Characterization of a New Cell Envelope Proteinase PrtP from

Sep 2, 2016 - The genome of Lactobacillus rhamnosus CGMCC11055 possesses two ... speculated that PrtP was a new type of CEPs from Lb. rhamnosus...
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Characterization of a New Cell Envelope Proteinase PrtP from Lactobacillus rhamnosus CGMCC11055 Tingting Guo,† Xudong Ouyang,† Yongping Xin,† Yue Wang,† Susu Zhang,† and Jian Kong*,† †

State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, P. R. China S Supporting Information *

ABSTRACT: Cell envelope proteinases (CEPs) play essential roles in lactic acid bacteria growth in milk and health-promoting properties of fermented dairy products. The genome of Lactobacillus rhamnosus CGMCC11055 possesses two putative CEP genes prtP and prtR2, and the PrtP displays the distinctive domain organization from PrtR2 reported. The PrtP was purified and biochemically characterized. The results showed that the optimal activity occurred at 44 °C, pH 6.5. pAmidinophenylmethylsulfonyl fluoride obviously inhibited enzymatic activity, suggesting PrtP was a member of serine proteinases. Under the optimal conditions, β-casein was a favorite substrate over αS1- and κ-casein, and 35 oligopeptides were identified in the β-casein hydrolysate, including the phosphoserine peptide and bioactive isoleucine-proline-proline. By analysis of the amino acid sequences of those oligopeptides, proline was the preferred residue at the breakdown site. Therefore, we speculated that PrtP was a new type of CEPs from Lb. rhamnosus. KEYWORDS: Lactobacillus rhamnosus, cell envelope proteinase, domain structure, β-casein hydrolysis, breakdown pattern

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INTRODUCTION Lactic acid bacteria (LAB) have a long history of use in dairy fermentation processes.1 Due to long time habitats in the protein-rich environment, numbers of genes for biosynthesis were lost, which made most of LAB nutritionally fastidious. Thus, they exist in nutritionally rich media or require an exogenous source of amino acids or oligopeptides for optimal growth.2 Since milk is poor in these low-molecular-weight compounds, LAB have evolved complex proteolytic systems to release free amino acids from casein, the most abundant protein in milk.3 In general, the exploitation of casein by LAB is initiated by the cell envelope proteinase (CEP) that degrades the protein into oligopeptides that are subsequently taken up by the cells via specific peptide transport systems for further degradation into oligopeptides and amino acids by a concerted action of various intracellular peptidases.3,4 In these cases, the CEPs play key roles in the LAB growth in milk. In addition, CEPs contribute to the organoleptic properties of the fermented dairy products and the bioactive peptides from milk protein,5,6 such as valine-proline-proline (VPP) and isoleucine-proline-proline (IPP), which inhibit the activity of angiotensin-I-converting enzyme (ACE) to regulate blood pressure.7 The CEPs from dairy starters, including Lactococcus lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. lactis, and Lb. helvetics have been extensively investigated because of their strong proteolytic activities in milk fermentation processes.8−13 Recently, the CEPs from probiotic lactobacilli, such as Lb. casei, Lb. paracasei, and Lb. rhamnosus, have also attracted great interest.14,15 Comparative genomics analysis revealed that the number of CEP genes in LAB varied from one to four in a strain-specific manner.4 The simultaneous presence of two or more CEPs could improve the efficiency of substrate breakdown.13 These CEPs could be encoded either by genome or plasmid and were originally synthesized in the cytoplasm as © XXXX American Chemical Society

prepro-proteinases of approximately 2,000 amino acid residues which were organized into one type of several functional domains, including a catalytic domain; an insert domain, possibly modulating the substrate specificity; a B domain involved in stabilizing the CEP activity; and a helix domain, positioning the other domains outside the bacterial cells.3,16 Caseins are divided into αS1-, αS2-, β-, and κ-caseins.17 CEPs have a strong preference for hydrolyzing caseins. On the basis of degradation patterns of caseins, two CEP specificity classes have been described in lactococci: CEPI and CEPIII.18 The favorite substrate of CEPI-type is β-casein, and, to a lesser extent, κ-casein, while the CEPIII-type is able to cleave three kinds of caseins equally.19 For lactobacilli, CEPI-, CEPIII-, the intermediate PI/PIII-type, and some novel type substrate specificities have been reported.11 Lb. rhamnosus is a commonly nonstarter lactobacillus strain in fermented dairy products, and more importantly, it is globally consumed as probiotics.20,21 Comparative genomic analysis revealed that the genes involved in biosynthesis of at least 15 amino acids were devoid in the Lb. rhamnosus genome (http://www.genome.jp/kegg-bin/show_pathway?lrh01230). Therefore, the CEPs are indispensable to allow the maximum growth of Lb. rhamnosus in milk. Based on five whole genomes available in GenBank, two types of CEP genes (prtP and prtR2) are annotated in Lb. rhamnosus. The PrtR2 has been genetically and biochemically characterized in Lb. rhamnosus BGT10, an isolate from human vaginal tissue.22 According to the sequence alignment, the PrtP and PrtR2 showed only 30% identity, suggesting that the PrtP might be a new group of CEPs from Lb. rhamnosus. However, little is known about the PrtP. Hence, Received: July 28, 2016 Revised: August 30, 2016 Accepted: September 2, 2016

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DOI: 10.1021/acs.jafc.6b03379 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

CATTTGGAAACAGATGCTAA-3′) and RT-16sR (5′-CAGTTACTCTGCCGACCATT-3′). Amino acid sequences were aligned using Clustal Omega. The phylogenetic tree of the PrtP and 16 LAB CEPs was constructed with MEGA 6.06. Proteinase Release and Purification. Cells grown in CDM for 14 h were harvested by centrifugation at 10,000g for 10 min at 4 °C, and then washed twice with 0.85% (w/v) saline supplemented with 10 mM CaCl2. The enzyme protein was extracted by suspending the cells in one-tenth of the original volume in 50 mM Tris-HCl (pH 7.5; buffer A) for 30 min at 37 °C. Then, the supernatant was recovered by centrifugation at 10,000g for 20 min at 4 °C. These operations were repeated three times, and the three supernatants were pooled and used as a crude enzyme extract. The crude enzyme extract was precipitated by 45% saturation with ammonium sulfate powder. The precipitate was collected by centrifugation at 10,000g for 10 min at 4 °C and dissolved in buffer A. After dialysis against buffer A for desalination, the enzyme in the solution was purified on a pre-equilibrated HiTrap Q HP column (GE) with a linear gradient of 0−1 M NaCl in buffer A. Fractions with proteinase activity were collected and further purified on a Sephadex G200 column (GE) with buffer A. Proteinase purity was analyzed by 12% SDS-PAGE and native-PAGE. For native-PAGE analysis, protein samples were separated on 12% SDS-PAGE gel containing 0.025% βcasein (w/v). After electrophoresis, the gel was washed with 2.7% (v/ v) Triton X-100 at room temperature for 90 min, and incubated for 12 h at 37 °C in reaction buffer containing 20 mM Tris-HCl, 13.5 mM CaCl2, 50 mM NaCl (pH 7.0). At the end of the incubation, the gel was stained with 0.1% Coomassie Brilliant Blue. Areas of caseinolytic activity appeared as clear zones against a dark blue background. The purified proteinase in a SDS-PAGE was blotted onto a polyvinylidene difluoride membrane, and its N-terminal amino acid sequence was determined by Edman degradation on a PPSQ-33A sequencer (Sangon Biotech, China). Protein Content Measurement and Enzyme Activity Assay. Protein concentration was measured using a BCA protein assay kit (Sangon Biotech, China) with bovine serum albumin as the standard protein. The activity of CEP was assessed by the chromogenic substrate succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (Sigma) according to a method described previously.24 One unit of proteinase activity was defined as the amount required to liberate 1 μmol of nitroaniline per minute. Specific activity was expressed in units of proteinase activity per mg protein. To analyze whole-cell proteinase activity, cells grown in CDM were harvested, washed, and resuspended as described for proteinase extraction to make a cell suspension. The level of cell lysis was determined by the release of lactate dehydrogenase (LDH), whose activity was detected according to a method described previously.25 Effects of pH (from 4.0 to 9.5), temperature (from 24 to 52 °C), various metal ions, p-amidinophenylmethylsulfonyl fluoride (PMSF), and chelators on the enzyme activity were measured. Purified enzyme solutions were incubated for 30 min at temperature intervals to assess the thermal stability of the enzyme. Caseinolytic Specificity and β-Casein Breakdown Pattern. As substrates, αS1-, β-, and κ-casein (Sigma) were respectively dissolved in 50 mM Tris-HCl (pH 6.5) at a concentration of 5 mg/mL. The casein was incubated with 1 μM PrtP at 44 °C. After incubation for the appropriate time, samples were analyzed by Tricine-SDS-PAGE.26 The products were separated by RP-HPLC (Shimaduz) on an XBridge BEH300 C18 reverse phase column (150 × 4.6 mm; Waters). The molecular masses of the β-casein hydrolysate were then determined by liquid chromatography−mass spectrometry (LC/MS). The sequences of these released peptides were identified using MASCOT MS/MS Ion Research tools and ExPASy tools. Nucleotide Sequence Accession Number. The GenBank accession number for the prtP nucleotide sequence reported in this study is KX061418.

the aim of this work was to purify PrtP protein from Lb. rhamnosus CGMCC11055 and to characterize its domain organization, enzymatic property, as well as substrate preference and cleavage pattern, so as to enable this strain to optimally grow in milk and produce bioactive health-beneficial peptides from milk.

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MATERIALS AND METHODS

Bacterial Strains, Media, and Growth Conditions. Lb. rhamnosus CGMCC11055 was isolated from a Chinese artisanal yoghurt sample collected from Xinjiang Province and deposited in the China General Microbiological Culture Collection Center. The strain was routinely grown anaerobically in MRS broth or agar at 37 °C, and was stored at −80 °C in MRS broth supplemented with 15% (v/v) glycerol. For determination of proteolytic activity, bacterial cells grown to the late exponential phase in MRS broth were harvested by centrifugation at 6,000g for 5 min, and washed twice in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and then resuspended in PBS buffer to the original volume. Then, the cell suspension was inoculated in a chemically defined medium (CDM; Table 1). The cell growth was monitored by the change of optical density at 600 nm (OD600).

Table 1. Composition of Chemically Defined Medium for Lb. rhamnosus CGMCC11055 Constituent

Concentration (g/L)

Glucose CH3COONa K2HPO4 MgSO4 MnSO4 FeSO4 Tween 80 L-Arginine L-Asparagine L-Cysteine L-Glutamic acid L-Isoleucine L-Phenylalanine L-Serine L-Threonine L-tryptophan L-Tyrosine L-Valine Pyridoxal

10 5 3 0.2 0.05 0.02 1 0.1 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.002

Cloning and Transcriptional Level of CEP Genes from Lb. rhamnosus CGMCC11055. The genes prtP and prtR2 were PCR amplified from the genomic DNA of Lb. rhamnosus CGMCC11055 with primers PrtP-F (5′-AACTATATCAAGCCAAGGTTTG-3′) and PrtP-R (5′-AACCATAGATTCATAACCGAG-3′) and primers PrtR2F (5′-TTTCTTAATACCATTTAAG-3′) and PrtR2-R (5′-TCCTTCCTTTCCTATTCAAC-3′), respectively, which were designed according to the five sequenced genomes of Lb. rhamnosus strains. The PCR products were sequenced by the Biosune Company (Shanghai, China). Transcriptional levels of prtP and prtR2 were detected after Lb. rhamnosus CGMCC11055 was cultivated in CDM to the early logarithmic phase (8 h) by real-time qPCR according to a previous method.23 The prtP and prtR2 transcripts were amplified with primers RT-PrtPF (5′-GCAATACTACCTGTTACG-3′) and PR-PrtPR (5′GACCAGTTGAATCATAATAAG-3′) and RT-PrtR2F (5′ATTAGCAGACGGCGACA-3′) and PR-PrtR2R (5′-GTGGAAGACTTGGTGACT-3′), respectively. The 16S rRNA gene transcript used as an internal standard was amplified with primers RT-16sF (5′B

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RESULTS CEP Activity Analysis of Lb. rhamnosus CGMCC11055. It was reported that the CEP activities of cells grown in the

peptide-rich medium MRS were remarkably reduced compared with those in a synthetic medium.27 Here, to assess the CEP activity of Lb. rhamnosus CGMCC11055, a specific CDM (Table 1) was adopted to cultivate this strain. As shown in Figure 1, the proteolytic activity of the cell suspension was the highest at the end of the exponential growth phase (14 h), and it began to decrease at the stationary phase. To eliminate the interference of the intracellular proteinases, the lactate dehydrogenase (LDH), an intracellular enzyme, was used as a control. It was found that the LDH activity was undetectable (data not shown) in cell suspension, confirming the proteolytic activity detected was due to the action of CEPs rather than intracellular proteinases. Cloning and Transcriptional Level Analysis of CEP Genes from Lb. rhamnosus CGMCC11055. To ensure the active expression of the two CEP genes annotated in the Lb. rhamnosus genome, DNA fragments containing prtP and prtR2 were PCR amplified from strain CGMCC11055 genome. The resultant two deduced amino acid sequences shared 98.8% and 99.6% identity with PrtPs and PrtR2s from the five genomesequenced Lb. rhamnosus strains, respectively (Figure S1 and Figure S2), indicating the CEP genes were conserved in Lb.

Figure 1. Cell growth (■), pH values (●), and CEP activity (white column) of Lb. rhamnosus CGMCC11055 grown in chemically defined medium (CDM). Error bars show standard deviations of three independent experiments.

Figure 2. Sequence analysis of PrtP from Lb. rhamnosus CGMCC11055. (A) Schematic diagram of the domain structure of the PrtP. The possible catalytic triad, Asp26, His90, and Ser429, is indicated using arrows. (B) Alignment of the putative LPxTG motif containing region of the PrtP with those found at the C-terminus of surface proteins of several Gram-positive bacteria strains. (C) Comparison of domain organization among the PrtP, PrtR2 from Lb. rhamnosus BGT10, PrtL from Lb. delbrueckii subsp. lactis CRL581, and PrtP from L. lactis SK11. (D) Phylogenetic map of the PrtP and 16 LAB CEPs. The tree was constructed using the maximum likelihood method with MEGA6.06. C

DOI: 10.1021/acs.jafc.6b03379 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Purification of PrtP from Lb. rhamnosus CGMCC11055 cultured in CDM. (A) SDS-PAGE (left) and native-PAGE (right) analysis of the crude enzyme protein precipitated by ammonium sulfate. (B) Purification of the PrtP by ion-exchange chromatography on a HiTrap Q HP column. Solid line, protein concentration; dotted line, relative activity; dashed line, NaCl concentration. (C) Final purification step of the PrtP by gel filtration chromatography using FPLC (AKTA purifier, GH Healthcare). The sample was applied to a Sephadex G200 column (10 × 300 mm) and eluted with 50 mM Tris-HCl (pH 7.5) at a flow rate of 0.4 mL/min. The PrtP peak is indicated by an arrow. (D) SDS-PAGE (left) and native-PAGE (right) analysis of the purified PrtP.

rhamnosus. Real-time qPCR showed that the CT values of prtP, prtR2, and the internal standard 16S rRNA gene were 20.02 ± 0.30, 32.37 ± 0.41, and 15.75 ± 0.12 with 1000-fold diluted cDNA of strain CGMCC11055 as template. According to the principle of real-time qPCR, prtP was normally transcribed, while prtR2 was not. Thus, we speculated that the CEP activity detected above mainly came from PrtP when strain CGMCC11055 was cultivated in CDM. Genetic Organization of PrtP. The open reading frame of prtP from strain CGMCC11055 was 5,922 bp in length and encoded an S8 proteinase precursor with 1,973 deduced amino acid residues. As shown in Figure 2A, the PrtP consisted of a 33-residues signal peptide sequence (Met−190-Ala−158), a propeptide (Ala−157-Ala−1), an S8 catalytic domain (Asn1Lys503), an A domain (Ala504-Ser917), a B domain (Thr918Pro1394), an H domain (Ala1395-Gln1609), a W domain (Pro1610Gly1697), and an AN domain (Thr1698-Ser1783). A 153-residues I domain (Thr227-Pro379) and a possible catalytic triad (Asp26, His90, and Ser429) were predicted in the catalytic domain. The AN domain contained the conserved sorting signal LPxTG motif (Figure 2B), suggesting the covalent attachment of the proteinase to the cell wall.3 Therefore, as shown in Figure 2C, the domain structure of PrtP differed from PrtR2 from Lb. rhamnosus BGT10 with a smaller B domain and without I, H domains and PrtL from Lb. delbrueckii subsp. lactis CRL581 without H, AN domains. PrtP from strain CGMCC11055 had the same domain organization as PrtP from L. lactis SK11, while the I domain composition of the two PrtPs was different

(81% sequence identity). Phylogenetic analysis (Figure 2D) indicated that PrtP from strain CGMCC11055 had a distant relationship with PrtR2 from Lb. rhamnosus BGT10, while a close relationship with PrtPs from L. lactis SK11 and Lb. casei BL23. Purification and Biochemical Characterization of PrtP. The CEP of strain CGMCC11055 was extracted using Ca2+free Tris-HCl buffer. The cell-free extracts were fractionated by ammonium sulfate, and the precipitate was analyzed by SDSPAGE and native-PAGE (Figure 3A). After the crude enzyme preparation was applied to ion-exchange chromatography, most of the proteinase activity was detected in the fraction eluted with 0.12 M NaCl (Figure 3B). The enzyme obtained from the Sephadex G200 column step (Figure 3C) showed a single protein band (over 170 kDa) in the SDS-PAGE and nativePAGE gels (Figure 3D). Approximately 9.2-fold purification was achieved, with a final recovery of 11.0% of the original activity. The first 15 amino acids at the N-terminus of purified proteinase were NSMANVQAVWSNYKY, matching the deduced amino acid sequence of PrtP from residues 191 to 205. These results confirmed that the purified proteinase was the mature form of PrtP from strain CGMCC11055. The PrtP displayed the highest proteolytic activity at pH 6.5, while the activity rapidly decreased at pH > 7.5 (Figure 4A). The optimal temperature for activity was 40−48 °C (Figure 4B). The enzyme was stable when incubated for 30 min at 30 and 40 °C at pH 6.5, while only 20% activity remained when incubated at 50 °C or higher (Figure 4C). D

DOI: 10.1021/acs.jafc.6b03379 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. Time course of hydrolysis of β-casein (A) and αS1-casein (B) by the PrtP using Tricine-SDS-PAGE analysis. M, protein marker. Lanes 0 to 4 show analysis of samples taken after addition of the PrtP for 0, 1, 2, 3, and 4 h, respectively.

Cu2+, Sr2+, Li+, and Sn2+ (Table 2). The chelators EDTA and EGTA also inhibited its activity. The activity was almost completely abolished by PMSF, a serine proteinase inhibitor, indicating that the PrtP was a member of the serine proteinases. Casein Hydrolysis. The hydrolytic ability of the PrtP to αS1-, β-, and κ-caseins was tested. β-Casein was degraded after 1 h incubation at the optimal conditions, and almost completely digested after 4 h; eight to ten protein bands in the hydrolysates could be visualized by Tricine-SDS-PAGE (Figure 5A). Compared with β-casein, αS1-casein was hydrolyzed at a lower rate, and only a small amount of oligopeptides was released (Figure 5B). No κ-casein hydrolysis was observed (data not shown). These results suggested that β-casein was the preferential substrate over αS1- and κ-caseins. β-Casein Breakdown Pattern. To study the breakdown pattern of β-casein by the PrtP, the hydrolysate was analyzed by RP-HPLC (Figure S3) and MS. 35 released oligopeptides with 3 to 16 amino acid residues were identified (Table 3), including a phosphoserine peptide and the bioactive isoleucine-prolineproline (IPP). Based on the amino acid sequences of those oligopeptides, cleavage sites of β-casein mediated by the PrtP were not concentrated at the N- or C-terminus, but dispersed throughout the β-casein sequence (Figure 6). Analysis of the amino acid residue frequencies at cleavage sites was shown in Table 4. The carbonyl position was almost occupied by a hydrophobic residue, while the amino position was more diverse, including hydrophobic and charged residues. Proline was the most frequent residue at cleavage sites at both the carbonyl and amino positions.

Figure 4. Effect of pH (A) and temperature (B) on the activity of the PrtP, and residual activity of the PrtP after 30 min incubation at temperatures ranging from 30 to 90 °C. Data are representative of three independent experiments.

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DISCUSSION Casein is the most abundant protein in milk.28 To adapt to the milk environment, LAB have to depend on the proteolytic

2+

The proteolytic activity of the PrtP was enhanced by Mg , Zn2+, Co2+, and Ca2+, and inhibited by K+, Ba2+, Fe2+, Mn2+,

Table 2. Effects of Metal Ions and Inhibitors on the Proteolytic Activity of PrtP Relative activity Metal ion Control Mg2+ K+ Ba2+ Zn2+ Fe2+ Co2+

1 mM (%) 100 109.1 ± 2.33 78.9 ± 1.89 82.3 ± 1.32 107.8 ± 2.11 81.5 ± 0.93 111.7 ± 1.46

2 mM (%) 100 125.4 ± 2.56 65.5 ± 1.48 69.2 ± 1.03 121.6 ± 2.18 56.6 ± 1.49 122.4 ± 2.55

Relative activity Metal ion 2+

Mn Cu2+ Ni2+ Ca2+ Sr2+ Li+ Sn2+

1 mM (%)

2 mM (%)

Inhibitor (1 mM)

Residual activity (%)

78.2 ± 1.34 76.9 ± 0.0.88 104.3 ± 2.16 105.5 ± 1.87 93.4 ± 1.35 89.2 ± 0.78 76.5 ± 1.43

55.8 ± 0.67 65.3 ± 1.22 109.5 ± 2.56 132.3 ± 2.71 90.2 ± 1.69 77.9 ± 1.02 54.3 ± 1.24

Control PMSF EDTA EGTA

100 12.4 ± 0.65 44.6 ± 0.41 51.5 ± 1.29

E

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Journal of Agricultural and Food Chemistry Table 3. Peptides Released from β-Casein by PrtP at 40°C

Table 4. Amino Acid Residue Frequencies at Cleavage Sites by PrtP on β-Casein

Mass (Da) Measured 977.50 759.38 977.50 977.50 954.39 1884.10 824.43 840.39 1884.10 1810.10 1720.03 325.66 868.57 408.25 1273.75 1047.46 768.95 909.90 484.23 1033.54 802.45 1290.60 1485.05 868.57 868.57 782.50 1033.54 759.38 802.56 1720.03 868.57 1033.54 1017.58 840.39 995.60

Expected 977.14 759.96 977.07 977.11 954.08 1884.03 823.05 839.07 1884.03 1810.14 1719.95 325.41 868.20 407.51 1273.51 1047.02 768.93 909.07 483.53 1032.33 802.01 1290.34 1484.68 868.16 868.16 781.07 1032.40 759.96 802.07 1719.95 868.13 1032.33 1016.29 839.08 995.28

Peptide sequence SEESITRI TRINKK QTEDELQN TEDELQNK NKIHPFAQ TQSLVYPFPGPIPNS SLVYPFP PFPGPIPN PNSLPQNIPPLTQTP LPQNIPPLTQTPVVVPP NIPPLTQTPVVVPP IPP PVVVPPFL FLQ PEVMGVSKVKEA VMGVSKVKEA PKHKEM PFTESQSL FTES VENLHLPLP LQSWMH FPPQSVLSLSQS QSVLSLSQSKVLPV QSKVLPVP SKVLPVPQ VLPVPQK PVPQKAVPYP QRDMPI PIQAFLL IQAFLLYQEPVLG IQAFLLY LLYQEPVLG LYQEPVLGP QEPVLGPV QEPVLGPVR

Position

amino acid

carbonyl site

amino acid

amino site

19−26 (1P) 24−29 40−47 41−48 48−54 55−69 57−63 61−68 67−81 70−86 73−86 74−76 81−88 87−89 90−101 92−101 104−109 118−125 119−122 130−138 140−145 157−168 160−173 167−174 168−175 170−176 172−181 182−187 186−192 187−199 188−193 191−199 192−200 194−201 194−202

Pro Gln Leu Ile Val Phe Ser Thr Asn

8 6 4 3 3 3 3 3 2

Pro Gln Leu Ser Lys Val Ile Asn Ala Gly Tyr Met His Arg

9 3 3 3 3 2 2 2 2 2 1 1 1 1

Lb. rhamnosus CGMCC11055, isolated from a Chinese artisanal yoghurt sample, showed CEP activity in the nitrogenlimited medium (Figure 1), suggesting that the CEP biosynthesis was modulated by the nitrogen source in the medium, as reported in other lactobacilli.24,29,30 Strain CGMCC11055 possesses two CEP genes, while only the prtP was transcribed in CDM, indicating that the CEP activity of strain CGMCC11055 was due to the action of PrtP. Although two CEP genes were also annotated in the genome, Lb. rhamnosus GG displayed no CEP activity.31 According to our results, the reason yielding this phenomenon might be silence of the CEP genes at the transcriptional level under nitrogen-limited conditions. The initially identified CEP from L. lactis and the subsequently characterized CEPs from lactobacilli were normally organized in several functional domains,16,19,29 so was the PrtP. The same domain organization was predicated in PrtP from strain CGMCC11055 and PrtP from L. lactis SK11, but they had different I domain compositions (81% sequence identity), which might yield different substrate specificity.16 The domain organization of PrtR2 from Lb. rhamnosus BGT10 was markedly different from the PrtP, indicating distinctive substrate specificity and enzymatic activity between the PrtP and the PrtR2.3,16 In addition, the sorting signal LPxTG motif was identified in the AN domain, suggesting covalent attachment of the PrtP to the cell envelope. 32 This phenomenon was further confirmed through the extraction of PrtP from strain CGMCC11055 cells by repeated treatment using a Ca2+-free buffer. Ca2+-free buffer was thought to induce the loss of weakly bound Ca2+ ions from CEP, leading to the exposure of a sequence that was highly susceptible to autoproteolytic attack.11,33 After multistep purification, the purified PrtP appeared as a single band in SDS-PAGE and native-PAGE gels in a mature form, suggesting that the enzyme consisted of a single subunit. The molecular mass of PrtP was >170 kDa, similar to that of PrtPs from L. lactis SK11, Wg2, and H2,19 while larger than that of PrtR2 from Lb. rhamnosus BGT10 and CEPs from Lb. rhamnosus OXY, Lb. delbrueckii subsp. lactis CRL581, Lb. helveticus CP790, and S. thermophilus CNRZ385.11,22,34−36 Enzymatic properties analysis indicated that the PrtP showed the optimal proteolytic activity at pH 6.5 and 40−48 °C, similar to the CEP from Lb. rhamnosus OXY.34 However, the optimum pH of some of the CEP purified from LAB, such as L. lactis

Figure 6. Location of the peptides released by hydrolysis by the PrtP in the primary structure of β-casein.

systems to degrade casein into oligopeptides or free amino acids for optimal growth.3,4,19 Here, a CEP, PrtP, from Lb. rhamnosus CGMCC11055 was identified, and genetically and biochemically characterized. The results showed that PrtP was a member of serine proteinases, and played main roles in bacterial growth. Moreover, it could hydrolyze β-casein to release 35 oligopeptides, including a phosphoserine peptide and the bioactive tripeptide IPP. All evidence further supported the robustness in milk as well as the probiotic properties of Lb. rhamnosus. F

DOI: 10.1021/acs.jafc.6b03379 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry LB12 and Lb. delbureckii subsp. lactis CRL 581, was in the range from 7 to 8.11,37 Moreover, the PrtP was more thermolabile than the CEP from L. lactis LB12;37 it retained only 20% activity after incubation at 50 °C for 30 min. The activity of the PrtP could be enhanced by Mg2+, Zn2+, Co2+, and Ca2+, indicating that this enzyme was a metallopeptidase. Moreover, its activity was inhibited by PMSF and chelators. Thereby, we hypothesized the PrtP was a member of serine proteinase and could bind the divalent cations. According to the I domain composition, different substrate specificity was speculated between PrtPs from L. lactis SK11 and strain CGMCC11055, which was confirmed by the experimental evidence. PrtP from L. lactis SK11 was classified as CEPIII-type, enabling degradation to three kinds of caseins.19 However, in the case of PrtP from strain CGMCC11055, βcasein could be completely hydrolyzed after incubation for 4 h, while the hydrolysis efficiency was much lower with αS1-casein as substrate, and no hydrolysis activity against κ-casein was observed. This substrate specificity was similar to that of PrtL from Lb. delbrueckii CRL 581,11 although the PrtL and PrtP from strain CGMCC11055 showed different domain organization. The reason might be that β-casein contained more unstructured domains than αS1- and κ-caseins, rendering it more easily accessible to cleavage than the others.38 The βcasein cleavage sites by PrtP from strain CGMCC11055 were diverse, as suggested for other CEPs from lactobacilli.15,24,38 This feature was thought to confer an advantage for supplying more oligopeptides or amino acids essential for bacterial maximum growth.38 In addition, breakdown sites were distributed along the whole β-casein sequence, which was different from PrtR2 from Lb. rhamnosus BGT10, as well as most of the previously described LAB CEPs preferentially targeted the C-terminus of β-casein.15 Thus, these properties could yield the differences between the texture and flavor of the final fermented products by the strains CGMCC11055 and BGT10. By comparative analysis of the amino acid sequences of the oligopeptides released, the breakdown site of the PrtP was a hydrophobic residue on the carbonyl position, particularly proline, while glutamine and (or) phenylalanine were favorite cleavage sites for PrtR2 from strain BGT10 and CEPs from Lb. helveticus, Lb. paracasei, and Lb. delbrueckii.15,24 Among the 35 oligopeptides obtained from the β-casein hydrolysate by the PrtP, most of them have not been identified before, especially oligopeptide f19-26 at the N-terminus of β-casein, which was identified for the first time. Therefore, a new cleavage site between two phosphorylated serine residues was found. Since the phosphorylated N-terminus of β-casein was resistant to hydrolysis,15 this result made a contribution to the knowledge about CEPs from lactobacilli. Moreover, oligopeptide f74-76 (IPP), known to lower the systolic blood pressure by inhibition of ACE,24,39 was also released by the action of PrtP, indicating Lb. rhamnosus CGMCC11055 was an ideal candidate to ferment milk with antihypertensive activity. In conclusion, this study provided knowledge about the CEP from a Lactobacillus species. The unique domain organization, caseinolytic specificity, and breakdown pattern of β-casein demonstrated that PrtP represented a new type of CEP from Lb. rhamnosus.

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Multiple sequence alignments, and RP-HPLC profiles of casein hydrolysate (PDF)

AUTHOR INFORMATION

Corresponding Author

*Jian Kong Mailing address: 27 Shanda Nanlu, Jinan 250100, P. R. China. E-mail: [email protected]. Tel: +86 531 88362318. Fax: +86 531 88565234. Funding

This work was supported by National Natural Science Foundation of China (31471715), Public Service Sectors (Agriculture) Special and Scientific Research Projects (201503134), and Project funded by China Postdoctoral Science Foundation (2015T80709). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ACE, angiotensin-converting enzyme; CDM, chemically defined medium; CEP, cell envelope proteinase; CGMCC, China General Microbiological Culture Collection Center; EDTA, ethylene diamine tetraacetic acid; EGTA, ethylene glycol tetraacetic acid; IPP, isoleucine-proline-proline; LAB, lactic acid bacteria; LDH, lactate dehydrogenase; LC/MS, liquid chromatography/mass spectrometry; PCR, polymerase chain reaction; PMSF, p-amidinophenylmethylsulfonyl fluoride; RP-HPLC, reverse phase high performance liquid chromatography; VPP, valine-proline-proline

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REFERENCES

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DOI: 10.1021/acs.jafc.6b03379 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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DOI: 10.1021/acs.jafc.6b03379 J. Agric. Food Chem. XXXX, XXX, XXX−XXX