A method for improving expression level of cutinase-anchor peptide fusion protein in escherichia coli

Abstract

The application discloses a method for improving expression level of cutinase-anchor peptide fusion protein in Escherichia coli, and belongs to the field of fermentation engineering. The application takes Escherichia coli E.coli C43(DE3) as a starting strain, and can obtain correctly folded fusion protein HiC-OMP25, and the HiC-OMP25 has good stability at 50 DEG C, can well adapt to the environment of PEA degradation, and can play a good degradation role. Further, the intracellular expression amount of the HiC-OMP25 can be increased by 2.18 times compared with a control by expressing disulfide bond isomerase in the recombinant cell; on the basis, the extracellular expression amount of the fusion protein HiC-OMP25 can be significantly increased by expressing sulfhydryl oxidase in the recombinant cell, and the extracellular enzyme activity reaches 135.43 U.mL ‑1 , which is increased by 2.14 times compared with the control. Thus, the method and the constructed recombinant Escherichia coli have great application prospect in the field of polymer degradation.

Description

Technical Field

[0001] This invention relates to a method for increasing the expression level of keratinase-anchored peptide fusion protein in Escherichia coli, belonging to the field of fermentation engineering. Background Technology

[0002] Adhesives are pollutants generated during waste paper recycling. They easily remain on paper and equipment surfaces, affecting product quality, causing equipment damage, and severely impacting the recycling efficiency of waste paper. Their control has become an urgent problem to be solved in improving waste paper utilization. The main methods for controlling adhesives include physical, chemical, and biological methods. Due to their environmental friendliness, high efficiency, and specificity, biological control methods can effectively degrade major adhesive components such as polyvinyl acetate (PVAc) and polyethyl acrylate (PEA), and are gradually becoming the development trend of adhesive control technology. Keratinase is a multifunctional enzyme capable of degrading various polyesters, and anchoring peptides are short peptides that can improve the anchoring effect on specific substances. Previous studies have shown that the fusion protein HiC-OMP25, composed of humicolainsolens keratinase and anchoring peptide OMP25, can significantly improve the degradation effect on adhesives.

[0003] The expression level of HiC-OMP25 in Escherichia coli is currently low, so there is an urgent need to increase its expression level to meet industrial needs. Summary of the Invention

[0004] This invention compared the expression levels and properties of HiC-OMP25 in different E. coli hosts. It was found that the enzyme activity of HiC-OMP25 was higher when E. coli BL21(DE3) was used as the host than when E. coli C43(DE3). However, further analysis of intracellular and extracellular HiC-OMP25 produced by different hosts for PEA degradation showed that extracellular HiC-OMP25 expressed when E. coli BL21(DE3) had almost no degradative effect on PEA, while both intracellular and extracellular HiC-OMP25 expressed when E. coli C43(DE3) had a good degradation effect on PEA. Based on this, the inventors used the BATH (Bacterial Adhesion To Hydrocarbons) method to detect changes in the hydrophobicity of the above-mentioned bacterial cells. The results showed that the hydrophobic adsorption rate of E. coli BL21(DE3) recombinantly expressing HiC-OMP25 (19.49%) was higher than that of E. coli BL21(DE3) containing the empty vector (2.27%), proving that expressing HIC-OMP25 can improve the hydrophobicity of the strain. It is speculated that this may be due to the aggregation or direct insertion of HIC-OMP25 into the cell membrane surface, leading to increased bacterial cell hydrophobicity. At the same time, due to the phospholipase activity of keratinase itself, cell membrane permeability can be enhanced. Therefore, the HiC-OMP25 expressed in E. coli BL21(DE3) failed to fold correctly and was secreted into the extracellular space, affecting the application of HiC-OMP25 in actual production.

[0005] After identifying the host cell, this invention further co-expresses disulfide isomerase or a combination of disulfide isomerase and thiol oxidase (Erv1p) with HiC-OMP25 in Escherichia coli C43(DE3) to enhance the expression level of HiC-OMP25.

[0006] The first objective of this invention is to provide a recombinant Escherichia coli expressing HiC-OMP25 in E. coli C43(DE3), the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] In one embodiment, the recombinant Escherichia coli also expresses the gene dsbc encoding the disulfide isomerase DsbC, or a disulfide isomerase derived from Homo sapiens or Arabidopsis thaliana.

[0008] Furthermore, the recombinant Escherichia coli also expresses the gene erv1p encoding thiol oxidation Erv1p, or thiol oxidase derived from Rhinopithecus bieti or Rattus norvegicus.

[0009] In one embodiment, the amino acid sequence encoding the disulfide isomerase DsbC is shown in SEQ ID NO.2.

[0010] In one embodiment, the amino acid sequence encoding the thiol oxidase Erv1p is shown in SEQ ID NO.5.

[0011] In one embodiment, the amino acid sequences of the disulfide isomerase derived from Homo sapiens or Arabidopsis thaliana are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0012] In one embodiment, the amino acid sequences of the thiol oxidases derived from Rhinopithecus bieti or Rattus norvegicus are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0013] In one implementation, the pET-20b(+) vector is used to express the nucleotide encoding HiC-OMP25.

[0014] In one embodiment, pET-24a(+) is used to express the gene dsbc encoding the disulfide bond isomerase DsbC, or pCDFDuet-1 is used to co-express the gene dsbc encoding the disulfide bond isomerase DsbC and the gene erv1p encoding thiol oxidation Erv1p.

[0015] A second objective of this invention is to provide a method for producing HiC-OMP25, wherein the method uses the recombinant Escherichia coli as the production strain to ferment and produce HiC-OMP25.

[0016] In one embodiment, the recombinant Escherichia coli is cultured in LB medium to obtain a seed culture, and then OD is... 600 =7.0±0.5% seed culture was inoculated into TB medium and incubated at 37℃ and 200 rpm. -1 After culturing for 2 hours, transfer to a 25℃ constant temperature incubator and culture for 36 hours.

[0017] A third objective of this invention is to provide a method for degrading PEA, wherein the method utilizes HiC-OMP25 obtained by fermentation of the recombinant Escherichia coli to degrade PEA.

[0018] In one embodiment, an enzyme solution containing HiC-OMP25 is added to a system containing PEA, and the reaction is carried out at a temperature of not less than 40°C and 100–150 rpm for 0–10 h.

[0019] Preferably, the reaction is carried out at a temperature not lower than 50°C.

[0020] In one embodiment, the enzyme solution containing HiC-OMP25 is preheated at a temperature of not less than 40°C for 10 minutes, and then added to the system containing PEA.

[0021] Preferably, the temperature is preheated at a temperature not lower than 50°C.

[0022] The fourth objective of this invention is to provide a method for increasing the extracellular expression level of HiC-OMP25.

[0023] In one embodiment, disulfide isomerase and thiol oxidase are simultaneously expressed in HiC-OMP25 expression cells.

[0024] In one embodiment, the amino acid sequence of the disulfide isomerase is shown in any one of SEQ ID NO. 2 to 4.

[0025] In one embodiment, the amino acid sequence of the thiol oxidase is as shown in any one of SEQ ID NO. 5 to 7.

[0026] A fourth object of the present invention is to provide the use of the recombinant Escherichia coli in the production of the fusion protein HiC-OMP25, keratinase, or polymer degradation.

[0027] The beneficial effects of this invention are:

[0028] This invention uses *Escherichia coli* C43(DE3) as the starting strain to obtain a correctly folded fusion protein, HiC-OMP25. This HiC-OMP25 exhibits good stability at 50°C, effectively adapting to the PEA degradation environment and demonstrating good degradation activity. Furthermore, expression of disulfide isomerase in recombinant cells increased the intracellular expression level of HiC-OMP25 by 2.18 times compared to the control. Based on this, expression of thiol oxidase in recombinant cells significantly increased the extracellular expression level of the HiC-OMP25 fusion protein, achieving an extracellular enzyme activity of 135.43 U·mL⁻¹. -1 It increased by 2.14 times compared to the control. Attached Figure Description

[0029] Figure 1 Image showing the activity of extracellular and intracellular fusion proteases in E. coli when co-expressing different chaperone proteins.

[0030] Figure 2The image shows the temperature stability of HiC-OMP25 at 50℃; "■" indicates the fermentation supernatant of BL21 / HiC-OMP25, and "●" indicates the fermentation supernatant of C43 / HiC-OMP25. Detailed Implementation

[0031] Enzyme activity assay:

[0032] Substrate: Prepare a solution of pNPB and acetonitrile miscible to a concentration of 50 mmol·L⁻¹. -1 The pNPB solution was sealed and stored at -20°C.

[0033] The activity of the fusion protein was determined using spectrophotometry. A 10 mmol·L⁻¹ solution was used. -1 Preheat the pH 8.0 Tris-HCl buffer to 37°C. Then, take 1.44 mL of buffer, 30 μL of enzyme solution, and 30 μL of substrate into a 0.5 cm quartz cuvette, shake to mix, and immediately place it in a visible spectrophotometer to measure the change in absorbance of the reaction system at 405 nm.

[0034] Enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of pNPB to produce 1 μmol of p-nitrophenol per minute at 37°C. One unit of enzyme activity (U) is defined as the amount of enzyme that catalyzes the hydrolysis of pNPB to produce 1 μmol of p-nitrophenol per minute.

[0035] LB medium contains 50 μg / mL, depending on the strain being cultured. -1 Amp, or containing 50 μg·mL -1 Amp and 15 μg·mL -1 Kana, or containing 50 μg·mL -1 Amp and 25 μg·mL -1 Sm.

[0036] Example 1: Using different host bacteria to target HIC-OMP25 expression

[0037] (1) Construction of recombinant strains

[0038] The recombinant plasmid was constructed by ligating the codon hic-omp25 encoding the HiC-OMP25 fusion protein (amino acid sequence as shown in SEQ ID NO.1) to the polyclonal restriction site of the pET-20b(+) vector, thus obtaining the recombinant plasmid pET-20b(+)-hic-omp25.

[0039] The recombinant plasmid pET-20b(+)-hic-omp25 was transformed into E. coli C43(DE3) and E. coli BL21(DE3), respectively. The colonies were cultured on LB agar plates containing the corresponding antibiotics until they grew to single colonies. Single colonies were picked and verified by PCR and sequencing. The ones that were verified correctly were the positive transformants, namely recombinant E. coli E43(DE3) / pET-20b(+)-hic-omp25 and E. coli BL21(DE3) / pET-20b(+)-hic-omp25.

[0040] (2) Recombinant strain expressing HIC-OMP25

[0041] The recombinant strain obtained was inoculated into 10 mL of LB liquid medium (containing the corresponding antibiotic) at an inoculation rate of 2‰, and incubated at 37℃ and 200 rpm. -1 After culturing for 8-12 hours under the specified conditions, the bacterial culture was transferred at a 5% inoculum to 50 mL of TB medium (containing the appropriate antibiotic), and then incubated at 37°C and 200 rpm. -1 After culturing for 2 hours, the culture was transferred to a 25°C constant temperature incubator and cultured for 36 hours. The resulting bacterial culture was then subjected to a process at 8000 rpm. -1 After centrifugation for 15 minutes, the supernatant was collected as the fermentation supernatant. The bacterial cells in the lower precipitate were resuspended in buffer solution and then ultrasonically disrupted using an ultrasonic cell disruptor at 8000 rpm. -1 Centrifuge for 15 minutes, and the supernatant is the intracellular supernatant.

[0042] The intracellular and extracellular cutinase activities of the recombinant bacteria were measured. The results showed that the extracellular enzyme activity of recombinant E. coli C43(DE3) / pET-20b(+)-hic-omp25 was 43.13 U / mL, and the intracellular enzyme activity was 39.28 U / mL; the extracellular enzyme activity of recombinant E. coli BL21(DE3) / pET-20b(+)-hic-omp25 reached 207.89 U / mL, and the intracellular enzyme activity was 62.32 U / mL.

[0043] (3) Treating PEA with HIC-OMP25 expressed by different host strains

[0044] The effects of extracellular and intracellular HIC-OMP25 on PEA degradation were determined. 0.5 mL of crude enzyme solution (equal to protein content) was preheated with 8.5 mL of 50 mM Tris-HCl buffer (pH 8.5) at 50 °C for 10 min, followed by the addition of 1 mL of 1% PEA-acetone solution. The mixture was then incubated in a 50 °C water bath at 150 rpm for 6 h. The turbidity of the reaction system was measured at 600 nm.

[0045] The results showed that the relative turbidity of PEA after degradation by HIC-OMP25 secreted into the extracellular and intracellular regions of E.coli C43(DE3) was about 23%, indicating that the degradation effect was consistent and that the proteins were well folded.

[0046] The turbidity change of HIC-OMP25 secreted extracellularly by recombinant E. coli BL21(DE3) / pET-20b(+)-hic-omp25 after its action on PEA was similar to that of the blank control without enzyme, indicating that it had almost no degradation effect on PEA. The relative turbidity of PEA after degradation by intracellular HIC-OMP25 was about 50%.

[0047] Example 2

[0048] The thermostability of HiC-OMP25 expressed extracellularly in two different host strains, E. coli BL21(DE3) and E. coli C43(DE3), was further determined.

[0049] When the crude enzyme solutions were placed at 50°C for 12 hours, the residual enzyme activity of HiC-OMP25 secreted and expressed in E. coli C43(DE3) was still close to 100%, but the residual enzyme activity of HiC-OMP25 secreted and expressed in E. coli BL21(DE3) was only 36%.

[0050] The results showed that HiC-OMP25 expressed using E. coli C43(DE3) as the host had good thermal stability, while the reaction temperature for PEA degradation is usually 50℃. Therefore, the expressed HiC-OMP25 could effectively degrade PEA at the reaction temperature.

[0051] Example 3

[0052] The codon sequence encoding the disulfide isomerase DsbC (amino acid sequence as shown in SEQ ID NO.2) was ligated to the polyclonal restriction site of the pET-24a(+) plasmid to construct the recombinant plasmid pET-24a(+)-dsbc; then, the codon sequence encoding the DsbC protein (amino acid sequence as shown in SEQ ID NO.2) and the codon sequence encoding the thiol oxidase Erv1p protein (amino acid sequence as shown in SEQ ID NO.5) were ligated to the polyclonal restriction site of pCDFDuet-1 to construct the recombinant plasmid pCDFDuet-1-erv1p-dsbc.

[0053] The recombinant plasmids pET-20b(+)-hic-omp25 and pET-24a(+)-dsbc obtained were co-transformed into E. coli C43(DE3) by thermal shock.

[0054] The recombinant plasmids pET-20b(+)-hic-omp25 and pCDFDuet-1-erv1p-dsbc obtained were co-transformed into E. coli C43(DE3) by thermal shock.

[0055] The recombinant strain obtained was inoculated into 10 mL of LB liquid medium (containing the corresponding antibiotic) at an inoculation rate of 2‰, and incubated at 37℃ and 200 rpm. -1 Incubate under the specified conditions for 8-12 hours, then add the bacterial culture (OD200). 600 Approximately 7.0) was transferred at a 5% inoculum to 50 mL of TB medium (containing the appropriate antibiotic), and then incubated at 37°C for 200 rpm. -1 After culturing for 2 hours, the culture was transferred to a 25°C constant temperature incubator and cultured for 36 hours. The resulting bacterial culture was then subjected to a process at 8000 rpm. -1 After centrifugation for 15 minutes, the supernatant was collected as the fermentation supernatant. The bacterial cells in the lower precipitate were resuspended in buffer solution and then ultrasonically disrupted using an ultrasonic cell disruptor at 8000 rpm. -1 Centrifuge for 15 minutes. The supernatant is the intracellular supernatant, and the precipitate at the bottom of the centrifuge tube is the cell wall disruption precipitate.

[0056] The enzyme activities of the fermentation supernatant and the cell wall disruption supernatant were measured, and the results are as follows: Figure 1 As shown in the figure. The results indicate that when HiC-OMP25 is co-expressed with DsbC, the extracellular expression level of HiC-OMP25 does not increase significantly, but the intracellular expression level increases by 2.18-fold to 124.77 U·mL. -1 This indicates that DsbC can increase the intracellular expression level of HiC-OMP25. Furthermore, when Erv1p, DsbC, and HiC-OMP25 were co-expressed in E. coli, most of the HiC-OMP25 was secreted extracellularly after 36 hours of fermentation, and the extracellular enzyme activity was 2.44 times higher than that of HiC-OMP25 expressed alone, reaching 135.43 U·mL⁻¹. -1 .

[0057] Example 4

[0058] The codon sequences encoding a disulfide isomerase from Homo sapiens (amino acid sequence shown in SEQ ID NO.3) and the codon sequences encoding a thiol oxidase from Rhinopithecus bieti (amino acid sequence shown in SEQ ID NO.6) were ligated to the polyclonal restriction site of pCDFDuet-1 to construct the recombinant plasmid pCDFDuet-1-erv1p-dsbc.

[0059] The recombinant plasmids pET-20b(+)-hic-omp25 and pCDFDuet-1-erv1p-dsbc obtained were co-transformed into E. coli C43(DE3) by thermal shock.

[0060] The recombinant strain obtained was inoculated into 10 mL of LB liquid medium (containing the corresponding antibiotic) at an inoculation rate of 2‰, and incubated at 37℃ and 200 rpm. -1 Incubate under the specified conditions for 8-12 hours, then add the bacterial culture (OD200). 600 Approximately 7.0) was transferred at a 5% inoculum to 50 mL of TB medium (containing the appropriate antibiotic), and then incubated at 37°C for 200 rpm. -1 After culturing for 2 hours, the culture was transferred to a 25°C constant temperature incubator and cultured for 36 hours. The resulting bacterial culture was then subjected to a process at 8000 rpm. -1 After centrifugation for 15 minutes, the supernatant was collected as the fermentation supernatant. The bacterial cells in the lower precipitate were resuspended in buffer solution and then ultrasonically disrupted using an ultrasonic cell disruptor at 8000 rpm. -1 Centrifuge for 15 minutes. The supernatant is the intracellular supernatant, and the precipitate at the bottom of the centrifuge tube is the cell wall disruption precipitate.

[0061] The enzyme activities in the fermentation supernatant and cell wall disruption supernatant were measured. The results showed that when Erv1p, DsbC, and HiC-OMP25 were co-expressed in E. coli, the extracellular enzyme activity after 36 h of fermentation culture was 1.25 times higher than that when HiC-OMP25 was expressed alone, reaching 88.48 U·mL⁻¹. -1 .

[0062] Example 5

[0063] The codon sequences encoding a disulfide isomerase from Arabidopsis thaliana (amino acid sequence as shown in SEQ ID NO. 4) and a thiol oxidase from Rattus norvegicus (amino acid sequence as shown in SEQ ID NO. 7) were ligated to the polyclonal restriction site of pCDFDuet-1 to construct the recombinant plasmid pCDFDuet-1-erv1p-dsbc.

[0064] The recombinant plasmids pET-20b(+)-hic-omp25 and pCDFDuet-1-erv1p-dsbc obtained were co-transformed into E. coli C43(DE3) by thermal shock.

[0065] The recombinant strain obtained was inoculated into 10 mL of LB liquid medium (containing the corresponding antibiotic) at an inoculation rate of 2‰, and incubated at 37℃ and 200 rpm. -1 Incubate under the specified conditions for 8-12 hours, then add the bacterial culture (OD200). 600 Approximately 7.0) was transferred at a 5% inoculum to 50 mL of TB medium (containing the appropriate antibiotic), and then incubated at 37°C for 200 rpm. -1 After culturing for 2 hours, the culture was transferred to a 25°C constant temperature incubator and cultured for 36 hours. The resulting bacterial culture was then subjected to a process at 8000 rpm. -1 After centrifugation for 15 minutes, the supernatant was collected as the fermentation supernatant. The bacterial cells in the lower precipitate were resuspended in buffer solution and then ultrasonically disrupted using an ultrasonic cell disruptor at 8000 rpm. -1 Centrifuge for 15 minutes. The supernatant is the intracellular supernatant, and the precipitate at the bottom of the centrifuge tube is the cell wall disruption precipitate.

[0066] The enzyme activities in the fermentation supernatant and cell wall disruption supernatant were measured. The results showed that when Erv1p, DsbC, and HiC-OMP25 were co-expressed in E. coli, the extracellular enzyme activity after 36 h of fermentation culture was 1.89 times higher than that when HiC-OMP25 was expressed alone, reaching 113.78 U·mL⁻¹. -1 .

[0067] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims. SEQUENCE LISTING <110> Jiangnan University Shandong Huatai Paper Co., Ltd. <120> A method to increase the expression level of keratinase-anchored peptide fusion protein in Escherichia coli <130> BAA220610A <160> 7 <170> PatentIn version 3.3 <210> 1 <211> 248 <212> PRT <213> Artificial sequence <400> 1 Met Asp Gln Leu Gly Ala Ile Glu Asn Gly Leu Glu Ser Gly Ser Ala 1 5 10 15 Asn Ala Cys Pro Asp Ala Ile Leu Ile Phe Ala Arg Gly Ser Thr Glu 20 25 30 Pro Gly Asn Met Gly Ile Thr Val Gly Pro Ala Leu Ala Asn Gly Leu 35 40 45 Glu Ser His Ile Arg Asn Ile Trp Ile Gln Gly Val Gly Gly Pro Tyr 50 55 60 Asp Ala Ala Leu Ala Thr Asn Phe Leu Pro Arg Gly Thr Ser Gln Ala 65 70 75 80 Asn Ile Asp Glu Gly Lys Arg Leu Phe Ala Leu Ala Asn Gln Lys Cys 85 90 95 Pro Asn Thr Pro Val Val Ala Gly Gly Tyr Ser Gln Gly Ala Ala Leu 100 105 110 Ile Ala Ala Ala Val Ser Glu Leu Ser Gly Ala Val Lys Glu Gln Val 115 120 125 Lys Gly Val Ala Leu Phe Gly Tyr Thr Gln Asn Leu Gln Asn Arg Gly 130 135 140 Gly Ile Pro Asn Tyr Pro Arg Glu Arg Thr Lys Val Phe Cys Asn Val 145 150 155 160 Gly Asp Ala Val Cys Thr Gly Thr Leu Ile Ile Thr Pro Ala His Leu 165 170 175 Ser Tyr Thr Ile Glu Ala Arg Gly Glu Ala Ala Arg Phe Leu Arg Asp 180 185 190 Arg Ile Arg Ala Met Ala Ile Ala Ala Gly Gly Thr Asn Pro Asn Pro 195 200 205 Asn Pro Asn Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Pro Pro 210 215 220 Asp Val Glu Gly Ile Gly Asp Val Asp Leu Val Asn Tyr Phe Glu Val 225 230 235 240 Gly Ala Thr Tyr Thr Phe Asn Lys 245 <210> 2 <211> 236 <212> PRT <213> Escherichia coli <400> 2 Met Lys Lys Gly Phe Met Leu Phe Thr Leu Leu Ala Ala Phe Ser Gly 1 5 10 15 Phe Ala Gln Ala Asp Asp Ala Ala Ile Gln Gln Thr Leu Ala Lys Met 20 25 30 Gly Ile Lys Ser Ser Asp Ile Gln Pro Ala Pro Val Ala Gly Met Lys 35 40 45 Thr Val Leu Thr Asn Ser Gly Val Leu Tyr Ile Thr Asp Asp Gly Lys 50 55 60 His Ile Ile Gln Gly Pro Met Tyr Asp Val Ser Gly Thr Ala Pro Val 65 70 75 80 Asn Val Thr Asn Lys Met Leu Leu Lys Gln Leu Asn Ala Leu Glu Lys 85 90 95 Glu Met Ile Val Tyr Lys Ala Pro Gln Glu Lys His Val Ile Thr Val 100 105 110 Phe Thr Asp Ile Thr Cys Gly Tyr Cys His Lys Leu His Glu Gln Met 115 120 125 Ala Asp Tyr Asn Ala Leu Gly Ile Thr Val Arg Tyr Leu Ala Phe Pro 130 135 140 Arg Gln Gly Leu Asp Ser Asp Ala Glu Lys Glu Met Lys Ala Ile Trp 145 150 155 160 Cys Ala Lys Asp Lys Asn Lys Ala Phe Asp Asp Val Met Ala Gly Lys 165 170 175 Ser Val Ala Pro Ala Ser Cys Asp Val Asp Ile Ala Asp His Tyr Ala 180 185 190 Leu Gly Val Gln Leu Gly Val Ser Gly Thr Pro Ala Val Val Leu Ser 195 200 205 Asn Gly Thr Leu Val Pro Gly Tyr Gln Pro Pro Lys Glu Met Lys Glu 210 215 220 Phe Leu Asp Glu His Gln Lys Met Thr Ser Gly Lys 225 230 235 <210> 3 <211> 420 <212> PRT <213> Homo sapiens <400> 3 Met Ala Pro Trp Pro Pro Lys Gly Leu Val Pro Ala Met Leu Trp Gly 1 5 10 15 Leu Ser Leu Phe Leu Asn Leu Pro Gly Pro Ile Trp Leu Gln Pro Ser 20 25 30 Pro Pro Pro Gln Ser Ser Pro Pro Pro Gln Pro His Pro Cys His Thr 35 40 45 Cys Arg Gly Leu Val Asp Ser Phe Asn Lys Gly Leu Glu Arg Thr Ile 50 55 60 Arg Asp Asn Phe Gly Gly Gly Asn Thr Ala Trp Glu Glu Glu Asn Leu 65 70 75 80 Ser Lys Tyr Lys Asp Ser Glu Thr Arg Leu Val Glu Val Leu Glu Gly 85 90 95 Val Cys Ser Lys Ser Asp Phe Glu Cys His Arg Leu Leu Glu Leu Ser 100 105 110 Glu Glu Leu Val Glu Ser Trp Trp Phe His Lys Gln Gln Glu Ala Pro 115 120 125 Asp Leu Phe Gln Trp Leu Cys Ser Asp Ser Leu Lys Leu Cys Cys Pro 130 135 140 Ala Gly Thr Phe Gly Pro Ser Cys Leu Pro Cys Pro Gly Gly Thr Glu 145 150 155 160 Arg Pro Cys Gly Gly Tyr Gly Gln Cys Glu Gly Glu Gly Thr Arg Gly 165 170 175 Gly Ser Gly His Cys Asp Cys Gln Ala Gly Tyr Gly Gly Glu Ala Cys 180 185 190 Gly Gln Cys Gly Leu Gly Tyr Phe Glu Ala Glu Arg Asn Ala Ser His 195 200 205 Leu Val Cys Ser Ala Cys Phe Gly Pro Cys Ala Arg Cys Ser Gly Pro 210 215 220 Glu Glu Ser Asn Cys Leu Gln Cys Lys Lys Gly Trp Ala Leu His His 225 230 235 240 Leu Lys Cys Val Asp Ile Asp Glu Cys Gly Thr Glu Gly Ala Asn Cys 245 250 255 Gly Ala Asp Gln Phe Cys Val Asn Thr Glu Gly Ser Tyr Glu Cys Arg 260 265 270 Asp Cys Ala Lys Ala Cys Leu Gly Cys Met Gly Ala Gly Pro Gly Arg 275 280 285 Cys Lys Lys Cys Ser Pro Gly Tyr Gln Gln Val Gly Ser Lys Cys Leu 290 295 300 Asp Val Asp Glu Cys Glu Thr Glu Val Cys Pro Gly Glu Asn Lys Gln 305 310 315 320 Cys Glu Asn Thr Glu Gly Gly Tyr Arg Cys Ile Cys Ala Glu Gly Tyr 325 330 335 Lys Gln Met Glu Gly Ile Cys Val Lys Glu Gln Ile Pro Glu Ser Ala 340 345 350 Gly Phe Phe Ser Glu Met Thr Glu Asp Glu Leu Val Val Leu Gln Gln 355 360 365 Met Phe Phe Gly Ile Ile Ile Cys Ala Leu Ala Thr Leu Ala Ala Lys 370 375 380 Gly Asp Leu Val Phe Thr Ala Ile Phe Ile Gly Ala Val Ala Ala Met 385 390 395 400 Thr Gly Tyr Trp Leu Ser Glu Arg Ser Asp Arg Val Leu Glu Gly Phe 405 410 415 Ile Lys Gly Arg 420 <210> 4 <211> 187 <212> PRT <213> Arabidopsis thaliana <400> 4 Met Phe Arg Leu Tyr Pro Asn Cys Ser Leu Pro Ser His Arg Pro Leu 1 5 10 15 Val Phe Leu Pro Arg Leu Pro Ser Arg Ser Leu Arg Cys Arg Ala Ala 20 25 30 Ala Asp Ile Pro Leu Gly Asp Gly Ile Arg Leu Pro Arg Glu Ala Asp 35 40 45 Ser Thr Ser Asp Thr Ala Arg Ser Arg Asp Val Ser Val Ala Ala Gly 50 55 60 Gly Asn Gly Glu Gly Ala Lys Trp Arg Lys Arg Arg Leu Leu Trp Ser 65 70 75 80 Lys Ser Gly Glu Ser Tyr Leu Val Asp Asp Gly Asp Ala Leu Pro Leu 85 90 95 Pro Met Thr Tyr Pro Asp Thr Ser Pro Val Ser Pro Asp Val Ile Asp 100 105 110 Arg Arg Leu Gln Cys Asp Pro Val Val Glu Asp Cys Arg Glu Val Val 115 120 125 Tyr Glu Trp Thr Gly Lys Cys Arg Ser Cys Gln Gly Ser Gly Thr Val 130 135 140 Ser Tyr Tyr Lys Lys Arg Gly Lys Glu Val Ile Cys Lys Cys Ile Pro 145 150 155 160 Cys Gln Gly Ile Gly Tyr Val Gln Lys Ile Thr Ser Arg Thr Asp Ile 165 170 175 Glu Val Met Glu Asp Leu Asp Asn Glu Pro Ser 180 185 <210> 5 <211> 189 <212> PRT <213> Saccharomyces cerevisiae <400> 5 Met Lys Ala Ile Asp Lys Met Thr Asp Asn Pro Pro Gln Glu Gly Leu 1 5 10 15 Ser Gly Arg Lys Ile Ile Tyr Asp Glu Asp Gly Lys Pro Cys Arg Ser 20 25 30 Cys Asn Thr Leu Leu Asp Phe Gln Tyr Val Thr Gly Lys Ile Ser Asn 35 40 45 Gly Leu Lys Asn Leu Ser Ser Asn Gly Lys Leu Ala Gly Thr Gly Ala 50 55 60 Leu Thr Gly Glu Ala Ser Glu Leu Met Pro Gly Ser Arg Thr Tyr Arg 65 70 75 80 Lys Val Asp Pro Pro Asp Val Glu Gln Leu Gly Arg Ser Ser Trp Thr 85 90 95 Leu Leu His Ser Val Ala Ala Ser Tyr Pro Ala Gln Pro Thr Asp Gln 100 105 110 Gln Lys Gly Glu Met Lys Gln Phe Leu Asn Ile Phe Ser His Ile Tyr 115 120 125 Pro Cys Asn Trp Cys Ala Lys Asp Phe Glu Lys Tyr Ile Arg Glu Asn 130 135 140 Ala Pro Gln Val Glu Ser Arg Glu Glu Leu Gly Arg Trp Met Cys Glu 145 150 155 160 Ala His Asn Lys Val Asn Lys Lys Leu Arg Lys Pro Lys Phe Asp Cys 165 170 175 Asn Phe Trp Glu Lys Arg Trp Lys Asp Gly Trp Asp Glu 180 185 <210> 6 <211> 144 <212> PRT <213> Rhinopithecus bieti <400> 6 Met Gly Asp Gly Arg Lys Arg Glu Gln Gly Asp Ala Gly Arg Pro Gly 1 5 10 15 Ala Gly Gly Ala Ala Arg Leu Tyr Arg Ala Gly Glu Asp Ala Val Trp 20 25 30 Val Leu Asp Ser Gly Ser Val Arg Gly Ala Thr Ala Asn Ser Ser Ala 35 40 45 Ala Trp Leu Val Gln Phe Tyr Ser Ser Trp Cys Gly His Cys Ile Gly 50 55 60 Tyr Ala Pro Thr Trp Arg Ala Leu Ala Gly Asp Val Arg Asp Trp Ala 65 70 75 80 Ser Ala Ile Arg Val Ala Ala Leu Asp Cys Met Glu Glu Lys Asn Gln 85 90 95 Ala Val Cys His Asp Tyr Asp Ile His Phe Tyr Pro Thr Phe Arg Val 100 105 110 Ser Ile Thr Gly Cys Leu Ser Val Glu Ile His Leu Leu Pro His Leu 115 120 125 Pro Gly Glu Tyr His Gly Val Ser Cys Met Leu Lys Phe Ala Thr Ser 130 135 140 <210> 7 <211> 139 <212> PRT <213> Rattus norvegicus <400> 7 Met Thr Asp Gly Thr Gly Asn His Ser Lys Gln Thr Arg Asp Asp Arg 1 5 10 15 Phe Ile Thr Glu Arg Asp Ile Lys Phe Arg Glu Asn Cys Pro Gln Asp 20 25 30 Arg Glu Glu Leu Gly His Asn Thr Trp Pro Phe Leu His Thr Leu Ala 35 40 45 Ser Tyr Tyr Leu Asp Met Pro Arg Pro Glu Gln Gln Gln Asp Met Ala 50 55 60 Gln Phe Met His Ile Phe Ser Lys Phe Tyr Pro Cys Glu Glu Cys Ala 65 70 75 80 Glu Asp Ile Arg Lys Arg Lys Gly Arg Ser Gln Pro Asp Thr Ser Thr 85 90 95 Arg Val Ser Phe Ser Gln Trp Leu Cys Cys Leu His Ser Glu Val Asn 100 105 110 Arg Lys Leu Gly Lys Pro Asp Phe Asp Cys Ser Lys Ile Asp Glu Leu 115 120 125 Trp Arg Glu Gly Trp Lys Asp Gly Ser Cys Asp 130 135

Claims

1. A recombinant *Escherichia coli* strain for producing the fusion protein HiC-OMP25, characterized in that, The fusion protein HiC-OMP25, with the amino acid sequence shown in SEQ ID NO.1, is expressed in Escherichia coli C43(DE3). It also expresses a disulfide isomerase, or co-expresses a disulfide isomerase and a thiol oxidase. The amino acid sequence of the disulfide isomerase is shown in any one of SEQ ID NO.2–4; the amino acid sequence of the thiol oxidase is shown in any one of SEQ ID NO.5–7.

2. The recombinant Escherichia coli according to claim 1, characterized in that, Nucleotides encoding HiC-OMP25 are expressed using expression vectors; and genes encoding disulfide isomerases are expressed using expression vectors, or genes encoding disulfide isomerases and thiol oxidases are co-expressed using expression vectors; the expression vectors include, but are not limited to, pET series, pRSFDuet, pCDFDuet, and pETDuet.

3. A method for producing HiC-OMP25, characterized in that, The method uses the recombinant Escherichia coli described in any one of claims 1 to 2 as the production strain to ferment and produce HiC-OMP25.

4. The method according to claim 3, characterized in that, The recombinant Escherichia coli was cultured in LB medium to obtain a seed culture, which was then inoculated into TB medium and cultured at 35–40°C and 150–200 rpm for 1–3 h. After that, the culture was transferred to a constant temperature incubator at 20–28°C and cultured for 30–40 h to obtain a fermentation broth. The fermentation broth was centrifuged and the supernatant was collected, which was the crude enzyme solution containing extracellular HiC-OMP25. The cells were then lysed and centrifuged to collect the supernatant, which was the crude enzyme solution containing intracellular HiC-OMP25.

5. A method for degrading PEA, characterized in that, The method involves using HiC-OMP25, obtained by fermentation of any one of the recombinant Escherichia coli according to claims 1 to 2, to degrade PEA.

6. The method according to claim 5, characterized in that, The crude enzyme solution containing HiC-OMP25 obtained by fermentation of the recombinant Escherichia coli according to any one of claims 1 to 2 is added to the system containing PEA and reacted at a temperature of not less than 40°C and 100 to 150 rpm.

7. The method according to claim 6, characterized in that, The crude enzyme solution containing HiC-OMP25 is first preheated at a temperature not lower than 40°C, and then added to the system containing PEA.

8. A method for increasing the extracellular expression level of HiC-OMP25, characterized in that, The disulfide isomerase and thiol oxidase are simultaneously expressed in HiC-OMP25 cells; the amino acid sequence of the disulfide isomerase is shown in any one of SEQ ID NO. 2 to 4; the amino acid sequence of the thiol oxidase is shown in any one of SEQ ID NO. 5 to 7.

9. The use of the recombinant Escherichia coli according to any one of claims 1 to 2 in the production of keratinase or the degradation of polymers.

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