CN110982808A

CN110982808A - Kex2 enzyme variants and methods for stable expression

Info

Publication number: CN110982808A
Application number: CN201911373353.5A
Authority: CN
Inventors: 辛中帅; 陈慧梅; 文良柱; 王玉刚; 朱亮
Original assignee: Wanxin Pharmaceutical Technology Suzhou Co Ltd
Current assignee: Wanxin Pharmaceutical Technology Suzhou Co Ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-04-10

Abstract

The invention discloses a Kex2 enzyme variant and a stable expression method, wherein the Kex2 enzyme variant is obtained by suitably truncating and point mutating a Kex2 amino acid sequence, can stably and efficiently secrete and express a target protein through a constitutive pichia pastoris expression system, and keeps good biological enzyme activity. The fermentation process is simple and efficient, low in cost and high in product activity, and can be suitable for commercial production.

Description

Kex2 enzyme variants and methods for stable expression

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a Kex2 enzyme variant and a stable expression method.

Technical Field

The Kex2 enzyme, a dibasic endopeptidase, is derived from Saccharomyces cerevisiae cerevisia, belongs to the subtilisin family, and is a calcium ion-dependent serine proteolytic enzyme. Kex2 specifically recognizes the double basic amino acid residue pair Lys-Arg or Arg-Arg in a protein or polypeptide, and acts to cleave the peptide bond from the carboxyl terminal (i.e., Arg-) of the second amino acid residue.

In yeast, the kex2 molecule is translated on the ribosome, via cleavage of the signal and precursor peptides, and through complex O-and N-linked glycosylation processes, into a viable protease molecule. Kex2, a precursor processing enzyme of eukaryotes, is capable of processing hormone precursors that mature many mammals in both in vivo and in vitro environments, and is important not only for the study of the entire family of precursor processing enzymes of higher organisms, but also in the field of recombinant protein production.

In previous researches, it is found that most of kex2 uses a methanol-induced yeast expression system, the protein expression induction process is complicated to operate, and the expression level is relatively low, which is not favorable for industrial production.

Disclosure of Invention

The invention aims to provide a Kex2 enzyme variant and a stable expression method, wherein the enzyme variant has good enzyme activity and can be suitable for a low-salt inorganic salt culture medium without yeast powder or peptone, the stable and efficient secretory expression, high-density fermentation preparation and purification of the Kex2 enzyme can be realized by the expression of the Kex2 enzyme variant by the method, and a final product with high yield and purity can be obtained.

In a first aspect of the invention, there is provided a method for stable and efficient secretory expression of a kex2 enzyme, the method comprising:

(1) providing a variant of the kex2 enzyme, with or without a His tag at the C-terminus of the variant; the variant is a 1-613 bit truncation, a 1-667 bit truncation, a 1-673 bit truncation, a 20-613 bit truncation, a 20-660 bit truncation, a 20-667 bit truncation or a 20-673 bit truncation according to the number of amino acid sequence bits of the full-length Kex2 shown in SEQ ID NO. 1;

(2) cloning a DNA sequence containing the variant of (1) onto a yeast expression vector to obtain a recombinant expression plasmid;

(3) transforming the recombinant expression plasmid containing (2) into yeast to obtain recombinant yeast;

(4) culturing the recombinant yeast of (3), fermenting and directly collecting fermentation supernatant;

(5) and (4) directly diluting the fermentation supernatant in the step (4) without special pretreatment, and purifying by ion exchange chromatography to obtain a variant of the kex2 enzyme.

In a preferred embodiment of the invention, the variant described in step (1) of the invention: based on the amino acid sequence number of the full-length Kex2 shown in SEQ ID NO.1, wherein the 225 th, 436 th and 503 th positions are mutated from K to A or P.

In a specific embodiment, the variant in step (1) of the present invention may be:

the 1 st to 613 th truncations of Kex2, and the number of amino acid sequence positions of the full-length Kex2 enzyme shown in SEQ ID NO.1 is counted, wherein the 225 th, 436 th and 503 th positions are mutated from K to A or P, and a His tag is added or not added at the C end of the truncations; or

A 1-667 th truncation of Kex2, and is calculated by the amino acid sequence number of the full-length Kex2 enzyme shown in SEQ ID NO.1, wherein the 225 th, 436 th and 503 th positions are mutated from K to A or P, and a His tag is added or not added at the C end of the truncation; or

The 1-673 th truncation of Kex2, and is counted by the amino acid sequence number of the full-length Kex2 enzyme shown in SEQ ID NO.1, wherein the 225 th, 436 th and 503 th positions are mutated from K to A or P, and meanwhile, a His tag is added or not added at the C end of the truncation; or

The 20 th to 613 th truncations of Kex2, and the number of the amino acid sequence bits of the full-length Kex2 enzyme shown in SEQ ID NO.1 is counted, wherein the 225 th, 436 th and 503 th bits are mutated from K to A or P, and meanwhile, a His tag is added or not added at the C end of the truncations; or

The 20 th-660 th truncation of Kex2, and the number of the amino acid sequence positions of the full-length Kex2 enzyme shown in SEQ ID NO.1 is counted, wherein the 225 th position, the 436 th position and the 503 th position are mutated from K to A or P, and meanwhile, a His tag is added or not added at the C end of the truncation; or

A 20-667 th truncation of Kex2, and is counted by the amino acid sequence number of the full-length Kex2 enzyme shown in SEQ ID NO.1, wherein the 225 th, 436 th and 503 th positions are mutated from K to A or P, and a His tag is added or not added at the C end of the truncation; or

The 20 th-673 rd truncated body of Kex2 is counted by the amino acid sequence number of the full-length Kex2 enzyme shown in SEQ ID NO.1, wherein the 225 th, 436 th and 503 th positions are mutated from K to A or P, and meanwhile, a His tag is added or not added at the C end of the truncated body.

The recombinant expression plasmid in the step (2) is suitable for pichia pastoris, and contains a DNA sequence of a kex2 variant.

The yeast used in step (2) of the present invention may be a yeast commonly used in the art, and is preferably pichia pastoris. The expression product is constitutively expressed.

The recombinant expression plasmid is transformed into yeast, and the recombinant yeast transformed with the recombinant expression plasmid is cultured, so that the kex2 enzyme can be expressed. The recombinant expression plasmid is linearized and then transformed into pichia pastoris. The transformation method for transforming the pichia pastoris can be used for preparing pichia pastoris competent cells by electrotransformation or a physicochemical method. Preferably, electrotransformation is selected.

The variant protein of the kex2 enzyme expressed by the method is soluble and secreted, so that the fermentation supernatant can be directly harvested to obtain the high-expression target protein.

The culture medium used for fermentation in the step (4) is a low-salt inorganic salt culture medium without yeast powder or peptone, and glycerol or glucose is used as a unique carbon source; the inventor finds that the variant of the invention can be applied to a low-salt inorganic salt culture medium without yeast powder or peptone, the expression of target protein can not be influenced by using the culture medium, and meanwhile, the efficiency of downstream protein purification can be effectively improved.

In a preferred embodiment, the fermentation in step (4) of the present invention uses a low-salt inorganic salt medium without yeast powder or peptone, which comprises: 4g to 4.5 g/L of magnesium sulfate heptahydrate, 5g to 6g/L of potassium sulfate, 0.3 g to 0.5g/L of calcium sulfate dihydrate, 30 g to 40g/L of glycerol, 10g to 11g/L of 85 percent phosphoric acid and 1g to 1.2g/L of 85 percent potassium hydroxide. The inventors found that the culture medium can improve protein expression, further effectively improve the efficiency of downstream protein purification, and the target protein is not easy to degrade.

The fermentation supernatant obtained in the step (4) of the invention does not need to carry out pretreatment steps such as molecular sieve desalination or large-volume dilution, and the like, and can be directly subjected to anion exchange chromatography only by diluting 2-3 times with pure water and controlling the conductivity to be not more than 2.5mS/cm, so as to obtain a purified product with the electrophoretic purity of more than 80%.

In a second aspect of the invention, there is also provided a variant of the Kex2 enzyme, which variant may be:

The present invention also provides polynucleotides encoding variants of the kex2 enzyme described herein.

The polynucleotide of the invention can be codon optimized according to the preference of pichia pastoris.

The invention also provides a recombinant expression plasmid comprising the polynucleotide, wherein the recombinant expression plasmid comprises the polynucleotide.

The invention also provides a host cell comprising the polynucleotide or recombinant expression plasmid, wherein the host cell comprises the recombinant expression plasmid or integrates the polynucleotide in the genome.

The invention also provides application of the Kex2 enzyme variant in proteolysis.

In another preferred embodiment, the recombinant expression plasmid we describe is a constitutive expression plasmid.

In another preferred embodiment, the host cell we describe is yeast, more preferably pichia pastoris. The expression product is constitutive expression, and glycerol or glucose is used as a unique carbon source to induce the high expression of the expression product.

In another preferred embodiment, the yeast described by us uses glycerol as sole carbon source to induce high expression of the expression product.

In another preferred example, the yeast expression product is soluble secretory expression, and the high expression target protein can be obtained by directly harvesting fermentation supernatant.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

The advantages of the invention over the prior art are:

(1) the invention obtains the variant of the kex2 enzyme by proper truncation and amino acid site mutation, the variant not only has higher enzyme activity, but also can be suitable for a low-salt inorganic salt culture medium without yeast powder or peptone, and the recombinant yeast is obtained by selecting a constitutive expression plasmid and transferring the plasmid into a yeast body, and can utilize glycerol as a carbon source and continuously and efficiently express target protein. Methanol induction is not used in the whole high-density fermentation process, so that the whole high-density fermentation process is simpler and safer; the whole fermentation period of the invention is about 70 hours, which is greatly shortened compared with the prior methanol-induced fermentation process.

(2) The inventor invents a low-salt basic fermentation culture medium without peptone and yeast powder, which not only reduces the fermentation cost, but also greatly facilitates the purification of downstream KEX2 enzyme, and also invents a whole set of high-density fermentation and purification technical route aiming at the product, and can meet the process flow of preparing commercial scale Kex2 enzyme.

(3) The inventor expresses the kex2 enzyme variant in pichia pastoris, selects a constitutive expression plasmid, can realize stable and efficient secretion expression of the kex2, and can well retain the biological activity of protein.

The definitions herein are:

as used herein, "isolated and purified" refers to the kex2 enzyme that is isolated and purified from the kex2 expression supernatant for good enzyme activity, yield and purity.

As used herein, "recombinant" refers to a protein, a genetically engineered vector, or a cell, etc., obtained (or prepared in large quantities) by means of genetic engineering.

As used herein, the term "expression cassette" refers to a gene expression system comprising the necessary elements for expression of a protein of interest (a kex2 truncation or variant of the invention), typically including the following elements: a signal peptide, a promoter, a gene sequence encoding a protein of interest, a terminator, which are operably linked. When considering the convenience of purification of the protein of interest, a purification tag comprising 6 × His may be added to the kex2 truncation or variant.

Drawings

FIG. 1 is a kex2 structure;

FIG. 2 shows the results of protein expression identification by sampling at 24h of culture;

FIG. 3 shows the results of protein expression identification by sampling at 48h of culture;

FIG. 4 shows the results of sampling and identifying protein expression at 72h of culture;

FIG. 5 shows the expression of a target protein in a fermenter during different periods of cultivation;

FIG. 6 is a chromatogram of a first purification method;

FIG. 7 shows the result of SDS-PAGE protein identification analysis in the first purification method;

FIG. 8 is a chromatogram of purification method two;

FIG. 9 shows the SDS-PAGE protein identification analysis of the second purification method;

figure 10 is the viability results of the purified samples.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and not to limit the scope of the present invention, and all simple modifications of the preparation method of the present invention based on the idea of the present invention are within the scope of the present invention. The following examples are experimental methods without specifying specific conditions, and generally follow the methods known in the art.

Kex2 truncation:

in the process of using pichia pastoris to express kex2 in a recombination way, the inventor finds that obvious differences exist between the protein expression amount and the protein activity of kex2 truncations with different sequence lengths. Therefore, the inventor designs a series of kex2 truncations, studies the expression condition of the kex2 truncations in pichia pastoris, and determines a plurality of kex2 truncations which can be used for experimental stable and efficient secretion expression in yeast and retain good enzyme activity through repeated research experiments.

Based on the above findings of the inventors, a truncation of kex2 is provided that removes the C-terminal transmembrane region and extracellular domain relative to wild-type kex2, or removes part or all of the C-terminal Ser/Thr-rich region, transmembrane region and extracellular domain relative to wild-type kex 2.

The nucleotide sequence encoding the kex2 truncation or variant is also encompassed by the present invention, and its polynucleotides are optimized by pichia pastoris-preferred codons.

The polynucleotide sequence of the kex2 truncation or variant of the invention can be obtained by PCR amplification, enzymatic cleavage, or gene synthesis methods. This sequence is then introduced into expression vectors and cells.

Expression system:

fermentation culture A yeast powder-containing peptone medium, a high-salt medium and a low-salt medium are described in comparison herein, and it is shown that the optimized low-salt medium is the best mode.

Inoculating 1-2% of glycerol bacteria stored in a freezing storage tube into an YPD culture medium, and performing primary seed culture for 20-24 h; inoculating 5-8% of the seed into an YPD culture medium, and performing secondary seed culture for 6-10 h; after two-stage amplification of the YPD culture medium, the YPD culture medium is inoculated into a tank medium at an inoculation ratio of 5-10%. Glycerol is supplemented after the fermentation tank is cultured for about 16-20 hours, the process is sampled, centrifuged and collected, and the target protein expression conditions at different periods are observed by running electrophoresis. Feeding the materials for about 50 hours, wherein the total time is about 70 hours, placing the materials in a tank, centrifuging to remove thalli, collecting supernatant, and storing at the temperature of minus 20 ℃. The inventor finally proves that the low-salt inorganic salt culture medium without yeast powder or peptone adopted by the invention is the optimal culture medium by comparing the tank base, the basic salt culture medium, the low-base salt culture medium and the rich nutrient culture medium, comparing the target protein expression condition of fermentation supernatant and the purification treatment effect.

The purification process comprises the following steps:

the supernatant obtained by centrifuging the fermentation broth to remove the precipitate is treated by an appropriate method and then separated and purified by anion exchange chromatography or hydrophobic chromatography. Anion exchange chromatography requires the ionic strength of the sample to be lower than the binding strength (G25 gel filtration desalination or direct dilution method can be adopted), Kex2 protease with the purity meeting the requirement can be obtained by using NaCl linear elution, and the concentration of NaCl in the eluent is lower without influencing the activity of the enzyme. Hydrophobic chromatography requires the addition of higher concentrations of (NH) to the sample₄)₂SO₄To achieve binding to the packing, a substantial concentration of (NH) is still present in the eluent₄)₂SO₄And the influence of the ultrafiltration desalting on the activity of the enzyme can be eliminated by adding one step of ultrafiltration desalting. The purity and the activity of Kex2 protease obtained by the two methods have no significant difference, and the invention preferably adopts anion exchange chromatography (Polar MC60Q) to separate and purify the sample.

The present invention relates to expression vectors comprising the genes encoding the kex2 truncations or variants of the invention, as well as host cells produced by genetic engineering using the vectors of the invention.

Example 1 design of Kex2 protease truncations or variants

Kex2 consists of 814 amino acid residues (SEQ ID NO.1), the entire molecule comprises a signal peptide (1-19aa), a precursor peptide (20-113aa residues), a catalytic domain (114-.

The 1 st to 613 th truncations of Kex2, calculated by the number of amino acid sequences of the full-length Kex2 enzyme, wherein the 225 th, 436 th and 503 th positions are mutated from K to A or P, and a His tag is added or not added at the C end of the truncations.

The 1-667 th truncation of Kex2, counted as the number of amino acid sequences of the full-length Kex2 enzyme, wherein the 225 th, 436 th and 503 th are mutated from K to A or P, and the C end of the truncation is added with or without His tag.

The 1 st-673 rd truncation of Kex2 is counted by the amino acid sequence number of the full-length Kex2 enzyme, wherein, the 225 th, 436 th and 503 th are mutated from K to A or P, and a His tag is added or not added at the C end of the truncation.

The 20 th to 613 th truncations of Kex2 are counted by the number of amino acid sequences of the full-length Kex2 enzyme, wherein the 225 th, 436 th and 503 th positions are mutated from K to A or P, and a His tag is added or not added at the C end of the truncations.

The 20 th-660 th truncation of Kex2 is counted by the amino acid sequence number of the full-length Kex2 enzyme, wherein, the 225 th, 436 th and 503 th are mutated from K to A or P, and meanwhile, a His tag label is added or not added at the C end of the truncation.

The 20-667 th truncation of Kex2, counted as the number of amino acid sequences of the full-length Kex2 enzyme, wherein the 225 th, 436 th and 503 th are mutated from K to A or P, and the C end of the truncation is added with or without His tag.

The 20 th-673 rd truncation of Kex2 is counted by the amino acid sequence number of the full-length Kex2 enzyme, wherein, the 225 th, 436 th and 503 th are mutated from K to A or P, and a His tag is added or not added at the C end of the truncation.

The polynucleotide sequence of the kex2 truncated body or variant is subjected to codon optimization according to pichia pastoris preference, and then is cloned and constructed on a pGAPZa A vector.

Example 2 construction of recombinant Pichia pastoris

The recombinant expression plasmid containing the kex2 truncation or variant was linearized with avll or BspH I enzyme and then recovered using TaKaRa MiniBEST DNA Fragment Purification Kit ver.4.0.

And transferring the constructed linear recombinant expression plasmid into a pichia pastoris X33 or gs115 competent cell by adopting an electrotransformation method, thereby obtaining the recombinant pichia pastoris.

The pGAPZa A expression plasmid contains a Zeocin antibiotic screening gene, and the recombinant pichia pastoris which can stably and efficiently secrete and express kex2 can be screened out by using Zeocin antibiotics with different concentrations.

YPD plates (1% Yeast Extraction, 2% Peptone, 2% glucose) with Zeocin concentration of 100. mu.g/ml were prepared for screening recombinant Pichia pastoris with stable and efficient secretory expression.

Example 3 high-Performance secretory expression of Kex2 protease

Well-growing single clones were randomly picked from YPD plates containing Zeocin antibiotics, inoculated into 3ml of YPD medium, cultured at 28-30 ℃ at 250-300rpm for about 24 hours, transferred to fresh YPD medium at 0.2% volume, cultured, and sampled at about 24 hours, 48 hours, and 72 hours, respectively, to identify protein expression results, as shown in FIGS. 2-4 (A: protein expression in host X33, 1-18 in the figure, respectively, for 1-18 picked clones; B: protein expression in host gs115, and 1-18 in the figure, respectively, for 1-18 picked clones).

Example 4 high Density fermentation of Kex2 protease on Low salt Medium without Yeast powder or peptone

First-stage seed: and (3) subpackaging 50mL of YPD culture medium in a shaking flask, sterilizing at 121 ℃ for 20min, cooling to room temperature, and aseptically inoculating 1-2% of Glycerin tube strain in an ultraclean workbench. Culturing at 30 ℃ and 250rpm for 16-24 h. The OD600 is 4-10.

Secondary seeds: 150mL of YPD culture medium is subpackaged in a 500mL shaking flask, sterilized at 121 ℃ for 20min, and inoculated with first-grade seeds in an ultra-clean workbench in an aseptic manner at an inoculation ratio of 5-8% after the temperature is reduced to room temperature. Culturing at 30 ℃ and 250rpm for 6-10 h. The OD600 was as long as about 10.

Preparing a fermentation tank: 2.0L of low salt inorganic salt medium material without yeast powder or peptone: 8g of magnesium sulfate heptahydrate, 10g of potassium sulfate, 1g of calcium sulfate dihydrate, 80g of glycerol, 22g of 85% phosphoric acid and 2g of 85% potassium hydroxide, wherein the volume is determined to be 1.8L by using purified water, and the mixture is poured into a fermentation tank. Sterilizing at 121 deg.C for 20 min. And cooling the fermentation tank to 30 ℃ by cooling water, adjusting the pH to 4.5 by using dilute ammonia water, adding 8.7mL of filter-sterilized PTM1 culture medium into a fire circle, and inoculating the second-level seeds with an inoculation ratio of 5-10%. 500g of glycerol, and the volume is up to 1.0L. After sterilization at 121 ℃ for 20min, the temperature was lowered to room temperature, and 12mL of filter-sterilized PTM1 medium was added as a supplement. By using a low-salt inorganic salt culture medium without yeast powder or peptone instead of a basic salt culture medium, the efficiency of downstream protein purification can be effectively improved without affecting the expression of target protein.

And (3) fermentation control: 30 ℃, 200rpm, 2L/min aeration, pH 5.0. Regulating the rotation speed and aerating according to the dissolved oxygen, and keeping the dissolved oxygen to be more than 30 percent. Sampling at fixed time to detect bacterial concentration. Culturing for about 20h, ending glycerol consumption in the culture medium, increasing the mutation of dissolved oxygen, starting glycerol feeding at a feeding speed of about 20mL/h, and properly adjusting the feeding speed to keep the dissolved oxygen at more than 30%. The process samples and centrifugates to collect the supernatant, and the expression of the target protein is observed in different periods after running electrophoresis, and the result is shown in FIG. 5 (the target protein expression is observed at 11 sampling points in 20-70 hours in a fermenter). Feeding for about 50h, and placing the strain into a tank when the concentration of the strain reaches about 450 g/L. Centrifuging at 9000rpm for 30min, removing thallus, and collecting supernatant. Subpackaging, and freezing at-20 deg.C.

Example 5 purification of Kex2 protease method one

And (3) processing of a sample: the ionic strength of the fermentation supernatant obtained by adopting the low-salt inorganic salt culture medium without yeast powder or peptone is low, the purification can be carried out on the column only by adding 10mM NaAc-HAc (pH 5.5) buffer solution with twice volume, and compared with the common culture medium which needs to be diluted by 10 times volume or is subjected to gel filtration desalination, the purification efficiency is greatly improved.

Preparation of buffer solution A: 20mM NaAc-HAc (pH 5.5).

Preparation of buffer B: 20mM NaAc-HAc +0.5M NaCl (pH 5.5).

Chromatography medium: polar MC60Q anion exchange packing was loaded into a Φ 25X 200mm column bed.

The purification method comprises the following steps: taking a linear flow rate of 120cm/h in the whole process, loading the diluted sample to a Polar MC60Q chromatographic column which is balanced by buffer solution A in advance, washing the sample by using the buffer solution A after the completion of the whole process, and then collecting eluent in stages according to chromatographic peak shapes by using a linear 40CV gradient elution mode of 0-100% B. A typical chromatogram is shown in FIG. 6.

And (3) detection: protein purity was identified by SDS-PAGE and analyzed by densitometric scanning of the electrophoresis gel, which was 60% (lane 3: pre-column sample) and 85% (lane 7: main peak after purification), respectively. And (4) storing qualified samples at-20 ℃ according to the electrophoresis result. The analysis results are shown in FIG. 7.

Example 6 purification of Kex2 protease II

And (3) processing of a sample: centrifuging the fermentation liquor, taking the supernatant, stirring while slowly adding (NH4)2SO 4-2.5M, placing in a chromatography cabinet at 2-8 ℃, fully stirring for 2 hours, and centrifuging to take the supernatant.

Preparation of buffer solution A: 20mM NaAc-HAc +2.5M (NH4)2SO4(pH 5.5).

Preparation of buffer B: 20mM NaAc-HAc (pH 5.5).

Chromatography medium: Phenyl-FF hydrophobic packing is filled into a column bed with the diameter of phi 25 multiplied by 200 mm.

The purification method comprises the following steps: taking a linear flow velocity of 120cm/h in the whole process, loading the processed sample to a Phenyl-FF chromatographic column which is well balanced by a buffer solution A in advance, washing the sample by using the buffer solution A after the completion, and then collecting eluent in stages according to chromatographic peak shapes by using a linear 20CV gradient elution mode of 0-100% B. A typical chromatogram is shown in FIG. 8.

And (3) detection: the purity of the protein was determined by SDS-PAGE and was 60% (lane 1: pre-column) and 83% (lane 3: Collection 2) before and after purification. According to the results of electrophoresis, the samples which were acceptable were desalted by ultrafiltration using 20mM NaAc-HAc (pH 5.5) buffer and stored at-20 ℃. The analysis results are shown in FIG. 9.

Example 7 detection of Kex2 enzyme Activity

Using Boc-Gln-Arg-Arg-pNA (Boc-QRR-pNA) as the substrate for the activity determination, the enzyme activity unit (U) is defined as: the amount of enzyme required to catalyze the conversion of 1. mu. mol of Boc-QRR-pNA to the product per minute at 25 ℃ and pH 8.0. The activity detection buffer solution is as follows: 50mM Tris-HCl, pH 8.0, 2mM Ca ²⁺100. mu. mol/L Boc-QRR-pNA. Adding appropriate amount of enzyme into 3ml substrate solution, recording absorbance A405 at wavelength of 405nm at intervals of 20s for 3min, and ensuring that the change of absorbance per minute is not more than 0.040. The enzyme activity was calculated by the following formula.

The enzyme activity U (mu mol/L) is △ A/min XF.

Wherein: TV is total reaction volume (mL), SV is sample volume (mL) added, L is optical path of cuvette, L is 1cm, epsilon is molar extinction coefficient of substance to be detected in the reaction system, epsilon is 1.02X 10⁴(L·mol^-1·cm^-1)。

In example 5, the activity of the purified sample (ion exchange elution peak and hydrophobic chromatography elution peak) is 15-20U/mg, and the enzyme activity is higher than that of commercial KEX2 enzyme. The enzyme activity was detected as shown in FIG. 10.

Sequence listing

<110> Ten thousand New medicine science and technology (Suzhou) Co., Ltd

<120> Kex2 enzyme variants and methods for stable expression

<160>1

<170>SIPOSequenceListing 1.0

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<213> Saccharomyces cerevisiae YJM1526(Saccharomyces cerevisiae YJM1526)

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Met Lys Val Arg Lys Tyr Ile Thr Leu Cys Phe Trp Trp Ala Phe Ser

1 5 10 15

Thr Ser Ala Leu Val Ser Ser Gln Gln Ile Pro Leu Lys Asp His Thr

20 25 30

Ser Arg Gln Tyr Phe Ala Val Glu Ser Asn Glu Thr Leu Ser Arg Leu

35 40 45

Glu Glu Met His Pro Asn Trp Lys Tyr Glu His Asp Val Arg Gly Leu

50 55 60

Pro Asn His Tyr Val Phe Ser Lys Glu Leu Leu Lys Leu Gly Lys Arg

65 70 75 80

Ser Ser Leu Glu Glu Leu Gln Gly Asp Asn Asn Asp His Ile Leu Ser

85 90 95

Val His Asp Leu Phe Pro Arg Asn Asp Leu Phe Lys Arg Leu Pro Val

100 105 110

Pro Ala Pro Pro Met Asp Ser Ser Leu Leu Pro Val Lys Glu Ala Glu

115 120 125

Asp Lys Leu Ser Ile Asn Asp Pro Leu Phe Glu Arg Gln Trp His Leu

130 135 140

Val Asn Pro Ser Phe Pro Gly Ser Asp Ile Asn Val Leu Asp Leu Trp

145 150 155 160

Tyr Asn Asn Ile Thr Gly Ala Gly Val Val Ala Ala Ile Val Asp Asp

165 170 175

Gly Leu Asp Tyr Glu Asn Glu Asp Leu Lys Asp Asn Phe Cys Ala Glu

180 185 190

Gly Ser Trp Asp Phe Asn Asp Asn Thr Asn Leu Pro Lys Pro Arg Leu

195 200 205

Ser Asp Asp Tyr His Gly Thr Arg Cys Ala Gly Glu Ile Ala Ala Lys

210 215 220

Lys Gly Asn Asn Phe Cys Gly Val Gly Val Gly Tyr Asn Ala Lys Ile

225 230 235 240

Ser Gly Ile Arg Ile Leu Ser Gly Asp Ile Thr Thr Glu Asp Glu Ala

245 250 255

Ala Ser Leu Ile Tyr Gly Leu Asp Val Asn Asp Ile Tyr Ser Cys Ser

260 265 270

Trp Gly Pro Ala Asp Asp Gly Arg His Leu Gln Gly Pro Ser Asp Leu

275 280 285

Val Lys Lys Ala Leu Val Lys Gly Val Thr Glu Gly Arg Asp Ser Lys

290295 300

Gly Ala Ile Tyr Val Phe Ala Ser Gly Asn Gly Gly Thr Arg Gly Asp

305 310 315 320

Asn Cys Asn Tyr Asp Gly Tyr Thr Asn Ser Ile Tyr Ser Ile Thr Ile

325 330 335

Gly Ala Ile Asp His Lys Asp Leu His Pro Pro Tyr Ser Glu Gly Cys

340 345 350

Ser Ala Val Met Ala Val Thr Tyr Ser Ser Gly Ser Gly Glu Tyr Ile

355 360 365

His Ser Ser Asp Ile Asn Gly Arg Cys Ser Asn Ser His Gly Gly Thr

370 375 380

Ser Ala Ala Ala Pro Leu Ala Ala Gly Val Tyr Thr Leu Leu Leu Glu

385 390 395 400

Ala Asn Pro Asn Leu Thr Trp Arg Asp Val Gln Tyr Leu Ser Ile Leu

405 410 415

Ser Ala Val Gly Leu Glu Lys Asn Ala Asp Gly Asp Trp Arg Asp Ser

420 425 430

Ala Met Gly Lys Lys Tyr Ser His Arg Tyr Gly Phe Gly Lys Ile Asp

435 440 445

Ala His Lys Leu Ile Glu Met Ser Lys Thr Trp Glu Asn Val Asn Ala

450455 460

Gln Thr Trp Phe Tyr Leu Pro Thr Leu Tyr Val Ser Gln Ser Thr Asn

465 470 475 480

Ser Thr Glu Glu Thr Leu Glu Ser Val Ile Thr Ile Ser Glu Lys Ser

485 490 495

Leu Gln Asp Ala Asn Phe Lys Arg Ile Glu His Val Thr Val Thr Val

500 505 510

Asp Ile Asp Thr Glu Ile Arg Gly Thr Thr Thr Val Asp Leu Ile Ser

515 520 525

Pro Ala Gly Ile Ile Ser Asn Leu Gly Val Val Arg Pro Arg Asp Val

530 535 540

Ser Ser Glu Gly Phe Lys Asp Trp Thr Phe Met Ser Val Ala His Trp

545 550 555 560

Gly Glu Asn Gly Val Gly Asp Trp Lys Ile Lys Val Lys Thr Thr Glu

565 570 575

Asn Gly His Arg Ile Asp Phe His Ser Trp Arg Leu Lys Leu Phe Gly

580 585 590

Glu Ser Ile Asp Ser Ser Lys Thr Glu Thr Phe Val Phe Gly Asn Asp

595 600 605

Lys Glu Glu Val Glu Pro Ala Ala Thr Glu Ser Thr Val Ser Gln Tyr

610615 620

Ser Ala Ser Ser Thr Ser Ile Ser Ile Ser Ala Thr Ser Thr Ser Ser

625 630 635 640

Ile Ser Ile Gly Val Glu Thr Ser Ala Ile Pro Gln Thr Thr Thr Ala

645 650 655

Ser Thr Asp Pro Asp Ser Asp Pro Asn Thr Pro Lys Lys Leu Ser Ser

660 665 670

Pro Arg Gln Ala Met His Tyr Phe Leu Thr Ile Phe Leu Ile Gly Ala

675 680 685

Thr Phe Leu Val Leu Tyr Phe Met Phe Phe Met Lys Ser Arg Arg Arg

690 695 700

Ile Arg Arg Ser Arg Ala Glu Thr Tyr Glu Phe Asp Ile Ile Asp Thr

705 710 715 720

Asp Ser Glu Tyr Asp Ser Thr Leu Asp Asn Gly Thr Ser Gly Ile Thr

725 730 735

Glu Pro Glu Glu Val Glu Asp Phe Asp Phe Asp Leu Ser Asp Glu Asp

740 745 750

His Leu Ala Ser Leu Ser Ser Ser Glu Asn Gly Asp Ala Glu His Thr

755 760 765

Ile Asp Ser Val Leu Thr Asn Glu Asn Pro Phe Ser Asp Pro Ile Lys

770 775780

Gln Lys Phe Pro Asn Asp Ala Asn Ala Glu Ser Ala Ser Asn Lys Leu

785 790 795 800

Gln Glu Leu Gln Pro Asp Val Pro Pro Ser Ser Gly Arg Ser

805 810

Claims

1. A method for stably and efficiently expressing a kex2 enzyme, the method comprising:

2. The method of claim 1, wherein the variant of (1): counting the amino acid sequence number of the full-length Kex2 shown in SEQ ID NO.1, wherein the 225 th position, the 436 th position and the 503 th position are mutated from K to A or P; preferably, the variant in step (1) is selected from the group consisting of:

3. The method of claim 1, wherein the yeast of step (2) is pichia pastoris.

4. The process according to claim 1, wherein the conversion process in step (3) is an electro-or physicochemical process, preferably an electro-conversion.

5. The method of claim 1, wherein the fermentation in step (4) is performed using a low-salt inorganic salt medium without yeast powder or peptone; preferred media components are: 4g to 4.5 g/L of magnesium sulfate heptahydrate, 5g to 6g/L of potassium sulfate, 0.3 g to 0.5g/L of calcium sulfate dihydrate, 30 g to 40g/L of glycerol, 10g to 11g/L of 85 percent phosphoric acid and 1g to 1.2g/L of 85 percent potassium hydroxide.

6. The method of claim 1, wherein the absence of a special pretreatment in step (4) is a pretreatment step without desalting with a molecular sieve or large-volume dilution; the dilution in the step (4) is 2 to 3 times that of pure water, and the conductivity of ion exchange is controlled to be not more than 2.5 mS/cm.

A variant of the Kex2 enzyme, wherein the variant is selected from the group consisting of:

8. A polynucleotide encoding a variant of the kex2 enzyme of claim 7.

9. A recombinant expression plasmid comprising the polynucleotide of claim 8.

10. A host cell comprising the recombinant expression plasmid of claim 9 or having the polynucleotide of claim 8 integrated into its genome.