微生物学报

Strains and plasmids	Description	Reference
Strains
Escherichia coli DH5α	The strain used for gene cloning
Pyrococcus yayanosii A1	The facultatively piezophilic derivative strain	Li et al.^[23]
Thermococcus kodakarensis TS559	Agmatine auxotrophic strain	Santangelo et al.^[21]
ΔPYCH_07700	PYCH_07700 deletion strain	This study
Plasmids
pTE1	AT.kodakarensis-E.coli (Tk-Ec) shuttle vector	Song et al.^[25]
pTE-Pyprol	pTE1:: P_gdh-PYCH_07700	This study
pTE-Pfprol	pTE1:: P_gdh-PF_1343	This study

Strains and plasmids	Description	Reference
Strains
Escherichia coli DH5α	The strain used for gene cloning
Pyrococcus yayanosii A1	The facultatively piezophilic derivative strain	Li et al.^[23]
Thermococcus kodakarensis TS559	Agmatine auxotrophic strain	Santangelo et al.^[21]
ΔPYCH_07700	PYCH_07700 deletion strain	This study
Plasmids
pTE1	AT.kodakarensis-E.coli (Tk-Ec) shuttle vector	Song et al.^[25]
pTE-Pyprol	pTE1:: P_gdh-PYCH_07700	This study
pTE-Pfprol	pTE1:: P_gdh-PF_1343	This study

Primer	Sequence (5′→3′)
0770-kod-F	CCTAATTTGGAGGGATGAACGTGAAAGATAGAATTAAAAGGCTC
0770-kod-R	TCAGTGATGATGATGATGATGATGATGATGATGATGATGTATCAGCTCCCGC
1343-kod-F	CCTAATTTGGAGGGATGAACATGAAAGAAAGACTTGAAAAATTAG
1343-kod-R	TCAGTGATGATGATGATGATGATGATGATGATGATGATGGAGTAGCTCTCTTTCGG
pTE1-F	CATCATCATCATCATCATCACTGAATCCATCACACTGGCGGCCG
pTE1-R	GTTCATCCCTCCAAATTAG

Primer	Sequence (5′→3′)
0770-kod-F	CCTAATTTGGAGGGATGAACGTGAAAGATAGAATTAAAAGGCTC
0770-kod-R	TCAGTGATGATGATGATGATGATGATGATGATGATGATGTATCAGCTCCCGC
1343-kod-F	CCTAATTTGGAGGGATGAACATGAAAGAAAGACTTGAAAAATTAG
1343-kod-R	TCAGTGATGATGATGATGATGATGATGATGATGATGATGGAGTAGCTCTCTTTCGG
pTE1-F	CATCATCATCATCATCATCACTGAATCCATCACACTGGCGGCCG
pTE1-R	GTTCATCCCTCCAAATTAG

Protein	T/℃	Hydrostatic pressure (MPa)	Specific enzyme activity (U/mg)
Pyprol	40	0.1	578
Pyprol	40	40.0	967
Pyprol	70	0.1	1 120
Pyprol	70	40.0	1 471
Pyprol	100	0.1	1 857
Pyprol	100	40.0	2 309
Pfprol	40	0.1	38
Pfprol	40	20.0	56
Pfprol	70	0.1	899
Pfprol	70	20.0	1 142
Pfprol	100	0.1	1 977
Pfprol	100	20.0	2 469

Protein	T/℃	Hydrostatic pressure (MPa)	Specific enzyme activity (U/mg)
Pyprol	40	0.1	578
Pyprol	40	40.0	967
Pyprol	70	0.1	1 120
Pyprol	70	40.0	1 471
Pyprol	100	0.1	1 857
Pyprol	100	40.0	2 309
Pfprol	40	0.1	38
Pfprol	40	20.0	56
Pfprol	70	0.1	899
Pfprol	70	20.0	1 142
Pfprol	100	0.1	1 977
Pfprol	100	20.0	2 469

Hydrostatic pressure (MPa)	K_m (mmol/L)	V_max (μmol/(min·mg))	k_cat (s⁻¹)	k_cat/K_m (L/(mmol·s))
0.1	2.5	2 722	2 238	895
40	2.4	3 100	2 481	1 034

Hydrostatic pressure (MPa)	K_m (mmol/L)	V_max (μmol/(min·mg))	k_cat (s⁻¹)	k_cat/K_m (L/(mmol·s))
0.1	2.5	2 722	2 238	895
40	2.4	3 100	2 481	1 034

来源于超嗜热古菌雅氏火球菌(Pyrococcus yayanosii) CH1耐热耐压脯肽酶的酶学性质研究

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张欢欢 ¹^,² , 陈柔珂 ¹ , 徐俊 ¹^,^*

微生物学报 | 研究报告 2024,64(5): 1494-1505

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微生物学报 | 研究报告 2024, 64(5): 1494-1505

来源于超嗜热古菌雅氏火球菌(Pyrococcus yayanosii) CH1耐热耐压脯肽酶的酶学性质研究

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张欢欢¹^,², 陈柔珂¹, 徐俊¹^,^*

作者信息

1 上海交通大学生命科学技术学院微生物代谢国家重点实验室, 上海 200240

2 上海交通大学海洋学院, 上海 200240

Characterization of a thermostable and piezotolerant prolidase from the hyperthermophilic archaeonPyrococcus yayanosii CH1

Huanhuan ZHANG¹^,², Rouke CHEN¹, Jun XU¹^,^*

Affiliations

1 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

2 School of Oceanography, Shanghai Jiao Tong University, Shanghai 200240, China

出版时间: 2024-05-04 doi: 10.13343/j.cnki.wsxb.20230697

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摘要

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【目的】脯肽酶是一种能从二肽(Xaa-Pro)的C末端水解脯氨酸或羟脯氨酸残基的肽酶。对深海来源的雅氏火球菌(Pyrococcus yayanosii) CH1基因组中PYCH_07700基因编码的蛋白Pyprol的体外酶学性质进行研究, 以期发现新型脯肽酶。【方法】在小宝岛热球菌(Thermococcus kodakarensis) TS559中异源表达Pyprol。使用二肽Met-Pro作为底物, 检测重组蛋白的脯肽酶活性。【结果】Pyprol的最适温度为100 ℃, 最适pH为6.0。Pyprol在与Co²⁺结合时活性最高, 最适的金属离子浓度为1.2 mmol/L。与P.furiosus来源的脯肽酶Pfprol相比,Pyprol在更宽的pH范围具有活性, 并且能够耐受更高浓度的金属离子。Pyprol是耐压蛋白, 最适静水压为40 MPa。与常压条件下相比, 40 MPa下,Pyprol在40、70和100 ℃均有更高的活性。【结论】来源于深海热液喷口的严格嗜压的超嗜热古菌P.yayanosii CH1的新型脯肽酶Pyprol具有热稳定和耐压特性。

关键词

超嗜热古菌 / 雅氏火球菌 / 脯肽酶 / 热稳定 / 耐压

Abstract

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[Objective] Prolidase is an enzyme that can hydrolyze proline or hydroxyproline residues from the C-terminal dipeptides (Xaa-Pro). A putative prolidase-encoding gene was identified in the genome ofPyrococcus yayanosii CH1 isolated from the deep sea. In this study, we characterized the enzymatic properties ofPyprol encoded byPYCH_07700in vitro, aiming to find a new prolidase. [Methods] Pyprol was heterologously expressed in the hyperthermophilic archaeonThermococcus kodakarensis TS559. The dipeptide Met-Pro was used as a substrate to test the prolidase activity of the purified recombinant protein. [Results] Pyprol showed the best performance at 100 ℃ and pH 6.0.Pyprol binding to Co²⁺ exhibited the maximum activity, and the optimal metal ion concentration was 1.2 mmol/L.Pyprol had catalytic activity in a wider pH range and can tolerate higher concentrations of metal ions than the prolidasePfprol fromP.furiosus.Pyprol was a piezotolerant protein with an optimal hydrostatic pressure of 40 MPa. It exhibited enhanced activities at 40, 70, and 100 ℃ under 40 MPa, compared with at the atmospheric pressure. [Conclusion] Pyprol is a novel thermostable and piezotolerant prolidase ofP.yayanosii CH1, which is an obligate piezophilic hyperthermophilic archaeon strain isolated from a deep-sea hydrothermal vent.

Key words

hyperthermophilic archaeon / Pyrococcus yayanosii / prolidase / thermostable / piezotolerant

引用本文

张欢欢, 陈柔珂, 徐俊. 来源于超嗜热古菌雅氏火球菌(Pyrococcus yayanosii) CH1耐热耐压脯肽酶的酶学性质研究. 微生物学报, 2024 , 64 (5) : 1494 -1505 . DOI: 10.13343/j.cnki.wsxb.20230697

Huanhuan ZHANG, Rouke CHEN, Jun XU. Characterization of a thermostable and piezotolerant prolidase from the hyperthermophilic archaeonPyrococcus yayanosii CH1[J]. Acta Microbiologica Sinica, 2024 , 64 (5) : 1494 -1505 . DOI: 10.13343/j.cnki.wsxb.20230697

正文

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Prolidase (EC 3.4.13.9) is a peptidase that specifically cleaves the C-terminal proline or hydroxyproline residues of dipeptides. Prolidase has been identified in various organisms to date, including humans^[1-2], bacteria^[3-4], and archaea^[5-7]. In prokaryotes, it is generally believed that prolidase is involved in the proline cycle^[8] and bacterial defense against toxins^[9]. In humans, prolidase is involved in the degradation of collagen^[10]. The activity of prolidase has been found to be abnormally increased in breast cancer^[11] and myeloproliferative neoplasms^[12]. In addition, the enzyme can also combine with the tumor suppressor p53^[13]. So human prolidase is also an important cancer marker. Prolidase has many applications in biotechnology. During cheese fermentation, prolidase can be added to increase the content of proline in the product^[14]. In addition, prolidase can be used as an antidote for organophosphorus compounds^[15].

Prolidase is a metalloenzyme, and metal ions contribute to stabilizing its structure and anchoring the substrate at the active site^[8]. Prolidase always contains two metal-binding cores, and it requires both metal cores to be occupied for full enzymatic activity^[16]. The metal-binding site amino acids (Asp-Asp-His-Glu-Glu) are highly conserved^[8].

The enzymatic properties of prolidase from hyperthermophilic archaea are different from those of other species. While prolidase from humans andEscherichia coli preferentially bind to Mn^2+[4,17], prolidases from hyperthermophilic archaea have the highest activity when binding Co^2+[5,7]. It is worth noting that a prolidasePfprol ofPyrococcus furious can also bind Fe²⁺ under anaerobic conditions^[5]. The optimum temperature of prolidases fromP.furious DSM3638 andP. horikoshii OT3 reached 100 ℃^[5,7], which is the highest among the prolidases studied. Moreover, prolidases fromPyrococcus species exhibit excellent thermal stability, for example, the prolidase fromP.furiosus DSM 3638 andP.horikoshii OT3 maintained their activity without significant loss when incubated at 100 ℃ for 12 h and 8 h, respectively^[5,7].

P.yayanosii CH1 was isolated from the sediment sample collected at a depth of 4 100 m in the Mid-Atlantic Ridge^[18]. The optimum growth temperature ofP.yayanosii CH1 is 98 ℃ and the optimum growth pressure is 52 MPa^[19]. So in this regard,P.yayanosii CH1 is an obligate piezophilic hyperthermophile and serves as an important model organism for studying the mechanisms of high-pressure adaptation in microorganisms^[20]. A putative prolidase encoding genePYCH_07700 was found in the genome ofP.yayanosii CH1. This study will present the results of the characterization of the enzymatic properties of the above-mentioned prolidase, namelyPyprol.Pyprol was obtained by heterologous overexpression inThermococcus kodakarensis TS559, which is an agmatine auxotroph strain^[21-22]. The effect of hydrostatic pressure on the enzymatic activity ofPyprol was investigated, which provides clues for expanding the application of this type of prolidase.

1 Materials and Methods

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1.1 Strains, plasmids, and culture conditions

The strains and plasmids utilized in this study are outlined inTable 1.E.coli DH5α was cultured in Luria-Bertani (LB) medium at 37 ℃.P.yayanosii A1 was cultivated under anaerobic at 95 ℃ in TRM medium^[23].T.kodakarensis TS559 strains were cultivated in the artificial seawater (ASW-YT) liquid medium supplemented with 1 mmol/L agmatine under anaerobic at 85 ℃.

1.2 Bioinformatics analysis

The amino acid sequence of the experimentally characterized prolidasePfprol (WP_011012489.1) ofP.furiosus was used as a query to BLAST against the genome sequence ofP.yayanosii CH1 (GenBank accession number: NC_015680.1). Putative prolidases includingPyprol (WP_013905514.1),Pfprol, and other homologous sequences were retrieved from GenBank. The amino acid sequences of obtained prolidases were aligned using ClustalX2 and visualized using ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/). The phylogenetic tree was constructed by the neighbor-joining (NJ) method using MEGA (version 7)^[24]. Bootstrap analysis was computed with 1 000 replicates.

1.3 Cloning of prolidase encoding genes

Using the genomic DNA ofP.yayanosii A1 as a template, the full-length sequence of genePYCH_07700 was amplified using primer 0770-kod-F/R (Table 2). Plasmid pTE1 was used as the template DNA and the primer pTE1-F/R (Table 2) was used in PCR to amplify the backbone of thisE.coli-T.kodakarensis shuttle vector^[25]. Using ClonExpress Ⅱ One Step Cloning Kit (Vazyme, China), genePYCH_07700 was ligated to the pTE1 plasmid and transformed into DH5α. The prolidase encoding genePF_1343 ofP.furiosus DSM3638 was cloned into pTE1 using same strategy with changes in PCR primers (1343-kod-F/R) and corresponding template DNA (Table 2). In order to purify the protein, a 12×His tag was added to the C-terminus ofPYCH_07700 andPF_1343, respectively. Plasmid constructed with correct insertion of either genePYCH_07700 orPF_1343 was confirmed by colony PCR and DNA-sequencing analysis.

Genetic manipulations ofT.kodakarensis were carried out under anaerobic conditions. The transformation ofT.kodakaraensis was performed as previously described^[25]. The host strainT.kodakarensis TS559 was cultivated in ASW-YT liquid medium supplemented with 1 mmol/L agmatine at 85 ℃ for 10 h, and the cells were harvested by centrifugation (6 500×g, 5 min). The harvested cells were resuspended in 200 μL of 0.1 mol/L CaCl₂ and kept on ice for 30 min. Then, 3 μg of plasmid was added to the cell suspension and incubated on ice for 1 h, followed by a heat shock at 85 ℃ for 45 s and incubation on ice for 10 min. The cell suspension was added to 5 mL ASW-YT liquid medium and incubated at 85 ℃ for 4 h. The culture was spread onto ASW-YT solid medium without agmatine and cultured at 85 ℃ until colonies were observed. The positive colonies were confirmed by colony PCR and DNA sequencing analysis.

1.4 Prolidase expression and purification inThermococcus kodakarensis

The recombinant strains were inoculated into ASW-YT medium and cultured at 85 ℃ for 15 h under anaerobic conditions. Cells were collected by centrifugation at 10 000×g for 5 min at room temperature. The cells were resuspended in 50 mmol/L Tris-HCl (pH 8.0) containing 0.5 mol/L NaCl and then crushed by sonication on ice. The supernatant was collected by centrifugation at 10 000×g for 30 min at 4 ℃. Proteins were purified using Ni-NTA 6FF Sefinose Resin Kit (Sangon, China). Imidazole in the buffer that was used to elute the overexpressed protein was removed using Millipore 10 kDa ultrafiltration tubes. The purified protein was finally stored in 50 mmol/L Tris-HCl (pH 8.0) containing 0.5 mol/L NaCl.

1.5 Prolidase activity assay

Prolidase activity was determined as previously described^[5]. The 50 μL reaction mixture contained 50 mmol/L MOPS buffer (pH 7.0), 200 mmol/L NaCl, 5% glycerol, 0.1 mg/mL BSA protein, and 1.2 mmol/L CoCl₂. After adding an appropriate amount of protein, react at 100 ℃ for 5 min to allow the protein to bind to the metal ion. Add Met-Pro at a final concentration of 10 mmol/L and react at 100 ℃ for 10 min. Add 50 μL of acetic acid to stop the reaction, then add 50 μL of 3% (W/V) ninhydrin solution to react at 100 ℃ for 10 min. After cooling to room temperature, use a microplate reader to measure the absorbance at 515 nm. The activity unit of prolidase is defined as the amount of enzyme that releases one micromole of proline per minute.

1.6 Influence of temperature, pH, and metal preference on enzyme activity

The optimum temperature ofPyprol was determined in the range of 40–100 ℃. The optimum pH ofPyprol was determined in the range of pH 4.0–8.0. The buffer used were 50 mmol/L of CH₃COOH-CH₃COONa (pH 4.0–5.0), NaH₂PO₄-Na₂HPO₄ (pH 6.0–7.0), Tris-HCl (pH 8.0). To determine the optimal metal ion ofPyprol,Pyprol was combined with Co²⁺, Mn²⁺, Zn²⁺, Ca²⁺, Cu²⁺, Ni²⁺, and Mg²⁺, respectively, and then reacted with the substrate. To evaluate the effect of metal ion concentration on the enzyme activity,Pyprol was combined with Co²⁺ at a final concentration of 0–6 mmol/L and then reacted with the substrate.

1.7 Influence of hydrostatic pressure on enzyme activity

To assess the effect of hydrostatic pressure onPyprol activity, prolidase activity assays were performed at 0.1, 10, 20, 30, 40, and 52 MPa at 100 ℃, respectively. To evaluate the effect of high hydrostatic pressure on prolidase activity at different temperatures, the prolidase activity under its optimum hydrostatic pressure was measured at 40, 70, and 100 ℃, respectively. High pressure was achieved and controlled by adding water through the hand-operated pump that was equipped with a pressure gauge^[26-27]. The pin-closure pressure vessels were used in this study (constructed by Nantong Feiyu Petroleum Technology Development Co., Ltd., China).

1.8 Influence of temperature and hydrostatic pressure on enzyme stability

The thermal stability ofPyprol was determined by incubating the assays at specific temperatures (80, 90, and 100 ℃) for different periods (1, 2, 3, 4, and 5 h), and measuring the residual activity. The high hydrostatic pressure stability ofPyprol was determined by incubating at 80 ℃ under 20 MPa and 40 MPa for 1 h, and the residual enzyme activity was determined.

1.9 Determination of enzyme kinetics constants

The enzyme reaction rate ofPyprol was determined in the Met-Pro concentration range of 1–10 mmol/L at 0.1 MPa and 40 MPa, respectively. The reaction was conducted under the condition of 50 mmol/L NaH₂PO₄-Na₂HPO₄ buffer (pH 6.0), 1.2 mmol/L CoCl₂, and 100 ℃. The results were fitted using the Michaelis-Menten equation in the data analysis and graphing software Origin.

2 Results

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2.1 Overexpression and purification of a prolidasePyprol fromPyrococcus yayanosii inThermococcus kodakarensis

InP.yayanosii CH1, the proteinPyprol encoded byPYCH_07700 is predicted to function as a prolidase. InPyprol, there are two conserved structural domains, namely creatinase_N located at the N-terminus, and peptidase_M24 situated at the C-terminus (Figure S1, data was deposited in the China National Microbiology Data Center, accession No.: NMDCX0000258). BLASTp analysis showed thatPyprol exhibited high similarity to prolidases fromP.furiosus DSM3638 andP.horikoshii OT3, with amino acid sequence similarities of 76% and 75%, respectively (Figure 1). Pyprol also exhibits a high structural similarity to the prolidase fromP.furiosus (Figure S2, data was deposited in the China National Microbiology Data Center, accession No.: NMDCX0000259). Multiple alignments ofPyprol with its homologous proteins revealed thatPyprol contained the conserved metal-binding sites (Asp-Asp-His-Glu-Glu). These results suggested thatPyprol was a putative prolidase.

To overexpress the prolidase fromP.yayanosii CH1 inT.kodakarensis TS559,PYCH_07700 gene fragment was cloned into a shuttle vector plasmid pTE1 in the downstream region of the glutamate dehydrogenase (P_gdh) promoter fromP.furiosus DSM3638 (Figure 2A). The recombinant strains containing plasmids pTE-Pyprol and pTE-Pfprol were cultured in ASW-YT medium at 85 ℃ for 15 h, and cells were harvested by centrifugation. The cells were crushed by sonication, and the supernatants were collected by centrifugation. The prolidases were purified by using nickel-charged resin. The purified proteins were analyzed using SDS-PAGE (Figure 2B), and the results showed that the molecular weight of the recombinant protein was approximately 43 kDa which was consistent with the theoretical relative molecular weight.

2.2 Optimal temperature, optimal pH, and metal ion preference ofPyprol

The activity ofPyprol was determined by measuring its ability to cleave the Met-Pro dipeptide substrate over a temperature range of 40–100 ℃ (Figure 3A). The results indicated that the optimal temperature forPyprol activity was 100 ℃, and there was a notable decrease in activity when the temperature dropped below 70 ℃. The activity ofPyprol was determined over a pH range of 4.0–8.0 (Figure 3B). The optimal pH forPyprol activity was found to be 6.0, and a significant decline in activity was observed at pH 4.0. WhenPyprol was bound to different metal ions (Figure 3C), significant variations in activity were observed.Pyprol exhibited the highest activity when bound to Co²⁺. It also retained 93% of the activity observed when bound to Co²⁺ when it was bound to Mn²⁺. However, the binding of Zn²⁺, Ca²⁺, Cu²⁺, Ni²⁺, and Mg²⁺ resulted in almost undetectable prolidase activity. The results demonstrated thatPyprol exhibited maximum activity when bound to 1.2 mmol/L Co²⁺, but even when bound to 0.6 mmol/L Co²⁺, it retained 81% of the maximum activity (Figure 3C). Additionally, we observed thatPyprol retained 29% activity even in the absence of additional metal ions, compared to that of when 1.2 mmol/L Co²⁺ was added. This may be attributed to the binding of trace amounts of Co²⁺ from the culture medium toPyprol.

2.3 Effect of hydrostatic pressure onPyprol activity

The activity ofPyprol was found to be higher under high hydrostatic pressure compared to atmospheric pressure. At 40 MPa,Pyprol exhibited the highest activity (Figure 3D). Furthermore, the extent of enhancement inPyprol activity varied at different temperatures under high hydrostatic pressure. At 40, 70, and 100 ℃, compared to 0.1 MPa,Pyprol activity increased by 67%, 31%, and 24%, respectively (Table 3). This indicated that high hydrostatic pressure has a significant impact onPyprol activity at lower temperatures, especially at 40 ℃. Notably, at 40 ℃ and 40 MPa, the specific activity ofPyprol reached 967 U/mg, which is close to the specific activity observed at 70 ℃ and 0.1 MPa (1 120 U/mg). Similarly, we observed that the activity ofPfprol was higher at 20 MPa compared to atmospheric pressure. Additionally, compared to 0.1 MPa, at 20 MPa, the enzyme activity ofPfprol at 40, 70, and 100 ℃ increased by 47%, 27%, and 24%, respectively.

2.4 Kinetic constants

We used Met-Pro as the substrate and determined the kinetic constants ofPyprol at 0.1 MPa and 40 MPa. The results revealed that theK_m values ofPyprol at 0.1 MPa and 40 MPa were similar, but at 40 MPa,Pyprol exhibited a higherV_max value compared to that at 0.1 MPa (Table 4). Thek_cat/K_m value ofPyprol at 40 MPa was 1.16 times higher than that at 0.1 MPa.

2.5 Thermal stability and high hydrostatic pressure stability

Pyprol was incubated under different temperatures and hydrostatic pressure conditions, followed by the measurement of prolidase activity. The results (Figure 4A) showed that after incubatingPyprol at 80 ℃ and 90 ℃ for 5 h, it retained 64% and 55% of its activity, respectively. However, after incubatingPyprol at 100 ℃ for 1 h, its activity decreased to only 41% (Figure 4A). On the other hand, after incubatingPyprol at 20 MPa and 40 MPa for 1 h, it still retained 88% and 78% of its activity, respectively (Figure 4B).

3 Discussion

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In this study, we reported the characterization of a prolidasePyprol fromP.yayanosii CH1. To the best of our knowledge, this is the first report about a prolidase that exhibits higher activity under high hydrostatic pressure than under atmospheric pressure.

Previous studies have found that the native prolidase (N-prol) purified fromP.furiosus and the recombinant prolidase (R-prol) expressed inE.coli showed no significant differences in enzymatic properties^[5]. However, when Met-Pro was used as the substrate, thek_cat/K_m value of R-prol was 1.64 times higher than that of N-prol^[5]. To avoid the host background effect, we chose hyperthermophilic archaeonT.kodakarensis, which is a close relative ofP.yayanosii CH1, as the surrogate host to overexpress the prolidasePyprol^[22]. As shown in a previous report, successful expression of the soluble hydrogenase I (SHⅠ) fromP.furiosus has been achieved inT.kodakarensis TS559^[25]. We anticipated that the prolidase obtained fromT.kodakarensis TS559 may exhibit enzymatic properties more similar to the native prolidase fromP.yayanosii.

For comparison, we also cloned the prolidase encoding gene (PF_1343) ofP.furiosus and overexpressed it inT.kodakarensis TS559 (Figure 2B). For bothPyprol andPfprol, the prolidase activity was higher under high hydrostatic pressure than at of under atmospheric pressure (Table 3). At 40 ℃ and 0.1 MPa, the specific enzyme activity ofPyprol (578 U/mg) is 15.21 fold of that ofPfprol (38 U/mg). This indicated that at 40 ℃,Pyprol has greater potential in practical applications.

A phylogenetic analysis ofPyprol indicates that prolidase from diverse hyperthermophiles, including that from three genus ofThermococcaceae,Sulfolobus,Archaeoglobus andThermotoga are located within the same branch (Figure 5). It is interesting that the prolidase fromT.barophilus MP, which was the first true hyperthermophilic piezophilic archaeon isolated^[28], showed close relationship with prolidases from thePyrococcus genus. It is commonly believed that enzyme activity decreases under high hydrostatic pressure^[29]. However, there were reports that proteases derived fromMethanocaldococcus jannaschii exhibited activity 3.4 times higher at 50 MPa 125 ℃ compared to 10 MPa 125 ℃^[30]. As well as that, we found that the activity ofPyprol at high hydrostatic pressure was significantly higher than that at atmospheric pressure. Moreover, we observed thatPfprol exhibits a 25% increase in enzyme activity at 20 MPa compared to 0.1 MPa (Table 3). It is indicated that the prolidase from a deep-sea microbe may have a potential similarity in their enzymatic properties.

P.yayanosii CH1 lacks a complete pathway for synthesizing proline^[31], indicating the need to acquire proline from the external environment. The unique cyclic structure of proline makes the peptide bonds surrounding the proline residue resistant to degradation, and prolidase is one of the few enzymes in organisms capable of degrading proline residues^[32]. The higher activity ofPyprol under high hydrostatic pressure compared to atmospheric pressure suggests its potential role in proline acquisition forP.yayanosii CH1 under high hydrostatic pressure conditions. We deletedPYCH_07700 inP.yayanosii, but no significant decrease in biomass was observed under high hydrostatic pressure (result not shown). Whether functional compensation by other enzymes capable of degrading proline residues inP.yayanosii exist or not will be the focus of future experiments.

基金

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国家重点研发计划(2020YFA0906800)
国家自然科学基金面上项目(41976085)

参考文献

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文献

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参考文献引证文献

排序方式：

[1]

LUPI A, DELLA TORRE S, CAMPARI E, TENNI R, CETTA G, ROSSI A, FORLINO A.Human recombinant prolidase from eukaryotic and prokaryotic sources. Expression, purification, characterization and long-term stability studies[J].The FEBS Journal,2006,273(23):5466-5478.

https://doi.org/10.1111/j.1742-4658.2006.05538.x

[2]

BESIO R, ALLEVA S, FORLINO A, LUPI A, MENEGHINI C, MINICOZZI V, PROFUMO A, STELLATO F, TENNI R, MORANTE S.Identifying the structure of the active sites of human recombinant prolidase[J].European Biophysics Journal,2010,39(6):935-945.

https://doi.org/10.1007/s00249-009-0459-4

[3]

FERNÁNDEZ-ESPLÁ MD, MARTÍN-HERNÁNDEZ MC, FOX PF.Purification and characterization of a prolidase fromLactobacillus casei subsp. casei IFPL 731[J].Applied and Environmental Microbiology,1997,63(1):314-316.

https://doi.org/10.1128/aem.63.1.314-316.1997

[4]

WEAVER J, WATTS T, LI PW, RYE HS.Structural basis of substrate selectivity ofE.coli prolidase[J].PLoS One,2014,9(10):e111531.

https://doi.org/10.1371/journal.pone.0111531

[5]

GHOSH M, GRUNDEN AM, DUNN DM, WEISS R, ADAMS MW.Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeonPyrococcus furiosus[J].Journal of Bacteriology,1998,180(18):4781-4789.

https://doi.org/10.1128/JB.180.18.4781-4789.1998

[6]

WILLINGHAM K, MAHER MJ, GRUNDEN AM, GHOSH M, ADAMS MW, FREEMAN HC, GUSS JM.Crystallization and characterization of the prolidase fromPyrococcus furiosus[J].Acta Crystallographica Section D,2001,57(Pt 3):428-430.

[7]

THERIOT CM, TOVE SR, GRUNDEN AM.Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeonPyrococcus horikoshii[J].Applied Microbiology and Biotechnology,2010,86(1):177-188.

https://doi.org/10.1007/s00253-009-2235-x

[8]

KITCHENER RL, GRUNDEN AM.Prolidase function in proline metabolism and its medical and biotechnological applications[J].Journal of Applied Microbiology,2012,113(2):233-247.

https://doi.org/10.1111/j.1365-2672.2012.05310.x

[9]

VYAS NK, NICKITENKO A, RASTOGI VK, SHAH SS, QUIOCHO FA.Structural insights into the dual activities of the nerve agent degrading organophosphate anhydrolase/prolidase[J].Biochemistry,2010,49(3):547-559.

https://doi.org/10.1021/bi9011989

[10]

MISIURA M, MILTYK W.Current understanding of the emerging role of prolidase in cellular metabolism[J].International Journal of Molecular Sciences,2020,21(16):5906.

https://doi.org/10.3390/ijms21165906

[11]

PALKA JA, PHANG JM.Prolidase in human breast cancer MCF-7 cells[J].Cancer Letters,1998,127(1/2):63-70.

[12]

NOWICKA J, NAHACZEWSKA W, OWCZAREK H, WOŹNIAK M.The decrease in prolidase activity in myeloproliferative neoplasms[J].Advances in Clinical and Experimental Medicine,2012,21(6):767-771.

[13]

YANG L, LI Y, BHATTACHARYA A, ZHANG YS.PEPD is a pivotal regulator of p53 tumor suppressor[J].Nature Communications,2017,8:2052.

https://doi.org/10.1038/s41467-017-02097-9

[14]

COURTIN P, NARDI M, WEGMANN U, JOUTSJOKI V, OGIER JC, GRIPON JC, PALVA A, HENRICH B, MONNET V.Accelerating cheese proteolysis by enrichingLactococcus lactis proteolytic system with lactobacilli peptidases[J].International Dairy Journal,2002,12(5):447-454.

https://doi.org/10.1016/S0958-6946(02)00022-5

[15]

THERIOT CM, DU XL, TOVE SR, GRUNDEN AM.Improving the catalytic activity of hyperthermophilicPyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures[J].Applied Microbiology and Biotechnology,2010,87(5):1715-1726.

https://doi.org/10.1007/s00253-010-2614-3

[16]

DU XL, TOVE S, KAST-HUTCHESON K, GRUNDEN AM.Characterization of the dinuclear metal center ofPyrococcus furiosus prolidase by analysis of targeted mutants[J].FEBS Letters,2005,579(27):6140-6146.

https://doi.org/10.1016/j.febslet.2005.09.086

[17]

BESIO R, BARATTO MC, GIOIA R, MONZANI E, NICOLIS S, CUCCA L, PROFUMO A, CASELLA L, BASOSI R, TENNI R, ROSSI A, FORLINO A.A Mn(Ⅱ)-Mn(Ⅱ) center in human prolidase[J].Biochimica et Biophysica Acta,2013,1834(1):197-204.

https://doi.org/10.1016/j.bbapap.2012.09.008

[18]

ZENG X, BIRRIEN JL, FOUQUET Y, CHERKASHOV G, JEBBAR M, QUERELLOU J, OGER P, CAMBON-BONAVITA MA, XIAO X, PRIEUR D.Pyrococcus CH1, an obligate piezophilic hyperthermophile: extending the upper pressure-temperature limits for life[J].The ISME Journal,2009,3(7):873-876.

https://doi.org/10.1038/ismej.2009.21

[19]

BIRRIEN JL, ZENG X, JEBBAR M, CAMBON-BONAVITA MA, QUÉRELLOU J, OGER P, BIENVENU N, XIAO X, PRIEUR D.Pyrococcus yayanosii sp. nov., an obligate piezophilic hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent[J].International Journal of Systematic and Evolutionary Microbiology,2011,61(12):2827-2881.

https://doi.org/10.1099/ijs.0.024653-0

[20]

LI Z, SONG QH, WANG YZ, XIAO X, XU J.Identification of a functional toxin-antitoxin system located in the genomic island PYG1 of piezophilic hyperthermophilic archaeonPyrococcus yayanosii[J].Extremophiles,2018,22(3):347-357.

https://doi.org/10.1007/s00792-018-1002-2

[21]

SANTANGELO TJ, CUBONOVÁ L, REEVE JN.Thermococcus kodakarensis genetics: TK1827-encoded beta-glycosidase, new positive-selection protocol, and targeted and repetitive deletion technology[J].Applied and Environmental Microbiology,2010,76(4):1044-1052.

https://doi.org/10.1128/AEM.02497-09

[22]

TAKEMASA R, YOKOOJI Y, YAMATSU A, ATOMI H, IMANAKA T.Thermococcus kodakarensis as a host for gene expression and protein secretion[J].Applied and Environmental Microbiology,2011,77(7):2392-2398.

https://doi.org/10.1128/AEM.01005-10

[23]

LI XG, FU L, LI Z, MA XP, XIAO X, XU J.Genetic tools for the piezophilic hyperthermophilic archaeonPyrococcus yayanosii[J].Extremophiles,2015,19(1):59-67.

https://doi.org/10.1007/s00792-014-0705-2

[24]

KUMAR S, STECHER G, TAMURA K.MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1870-1874.

https://doi.org/10.1093/molbev/msw054

[25]

SONG YH, LIU MX, XIE LP, YOU C, SUN JS, ZHANG YPJ.A recombinant 12-his taggedPyrococcus furiosus soluble[NiFe]-hydrogenase I overexpressed inThermococcus kodakarensis KOD1 facilitates hydrogen-poweredin vitro NADH regeneration[J].Biotechnology Journal,2019,14(4):e1800301.

https://doi.org/10.1002/biot.201800301

[26]

LI SK, XIAO X, LI JY, LUO JX, WANG FP.Identification of genes regulated by changing salinity in the deep-sea bacteriumShewanella sp. WP3 using RNA arbitrarily primed PCR[J].Extremophiles,2006,10(2):97-104.

https://doi.org/10.1007/s00792-005-0476-x

[27]

YAYANOS AA, van BOXTEL R.Coupling device for quick high-pressure connections to 100 MPa[J].Review of Scientific Instruments,1982,53(5):704-705.

https://doi.org/10.1063/1.1137011

[28]

MARTEINSSON VT, BIRRIEN JL, REYSENBACH AL, VERNET M, MARIE D, GAMBACORTA A, MESSNER P, SLEYTR UB, PRIEUR D.Thermococcus barophilus sp. nov., a new barophilic and hyperthermophilic archaeon isolated under high hydrostatic pressure from a deep-sea hydrothermal vent[J].International Journal of Systematic Bacteriology,1999,49(2):351-359.

[29]

EISENMENGER MJ, REYES-DE-CORCUERA JI.High pressure enhancement of enzymes: a review[J].Enzyme and Microbial Technology,2009,45(5):331-347.

https://doi.org/10.1016/j.enzmictec.2009.08.001

[30]

MICHELS PC, CLARK DS.Pressure-enhanced activity and stability of a hyperthermophilic protease from a deep-sea methanogen[J].Applied and Environmental Microbiology,1997,63(10):3985-3991.

https://doi.org/10.1128/aem.63.10.3985-3991.1997

[31]

XU J, LIU LP, XU MJ, OGER P, WANG FP, JEBBAR M, XIAO X.Complete genome sequence of the obligate piezophilic hyperthermophilic archaeonPyrococcus yayanosii CH1[J].Journal of Bacteriology,2011,193(16):4297-4298.

https://doi.org/10.1128/JB.05345-11

[32]

CUNNINGHAM DF, O'CONNOR B.Proline specific peptidases[J].Biochimica et Biophysica Acta,1997,1343(2):160-186.

2024年第64卷第5期

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doi: 10.13343/j.cnki.wsxb.20230697

接收时间：2023-11-14
首发时间：2026-03-19
出版时间：2024-05-04

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收稿日期：2023-11-14
录用日期：2024-02-19

基金

National Key Research and Development Program of China(2020YFA0906800)

国家重点研发计划(2020YFA0906800)

National Natural Science Foundation of China (General Program)(41976085)

国家自然科学基金面上项目(41976085)

国家自然科学基金面上项目(42276091)

作者信息

1 上海交通大学生命科学技术学院微生物代谢国家重点实验室, 上海 200240

2 上海交通大学海洋学院, 上海 200240

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Strains and plasmids	Description	Reference
Strains
Escherichia coli DH5α	The strain used for gene cloning
Pyrococcus yayanosii A1	The facultatively piezophilic derivative strain	Li et al.^[23]
Thermococcus kodakarensis TS559	Agmatine auxotrophic strain	Santangelo et al.^[21]
ΔPYCH_07700	PYCH_07700 deletion strain	This study
Plasmids
pTE1	AT.kodakarensis-E.coli (Tk-Ec) shuttle vector	Song et al.^[25]
pTE-Pyprol	pTE1:: P_gdh-PYCH_07700	This study
pTE-Pfprol	pTE1:: P_gdh-PF_1343	This study

Strains and plasmids

Description

Reference

Strains

Escherichia coli DH5α

The strain used for gene cloning

Pyrococcus yayanosii A1

The facultatively piezophilic derivative strain

Li et al.^[23]

Thermococcus kodakarensis TS559

Agmatine auxotrophic strain

Santangelo et al.^[21]

ΔPYCH_07700

PYCH_07700 deletion strain

This study

Plasmids

pTE1

AT.kodakarensis-E.coli (Tk-Ec) shuttle vector

Song et al.^[25]

pTE-Pyprol

pTE1:: P_gdh-PYCH_07700

This study

pTE-Pfprol

pTE1:: P_gdh-PF_1343

This study

Primer	Sequence (5′→3′)
0770-kod-F	CCTAATTTGGAGGGATGAACGTGAAAGATAGAATTAAAAGGCTC
0770-kod-R	TCAGTGATGATGATGATGATGATGATGATGATGATGATGTATCAGCTCCCGC
1343-kod-F	CCTAATTTGGAGGGATGAACATGAAAGAAAGACTTGAAAAATTAG
1343-kod-R	TCAGTGATGATGATGATGATGATGATGATGATGATGATGGAGTAGCTCTCTTTCGG
pTE1-F	CATCATCATCATCATCATCACTGAATCCATCACACTGGCGGCCG
pTE1-R	GTTCATCCCTCCAAATTAG

Primer

Sequence (5′→3′)

0770-kod-F

CCTAATTTGGAGGGATGAACGTGAAAGATAGAATTAAAAGGCTC

0770-kod-R

TCAGTGATGATGATGATGATGATGATGATGATGATGATGTATCAGCTCCCGC

1343-kod-F

CCTAATTTGGAGGGATGAACATGAAAGAAAGACTTGAAAAATTAG

1343-kod-R

TCAGTGATGATGATGATGATGATGATGATGATGATGATGGAGTAGCTCTCTTTCGG

pTE1-F

CATCATCATCATCATCATCACTGAATCCATCACACTGGCGGCCG

pTE1-R

GTTCATCCCTCCAAATTAG

Protein	T/℃	Hydrostatic pressure (MPa)	Specific enzyme activity (U/mg)
Pyprol	40	0.1	578
Pyprol	40	40.0	967
Pyprol	70	0.1	1 120
Pyprol	70	40.0	1 471
Pyprol	100	0.1	1 857
Pyprol	100	40.0	2 309
Pfprol	40	0.1	38
Pfprol	40	20.0	56
Pfprol	70	0.1	899
Pfprol	70	20.0	1 142
Pfprol	100	0.1	1 977
Pfprol	100	20.0	2 469

Protein

T/℃

Hydrostatic pressure (MPa)

Specific enzyme activity (U/mg)

Pyprol

0.1

578

Pyprol

40.0

967

Pyprol

0.1

1 120

Pyprol

40.0

1 471

Pyprol

100

0.1

1 857

Pyprol

100

40.0

2 309

Pfprol

0.1

Pfprol

20.0

Pfprol

0.1

899

Pfprol

20.0

1 142

Pfprol

100

0.1

1 977

Pfprol

100

20.0

2 469

Hydrostatic pressure (MPa)	K_m (mmol/L)	V_max (μmol/(min·mg))	k_cat (s⁻¹)	k_cat/K_m (L/(mmol·s))
0.1	2.5	2 722	2 238	895
40	2.4	3 100	2 481	1 034

Hydrostatic pressure (MPa)

K_m (mmol/L)

V_max (μmol/(min·mg))

k_cat (s⁻¹)

k_cat/K_m (L/(mmol·s))

0.1

2.5

2 722

2 238

895

2.4

3 100

2 481

1 034