtering the reaction solution, and recovering the immobilized Lys-C.
The cleavage efficiency was determined by means of a C18 column (diameter 4.6mm, length 200mm, particle size 5 μm). Mobile phase A: 0.05% trifluoroacetic acid-water, mobile phase B: 0.05% trifluoroacetic acid-acetonitrile; elution gradient: 20% B-35B, 30min, flow rate 1 mL/min. The reaction solution (20. mu.L) was poured into a chromatograph, and the column temperature was 30 ℃ and the wavelength was 214nm, and a chromatogram was recorded, as shown in FIG. 8. The enzyme digestion efficiency is calculated to be 85.45% (by adopting an area normalization method, the enzyme digestion efficiency is calculated according to the proportion of the peak area of the enzyme digestion product M30 to the total area). Compared with free Lys-C enzyme digestion, the enzyme digestion efficiency has no obvious difference.
Precipitating the enzyme digestion reaction solution, dissolving wet precipitated powder in 20mL of 100mM boric acid solution, adding 20mL of DMF and 40mL of ethanol, adding tert-butyl ether threonine tert-butyl ester (threonine ester), adjusting the pH value to 8.0 by using sodium hydroxide or boric acid, adding 1.2g of immobilized Lys-C, stirring and reacting at 30 ℃ for 16h, filtering the reaction solution, and recovering the immobilized Lys-C.
The transpeptidation efficiency was determined using a C4 column (diameter 4.6mm, length 200mm, particle size 5 μm). Mobile phase A: 0.1% trifluoroacetic acid-water, mobile phase B: 0.1% trifluoroacetic acid-acetonitrile; elution gradient: 25% B-40% B, 30min, flow rate 1 mL/min. The reaction solution (20. mu.L) was poured into a chromatograph, and the column temperature was 30 ℃ and the wavelength was 214nm, and a chromatogram was recorded, as shown in FIG. 9. The transpeptidation efficiency is calculated to be 75.14% (by adopting an area normalization method, the enzyme digestion efficiency is calculated according to the proportion of the peak area of the transpeptidation product threonine aspartate ester to the total area).
And (3) carrying out reverse chromatography on the transpeptidation reaction solution, collecting the threonine ester fraction of the insulin aspart, precipitating, washing, filtering and drying to obtain the threonine ester of the insulin aspart. The mass spectrometry results are shown in FIG. 10, and the molecular weight of the product obtained by the immobilized Lys-C enzyme digestion and transpeptidation is consistent with the theoretical molecular weight.
And (3) carrying out deprotection group reaction on the harvested insulin aspart threonine ester dry powder, wherein the mass-to-volume ratio of the insulin aspart threonine ester dry powder to TFA is 5 g: 1ml, reaction scheme as shown in FIG. 11.
After the reaction, the insulin aspart fraction can be obtained through reverse chromatography, and the insulin aspart can be obtained through precipitation, washing, filtering and drying.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (10)
1. A method of preparing a modified epoxy vehicle, comprising the steps of:
1) mixing D-glutamic acid-1-tert-butyl ester with an epoxy carrier to obtain the D-glutamic acid-1-tert-butyl ester modified epoxy carrier, wherein the mass molar ratio of the epoxy carrier to the D-glutamic acid-1-tert-butyl ester is 1 g: 0.05-0.1M, and the reaction scheme in the step 1) is as follows:
2) mixing the D-glutamic acid-1-tert-butyl ester modified epoxy carrier obtained in the step 1) with an amino acid carboxyl activating agent to obtain an activated D-glutamic acid-1-tert-butyl ester modified epoxy carrier, namely the modified epoxy carrier, wherein the mass molar ratio of the D-glutamic acid-1-tert-butyl ester modified epoxy carrier to the amino acid carboxyl activating agent in the step 2) is 1 g: 0.05-0.1M; the amino acid carboxyl activating agent is selected from one or more of TSTU, DIPCDI, DCC, HOBt, PfP lipid and CDI.
2. The method of claim 1, wherein step 1) further comprises one or more of the following features:
a) step 1) carrying out a reaction in an organic solvent;
b) the concentration of the D-glutamic acid-1-tert-butyl ester is 0.05-0.1 mol/L;
c) the reaction temperature is 32-40 ℃;
d) the reaction time is 10-14 h;
e) and washing and/or sealing the product after the reaction of the D-glutamic acid-1-tert-butyl ester and the epoxy carrier is finished.
3. The method of claim 1, wherein step 2) further comprises one or more of the following features:
a) step 2) carrying out reaction in an organic solvent;
b) the concentration of the amino acid carboxyl activating agent is 0.05-0.1 mol/L;
c) the reaction temperature is 22-28 ℃;
d) the reaction time is 6-10 h;
e) washing with an organic solvent after the reaction is finished;
f) the modified epoxy carrier obtained in the step 2) is an epoxy carrier modified by succinimidyl activated D-glutamic acid-1-tert-butyl ester, and the reaction schematic diagram is as follows:
4. a modified epoxy carrier, wherein the modified epoxy carrier is obtained by the method of any one of claims 1 to 3.
5. A process for the preparation of immobilized Lys-C, comprising admixing Lys-C with the modified epoxy carrier of claim 4 to provide immobilized Lys-C.
6. The method of claim 5, further comprising one or more of the following features:
a) the mass ratio of the modified epoxy carrier to Lys-C is 1 g: 0.001-0.1 g;
b) carrying out reaction in a fixed buffer solution;
c) the concentration of Lys-C is 0.1-100 mg/mL;
d) the reaction temperature is 20-30 ℃;
e) the pH value of the reaction system is 8.5-9.5;
f) the reaction time is 20-40 min;
g) after the reaction is finished, filtering and collecting a product, and washing the product by using a washing buffer solution to finally obtain immobilized Lys-C;
h) the reaction scheme is as follows:
7. an immobilized Lys-C, wherein said immobilized Lys-C is obtained by the method of any one of claims 5 to 6.
8. Use of immobilized Lys-C according to claim 7 for the preparation of an insulin analogue intermediate selected from B30 threonine-deleted human insulin and B30 threonine-deleted insulin aspart.
9. A preparation method of an insulin analogue intermediate is characterized in that the preparation method comprises the steps of mixing and reacting an insulin precursor protein with the immobilized Lys-C of claim 7, and after the reaction is finished, respectively recovering the immobilized Lys-C and enzyme digestion products, wherein the enzyme digestion products are the insulin analogue intermediate, the insulin analogue intermediate is selected from B30 threonine-deleted human insulin and B30 threonine-deleted insulin aspart, and the insulin precursor protein comprises a human insulin precursor protein and an insulin aspart precursor protein.
10. The method of claim 9, further comprising one or more of the following features:
a) the mass ratio of the insulin precursor protein to the immobilized Lys-C is 50: 1-3;
b) the reaction temperature is 25-37 ℃;
c) the reaction pH is 8.5-10.5;
d) the reaction time is 8-16 h.
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| WO1987006007A1 (en) * | 1986-03-28 | 1987-10-09 | Toray Industries, Inc. | Immobilized physiologically active material |
| EP0562373A3 (en) * | 1992-03-23 | 1994-05-25 | Siemens Ag | Immobilisation of biochemical substances |
| DE4342770A1 (en) * | 1993-12-15 | 1995-07-06 | Boehringer Mannheim Gmbh | Carrier-fixed enzymes |
| WO2006114130A1 (en) * | 2005-04-26 | 2006-11-02 | Agilent Technologies, Inc. | Enzymes with modified amino acids |
| RU2571857C2 (en) * | 2008-03-18 | 2015-12-20 | Ново Нордиск А/С | Acylated insulin analogues stabilised with respect to proteases |
| GB201002824D0 (en) * | 2010-02-19 | 2010-04-07 | Temasek Polytechnic | A method of preparing a substrate for immobilization of functional substances thereon and the substrate obtained therefrom |
| CN102382809A (en) * | 2011-10-28 | 2012-03-21 | 南京工业大学 | Flexible immobilized enzyme of comb epoxy polymer carrier |
| CN102559648B (en) * | 2012-01-13 | 2014-04-02 | 南京工业大学 | Immobilized enzyme using modified epoxy resin as carrier and preparation method and application thereof |
| CN102703416B (en) * | 2012-06-26 | 2014-10-01 | 沧州宝恩吸附材料科技有限公司 | Preparation method of immobilized enzyme carrier |
| CN103667241B (en) * | 2012-09-18 | 2015-08-26 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of hair-like hydrophilic polymer hydridization magnetic-particle immobilization proteinase and preparation method thereof |
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| CN103710333A (en) * | 2013-12-21 | 2014-04-09 | 华中科技大学 | Carrier for immobilization as well as preparation method thereof and immobilized beta-glucosaccharase |
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| CN107779445B (en) * | 2016-08-25 | 2021-02-19 | 上海凯赛生物技术股份有限公司 | Immobilized lysine decarboxylase, preparation thereof, preparation method of 1, 5-pentanediamine and product |
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| CN110669753A (en) * | 2019-11-05 | 2020-01-10 | 杭州师范大学 | Multi-point immobilization method based on unnatural amino acid modified enzyme |
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