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Abstract Increasing amounts of antibiotic resistant bacteria have been an emergency problem. Antimicrobial peptides are promising antibiotic alternatives for broadspectrum antimicrobial activity and nearly no drug resistance. Natto peptide was a new antimicrobial peptide which consisted of 45 amino acids. In this study， to improve the antimicrobial activity of Natto peptide， three repeats of encoding sequences were synthesized and cloned into a pET28a （+） expression vector， and expressed in Escherichia coli as a soluble protein. Unexpectedly， the purified 3×Natto peptide exhibited antimicrobial activity against Listeria monocytogenes （50 μg/ml）and Salmonella enteriditis （30 μg/ml）. Furthermore， the antibacterial spectrum of 3×Natto peptide was not affected by temperature， pH value and proteinase digestion. Taken together， this was the first study proving that 3×Natto peptide could be produced in E. coli as a kind of water dissolve protein， and has great potential for commercial application in the future.
Key words Antimicrobial peptide; Antimicrobial activity; Escherichia coli; Heterogenous expression; Natto peptide
Antimicrobial peptides （AMPs）， which are promising antibiotic alternatives， have broadspectrum antimicrobial activities againstfungi， viruses， parasites， bacteria and many antibioticresistantstrains[1-4]. Natto is a kind of common Japanese fermentation food， which was considered as a functional food because it is highly effective in dissolving blood clots， lowering blood pressure， inhibiting proliferation of microorganisms and tumor cells[5-10]. Recently， a new peptide isolated from soybean fermentation products inhibited tumor cell proliferation and exhibited antibacterial activity against Streptococcus pneumoniae and Bacillus subtilis[8]. This Natto peptide consists of 45 amino acids and its molecular weight is about 5 kDa. According to structure analysis， the peptide is with considerable αhelix and similar to the typical cathelicidin family antimicrobial peptides. As we know， the cathelicidin antimicrobial peptides not only possess broad antimicrobial activity against bacteria， fungi， and enveloped viruses but also possess angiogenesis and wound healing function[11-14].
Compared with traditional isolation methods， recombinant expression is a promising way to produce in largescale manner. Due to easy DNA manipulation， low cultivation cost， high expression level and less production time， Escherichia coli is favorable for protein expression. In this study， E. coli was used as a host to express the multimeric Natto peptide containing three copies of Natto peptide. After being expressed in E. coli， the tandem multimers were purified by NiNTA affinity column and the antibacterial potency and stability were then analyzed. Materials and Methods
Bacteria， plasmids and biochemical regents
E. coli XL1blue and Rosetta （DE3） strains were used for amplifying plasmids and expressing protein respectively. Salmonella enteriditis （ATCC 10467）， S. aureus （ATCC 25923）， Listeria monocytogenes （ATCC 21633）， E. coli O157 （ATCC 35150）， were purchased from American Type Culture Collection. pET28a （+） vector was used to carry out Natto peptide expression. All the biochemical regents used for vector construction and protein purification were purchased from Thermo fisher and GE Healthcare.
Construction of plasmids
The 3×Natto peptide encoding sequences were optimized for expression in E. coli and synthesized by Genewiz （Suzhou， China）. The sequences had an EcoRI restriction enzyme site and a XhoI restriction enzyme site located at 5′ and 3′end， respectively. At first， the pET28a （+） vector and 3×Natto peptide fragment were digested by EcoRI and XhoI restriction enzymes， respectively. Then the DNA fragments were purified by gel extraction kit and ligated by T4 ligase， resulting in a pET28a3×Natto peptide vector.
Expression and affinity purification of 6×His3×Natto peptide
A single colony of E. coli Rosetta （DE3） containing pET28a3×Natto peptide vector was picked up and cultured in 200 ml LB medium containing kanamycin （120 μg/ml） at 37 ℃ with shaking at 220 rpm for 12 h incubation. 50 ml of start culture were transferred into 1 L of medium and incubated at 37 ℃ with shaking at 220 rpm. Cells harvested from 1 L of culture were resuspended in 20 ml of lysis buffer （50 mM Hepes， 0.5 M NaCl， 5 mMMgCl2， 1 mM PMSF， 10% glycerol， 20 mM imidazole， 4 mM βmercaptoethanol， 1 mg/ml lysozyme， pH 7.4） and incubated on ice for 15 min. The cell lysate was sonicated by a Soniprep （JY96IIN， Scientz， Ningbo， China） and centrifuged with 16 000 g for 10 min at 4 ℃ to discard the insoluble materials. The supernatant protein was mixed with equilibrated Ni balance column at 4 ℃ for 6 h， then washed with lysis buffer extensively and eluted with elution buffer （containing 500 mM imidazole）.
SDSPAGE analysis
The SDSPAGE analysis procedure was the same as described by Dong[15].
Minimum inhibition concentration assay
A variety of 3×Natto peptide concentration （10-120 μg/ml） were diluted with PBS. The 96well plate were added 20 μl of each dilution per well. The midlog tested bacteria （2×105-8×105 CFU/ml） were distributed to the 96well plate with 100 μl of suspension per well. Then the plate was incubated 12 h with 220 rpmat 37 ℃. The absorbance at 600 nm related to the bacteria growth was measured by Thermo Fisher Multiskan FC ELISA reader. Inhibition zone assay
The inhibition zone assay was used to test the antimicrobial stability of 3×Natto peptide on S. enteriditis （ATCC 10467） with different environmental conditions as described by Dong[15].
Results
Construction of the pET28a3×Natto peptide vector
The 3×Natto peptide consisted of three copies of Natto peptide encoding sequences （Fig. 1A）. The constructed vector of pET28a （+） contained an Nterminal 6×His tag was shown in Fig. 1B.
Expression and purification of 3×Natto peptide
As shown in Fig. 2A， a 18 kDa protein was expressed in the cells as a soluble form. After sonication and NiNTA affinity purification， the eluted fraction was analyzed by SDSPAGE method. A single band was visualized as we expected （Fig. 3C）.
Determination of the antibacterial spectrum of 3×Natto peptide
The antibacterial spectrums of 3×Natto peptide were determined by minimal growth inhibition concentration （MIC） assays and displayed in Table 1. The lowest MIC of 3×Natto peptide was 30 μg/ml for S. enteriditis （ATCC 10467）. The MICs of recombinant 3×Natto peptide against the other Gram positive strains were 50 μg/ml for L. monocytogenes （ATCC 221633）.
Tolerance of 3×Natto peptide to temperature， pH and proteinase digestion
The 3×Natto peptide which was treated with different temperatures （4， 25， 37， 65 and 90 ℃） showed no effect on the antibacterial activity when the inhibition zones had no difference （Fig. 3A）. As shown in Fig. 3B， the 3×Natto peptide bioactivity was not affected by different pH values （2， 4， 6， 8 and 10） treatments. To analyze what will happen if protease was treat on the 3×Natto peptide， we used papain， pepsin， proteinase K and trypsin to digest the 3×Natto peptide. The results showed the 3×Natto peptide was resistant to proteinase digestion （Fig. 3C）.
Bin DONG. Expression and Purification of Multimeric Natto Peptide in Escherichia coli and Initial Characterization of Its Antimicrobial Activity and Stability
Discussion
3×Natto peptide was constructed to the pET28a （+） expression cassette which used T7 as a strong promoter to initial the transcription and T7Ter as a terminator to stop the transcription. Our results proved that 3×Natto peptide could be expressed in E. coli as an expected molecular weight about 18 kDa for the first time. Since the preparation of Natto peptide from fermentation soybeans by B. subtilis is a high cost and low efficiency way in industrial application. As the fermentation peptide only showed a narrow and weak antimicrobial activity against S. pneumoniae and B. subtilis[9]， therefore we focus on the prokaryotes E. coli bioreactor for largescale preparation of Natto peptide. Many E. coliderived antimicrobial peptides such as hybrid AL32P113[16]and hG31PP113[17]have also been expressed successfully. These results suggested that E. coli might be a suitable host for 3×Natto peptide except for small molecular weight of proteins. To further explore the application of 3×Natto peptide in various environmental conditions， heat treatment， extreme pH change and proteinase digestion were tested on the 3×Natto peptide. The results indicated that the 3×Natto peptide had the potential to be used as a commercial antibiotic alternatives and food preservative in diverse environments.
In conclusion， Natto peptide was successfully expressed with a tandem multimer in E. coli as a soluble protein and exhibited a strong antibacterial activity against L. monocytogenes and S. enteriditis and the antibacterial potency of 3×Natto peptide was not affected by temperature， pH value and proteinase digestion.
References
[1] CHU HL， YIP BS， CHEN KH， et al. Novel antimicrobial peptides with high anticancer activity and selectivity[J]. PLos One， 2015， 10， e0126390.
[2] CONLON JM， SONNEVEND A. Antimicrobial peptides in frog skin secretions[J]. Methods Mol Biol， 2010（618）： 3-14.
[3] LI C， BLENCKE HM， PAULSEN V， et al. Powerful workhorses for antimicrobial peptide expression and characterization[J]. Bioeng Bugs， 2010（1）： 217-220.
[4] RADEK K， GALLO R. Antimicrobial peptides： natural effectors of the innate immune system[J]. Semin Immunopathol， 2007（29）： 27-43.
[5] YAMASHITA T， ODA E， GIDDINGS JC， et al. The effect of dietary Bacillus natto productive protein on in vivo endogenous thrombolysis[J]. Pathophysiol Haemost Thromb， 2003（33）： 138-143.
[6] KIM JY， GUM SN， PAIK JK， et al. Effects of nattokinase on blood pressure： a randomized， controlled trial[J]. Hypertens Res， 2008（31）： 1583-1588.
[7] MURATA D， SAWANO S， OHIKE T， et al. Isolation of antifungal bacteria from Japanese fermented soybeans， natto[J]. J Environ Sci （China）， 2013， 25（Suppl 1）： 127-131.
[8] KITAGAWA M， SHIRAISHI T， YAMAMOTO S， et al. Novel antimicrobial activities of a peptide derived from a Japanese soybean fermented food， Natto， against Streptococcus pneumoniae and Bacillus subtilis group strains[J]. AMB Express， 2017， 7（1）： 127.
[9] CAO XH， LIAO ZY， WANG CL， et al. Evaluation of a lipopeptide biosurfactant from Bacillus natto TK1 as a potential source of antiadhesive， antimicrobial and antitumor activities[J]. Braz J Microbiol， 2009（40）： 373-379.
[10] TAKAHASHI C， KIKUCHI N， KATOU N， et al. Possible antitumourpromoting activity of components in Japanese soybean fermented food， Natto： effect on gap junctional intercellular communication[J]. Carcinogenesis， 1995（16）： 471-476. [11] MATTAR EH， ALMEHDAR HA， YACOUB HA， et al. Antimicrobial potentials and structural disorder of human and animal defensins[J]. Cytokine Growth Factor Rev， 2016（28）： 95-111.
[12] SANG Y， BLECHA F. Antimicrobial peptides and bacteriocins： alternatives to traditional antibiotics[J]. Anim Health Res Rev， 2008（9）： 227-235.
[13] SUAREZCARMONA M， HUBERT P， DELVENNE P， et al. Defensins： "Simple" antimicrobial peptides or broadspectrum molecules[J] Cytokine Growth Factor Rev， 2015（26）： 361-370.
[14] GRONBERG A， ZETTERGREN L， AGREN MS. Stability of the cathelicidin peptide LL37 in a nonhealing wound environment[J]. Acta Derm Venereol， 2011（91）： 511-515.
[15] DONG B， CHENG RQ， LIU QY， et al. Multimer of the antimicrobial peptide Mytichitin——A expressed in， Chlamydomonas reinhardtii， exerts a broader antibacterial spectrum and increased potency[J]. Journal of Bioscience and Bioengineering， 2017： 125（2）： 175–179.
[16] MANLIKA W， SANTHASIRI O， AMORNRAT I， et al. Expression in Escherichia coli of novel recombinant hybrid antimicrobial peptide AL32P113 with enhanced antimicrobial activity， in vitro[J]. Gene， 2018（671）： 1-9.
[17] KUANGTING C， CHIHLUNG W， BAKSAU Y， et al. High level expression and purification of the clinically active antimicrobial peptide P113 in Escherichia coli[J]. Molecules， 2018， 23（4）：1-11.
Key words Antimicrobial peptide; Antimicrobial activity; Escherichia coli; Heterogenous expression; Natto peptide
Antimicrobial peptides （AMPs）， which are promising antibiotic alternatives， have broadspectrum antimicrobial activities againstfungi， viruses， parasites， bacteria and many antibioticresistantstrains[1-4]. Natto is a kind of common Japanese fermentation food， which was considered as a functional food because it is highly effective in dissolving blood clots， lowering blood pressure， inhibiting proliferation of microorganisms and tumor cells[5-10]. Recently， a new peptide isolated from soybean fermentation products inhibited tumor cell proliferation and exhibited antibacterial activity against Streptococcus pneumoniae and Bacillus subtilis[8]. This Natto peptide consists of 45 amino acids and its molecular weight is about 5 kDa. According to structure analysis， the peptide is with considerable αhelix and similar to the typical cathelicidin family antimicrobial peptides. As we know， the cathelicidin antimicrobial peptides not only possess broad antimicrobial activity against bacteria， fungi， and enveloped viruses but also possess angiogenesis and wound healing function[11-14].
Compared with traditional isolation methods， recombinant expression is a promising way to produce in largescale manner. Due to easy DNA manipulation， low cultivation cost， high expression level and less production time， Escherichia coli is favorable for protein expression. In this study， E. coli was used as a host to express the multimeric Natto peptide containing three copies of Natto peptide. After being expressed in E. coli， the tandem multimers were purified by NiNTA affinity column and the antibacterial potency and stability were then analyzed. Materials and Methods
Bacteria， plasmids and biochemical regents
E. coli XL1blue and Rosetta （DE3） strains were used for amplifying plasmids and expressing protein respectively. Salmonella enteriditis （ATCC 10467）， S. aureus （ATCC 25923）， Listeria monocytogenes （ATCC 21633）， E. coli O157 （ATCC 35150）， were purchased from American Type Culture Collection. pET28a （+） vector was used to carry out Natto peptide expression. All the biochemical regents used for vector construction and protein purification were purchased from Thermo fisher and GE Healthcare.
Construction of plasmids
The 3×Natto peptide encoding sequences were optimized for expression in E. coli and synthesized by Genewiz （Suzhou， China）. The sequences had an EcoRI restriction enzyme site and a XhoI restriction enzyme site located at 5′ and 3′end， respectively. At first， the pET28a （+） vector and 3×Natto peptide fragment were digested by EcoRI and XhoI restriction enzymes， respectively. Then the DNA fragments were purified by gel extraction kit and ligated by T4 ligase， resulting in a pET28a3×Natto peptide vector.
Expression and affinity purification of 6×His3×Natto peptide
A single colony of E. coli Rosetta （DE3） containing pET28a3×Natto peptide vector was picked up and cultured in 200 ml LB medium containing kanamycin （120 μg/ml） at 37 ℃ with shaking at 220 rpm for 12 h incubation. 50 ml of start culture were transferred into 1 L of medium and incubated at 37 ℃ with shaking at 220 rpm. Cells harvested from 1 L of culture were resuspended in 20 ml of lysis buffer （50 mM Hepes， 0.5 M NaCl， 5 mMMgCl2， 1 mM PMSF， 10% glycerol， 20 mM imidazole， 4 mM βmercaptoethanol， 1 mg/ml lysozyme， pH 7.4） and incubated on ice for 15 min. The cell lysate was sonicated by a Soniprep （JY96IIN， Scientz， Ningbo， China） and centrifuged with 16 000 g for 10 min at 4 ℃ to discard the insoluble materials. The supernatant protein was mixed with equilibrated Ni balance column at 4 ℃ for 6 h， then washed with lysis buffer extensively and eluted with elution buffer （containing 500 mM imidazole）.
SDSPAGE analysis
The SDSPAGE analysis procedure was the same as described by Dong[15].
Minimum inhibition concentration assay
A variety of 3×Natto peptide concentration （10-120 μg/ml） were diluted with PBS. The 96well plate were added 20 μl of each dilution per well. The midlog tested bacteria （2×105-8×105 CFU/ml） were distributed to the 96well plate with 100 μl of suspension per well. Then the plate was incubated 12 h with 220 rpmat 37 ℃. The absorbance at 600 nm related to the bacteria growth was measured by Thermo Fisher Multiskan FC ELISA reader. Inhibition zone assay
The inhibition zone assay was used to test the antimicrobial stability of 3×Natto peptide on S. enteriditis （ATCC 10467） with different environmental conditions as described by Dong[15].
Results
Construction of the pET28a3×Natto peptide vector
The 3×Natto peptide consisted of three copies of Natto peptide encoding sequences （Fig. 1A）. The constructed vector of pET28a （+） contained an Nterminal 6×His tag was shown in Fig. 1B.
Expression and purification of 3×Natto peptide
As shown in Fig. 2A， a 18 kDa protein was expressed in the cells as a soluble form. After sonication and NiNTA affinity purification， the eluted fraction was analyzed by SDSPAGE method. A single band was visualized as we expected （Fig. 3C）.
Determination of the antibacterial spectrum of 3×Natto peptide
The antibacterial spectrums of 3×Natto peptide were determined by minimal growth inhibition concentration （MIC） assays and displayed in Table 1. The lowest MIC of 3×Natto peptide was 30 μg/ml for S. enteriditis （ATCC 10467）. The MICs of recombinant 3×Natto peptide against the other Gram positive strains were 50 μg/ml for L. monocytogenes （ATCC 221633）.
Tolerance of 3×Natto peptide to temperature， pH and proteinase digestion
The 3×Natto peptide which was treated with different temperatures （4， 25， 37， 65 and 90 ℃） showed no effect on the antibacterial activity when the inhibition zones had no difference （Fig. 3A）. As shown in Fig. 3B， the 3×Natto peptide bioactivity was not affected by different pH values （2， 4， 6， 8 and 10） treatments. To analyze what will happen if protease was treat on the 3×Natto peptide， we used papain， pepsin， proteinase K and trypsin to digest the 3×Natto peptide. The results showed the 3×Natto peptide was resistant to proteinase digestion （Fig. 3C）.
Bin DONG. Expression and Purification of Multimeric Natto Peptide in Escherichia coli and Initial Characterization of Its Antimicrobial Activity and Stability
Discussion
3×Natto peptide was constructed to the pET28a （+） expression cassette which used T7 as a strong promoter to initial the transcription and T7Ter as a terminator to stop the transcription. Our results proved that 3×Natto peptide could be expressed in E. coli as an expected molecular weight about 18 kDa for the first time. Since the preparation of Natto peptide from fermentation soybeans by B. subtilis is a high cost and low efficiency way in industrial application. As the fermentation peptide only showed a narrow and weak antimicrobial activity against S. pneumoniae and B. subtilis[9]， therefore we focus on the prokaryotes E. coli bioreactor for largescale preparation of Natto peptide. Many E. coliderived antimicrobial peptides such as hybrid AL32P113[16]and hG31PP113[17]have also been expressed successfully. These results suggested that E. coli might be a suitable host for 3×Natto peptide except for small molecular weight of proteins. To further explore the application of 3×Natto peptide in various environmental conditions， heat treatment， extreme pH change and proteinase digestion were tested on the 3×Natto peptide. The results indicated that the 3×Natto peptide had the potential to be used as a commercial antibiotic alternatives and food preservative in diverse environments.
In conclusion， Natto peptide was successfully expressed with a tandem multimer in E. coli as a soluble protein and exhibited a strong antibacterial activity against L. monocytogenes and S. enteriditis and the antibacterial potency of 3×Natto peptide was not affected by temperature， pH value and proteinase digestion.
References
[1] CHU HL， YIP BS， CHEN KH， et al. Novel antimicrobial peptides with high anticancer activity and selectivity[J]. PLos One， 2015， 10， e0126390.
[2] CONLON JM， SONNEVEND A. Antimicrobial peptides in frog skin secretions[J]. Methods Mol Biol， 2010（618）： 3-14.
[3] LI C， BLENCKE HM， PAULSEN V， et al. Powerful workhorses for antimicrobial peptide expression and characterization[J]. Bioeng Bugs， 2010（1）： 217-220.
[4] RADEK K， GALLO R. Antimicrobial peptides： natural effectors of the innate immune system[J]. Semin Immunopathol， 2007（29）： 27-43.
[5] YAMASHITA T， ODA E， GIDDINGS JC， et al. The effect of dietary Bacillus natto productive protein on in vivo endogenous thrombolysis[J]. Pathophysiol Haemost Thromb， 2003（33）： 138-143.
[6] KIM JY， GUM SN， PAIK JK， et al. Effects of nattokinase on blood pressure： a randomized， controlled trial[J]. Hypertens Res， 2008（31）： 1583-1588.
[7] MURATA D， SAWANO S， OHIKE T， et al. Isolation of antifungal bacteria from Japanese fermented soybeans， natto[J]. J Environ Sci （China）， 2013， 25（Suppl 1）： 127-131.
[8] KITAGAWA M， SHIRAISHI T， YAMAMOTO S， et al. Novel antimicrobial activities of a peptide derived from a Japanese soybean fermented food， Natto， against Streptococcus pneumoniae and Bacillus subtilis group strains[J]. AMB Express， 2017， 7（1）： 127.
[9] CAO XH， LIAO ZY， WANG CL， et al. Evaluation of a lipopeptide biosurfactant from Bacillus natto TK1 as a potential source of antiadhesive， antimicrobial and antitumor activities[J]. Braz J Microbiol， 2009（40）： 373-379.
[10] TAKAHASHI C， KIKUCHI N， KATOU N， et al. Possible antitumourpromoting activity of components in Japanese soybean fermented food， Natto： effect on gap junctional intercellular communication[J]. Carcinogenesis， 1995（16）： 471-476. [11] MATTAR EH， ALMEHDAR HA， YACOUB HA， et al. Antimicrobial potentials and structural disorder of human and animal defensins[J]. Cytokine Growth Factor Rev， 2016（28）： 95-111.
[12] SANG Y， BLECHA F. Antimicrobial peptides and bacteriocins： alternatives to traditional antibiotics[J]. Anim Health Res Rev， 2008（9）： 227-235.
[13] SUAREZCARMONA M， HUBERT P， DELVENNE P， et al. Defensins： "Simple" antimicrobial peptides or broadspectrum molecules[J] Cytokine Growth Factor Rev， 2015（26）： 361-370.
[14] GRONBERG A， ZETTERGREN L， AGREN MS. Stability of the cathelicidin peptide LL37 in a nonhealing wound environment[J]. Acta Derm Venereol， 2011（91）： 511-515.
[15] DONG B， CHENG RQ， LIU QY， et al. Multimer of the antimicrobial peptide Mytichitin——A expressed in， Chlamydomonas reinhardtii， exerts a broader antibacterial spectrum and increased potency[J]. Journal of Bioscience and Bioengineering， 2017： 125（2）： 175–179.
[16] MANLIKA W， SANTHASIRI O， AMORNRAT I， et al. Expression in Escherichia coli of novel recombinant hybrid antimicrobial peptide AL32P113 with enhanced antimicrobial activity， in vitro[J]. Gene， 2018（671）： 1-9.
[17] KUANGTING C， CHIHLUNG W， BAKSAU Y， et al. High level expression and purification of the clinically active antimicrobial peptide P113 in Escherichia coli[J]. Molecules， 2018， 23（4）：1-11.