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Efficient extracellular expression of metalloprotease for Z-aspartame synthesis Fucheng Zhu, Feng Liu, Bin Wu, and Bingfang He J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04164 • Publication Date (Web): 02 Dec 2016 Downloaded from http://pubs.acs.org on December 5, 2016

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Journal of Agricultural and Food Chemistry

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Efficient extracellular expression of metalloprotease for

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Z-aspartame synthesis

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Fucheng Zhu†, Feng Liu†, Bin Wu†, Bingfang He†‡*

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†

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No. 30 Puzhu South Road, Nanjing, 211816, China.

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‡

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Road, Nanjing, 211816, China.

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,

School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 Puzhu South

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*

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E-mail: [email protected]; Phone: 86-25-58139902; Fax: 86-25-58139902

Author to whom correspondence should be addressed

1

ACS Paragon Plus Environment


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ABSTRACT

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Metalloprotease PT121 and its mutant Y114S (Tyr114 was substituted into Ser)

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are effective catalysts for synthesis of Z-aspartame (Z-APM). In this study, selection

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of a suitable signal peptide for improving expression and extracellular secretion of

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protease PT121 and Y114S by Escherichia coli was presented. Co-inducers

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containing IPTG and arabinose were used to promote the proteases production and

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cell growth. Under optimal conditions, the expression levels of PT121 and Y114S

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reached over 500 mg/L, and the extracellular activity of PT121/Y114S accounted for

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87%/82% of the total activity of proteases. Surprisingly, purer protein was obtained in

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the supernatant, because arabinose reduced the cell membrane permeability and

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avoided cell lysis. Comparison of Z-APM synthesis and caseinolysis between protease

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PT121 and Y114S showed that mutant Y114S presented remarkably higher activity of

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Z-APM synthesis and considerably lower activity of caseinolysis. The significant

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difference in substrate specificity render these enzymes promising biocatalysts.

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KEYWORDS: Extracellular expression, metalloprotease, Escherichia coli，arabinose,

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Z-aspartame

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INTRODUCTION Aspartame (L-aspartyl-L-phenylalanine methyl ester, APM), is a protected

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dipeptide sweetener, which is 200 times sweeter than sucrose.1 Since its approval,

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APM has been widely used as a low calorie sweetener in soft drinks and food

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products.2 More than 19,000 metric tons of APM are produced every year, making it

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the most highly synthesized peptide in the world.3 APM can be easily obtained by

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deprotection of carboxybenzyl from Z-aspartame

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(N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester, Z-APM).4 Members of

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metalloprotease family M4 (also known as the thermolysin family) such as

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thermolysin and pseudolysin, as efficient biocatalysts, were utilized to catalyze the

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synthesis of Z-APM from N-carbobenzoxy-L-aspartic acid (Cbz-Asp) and

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L-phenylalanine methyl ester (Phe-OMe).5 A previous study developed mutant Y114S,

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which can efficiently catalyze the synthesis of Z-APM, by site-directed mutagenesis

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of protease PT121 from Pseudomonas aeruginosa.6 However, due to the limited

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expression level, these metalloproteases become one of the limiting factors in Z-APM

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enzymatic preparation.

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Various alkaline proteases have been produced in industrial scale.7 However, to

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the best of our knowledge, the expression levels of many thermolysin family

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metalloproteases, such as thermolysin-like protease and pseudolysin, are still limited.

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In Bacillus subtilis and Pichia pastoris host, extracellular expression of thermolysin

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family proteases can reach 10–50 mg/L.8-10 As the most commonly used host,

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Escherichia coli is also utilized to express thermolysin family metalloproteases, but

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most of these enzymes are expressed either in intracellular form, ranging from 6 mg/L

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to 20 mg/L,11-13 or in inclusion body form.14 Because of its high proteolysis, matured

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proteases located inside cells could cause recombinant cell growth inhibition, or even 3



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cell lysis. Targeting the protein to the culture medium may overcome this obstacle.15

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Additionally, extracellular expression may facilitate downstream processing and

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achieve higher production.16 Previously, we have demonstrated the expression of

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metalloprotease PT121 and its mutant Y114S with improved activity of Z-APM

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synthesis.6 However, expressed PT121 proteases were mainly obtained as intracellular

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form and the expression level remained low. Therefore, exploring effective approach

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for improving extracellular expression is necessary.

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Many approaches have been reported to facilitate extracellular secretion of

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proteins in E. coli. These studies have included almost every aspect of the secretion

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process, such as utilization of different secretion pathways,17 construction of leaky

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strains,18 co-expression of the key secretion components,19 as well as optimization of

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culture conditions.20 Nevertheless, extracellular expression of the recombinant

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proteins in E. coli is still a daunting and challenging task. Signal peptide plays an

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important role in the expression and secretion of proteins.21 The nucleotide sequence

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encoding signal peptide affects 5′mRNA secondary structure of the translation

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initiation region (TIR), which was confirmed to play a crucial role in the efficiency of

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gene expression.22 The stability of mRNA secondary structure is quantified as the

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value of minimal fold free energy (∆G). By increasing the ∆G of TIR from position –

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35 to +36, the human tumor necrosis factor α and extracellular domain of Her2/neu

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protein were over expressed in E. coli.23 Moreover, because the expressed protease

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has toxic effect on cell due to high proteolytic activity, promoting cell growth is

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another key factor for protease expression. Increasing evidence indicates that

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arabinose promotes cell growth for overexpression of penicillin acylase.24 Xu et al.

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reported that the arabinose efficiently reduced the amount of inclusion bodies and

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significantly improved the activity of penicillin acylase.25 To our best knowledge, no 4


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study has reported on arabinose-induced expression of protease. In the present study, replacement of signal peptide was applied to promote the

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extracellular expression of metalloprotease PT121 and its mutant Y114S. Arabinose

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supplementation significantly increased the expression level of proteases and

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improved the purity of secreted proteases. Possible mechanisms are also discussed.

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Additionally, the substrate specificity of PT121 and Y114S in the catalyzed synthesis

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of Z-APM and caseinolytic activity was investigated, and rationalized by assessing

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kinetic parameters and conducting molecular dynamic simulation.

88 89

MATERIALS AND METHODS

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Chemicals, Reagents, and Materials

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N-carbobenzoxy-L-aspartic acid (Cbz-Asp) and L-phenylalanine methyl ester

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(Phe-OMe) were obtained from GL Biochem Co., Ltd (GLS, Shanghai, China).

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O-nitrophenyl-β-D-galactopyranoside (ONPG) and N-phenyl-α-naphthylamine (NPN)

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were purchased from Aladdin Co., Ltd (Aladdin, Shanghai, China). All primers were

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synthesized at Invitrogen (Invitrogen, Shanghai, China). DNA polymerase, restriction

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enzymes, and T4 DNA ligase were purchased from Takara (Takara, Dalian, China).

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Molecular Techniques

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All primers used in this study are shown in Table S1. Genomic DNA of P.

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aeruginosa PT121 was used as template for PCR.26 The 1497 bp fragment of gene

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lasB was amplified with cds-F and cds-R as the nucleotide primers. The amplified

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DNA product was inserted into the pMD-18T vector (Takara, Dalian, China). The

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recombinant plasmid was used as a template for PCR. The 1425 bp fragment

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containing the gene encoding the pro-peptide and mature peptide was amplified using

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the primers lasB-F1 and lasB-R1. Thus, the original pelB signal sequence of pET-22b 5



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(Takara, Dalian, China) was retained. The product of PCR was digested with

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restriction enzymes Nco I and BamH I and then ligated into pET-22b. Primers lasB-F2

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and lasB-R2 were used to obtain the DNA product with its native signal peptide by

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PCR. The product was digested with the restriction enzymes Nde I and BamH I and

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then ligated into pET-22b. As a result, the native signal peptide of metalloprotease

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PT121 was retained. The nucleotide sequence of signal peptide ompA was fused to

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the lasB gene without its native signal peptide by overlap PCR using the primer-rF1,

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primer-rR1, primer-rF2 and primer-rR2. Then the constructed fragment was cloned

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into vector pET-22b. The recombinant pET-22b harboring the inserted gene sequence

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was transformed into E. coli BL21 (DE3). Mutant Y114S was generated using the

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QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA), and the

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recombinant plasmid was transformed into E. coli BL21. The correct clones were

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selected for DNA sequencing (GenScript, Nanjing, China).

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Medium and Culture Conditions

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The transformed BL21 was inoculated in 5 mL of LB medium and grown with

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shaking at 37 °C overnight to serve as the seed. A 2% (v/v) concentration of the

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inoculum was inoculated in 50 mL of LB medium containing 100 µg/mL ampicillin

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and then incubated at 37 °C until an optical density at 600 nm (OD600) reached 1.0.

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Then, 10 µM isopropyl thio-β-D-galactopyranoside (IPTG) was added to induce

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protein expression at 30 °C for 24 h. The E. coli transformed with pET-22b was taken

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as the control group for analysis.

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The pooled supernatant was dialyzed against 50 mM Tris-HCl (pH 8.0) buffer to

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remove salt and other small molecules, and then the sample was submitted for

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assessment of its casein hydrolysis activity. The purity and molecular weight of the

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target protein were measured using SDS-PAGE with 12.5% separating gel as 6


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described by Laemmli.27

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Purification of Protease

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The protease purification was performed as described by Tang et al.28 with slight

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modification. The culture supernatant was harvested by centrifugation at 12,000×g

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and 4 °C for 10 min and loaded onto a Phenyl Sepharose column (GE Healthcare,

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Uppsala, Sweden), which was equilibrated with 50 mM Tris-HCl (pH 8.0) containing

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1.0 M NaCl. After binding, the column was eluted with 50 mM Tris–HCl (pH 8.0) at

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a flow rate of 1.0 mL/min. The absorbance of eluent at 280 nm was measured, and the

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active fractions were collected and dialyzed for further research. Bradford’s method

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was used for assaying the protein concentration.

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Assay of Caseinolytic Activity

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The activity of the supernatant toward casein was measured as previously

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described.26 Two milliliters of the diluted protease were added to 2 mL Tris-HCl

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buffer (50 mM, pH 8.0) containing 2% (w/v) casein. The reaction mixture was

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incubated at 40 °C for 10 min and terminated by adding 4.0 mL of TCA mixture

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containing trichloroacetic acid (0.11 M), sodium acetate (0.22 M) and acetic acid

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(0.33 M). The mixture was then centrifuged at 12,000×g for 10 min. The absorbance

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at 280 nm of the reaction supernatant was measured against a blank control. The

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amount of enzyme that hydrolyzes casein and produces 1 µg of tyrosine per min is

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defined as one unit (U) of protease activity. All above assays were performed in

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triplicates for calculating the mean and standard deviation.

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Effect of IPTG and Arabinose on Expression of Proteases

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To examine the effect of IPTG and arabinose on expression of protease PT121

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and Y114S, IPTG (0-100 µM) and arabinose (0–5.0 mg/mL) were applied to induce

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the expression of proteases, respectively. The flask cultures were further shaken at 30 7



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°C for another 24 h. Then, the cultures were centrifuged at 4 °C and 12,000×g for 10

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min. The supernatant of culture was assayed for extracellular caseinolytic activity.

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The cell pellet was resuspended in Tris-HCl buffer (50 mM, pH 8.0). The cell

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suspension was sonicated for 5 min by using ultrasonic processor, and then

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centrifuged at 12,000×g and 4 °C for 5 min. The supernatant of cell extracts was

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assayed for intracellular protease activity. All above assays were performed in

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triplicates for calculating the mean and standard deviation.

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Determination of Cell Lysis

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Cell lysis was determined as the percentage of extracellular activity versus the

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total activity containing extracellular and intracellular portion by using

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β-galactosidase as the reporter protein.18, 29 Activity of β-galactosidase was assayed as

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previously reported.30

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Assay of Membrane Permeability

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Permeability of the inner membrane was determined by measuring the influx of

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ONPG, a substrate of cytosolic β-galactosidase. β-galactosidase localized within the

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cytoplasm can hydrolyze ONPG that passes the inner membrane and resulting in the

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absorbance at 420 nm. Thus, increase rate of the absorbance indicates the

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permeability of inner membrane. In this study, the permeability of inner membrane

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was assayed by measuring the access of ONPG to the cytoplasm in accordance with a

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previously reported method with minor modifications.31 The recombinant E. coli

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BL21 containing the constructed plasmid was obtained by centrifugation (12,000×g),

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rinsed once, and then suspended in 10 mM sodium phosphate buffer (pH 7.4) to an

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OD600 of 0.15. ONPG with final concentration of 100 µg/mL was added to an ELISA

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plate containing 200 µL of the cell suspension. Substrate cleavage by β-galactosidase

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was measured by light absorption every 3 min at 420 nm in a microplate reader 8


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(BioTek, Vermont, USA).

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The hydrophobic fluorescent probe NPN was used as an indicator of outer

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membrane integrity. NPN has a low fluorescence quantum yield in aqueous solution

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but its fluorescence is strong in the hydrophobic environment of cell membrane.

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Normally, NPN is excluded from the lipopolysaccharide layer of the outer membrane

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but can enter at points where membrane integrity is compromised. Thus, the

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permeability of outer membrane can be indicated by fluorescence value and its

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increased rate. Outer membrane permeability was measured according to previously

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report with minor modifications .31 NPN with final concentration of 10 µM was added

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to a quartz cuvette containing 3 mL of cell suspension. Fluorescence absorption was

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assayed using a spectrofluorometer Shimadzu RF-1501 (Shimadzu, Kyoto, Japan)

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with 5 nm of slit widths. The emission and excitation wavelengths were set to 420 and

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350 nm, respectively.

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Kinetic Constants of Recombinant PT121 and Y114S

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The initial rate of tyrosine formation, presented as the initial rate of caseinolysis,

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were determined at a fixed concentration of purified enzyme (0.1 mg/mL) and at

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different concentrations of casein (0.5–10.0 mg/mL) for 5 min. All above assays were

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performed in triplicates for calculating the mean and standard deviation. The

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determined value was plotted by non-linear fitting using origin 9.0.

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To measure the kinetic parameters of the synthetic reaction for Z-APM, various

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Cbz-Asp and Phe-OMe concentrations of 10–100 mM at a fixed concentration 500

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mM Phe-OMe and 100 mM Cbz-Asp were used as substrates, respectively. Purified

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enzyme (0.9 µM) was added into the reaction mixture, and the reaction system was

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shaken at 37 °C and 180 rpm for 1 h. All samples were analyzed by high-performance

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liquid chromatography (HPLC) as previous reported.6 The kinetic constants KM were 9



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calculated from Lineweaver-Burk plots. We calculated the KM1 of Cbz-Asp (ZD) and KM2 of Phe-OMe (FM) respectively.

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Random rapid equilibration mechanism was applied to calculate the kinetic constants

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of Z-APM synthesis according to previous report.32 Initial reaction rate v0 of Z-APM

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synthesis was obtained at 100 mM Cbz-Asp and 500 mM Phe-OMe. The kcat would be

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obtained by the following equation.

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212 213

v0 =

[E]0 [ZD]0 [FM]0 M1 M2 M1 [FM]0 M2 [ZD]0 [ZD]0 [FM]0

RNA Secondary Structure Prediction The secondary structure and Gibbs free energy (∆G) of the mRNA TIR were

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predicted and calculated at online server

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(http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form).

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Molecular Simulation

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The crystal structure (PDB code: 1EZM) determined by Thayer et al.33 was used

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for the molecular modeling of PT121 and Y114S. The 3D structure of Phe-OMe was

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stripped from the crystal structure of thermolysin (PDB code: 3QGO). Molecular

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docking of the substrate was performed using LeDock (http://lephar.com). The

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software in optimizations of the ligand pose (position and orientation) and its rotatable

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bonds is based on simulated annealing as well as genetic algorithm.34 The protein was

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automatically removed all crystal waters and added polar hydrogen atoms by LePro

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module in the docking suite LeDock. All catalytic residues were included in the active

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pocket. Molecular dynamic simulation was performed using Amber 12 with the

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Amber 99SB force field.35 Ligand topology was generated using the Leap Program.

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Sodium ions were added to maintain system neutrality. The neutral system was

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subjected to energy minimization with the steepest descent of 1000 steps and 10


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conjugate gradient of 2000 steps. Then, the 50,000 steps position-restrained dynamic

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simulation was applied to system at 300 K. Finally, MD was performed for 40-ns at

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300 K. Postprocessing and analysis were performed using standard Amber tools and

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customized scripts.

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RESULTS AND DISCUSSION

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Effect of Signal Peptides on Expression of PT121 and Y114S

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The effect of signal peptides (pelB, ompA and its native signal peptide) on the

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expression of PT121 and Y114S was studied. The recombinant E. coli cells harboring

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Sp-PT121 (BL21/Sp-PT121), ompA-PT121 (BL21/ompA-PT121), pelB-PT121

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(BL21/pelB-PT121) and pelB-Y114S (BL21/pelB-Y114S) were induced for 24 h. In

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Fig. 1, SDS-PAGE shows that most of the protease PT121 and Y114S were

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extracellularly expressed by using pelB or ompA as signal peptide, whereas most of

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the target proteins were intracellularly expressed for Sp-PT121 constructs. The culture

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supernatant protease activity of recombinant BL21/Sp-PT121 only reached 61.6±9.6

244

U/mL, which accounted for 10% of the total activity. By comparison, the signal

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peptides pelB/ompA effectively promoted the extracellular secretion of protease

246

PT121, and the highest expression of protease PT121 using pelB as signal peptide was

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observed as shown in Fig. 1. The protease activities of pelB-PT121 and pelB-Y114S

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in the culture supernatant reached 5200±230 and 2700±170 U/mL, respectively,

249

which constituted 91.5% and 88.4% of the total activity. Even though the

250

concentrations of PT121 and Y114S in supernatant were almost the same, the

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caseinolytic activity of Y114S was only half that of the PT121. This might be due to

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the difference in substrate specificity between protease PT121 and Y114S.

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Signal peptide not only promoted extracellular secretion but also affected the 11



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5′mRNA secondary structure. In the present study, the secondary structures of the

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mRNA TIR (from nucleotide points −35 to +36) of three constructs named TIR-pelB

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(pelB signal peptide), TIR-ompA (ompA signal peptide) and TIR-Sp (native signal

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peptide of protease PT121) were predicted theoretically. The essential architecture of

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the TIR is shown in Fig. 2. The ∆G values of mRNA TIR in TIR-pelB, TIR-ompA

259

and TIR-Sp were −9.5, −9.7 and −10.5 kcal/mol, respectively. Previous studies

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demonstrated that increasing the ∆G of TIR increases protein expression.36, 37 In

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addition, the start AUG codon was demonstrated as a key limiting factor for protein

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expression in E. coli.38 The AUG start codon in TIR-pelB and TIR-ompA were fully

263

exposed, compared with that in TIR-Sp. The exposure of initiator AUG was

264

speculated to reduce the blockade element for gene translation and to improve protein

265

expression. Therefore, the pelB signal peptides were more suitable for secretory

266

expression of protease PT121 and Y114S. To our knowledge, this study is the first

267

report on the extracellular expression of thermolysin-like protease from P. aeruginosa

268

in an E. coli system. The strategies described herein may be feasible for rational

269

selection of a suitable signal peptide for efficient protein expression.

270

Effect of Inducers (IPTG and arabinose) on Expression of Protease PT121

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Mutant Y114S exerts low effect on protein expression. Thus, protease PT121 was

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selected as a model to investigate. IPTG is commonly used as an inducer of protein

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expression regulated by T7 promoter, and arabinose was also able to induce penicillin

274

acylase expression regulated by T7 promoter in E. coli.25 The effects of IPTG and

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arabinose on the expression of protease PT121 were investigated.

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As the concentration of IPTG increased, the expression level increased and a

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maximum value was obtained at 10 µM (Table 1). Further increase of IPTG

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concentration beyond 10 µM significantly decreased the expression level of protease. 12


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On the other hand, the cell density (OD600) significantly decreased even when 5 µM

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IPTG was added. High concentrations of IPTG negatively influenced the expression

281

of the target protein. This result may be attributed to the fact that overexpression of

282

target protease inhibits cell growth or even causes cell lysis.39, 40 Thus, lowering the

283

concentration of IPTG could promote protease expression and cell growth.

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Arabinose has been applied to induce the expression of penicillin acylase,24 but

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whether arabinose can induce protease expression is unclear. In this study, the effects

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of arabinose concentration on cell growth and protease production were investigated

287

(Table 1). The extracellular protease activity and cell density initially increased with

288

increasing arabinose concentration, and then decreased when this concentration

289

exceeded 3.0 g/L. The maximum protease activity achieved 2200 U/mL. Although the

290

protease activity in the arabinose-induced culture was lower than that in the

291

IPTG-induced culture, the cell density was over twofold that when IPTG was used as

292

an inducer. The result indicates that arabinose not only enhances cell growth but also

293

induces protease PT121 expression.

294

Conventionally, cell growth is severely inhibited by protease expression because

295

of its high proteolytic activity.41 To overcome these deficiencies, arabinose and

296

low-concentration IPTG (10 µM) were used as co-inducers for protease expression.

297

As shown in Fig. 3, the supernatant activity and total activity increased with

298

increasing arabinose concentration. The optimum arabinose concentration was 3.0

299

mg/mL, and the total activity reached 11900±500 U/mL, which was 2.1-fold that of

300

the arabinose-free culture. Significantly, the extracellular protease reached 10300±460

301

U/mL (468 mg/L). Similarly, the total activity/OD600 increased when the arabinose

302

concentration was increased from 0.0 mg/mL to 3.0 mg/mL, but remarkably

303

decreased when the concentration was further increased to 4 mg/mL. The maximum 13



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total activity/OD600 was achieved at 3.0 mg/mL arabinose and was 1.7-fold that

305

without arabinose addition. The OD600 was 1.2-fold that without arabinose addition.

306

The production of mutant Y114S was also determined under the same conditions. The

307

total and extracellular activity of Y114S reached 6600±240 U/mL and 5400±200

308

U/mL, respectively, both of which were almost 2.0-fold that of the arabinose-free

309

cultivation. Interestingly, SDS-PAGE gel electrophoresis showed that the proteases in

310

the culture supernatant added with 3.0 g/L arabinose was purer than that in the

311

arabinose-free culture (Fig. 4). In particular, the specific activity of protease PT121 in

312

culture supernatant with 3.0 g/L arabinose was 22,000 U/mg, which was almost equal

313

to that of purified native protease PT121.28 These results suggest that arabinose may

314

prevent cell lysis by decreasing the leakage of intracellular proteins, so that improve

315

the purity of target protease in the culture supernatant. Arabinose enhances the

316

production of penicillin acylase by mediating the processing and correct folding of

317

this enzyme.25, 42 However, the decrease of the leakage of intracellular proteins in E.

318

coli by arabinose has not been reported.

319

In early reports, the full length of the lasB gene with the native signal sequence

320

integrated into vector pUC19 was intracellularly expressed in E. coli JM109 with 23.7

321

U/mL.13 Odunuga et al. lasB gene that only codes for the mature pseudolysin into

322

pET28a and achieved the yield of 40 mg/L in E. coli BL21 as inclusion bodies

323

followed by refolding.43 In P. pastoris, Pseudomonas aeruginosa elastase was

324

successfully expressed and secreted into cultures with 330 U/mL, and the expressed

325

protease was glycosylated.10 In this study, addition of arabinose significantly

326

promoted expression of protease PT121 and kept cells from leakage. High-purity

327

extracellular protein would considerably simplify the process of purification. This

328

study may provide a useful strategy in expressing proteases. 14


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329 330

Determination of Cell Lysis and Membrane Permeability Extracellular expression of protease may cause hydrolysis of membrane proteins

331

causing cell lysis. To explore this possibility, β-galactosidase was taken as the

332

reporter protein. β-Galactosidase is a cytoplasmic protein, and its level in the culture

333

medium is very low under normal conditions; thus, the presence of extracellular

334

β-galactosidase activity would be an indication of cell lysis.18, 29 In this study, 91.5%

335

of the total expressed protease PT121 and 13.6% of the total expressed

336

β-galactosidase were found in the arabinose-free culture medium. However, 86.9% of

337

the total expressed protease PT121 and less than 1% of the total β-galactosidase were

338

found in the arabinose-supplemented culture medium, and this amount of

339

β-galactosidase was similar to that of the control E. coli cells harboring pET-22b

340

(BL21/pET22b) without the target gene inserted. The addition of arabinose reduced

341

the percentage of β-galactosidase in the culture supernatant, but the percentage of

342

target protease in the culture supernatant remained almost the same. These results

343

suggest that the presence of extracellular protease is not due to cell lysis. Additionally,

344

supplementation of arabinose could prevent cell lysis.

345

We analyzed cell membrane permeability to further explore the mechanism, by

346

which arabinose prevents cell lysis and increases the purity of the recombinant

347

enzyme. As shown in Fig. 5, under the arabinose-free supplemented culture, the inner

348

and outer membranes permeability of BL21/pelB-PT121 were significantly higher

349

than those of the control BL21/pET22b, which may be the result of hydrolysis by

350

expressed protease. However, when BL21/pelB-PT121 were cultured in

351

arabinose-supplemented LB medium at the inductive phase, inner membrane

352

permeability significantly decreased to a level even lower than that of BL21/pET22b

353

in the culture without addition of arabinose. The outer membrane permeability also 15



Page 16 of 35

354

decreased when arabinose was added. However, the outer membrane still maintained

355

its higher permeability even when arabinose was added. Proteases of the thermolysin

356

family are folded and matured in periplasmic space, and then a correctly folded

357

mature protease is secreted.44 The higher permeability of the outer membrane

358

promoted the secretion of the recombinant protease located in periplasmic space into

359

culture. The inner membrane maintained its lower permeability with the addition of

360

arabinose, and this phenomenon might have two benefits. One is prevention of

361

cytoplasmic protein from leaking out of the cytoplasm to express purer secreted

362

proteases; the other is maintenance of bacterial growth.

363

Mutant Y114S with efficient Synthesis of Z-APM and lower Caseinolysis

364

The activities of Z-APM synthesis and caseinolysis were compared between

365

protease PT121 and its mutant Y114S with the same concentrations of proteins. As

366

shown in Fig. 6, protease PT121 presented higher initial reaction rate of caseinolysis

367

than that of Y114S. The specific activity of PT121 on caseinolysis was 22,000 U/mg,

368

which was almost two fold that of Y114S (12,000 U/mg). The finding results may

369

demonstrate that PT121 has higher hydrolysis activity compared with Y114S.

370

Interestingly, mutant Y114S facilitates efficient synthesis of Z-APM. As shown

371

in Table. 2, v0 and kcat of the Y114S in Z-APM synthesis were 8.2-fold and 8.5-fold

372

those of PT121, respectively, while both KM1 and KM2 of mutant Y114S were slightly

373

declined compared with that of PT121. Mutant Y114S showed remarkably higher

374

activity of Z-APM synthesis, and considerably decreased the activity of caseinolysis.

375

This difference may be the result of alteration in substrate specificity. In the present

376

study, the metalloprotease presented efficient peptide synthesis through site-directed

377

mutagenesis, which may illustrate that the mutation site Tyr114 is a key residue for

378

mediating the substrate specificity of protease PT121. 16


Page 17 of 35


379

To rationalize this difference between PT121 and Y114S, molecular docking was

380

applied to obtain the enzyme–substrate complex as shown in Fig. S1. Molecular

381

dynamic simulation was used to calculate the binding free energies of two enzymes

382

toward Phe-OMe, and the binding free energies were −20.23 and −23.34 kcal/mol for

383

PT121 and Y114S, respectively, by sampling the last 5-ns of trajectories. This result

384

illustrates that mutant Y114S has stronger binding ability to Phe-OMe than that of

385

PT121. Previous studies also reported only one mutation that considerably affects

386

substrate specificity.45 Protease PT121 and its mutant Y114S might be useful in the

387

degradation and engineering of food proteins46 and in the synthesis of bioactive

388

peptides, such as aspartame, respectively. Further work will be conducted to engineer

389

the protease PT121 and Y114S for enhancing yield in the synthesis of other active

390

peptides.

391

In summary, we demonstrated that replacement of the signal peptide promotes

392

the extracellular expression of metalloprotease PT121 and its mutant Y114S. The

393

addition of arabinose significantly promoted the expression of proteases and improved

394

the purity of the proteases in E. coli. In addition, the residue 114 has been well

395

characterized as a key residue to alter substrate specificity. Molecular simulation

396

suggested that residue 114 plays a positive role in substrate binding.

397

ACKNOWLEDGEMENTS

398

This research was supported by the National Natural Science Foundation of

399

China (21376119, 21506099, 81503012) and Specialized Research Fund for the

400

Doctoral Program of Higher Education (20123221130001). We also acknowledge the

401

fund sponsored by the Research Innovation Program for College Graduates of Jiangsu

402

Province (KYZZ_0228).

403

ASSOCIATED CONTENT 17



Page 18 of 35

404

Supporting Information

405

Table S1. Primers used in this study.

406

Figure S1. (A) 3D structure of Y114S-Phe-OMe complex interaction; (B) structure of

407

Phe-OMe in the active center; (C) RMSDs of the Cα atoms for PT121 and Y114S

408

over all trajectories at 300 K (40 ns).

409

The Supporting data is available free of charge on the ACS Publications website.

410 411

AUTHOR INFORMATION

412

Corresponding Author

413

*Corresponding author: Bingfang He. Current address: No. 30 Puzhu South Road,

414

Nanjing 211816, China. Tel/Fax: +86-25-58139902. E-mail:

415

[email protected]

416

18


Page 19 of 35


417

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560

24


Page 25 of 35


561

Legends for Figures

562 563

Figure 1. (A) SDS-PAGE analysis of proteases in the culture supernatant and cell

564

extracted supernatant. Lane M, protein marker; lanes 1-10, extracellular fraction and

565

intracellular fraction of BL21/pET22b (lanes1 and 2), BL21/Sp-PT121 (lanes 3 and 4),

566

BL21/pelB-PT121 (lanes 5 and 6), BL21/pelB-Y114S (lanes 7 and 8) and

567

BL21/ompA-PT121 (lanes 9 and 10); (B) Extracellular and intracellular activities of

568

BL21/pET22b, BL21/Sp-PT121, BL21/pelB-PT121, BL21/pelB-Y114S and

569

BL21/ompA-PT121. The values are the means ± standard deviation from three

570

independent experiments.

571 572

Figure 2. Predicted secondary structure and minimum free energy of the 5′mRNA

573

translation initiation region (TIR), including 36 bases upstream of the start codon and

574

35 bases downstream of the start codon. The start codon AUG was labeled in red.

575 576

Figure 3. Synergistic effect of IPTG and arabinose on protease PT121 production.

577

Extracellular activity filled with grid, total activity filled with black, total

578

activity/OD600 filled with light gray, and pH of supernatant filled with slash. The

579

results are expressed as the means ± standard deviation from three independent

580

experiments.

581 582

Figure 4. SDS-PAGE analysis of proteins in the culture supernatant. Lane M, protein

583

marker; lanes 1 and 3: culture supernatant of BL21/pelB-PT121 and

584

BL21/pelB-Y114S induced by IPTG, lanes 2 and 4: culture supernatant of

585

BL21/pelB-PT121 and BL21/pelB-Y114S induced by IPTG and arabinose. 25



Page 26 of 35

586

Figure 5. Permeability of the inner (A) and outer (B) membranes of E. coli

587

BL21(DE3) containing target plasmid. ■, cultivation of BL21/pET22b added with

588

arabinose; □, cultivation of BL21/pET22b; ▲, cultivation of BL21/pelB-PT121 added

589

with arabinose; and △, cultivation of BL21/pelB-PT121.

590 591

Figure 6. Caseinolytic initial rate at various of casein concentration for PT121(■) and

592

Y114S (●). Experiments were carried out in triplicate, and the results expressed as the

593

means ± standard deviation from three independent experiments.

594

26


Page 27 of 35


Table 1. Expression of protease PT121 under various inducer conditionsa

IPTG (µM)

Arabinose (g/L)

Inducer 0

5

10

50

100

0.5

1

2

3

4

Protease activity

210±6

4900±150

5600±270

4800±150

4100±120

550±17

660±18

1500±45

2200±67

670±17

OD600

5.4±0.2

2.7±0.2

2.6±0.3

2.6±0.2

2.5±0.3

6.8±0.3

7.2±0.3

6.0±0.3

5.3±0.2

5.0±0.1

Protease activity/OD600

39±1.2

1800±50

2100±60

1800±50

1600±30

81±2.5

91±3.2

250±6

420±13

130±3.9

Experiments

were

performed

a

in

triplicate

and

presented

as

the

means

±

standard

deviation.

27



Page 28 of 35

Table 2. Kinetic parameters of Z-APM synthesis of protease PT121 and mutant Y114Sa

v0 (µM·s-1)

KM1 (mM)

KM2 (mM)

kcat (s-1)

Mutant Y114S

7.4 ± 1.2

46 ± 3.2

32 ± 2.1

12.8 ± 0.8

Protease PT121

0.9 ± 0.1

48 ± 3.1

38 ± 2.8

1.5 ± 0.2

Enzymes

a

Z-APM synthesis reaction was carried out in aqueous solvent at 37 °C and pH 6.0 for 60 min using various concentrations (10-100 mM) of Cbz-Asp and Phe-OMe

at fixed 500 mM Phe-OMe and 100 mM Cbz-Asp, respectively. The values are expressed the means ± standard deviation.

28


Page 29 of 35


Figure 1. (A) SDS-PAGE analysis of proteases in the culture supernatant and cell extracted supernatant. Lane M, protein marker; lanes 1-10, extracellular fraction and intracellular fraction of BL21/pET22b (lanes1 and 2), BL21/Sp-PT121 (lanes 3 and 4), BL21/pelB-PT121 (lanes 5 and 6), BL21/pelB-Y114S (lanes 7 and 8) and BL21/ompA-PT121 (lanes 9 and 10); (B) Extracellular and intracellular activities of BL21/pET22b, BL21/Sp-PT121, BL21/pelB-PT121, BL21/pelB-Y114S and BL21/ompA-PT121. The values are the means ± standard deviation from three independent experiment. Figure 1 100x110mm (300 x 300 DPI)



Figure 2. Predicted secondary structure and minimum free energy of the 5′mRNA translation initiation region (TIR), including 36 bases upstream of the start codon and 35 bases downstream of the start codon. The start codon AUG was labeled in red. Figure 2 100x80mm (300 x 300 DPI)


Page 30 of 35

Page 31 of 35


Figure 3. Synergistic effect of IPTG and arabinose on protease PT121 production. Extracellular activity filled with grid, total activity filled with black, total activity/OD600 filled with light gray, and pH of supernatant filled with slash. The results are expressed as the means ± standard deviation from three independent experiment. Figure 3 85x59mm (300 x 300 DPI)



Figure 4. SDS-PAGE analysis of proteins in the culture supernatant. Lane M, protein marker; lanes 1 and 3: culture supernatant of BL21/pelB-PT121 and BL21/pelB-Y114S induced by IPTG, lanes 2 and 4: culture supernatant of BL21/pelB-PT121 and BL21/pelB-Y114S induced by IPTG and arabinose. Figure 4 70x69mm (300 x 300 DPI)


Page 32 of 35

Page 33 of 35


Figure 5. Permeability of the inner (A) and outer (B) membranes of E. coli BL21(DE3) containing target plasmid. ■, cultivation of BL21/pET22b added with arabinose; □, cultivation of BL21/pET22b; ▲, cultivation of BL21/pelB-PT121 added with arabinose; and △, cultivation of BL21/pelB-PT121. Figure 5 80x121mm (300 x 300 DPI)



Figure 6. Caseinolytic initial rate at various of casein concentration for PT121(■) and Y114S (●). Experiments were carried out in triplicate, and the result expressed as the means ± standard deviation from three independent experiment. Figure 6 80x64mm (300 x 300 DPI)


Page 34 of 35

Page 35 of 35


TOC graphic TOC graphic 120x46mm (300 x 300 DPI)


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