CN107937372B

CN107937372B - Nattokinase with improved acid resistance

Info

Publication number: CN107937372B
Application number: CN201711376850.1A
Authority: CN
Inventors: 周哲敏; 刘中美; 周丽; 崔文璟; 郭军玲; 赵菡
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2017-12-19
Filing date: 2017-12-19
Publication date: 2020-03-24
Anticipated expiration: 2037-12-19
Also published as: CN107937372A

Abstract

The invention discloses nattokinase with improved acid resistance, belonging to protein engineering. Under the acidic condition, the stability of the natto kinases Q59E-Y217K-N218D and Y217K obtained by the invention is greatly improved compared with that of wild enzymes Q59E-Y217K-N218D and Y217K. The natto kinase mutants Q59E-Y217K-N218D and Y217K provided by the invention can better cope with the extreme acid environment of the stomach, and the application value of the natto kinase as an oral medicine for treating cardiovascular and cerebrovascular diseases is improved.

Description

Nattokinase with improved acid resistance

Technical Field

The invention relates to a nattokinase mutant with improved acid resistance, belonging to protein engineering.

Background

Nattokinase (NK, EC3.4.21.62) is a serine protease produced by Bacillus natto in the fermentation process of traditional food natto in Japan, has high-efficiency thrombolytic effect compared with commercial thrombolytic drugs, and is the most potential fibrinolytic protease. Therefore, the development of the enzyme as an orally-taken medicine has wide application prospect, but the industrial application of the enzyme is severely limited due to the extremely acidic environment of the gastric tract. The existing nattokinase has poor acid resistance, so that a nattokinase mutant with improved acid resistance is urgently needed to adapt to the extreme acid environment of the gastric canal and promote the development and application of the nattokinase as a thrombolytic drug.

Disclosure of Invention

The first purpose of the invention is to provide a nattokinase mutant with improved acid resistance, wherein the mutant is obtained by mutating a plurality of amino acid sites of nattokinase with an amino acid sequence shown as SEQ ID NO. 1.

In one embodiment of the invention, the mutant is obtained by mutating one or more amino acid positions 59, 217 and 218 of nattokinase shown as SEQ ID NO.1 in amino acid sequence.

In one embodiment of the invention, the mutant is obtained by mutating 217 th tyrosine of nattokinase shown as SEQ ID NO.1 into lysine.

In one embodiment of the invention, the mutant is a combined mutant Q59E-Y217K-N218D obtained by mutating glutamine 59 to glutamic acid, tyrosine 217 to lysine and asparagine 218 to aspartic acid of nattokinase shown in SEQ ID NO. 1.

The second purpose of the invention is to provide a recombinant bacterium for expressing the nattokinase mutant.

In one embodiment of the present invention, the method for constructing the recombinant bacterium comprises: the gene of the nattokinase mutant is connected to a vector pET24a, and a recombinant bacterium is constructed by using a host of Escherichia coli BL 21.

The third purpose of the invention is to provide a method for expressing a nattokinase mutant, which comprises the following specific steps:

(1) the recombinant bacteria are inoculated in LB culture medium containing kanamycin and are shaken overnight at the temperature of 35-38 ℃ and the speed of 150-.

(2) Inoculating the seed liquid into an expression culture medium containing kanamycin at a concentration of 1-2% by mass, and performing shaking culture at 35-38 deg.C and 150-₆₀₀When the temperature is 0.6-0.8, adding inducer IPTG, and inducing the expression of the nattokinase mutant for 18-24h at 15-18 ℃.

In one embodiment of the invention, the concentration of kanamycin is 80-120. mu.g/mL.

In one embodiment of the invention, the inducer is IPTG to 0.05-0.2 mM.

In one embodiment of the invention, the inducer IPTG is to 0.1mM

In one embodiment of the present invention, the expression medium is TB medium, and the TB medium comprises: tryptone 10-14g/L, yeast extract 20-26g/L, glycerin 3-8g/L, KH₂PO₄2.0-2.6g/L，K₂HPO₄·3H₂O15.8-16.8g/L，CaCl₂0.15-0.25g/L, and the balance of water.

In one embodiment of the invention, the purification process of nattokinase comprises the following steps: crushing the recombinant thallus, collecting supernatant, membrane filtering the supernatant, and separating with nickel column centrifugal column at 2-6 deg.c to obtain natto kinase mutant pure enzyme liquid.

The invention has the beneficial effects that: the nattokinase mutant Q59E-Y217K-N218D provided by the invention has better acid stability, particularly, after the nattokinase mutant is treated for 4 hours under the condition of pH4.0, about 25% of enzyme activity of wild type WT is remained, 67% of enzyme activity of the mutant Y217K is kept, about 80% of enzyme activity of the mutant Q59E-Y217K-N218D can be still kept, and the acid resistance of the nattokinase is greatly improved. Therefore, the nattokinase mutant Y217K, Q59E-Y217K-N218D provided by the invention are more suitable for oral administration and can better cope with the extreme acid environment of the stomach tract.

Drawings

FIG. 1 residual enzyme activity after 4h treatment of wild enzyme (WT) and mutant at pH 4.0.

Detailed Description

The LB medium comprises the following components: peptone 8-12g/L, yeast extract 4-6g/L, NaCl 8-12g/L

The TB culture medium comprises the following components: tryptone 10-14g/L, yeast extract 20-26g/L, glycerin 3-8g/L, KH₂PO₄2.0-2.6g/L，K₂HPO₄·3H₂O 15.8-16.8g/L，CaCl₂0.15-0.25g/L

Definition of enzyme activity: the amount of enzyme required to convert 1. mu. mol of suc-AAPF-pNA to 1. mu. mol of pNA within 1min at 25 ℃ and pH8.0 at a substrate concentration of 0.4mM is defined as one unit of enzyme activity.

The enzyme activity determination method comprises the following steps: Tris-HCl buffer (containing 0.1mM CaCl) at 100mM pH8.0₂) Taking artificially synthesized suc-AAPF-pNA as a substrate, reacting for 3min at 25 ℃, and determining the concentration of generated p-nitroaniline (pNA) according to the absorption value of 405nm to determine the enzyme activity.

Determination of pH stability: the residual enzyme activity was determined after the wild enzyme WT and the mutant were retained in a buffer solution of pH4 at 4 ℃ for 4 hours, and the highest enzyme activity was defined as 100%, to obtain a pH stability histogram.

Example 1 construction of Nattokinase Single and double mutants

(1) Construction of Single mutant Q59E

Using pET24a-pro-NK as template and the primers shown in Table 1, the recombinant vector carrying the gene encoding Q59E mutant was obtained by PCR under the conditions shown in Table 5, and plasmid pET24a-pro-NK/Q59E was obtained.

TABLE 1 mutant Q59E primer

Analyzing and identifying the PCR product by 1.0% agarose gel electrophoresis; purifying the PCR product with DNA purification kit, and finally using Quichcut^TmDpnI is digested at 37 ℃ for 5h to remove the methylated template plasmid, transferred into BL21 escherichia coli host, and the recombinant strain is named as BL21/pET24 a-pro-NK/Q59E.

(2) Construction of Single mutant Y217K

Using pET24a-pro-NK as template and the primers shown in Table 2, the recombinant vector carrying the gene encoding Y217K mutant was obtained by PCR under the conditions shown in Table 5, and plasmid pET24a-pro-NK/Y217K was obtained.

TABLE 2 mutant Y217K primer

Analyzing and identifying the PCR product by 1.0% agarose gel electrophoresis; purifying the PCR product with DNA purification kit, and finally using Quichcut^TmDpnI is digested at 37 ℃ for 5h to remove the methylated template plasmid, transferred into BL21 escherichia coli host, and the recombinant strain is named as BL21/pET24 a-pro-NK/Y217K.

(3) Construction of Single mutant N218D

Using pET24a-pro-NK as template and the primers shown in Table 3, the recombinant vector carrying the N218D mutant gene was obtained by PCR under the conditions shown in Table 5, and plasmid pET24a-pro-NK/N218D was obtained.

TABLE 3 mutant N218D primer

Analyzing and identifying the PCR product by 1.0% agarose gel electrophoresis; purifying the PCR product with DNA purification kit, and finally using Quichcut^TmDpnI is digested at 37 ℃ for 5h to remove the methylated template plasmid, transferred into BL21 escherichia coli host, and the recombinant strain is named as BL21/pET24 a-pro-NK/N218D.

(4) Construction of double mutant Q59E-N218D

Using pET24a-pro-NK as a template, and using primers shown in Table 1 to obtain a recombinant vector carrying a gene encoding a Q59E mutant by PCR under the conditions shown in Table 5; pET24a-pro-NK/Q59E plasmid was extracted using a plasmid miniprep according to the procedures described in the specification, and then a recombinant vector carrying a gene encoding Q59E-N218D double mutant was obtained by PCR using pET24a-pro-NK/Q59E as a template and primers shown in Table 3 under the conditions shown in Table 5, and the plasmid was named pET24 a-pro-NK/Q59E-N218D.

Analyzing and identifying the PCR product by 1.0% agarose gel electrophoresis; purifying the PCR product with DNA purification kit, and finally using Quichcut^TmDpnI is digested at 37 ℃ for 5h to remove the methylated template plasmid, transferred into BL21 Escherichia coli host, and the recombinant strain is named as BL21/pET24 a-pro-NK/Q59E-N218D.

Example 2 construction of Nattokinase triple mutant

Using pET24a-pro-NK as a template, and using primers shown in Table 1 to obtain a recombinant vector carrying a gene encoding a Q59E mutant by PCR under the conditions shown in Table 5; pET24a-pro-NK/Q59E plasmid was extracted using a plasmid miniprep according to the procedures described in the specification, and then PCR was performed using pET24a-pro-NK/Q59E as a template and primers shown in Table 4 under the conditions shown in Table 5 to obtain a plasmid carrying the gene encoding three mutants of Q59E-Y217K-N218D, which was named pET24 a-pro-NK/Q59E-Y217K-N218D.

TABLE 4 mutant Q59E-Y217K-N218D primer

TABLE 5 Whole plasmid PCR amplification reaction System

The PCR amplification reaction conditions are as follows:

analyzing and identifying the PCR product by 1.0% agarose gel electrophoresis; purifying the PCR product with DNA purification kit, andpost-use Quichcut^TmDpnI is digested at 37 ℃ for 5h to remove the methylated template plasmid, transferred into BL21 escherichia coli host, and the recombinant strain is named as BL21/pET24 a-pro-NK/Q59E-Y217K-N218D.

Example 3 expression of Nattokinase mutant

(1) Recombinant Escherichia coli BL21/pET24a-pro-NK/Q59E-Y217K-N218D, BL21/pET24a-pro-NK/Q59E, BL21/pET24a-pro-NK/Y217K, BL21/pET24a-pro-NK/N218D, BL21/pET24a-pro-NK/Q59E were inoculated in 5mL LB medium with 100. mu.g/mL kanamycin concentration, and cultured overnight at 37 ℃ with shaking at 200 r/min.

(2) Inoculating the seed solution with an inoculum size of 1% by mass into 100mL TB expression medium containing kanamycin at 100. mu.g/mL, and culturing at 37 deg.C under shaking at 200r/min to OD₆₀₀When the concentration is 0.6-0.8, adding inducer IPTG to 0.1mM, inducing at 18 ℃ for 20h, collecting thalli, and centrifuging at the rotating speed of 5000g to collect the thalli.

(3) The recombinant cells were dissolved in 20mL of binding buffer (50 mmol/LNa)₂HPO₄、50mmol/LNaH₂PO₄500mmol/L NaCl, 20mmol/L imidazole), sonicated, 13000g centrifuged for 25min, and the supernatant filtered through a 0.22 μm filter. The column was equilibrated with 10 column volumes of binding buffer, the non-specifically adsorbed proteins were washed off with 5 column volumes of elution buffer containing 20mmol/L imidazole, the proteins were eluted with 50 and 100mmol/L imidazole of elution buffer, respectively (repeated elution), and the samples were collected and characterized by SDS-PAGE analysis.

Example 4 analysis of pH stability of Nattokinase Single and double mutants

Determination of pH stability: the wild enzyme WT, the single mutant, the double mutant and the triple mutant are reserved in a buffer solution of pH4 at 4 ℃ for 4h, and then the residual enzyme activity is measured at pH8, as shown in figure 1, the stability of the mutant Q59E-Y217K-N218D and the mutant Y217K is greatly improved compared with the wild enzyme under the acidic condition.

Example 5 kinetic analysis of Single and double mutants of Nattokinase

Determination of kinetic constants: artificially synthesized tetrapeptide suc-AAPF-pNA is taken as a substrate, and the concentration range of the substrate is 5 multiplied by 10^-5To 10^-3And M, calculating the enzymatic reaction rate under each substrate concentration according to the absorption value of 405nm by taking p-nitroaniline as a standard, and fitting by using software GraphPad Prism 5 to obtain Km and Vmax. The mutant Q59E-Y217K-N218D has catalytic efficiency (kcat/Km) and specific enzyme activity which are improved by about 30 percent compared with wild enzyme.

TABLE 6 kinetic analysis of the enzymes

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

Sequence listing

<110> university of south of the Yangtze river

<120> nattokinase with improved heat stability

<160>2

<170>PatentIn version 3.3

<210>1

<211>275

<212>PRT

<213> Artificial Synthesis

<400>1

Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu

1 5 10 15

His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp

20 25 30

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

35 40 45

Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His

50 55 60

Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly

65 70 75 80

Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu

85 90 95

Asp Ser Thr Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu

100 105 110

Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly

115 120 125

Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser

130 135 140

Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly

145 150 155 160

Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala

165 170 175

Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val

180 185 190

Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr

195 200 205

Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr

210 215 220

Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr

225 230 235 240

Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr

245 250 255

Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala

260 265 270

Ala Ala Gln

275

<210>2

<211>828

<212>DNA

<213> Artificial Synthesis

<400>2

gcgcaatctg ttccttatgg catttctcaa attaaagcgc cggctcttca ctctcaaggc 60

tacacaggct ctaacgtaaa agtagctgtt atcgacagcg gaattgactc ttctcatcct 120

gacttaaacg tcagaggcgg agcaagcttc gttccttctg aaacaaaccc ataccaggac 180

ggcagttctc acggtacgca tgtcgccggt acgattgccg ctcttaataa ctcaatcggt 240

gttctgggcg tagcgccaag cgcatcatta tatgcagtaa aagtgcttga ttcaacagga 300

agcggccaat atagctggat tattaacggc attgagtggg ccatttccaa caatatggat 360

gttatcaaca tgagccttgg cggacctact ggttctacag cgctgaaaac agtagttgat 420

aaagcggttt ccagcggtat cgtcgttgct gccgcagccg gaaacgaagg ttcatccgga 480

agcacaagca cagtcggcta ccctgcaaaa tatccttcta ctattgcagt aggtgcggta 540

aacagcagca accaaagagc ttcattctcc agcgtaggtt ctgagcttga tgtaatggct 600

cctggcgtgt ccatccaaag cacacttcct ggaggcactt acggcgctta taacggaacg 660

tccatggcga ctcctcacgt tgccggagca gcagcgctaa ttctttctaa gcacccgact 720

tggacaaacg cgcaagtccg tgatcgttta gaaagcactg caacatatct tggaaactct 780

ttctactatg gaaaagggtt aatcaacgta caagcagctg cacaataa 828

<210>3

<211>30

<212>DNA

<213> Artificial Synthesis

<400>3

gaaacaaacc catacgagga cggcagttct 30

<210>4

<211>30

<212>DNA

<213> Artificial Synthesis

<400>4

gtgagaactg ccgtcctcgt atgggtttgt 30

<210>5

<211>30

<212>DNA

<213> Artificial Synthesis

<400>5

ggcacttacg gcgctaagaa cggaacgtcc 30

<210>6

<211>32

<212>DNA

<213> Artificial Synthesis

<400>6

ccatggacgt tccgttctta gcgccgtaag tg 32

<210>7

<211>30

<212>DNA

<213> Artificial Synthesis

<400>7

acttacggcg cttatgacgg aacgtccatg 30

<210>8

<211>30

<212>DNA

<213> Artificial Synthesis

<400>8

cgccatggac gttccgtcat aagcgccgta 30

Claims

1. A nattokinase mutant with improved stability, which is characterized in that the mutant is (1) or (2):

(1) mutating 217 th tyrosine of nattokinase shown in SEQ ID NO.1 as amino acid sequence into lysine;

(2) the 59 th glutamine of nattokinase shown in SEQ ID NO.1 in amino acid sequence is mutated into glutamic acid, and the 217 th tyrosine is mutated into lysine and the 218 th asparagine is mutated into aspartic acid, so that the combined mutant Q59E-Y217K-N218D is obtained.

2. A recombinant bacterium which expresses the mutant of claim 1.

3. The method for constructing the recombinant strain of claim 2, wherein the method comprises the following steps: the gene of the nattokinase mutant is connected to a vector pET24a, and Escherichia coli BL21 is used as a host to construct a recombinant bacterium.

4. The method for expressing the nattokinase mutant by the recombinant bacteria as claimed in claim 2 is characterized by comprising the following specific steps:

(1) inoculating the recombinant bacteria to an LB culture medium containing kanamycin, and performing shaking overnight culture at 35-38 ℃ and 150-;

(2) inoculating the seed liquid into an expression culture medium containing kanamycin at a mass fraction of 1-2%, and performing shake culture at 35-38 deg.C and 150-₆₀₀When the temperature is 0.6-0.8, adding inducer IPTG, and inducing the expression of the nattokinase mutant for 18-24h at 15-18 ℃.

5. The method of claim 4, wherein the inducer IPTG is at a concentration of 0.05-0.2 mM.

6. The method of claim 4, wherein the step of removing the metal oxide layer comprises removing the metal oxide layer from the metal oxide layerThe expression medium is a TB medium, and the TB medium comprises the following components: tryptone 10-14g/L, yeast extract 20-26g/L, glycerin 3-8g/L, KH₂PO₄2.0-2.6g/L，K₂HPO₄·3H₂O 15.8-16.8g/L，CaCl₂0.15-0.25g/L, and the balance of water.

7. Nattokinase mutant obtainable by a method according to any one of claims 4 to 6.

8. Use of the nattokinase mutant according to claim 7 in the manufacture of a medicament for the treatment of cardiovascular and cerebrovascular diseases.