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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (3): 89-96    DOI: 10.13523/j.cb.20180312
综述     
赖氨酰内肽酶特性及其表达、应用的研究进展
曾杰()
江苏恒瑞医药股份有限公司 国家靶向药物工程技术研究中心 连云港 222047
Advances in Study of Properties, Recombinant Expression and Applications of Lysyl Endopeptidase
Jie ZENG()
Jiangsu Hengrui Medicine Co. Ltd., National Engineering Technology Research Center of Targeted Drugs, Lianyungang 222047, China
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摘要：

赖氨酰内肽酶是一种重要的工具酶,广泛应用于科学研究及工业化生产。目前市场上的赖氨酰内肽酶大多是从天然微生物中提取获得,其高昂的价格限制了其广泛运用,重组表达能够解决产量的难题。首次对赖氨酰内肽酶进行了综述,包括赖氨酰内肽酶的来源、结构、功能性质及其主要应用,并重点总结了近年来的重组表达进展,同时对今后的研究方向进行了展望。
关键词： 赖氨酰内肽酶;  Lys-C;  无色杆菌蛋白酶I;  铜绿假单胞菌蛋白酶IV    
Abstract:

Lysyl endopeptidase is an important enzyme as a tool for biotechnological purposes and industrial production. However, its applications are obstructed by its high costs of production, due to the current lysyl endopeptidase products are extracted from the native bacteria with very low yields. Nevertheless, the recombinant expression system have solved this problem perfectly. A review of recent progress in lysyl endopeptidase for the first time was presented. The origins, structure and function of lysyl endopeptidase were mainly focused on. Furthermore, the successful expression of recombinant lysyl endopeptidase were aslo summarized and analyzed. In addition, the potential perspective of lysyl endopeptidase for future research were further discussed.
Key words: Lysyl endopeptidase    Lys-C    Achrornobacter protease I    Pseudomonas aeruginosa    Protease IV
收稿日期: 2017-10-03 出版日期: 2018-04-04
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曾杰

引用本文:

曾杰. 赖氨酰内肽酶特性及其表达、应用的研究进展[J]. 中国生物工程杂志, 2018, 38(3): 89-96.

Jie ZENG. Advances in Study of Properties, Recombinant Expression and Applications of Lysyl Endopeptidase. China Biotechnology, 2018, 38(3): 89-96.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180312    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I3/89

图1  赖氨酰内肽酶的晶体结构[15]
Substrate Km/ Kcat [mmol/L(L/s)]
API (A. lyticus)[1,16]		 Lys-C (Lysobacter sp.
IB-9374) [2]		 Protease IV
(P. aeruginosa) [5]
Tos-Gly-Pro-Lys-pNA — — 4.4
Tos-Gly-Pro-Arg-pNA — — 0
Val-Leu-Lys- pNA — — 2.6
Lys-pNA 1.75 1 0
Arg-pNA 0 — —
Bz-Arg-pNA 0 — —
Bz-Lys-pNA 12.3 42 —
Ac-Lys-pNA — 115 —
Bz-Lys-OMe 2480 29 000 —
Tos-Lys-OMe 5700 14 600 —
Tos-Arg-OMe 0.14 — —
Bz-Orn-OMe 10.4 13 —
表1  赖氨酰内肽酶的底物特异性
性质 Lys-C (A.lyticus, L.enzymogenes) Protease IV (P.aeruginosa)
等电点 6.8~6.9[2,12] 8.7[3, 5]
最适温度 50℃[2] 45℃[5]
最适pH pH8.5~10.7[2,12], pH9[15] pH10.0[5]
耐盐性 5~7mol/L 尿素、0.1% SDS[12,15] 8mol/L 尿素[3]
抑制酶活条件 NaCl,盐酸胍[8] ≥2mol/L NaCl或pH<4[3]
抑制剂 TLCK(N-Tosyl-L-lysine chloromethyl ketone),
PMSF(phenylmethanesulfonyl fluoride)[12]		 DTT, 2-巯基乙醇[3, 5]; TLCK(totally abolish),
PMSF(partially inhibit)[5]
表2  赖氨酰内肽酶的特征
来源 产酶菌株 天然表达产量 酶比活
无色杆菌 A. lyticus M497-1[1] 32U/L[2] 9.1U/mg[3]
溶杆菌 Lysobacter sp. IB-9374[2] 183U/L[2]
L. enzymogenes ATCC 29487[5] 15U/L[2];5.6mg/L (折合50.96U/L)[7]
铜绿假单胞菌[15] P. aeruginosa PAO1 12.05U/(L·OD600) 14.6U/mg[3],52~89U/mg[4]
P. aeruginosa PA103-29 NR
P. aeruginosa 6294 28.01U/(L·OD600)
表3  赖氨酰内肽酶的天然表达
来源 宿主菌 表达载体 表达产物 产量 文献
A. lyticus M497-1 E.coli pKK233-2 酶原(未基因优化) 0.48mg/L(a) [21]
A. lyticus M497-1 E. coli JA221 pKYN200 酶原变体H210S、W169F 0.2~0.4mg/L(b) [24]
L. enzymogenes ATCC 27796 E. coli JM109 pJOE 酶原MGSK-Lys-C (560±33)U /L(d) [7]
P. aeruginosa E. coli BL21(DE3) pET32a 融合酶原Trx-pro-protease IV 36.6mg/L(b)
1.7g/L(c)		 [11]
[3]
A. lyticus P. pastoris KM71H pPICZαA 酶原变体K30R NR [15]
A. lyticus P. pastoris X-33 pPICZαA 成熟肽酶 NR [23]
Table 4  Recombinant expression of lysyl endopeptidase
[1] Masaki T, Nakamura K, Isono M , et al. A new proteolytic enzyme from Achromobacter lyticus M497-I. Agricultural and Biological Chemistry, 1978,42(7):1443-1445.
doi: 10.1271/bbb1961.42.1443
[2] Chohnan S, Nonaka J, Teramoto K , et al. Lysobacter strain with high lysyl endopeptidase production. Federation of European Materials Societies Microbiology Letters, 2002,213(1):13-20.
doi: 10.1111/j.1574-6968.2002.tb11279.x pmid: 12127482
[3] Zhao M Z, Cai M, Wu F L , et al. Recombinant expression,refolding,purification and characterization of Pseudomonas aeruginosa protease IV in Escherichia coli. Protein Expression and Purification, 2016,126:69-76.
doi: 10.1016/j.pep.2016.05.019 pmid: 27260967
[4] Elliott B W, Cohen C . Isolation and characterization of a lysine-specific protease from Pseudomonas aeruginosa. Journal of Biological Chemistry, 1986,261(24):11259-11265.
[5] Engel L S, Hill J M, Caballero A R , et al. Protease IV, a Unique extracellular protease and virulence factor from Pseudomonas aeruginosa. Journal of Biological Chemistry, 1998,273(27):16792-16797.
doi: 10.1074/jbc.273.27.16792 pmid: 9642237
[6] Jekel P A, Weijer W J, Beintema J J . Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis. Analytical Biochemistry, 1983,134(2):347-354.
doi: 10.1016/0003-2697(83)90308-1 pmid: 6359954
[7] Stressler T, Eisele T, Meyer S , et al. Heterologous expression and pro-peptide supported refolding of the high specific endopeptidase Lys-C. Protein Expression and Purification, 2016,118:31-38.
doi: 10.1016/j.pep.2015.09.024 pmid: 26431800
[8] Kuhlman P A, Chen R J, Alcantara J , et al. Rapid purification of Lys-C from Lysobacter enzymogenes cultures. BioProcess Techinical, 2009, 28-38.
[9] Oh J, Li X H, Kim S K , et al. Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa. Scientific Reports, 2017,7(1):4416.
doi: 10.1038/s41598-017-03733-6 pmid: 28667333
[10] Caballero A, Thibodeaux B, Marquart M , et al. Pseudomonas keratitis: protease IV gene conservation,distribution,and production relative to virulence and other Pseudomonas proteases. Investigative Ophthalmology & Visual Science, 2004,45(2):522-530.
doi: 10.1167/iovs.03-1050 pmid: 14744894
[11] 赵明治, 徐平, 吴飞林 , 等. 一种赖氨酰肽链内肽酶的原核重组表达与制备方法. 中国,CN105950593A, 2016-09-21. 一种赖氨酰肽链内肽酶的原核重组表达与制备方法. 中国,CN105950593A,2016-09-21. .
Zhao M Z, Xu P, Wu F L , et al. A Process for Preparation of Lysyl Endopeptidase by Prokaryotic Recombinant Expression: China,CN105950593A, 2016-09-21. A Process for Preparation of Lysyl Endopeptidase by Prokaryotic Recombinant Expression: China,CN105950593A,2016-09-21. .
[12] Tsunasawa S, Masaki T, Hirose M , et al. The primary structure and structural characteristics of Achromobacter lyticus protease I,a lysine-specific serine protease. Journal of Biological Chemistry, 1989,264(7):3832-3839.
pmid: 2492988
[13] Traidej M, Marquart M E, Caballero A R , et al. Identification of the active site residues of Pseudomonas aeruginosa protease IV - Importance of enzyme activity in autoprocessing and activation. Journal of Biological Chemistry, 2003,278(4):2549-2553.
doi: 10.1074/jbc.M208973200
[14] Ohnishi Y, Yamada T, Kurihara K , et al. Neutron and X-ray crystallographic analysis of Achromobacter protease I at pD 8.0:protonation states and hydration structure in the free-form. Biochimica et Biophysica Acta, 2013,1834(8):1642-1647.
doi: 10.1016/j.bbapap.2013.05.012 pmid: 23714114
[15] Asztalos P, Müller A , H?lke W,et al. Atomic resolution structure of a lysine-specific endoproteinase from Lysobacter enzymogenes suggests a hydroxyl group bound to the oxyanion hole. Acta crystallographica. Section D, Biological Crystallography, 2014,70(7):1832-1843.
doi: 10.1107/S1399004714008463 pmid: 25004961
[16] Masaki T, Fujihashi T, Nakamura K , et al. Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1. II. specificity and inhibition studies of Achromobacter protease I. Biochimica et Biophysica Acta, 1981,660(1):51-55.
doi: 10.1016/0005-2744(81)90107-8 pmid: 6168293
[17] Tizon R U, Serrano A E, Traifalgar R F . Effects of pH on amylase,cellulase and protease of the Angelwing clam,Pholas orientalis. European Journal of Experimental Biology, 2012,2(6):2280-2285.
[18] Shiraki K, Sakiyama F . Histidine 210 mutant of a trypsin-type Achromobacter protease I shows broad optimum pH range. Journal of Bioscience and Bioengineering, 2002,93(3):331-333.
doi: 10.1016/S1389-1723(02)80038-X pmid: 16233210
[19] Shiraki K, Norioka S, Li S , et al. Electrostatic role of aromatic ring stacking in the pH-sensitive modulation of a chymotrypsin-type serine protease,Achromobacter protease I. European Journal of Biochemistry, 2002,269(16):4152-4158.
doi: 10.1046/j.1432-1033.2002.03110.x pmid: 12180992
[20] Conibear T C, Willcox M D, Flanagan J L , et al. Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates. Journal of Medical Microbiology, 2012,61(2):180-190.
doi: 10.1099/jmm.0.034561-0 pmid: 21921113
[21] Ohara T, Makino K, Shinagawa H , et al. Cloning,nucleotide sequence,and expression of Achromobacter protease I gene. Journal of Biological Chemistry, 1989,264(34):20625-20631.
[22] Srinivasa B K, Pulicherla K K, Antony A , et al. Cloning and expression of recombinant human GMCSF from Pichia pastoris GS115--a progressive strategy for economic production. American Journal of Therapeutics, 2014,21(6):462-469.
doi: 10.1097/MJT.0000000000000040 pmid: 24531404
[23] 马妍, 付志成, 范开 . 重组赖氨酸内肽酶构建表达及活性方法研究. 重庆理工大学学报(自然科学版), 2015,29(4):53-59.
Ma Y, Fu Z C, Fan K . Research of recombinant lysine endopetidase construction expression and activity. Journal of Chongqing University of Technology(Natural Science), 2015,29(4):53-59.
[24] Ito L, Shiraki K, Uchida T , et al. Crystallization and preliminary crystallographic analysis of Achromobacter protease I mutants. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2010,66(11):1531-1532.
doi: 10.1107/S1744309110037759 pmid: 3001667744104202110241198099570
[25] Achour B, Barber J . The activities of Achromobacter lysyl endopeptidase and Lysobacter lysyl endoproteinase as digestive enzymes for quantitative proteomics. Rapid Communications in Mass Spectrometry, 2013,27(14):1669-1672.
doi: 10.1002/rcm.6612 pmid: 23765612
[26] Guo Z G, Cheng J, Sun H D , et al. A qualitative and quantitative evaluation of the peptide characteristics of microwave- and ultrasound-assisted digestion in discovery and targeted proteomic analyses. Rapid Communications in Mass Spectrometry, 2017,31(16):1353-1362.
doi: 10.1002/rcm.7913 pmid: 28557149
[27] Paulo J A, Mancias J D, Gygi S P . Proteome-wide protein expression profiling across five pancreatic cell lines. Pancreas, 2017,46(5):690-698.
doi: 10.1097/MPA.0000000000000800 pmid: 28375945
[28] Kishimoto T, Kondo J, Takai I T , et al. Accurate mass comparison coupled with two endopeptidases enables identification of protein termini. Proteomics, 2011,11(3):485-489.
doi: 10.1002/pmic.201000537 pmid: 21268277
[29] Guan X, Brownstein N C, Young N L , et al. Ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry for peptide de novo amino acid sequencing for a seven-protein mixture by paired single-residue transposed Lys-N and Lys-C digestion. Rapid Communications in Mass Spectrometry, 2017,31(2):207-217.
doi: 10.1002/rcm.v31.2
[30] Raijmakers R, Neerincx P, Mohammed S , et al. Cleavage specificities of the brother and sister proteases Lys-C and Lys-N. Chemical Communications (Cambridge,England), 2010,46(46):8827-8829.
doi: 10.1039/c0cc02523b pmid: 20953479
[31] Brownstein N C, Guan X, Mao Y , et al. Paired single residue-transposed Lys-N and Lys-C digestions for label-free identification of N-terminal and C-terminal MS/MS peptide product ions: ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry for peptide de novo sequencing. Rapid Communications in Mass Spectrometry, 2015,29(7):659-666.
doi: 10.1002/rcm.7137
[32] Tofteng A P, Jensen K J , Sch?ffer L,et al. Total synthesis of desB30 insulin analogues by biomimetic folding of single-chain precursors. A European Journal of Chemical Biology, 2008,9(18):2989-2996.
doi: 10.1002/cbic.200800430 pmid: 19035371
[33] Maiorino M I, Petrizzo M, Capuano A , et al. The development of new basal insulins:is there any clinical advantage with their use in type 2 diabetes. Expert Opinion on Biological Therapy. 2014,14(6):799-808.
doi: 10.1517/14712598.2014.895812 pmid: 24673155
[34] 夏晶 . 一种甘精胰岛素及其类似物的制备方法: 中国,CN 102816785 A, 2012-12-12. 一种甘精胰岛素及其类似物的制备方法: 中国,CN 102816785 A,2012-12-12. .
Xia J. A Process for Preparation of Insulin Glargine and Analogues: China,CN 102816785 A, 2012-12-12. A Process for Preparation of Insulin Glargine and Analogues: China,CN 102816785 A,2012-12-12. .
[35] Partha H, Srikanth G S, Suma S , et al. A Process for Preparation of Insulin Compounds: International,WO2010016069, 2010-02-11. A Process for Preparation of Insulin Compounds: International,WO2010016069,2010-02-11. .
[36] Cao H, Deterding L J, Blackshear P J . Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry. Public Library of Science One, 2014,9(7):e100977.
doi: 10.1371/journal.pone.0100977 pmid: 4091943
[37] Imamura H, Sugiyama N, Wakabayashi M , et al. Large-scale identification of phosphorylation sites for profiling protein kinase selectivity. Journal of Proteome Research, 2014,13(7):3410-3419.
doi: 10.1021/pr500319y pmid: 24869485
[38] Cheng Y, Chen Y, Yu C . Fast and efficient non-reduced Lys-C digest using pressure cycling technology for antibody disulfide mapping by LC-MS. Journal of Pharmaceutical and Biomedical Analysis, 2016,129:203-209.
doi: 10.1016/j.jpba.2016.07.002 pmid: 27429370
[39] Du Y, Wang F, May K , et al. LC-MS analysis of glycopeptides of recombinant monoclonal antibodies by a rapid digestion procedure. Journal of Chromatography B, 2012,907:87-93.
doi: 10.1016/j.jchromb.2012.09.004 pmid: 23036907
[40] 宋智心, 田恩冰, 马怀安 , 等. 探讨Lys-C胰蛋白酶对于质谱法测定糖化血红蛋白的应用价值. 国际检验医学杂志, 2013,34(3):257-259.
doi: 10.3969/j.issn.1673-4130.2013.03.001
Song Z X, Tian E B, Ma H A , et al. Application value of Lys-C trypsin for the detection of HbA 1c by using mass spectrometric method. International Journal of Laboratory Medicine, 2013,34(3):257-259.
doi: 10.3969/j.issn.1673-4130.2013.03.001
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