Abstract
This study aims to explore the potential aminopeptidases of Lysinibacillus sphaericus based on the unique metabolic characteristics of this species which cannot metabolize carbohydrates and may have a strong ability to metabolize amino acids. Fifteen peptidase-encoding genes predicted in L. sphaericus C3-41 have been heterologously expressed in Escherichia coli BL21, and of these genes, only Amp0279 shows a high ability to hydrolyze L-leucine-4-nitroanilide (Leu-pNA). Phylogenetic analysis, 3D-structure modeling, and enzyme assays indicated that Amp0279 should be a novel Co2+-dependent aminopeptidase belonging to the M29 family. The optimal conditions of Amp0279 were determined to be 50 °C and pH 8.0 with the addition of 100 μM Co2+, and under this condition, the specific activity of Amp0279 matched that of Flavourzyme® (3.54 × 104 vs. 3.37 × 104 U/mg for the protein ingredient of Flavourzyme®). Amp0279 is mainly expressed in the middle sporulation phase in wild-type L. sphaericus or in Bacillus subtilis under the control of the sporulation-dependent strong promoter pcry8E, which is carried by the recombinant vector pHT315-8E21b. Furthermore, the secretory expression systems based on B. subtilis and Corynebacterium glutamicum were used to enhance the soluble expression of Amp0279. Obvious expression and enzymatic activity were detected from the crude supernatant media of both host bacteria without further concentration and purification. Moreover, expression can occur in the vegetative phase in B. subtilis under the control of the Pgrac promoter.
Key points
• A novel Co 2+ -dependent leucyl aminopeptidase Amp0279 originating from L. sphaericus was characterized.
• The activity of Amp0279 as a leucyl aminopeptidase matches that of Flavourzyme® under optimal conditions.
• B. subtilis– and C. glutamicum–based expression systems are built to promote secretory (soluble) Amp0279 expression.
This is a preview of subscription content, log in via an institution to check access.
taken from WB800N (pHT315-8E21b-amp0279). + , positive control using purified Amp0279 expressed in BL21 (pET28a-amp0279). Lanes 1–8 are the samples taken at 0 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h, respectively. The samples were precipitated bacterial cells treated with loading buffer
taken from the supernatant of WB800N (pHT43) before and after induction. Due to the fused His_tag, the hybridization band in the positive control was larger than the expressed Amp0279 in B. subtilis. b Lanes 1 and 2 were the supernatant taken from CGMCC1.15647 (pXMJ19-5.7) and CGMCC1.15647 (pXMJ19-5.7-amp0279); lanes 3 and 4 were the supernatant after sonication and centrifugation from CGMCC1.15647 (pXMJ19-5.7) and CGMCC1.15647 (pXMJ19-5.7-amp0279); and lanes 5 and 6 were cell debris after sonication and centrifugation from CGMCC1.15647 (pXMJ19-5.7) and CGMCC1.15647 (pXMJ19-5.7-amp0279), respectively
Similar content being viewed by others
References
Berry C (2012) The bacterium, Lysinibacillus sphaericus, as an insect pathogen. J Invertebr Pathol 109(1):1–10. https://doi.org/10.1016/j.jip.2011.11.008
Cheng C, Wang X, Dong Z, Shao C, Yang Y, Fang W, Fang C, Wang H, Yang M, Jiang L, Zhou X, Song H (2015) Aminopeptidase T of M29 family acts as a novel intracellular virulence factor for Listeria monocytogenes infection. Sci Rep 5:17370. https://doi.org/10.1038/srep17370
Cohen SN, Chang AC, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69(8):2110–2114. https://doi.org/10.1073/pnas.69.8.2110
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797. https://doi.org/10.1093/nar/gkh340
European Food Safety A (2009) Assessment of the safety of cobalt(II) chloride hexahydrate added for nutritional purposes as a source of cobalt in food supplements and the bioavailability of cobalt from this source. EFSA J 7(5):1066. https://doi.org/10.2903/j.efsa.2009.1066
Fernandez-Espla MD, Rul F (1999) PepS from Streptococcus thermophilus. A new member of the aminopeptidase T family of thermophilic bacteria. Eur J Biochem 263(2):502–10. https://doi.org/10.1046/j.1432-1327.1999.00528.x
Field KL, Bachmanov AA, Mennella JA, Beauchamp GK, Kimball BA (2009) Protein hydrolysates are avoided by herbivores but not by omnivores in two-choice preference tests. PLoS ONE 4(1):e4126. https://doi.org/10.1371/journal.pone.0004126
Finley BL, Monnot AD, Paustenbach DJ, Gaffney SH (2012) Derivation of a chronic oral reference dose for cobalt. Regul Toxicol Pharmacol 64(3):491–503. https://doi.org/10.1016/j.yrtph.2012.08.022
Gaur VK, Bajaj A, Regar RK, Kamthan M, Jha RR, Srivastava JK, Manickam N (2019) Rhamnolipid from a Lysinibacillus sphaericus strain IITR51 and its potential application for dissolution of hydrophobic pesticides. Bioresour Technol 272:19–25. https://doi.org/10.1016/j.biortech.2018.09.144
Giesler L, Linke D, Rabe S, Appel D, Berger RG (2013) Hydrolysis of wheat gluten by combining peptidases of Flammulina velutipes and electrodialysis. J Agric Food Chem 61(36):8641–8649. https://doi.org/10.1021/jf401716m
Gonzales T, Robert-Baudouy J (1996) Bacterial aminopeptidases: properties and functions. FEMS Microbiol Rev 18(4):319–344. https://doi.org/10.1111/j.1574-6976.1996.tb00247.x
Hanif MU, Gul R, Hanif MI, Hashmi AA (2017) Heterologous secretory expression and characterization of dimerized bone morphogenetic protein 2 in Bacillus subtilis. Biomed Res Int 2017:9350537. https://doi.org/10.1155/2017/9350537
Hu X, Fan W, Han B, Liu H, Zheng D, Li Q, Dong W, Yan J, Gao M, Berry C, Yuan Z (2008) Complete genome sequence of the mosquitocidal bacterium Bacillus sphaericus C3–41 and comparison with those of closely related Bacillus species. J Bacteriol 190(8):2892–2902. https://doi.org/10.1128/JB.01652-07
Javier EV, Jenny D (2016) Adsorption of toxic metals and control of mosquitos-borne disease by Lysinibacillus sphaericus: dual benefits for health and environment. Biomed Environ Sci 29(3):187–196. https://doi.org/10.3967/bes2016.023
Kallscheuer N, Vogt M, Stenzel A, Gätgens J, Bott M, Marienhagen J (2016) Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones. Metab Eng 38:47–55. https://doi.org/10.1016/j.ymben.2016.06.003
Kanwal S, Abeysinghe S, Srisaisup M, Boonserm P (2021) Cytotoxic effects and intracellular localization of Bin toxin from Lysinibacillus sphaericus in human liver cancer cell line. Toxins (Basel) 13(4):228. https://doi.org/10.3390/toxins13040288
Lei F, Zhao Q, Sun-Waterhouse D, Zhao M (2017) Characterization of a salt-tolerant aminopeptidase from marine Bacillus licheniformis SWJS33 that improves hydrolysis and debittering efficiency for soy protein isolate. Food Chem 214:347–353. https://doi.org/10.1016/j.foodchem.2016.07.028
Leyssens L, Vinck B, Van Der Straeten C, Wuyts F, Maes L (2017) Cobalt toxicity in humans-A review of the potential sources and systemic health effects. Toxicology 387:43–56. https://doi.org/10.1016/j.tox.2017.05.015
Li J, Huang C, Zheng D, Wang Y, Yuan Z (2012) CcpA-mediated enhancement of sugar and amino acid metabolism in Lysinibacillus sphaericus by NMR-based metabolomics. J Proteome Res 11(9):4654–4661. https://doi.org/10.1021/pr300469v
Liu B, Du X, Zhou L, Kenji H, Su W, Cao M (2008a) Purification and characterization of a leucine aminopeptidase from the skeletal muscle of common carp (Cyprinus carpio). Food Chem 108(1):140–147. https://doi.org/10.1016/j.foodchem.2007.10.055
Liu YH, Lu FP, Li Y, Yin XB, Wang Y, Gao C (2008b) Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600. Appl Microbiol Biotechnol 78(1):85–94. https://doi.org/10.1007/s00253-007-1287-z
Lozano LC, Dussán J (2013) Metal tolerance and larvicidal activity of Lysinibacillus sphaericus. World J Microbiol Biotechnol 29(8):1383–1389. https://doi.org/10.1007/s11274-013-1301-9
Luo W, Liu C, Zhang R, He J, Han B (2014) Anticancer activity of binary toxins from Lysinibacillus sphaericus IAB872 against human lung cancer cell line A549. Nat Prod Commun 9(1):107–10
Matsushita-Morita M, Furukawa I, Suzuki S, Yamagata Y, Koide Y, Ishida H, Takeuchi M, Kashiwagi Y, Kusumoto KI (2010) Characterization of recombinant prolyl aminopeptidase from Aspergillus oryzae. J Appl Microbiol 109(1):156–165. https://doi.org/10.1111/j.1365-2672.2009.04641.x
McDonald JK (1985) An overview of protease specificity and catalytic mechanisms: aspects related to nomenclature and classification. Histochem J 17(7):773–785. https://doi.org/10.1007/bf01003313
Mitea C, Havenaar R, Drijfhout JW, Edens L, Dekking L, Koning F (2008) Efficient degradation of gluten by a prolyl endoprotease in a gastrointestinal model: implications for coeliac disease. Gut 57(1):25–32. https://doi.org/10.1136/gut.2006.111609
Motoshima H, Minagawa E, Tsukasaki F, Kaminogawa S (1997) Cloning of genes of the aminopeptidase T family from Thermus thermophilus HB8 and Bacillus stearothermophilus NCIB8924: apparent similarity to the leucyl aminopeptidase family. Biosci Biotechnol Biochem 61(10):1710–1717. https://doi.org/10.1271/bbb.61.1710
Nandan A, Nampoothiri KM (2020) Therapeutic and biotechnological applications of substrate specific microbial aminopeptidases. Appl Microbiol Biotechnol 104(12):5243–5257. https://doi.org/10.1007/s00253-020-10641-9
Naureen Z, Rehman NU, Hussain H, Hussain J, Gilani SA, Al Housni SK, Mabood F, Khan AL, Farooq S, Abbas G, Harrasi AA (2017) Exploring the potentials of Lysinibacillus sphaericus ZA9 for plant growth promotion and biocontrol activities against phytopathogenic fungi. Front Microbiol 8:1477. https://doi.org/10.3389/fmicb.2017.01477
Ng DHC, Klassen J, Embleton ND, McGuire W (2017) Protein hydrolysate versus standard formula for preterm infants. Cochrane Database Syst Rev 10:CD012412. https://doi.org/10.1002/14651858.CD012412.pub2
Odintsov SG, Sabała I, Bourenkov G, Rybin V, Bochtler M (2005) Staphylococcus aureus aminopeptidase S is a founding member of a new peptidase clan. J Biol Chem 280(30):27792–27799. https://doi.org/10.1074/jbc.M502023200
Oei C, Hindley J, Berry C (1992) Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains. J Gen Microbiol 138(7):1515–1526. https://doi.org/10.1099/00221287-138-7-1515
Panja S, Saha S, Jana B, Basu T (2006) Role of membrane potential on artificial transformation of E. coli with plasmid DNA. J Biotechnol 127(1):14–20. https://doi.org/10.1016/j.jbiotec.2006.06.008
Peng F, Wang X, Sun Y, Dong G, Yang Y, Liu X, Bai Z (2017) Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system. Microb Cell Fact 16(1):201. https://doi.org/10.1186/s12934-017-0814-6
Qin Q, Tang C, Wu J, Chen S, Yan Z (2021) A dual-functional aminopeptidase from Streptomyces canus T20 and its application in the preparation of small rice peptides. Int J Biol Macromol 167:214–222. https://doi.org/10.1016/j.ijbiomac.2020.11.175
Rahman A, Nahar N, Nawani NN, Jass J, Desale P, Kapadnis BP, Hossain K, Saha AK, Ghosh S, Olsson B, Mandal A (2014) Isolation and characterization of a Lysinibacillus strain B1-CDA showing potential for bioremediation of arsenics from contaminated water. J Environ Sci Health A Tox Hazard Subst Environ Eng 49(12):1349–1360. https://doi.org/10.1080/10934529.2014.928247
Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD (2018) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 46(D1):D624-d632. https://doi.org/10.1093/nar/gkx1134
Sierra EM, Pereira MR, Maester TC, Gomes-Pepe ES, Mendoza ER, Lemos EGM (2017) Halotolerant aminopeptidase M29 from Mesorhizobium SEMIA 3007 with biotechnological potential and its impact on biofilm synthesis. Sci Rep 7(1):10684. https://doi.org/10.1038/s41598-017-10932-8
Ta HM, Bae S, Han S, Song J, Ahn TK, Hohng S, Lee S, Kim KK (2013) Structure-based elucidation of the regulatory mechanism for aminopeptidase activity. Acta Crystallogr D Biol Crystallogr 69(Pt 9):1738–1747. https://doi.org/10.1107/s0907444913012651
Tang AX, Liu H, Liu YY, Li QY, Qing YM (2017) Purification and characterization of a novel beta-cypermethrin-degrading aminopeptidase from Pseudomonas aeruginosa GF31. J Agric Food Chem 65(43):9412–9418. https://doi.org/10.1021/acs.jafc.7b03288
Tang W, Li Z, Li C, Yu X, Wang F, Wan X, Wang Y, Ma L (2016) High-level expression and characterization of the Bacillus subtilis subsp. subtilis str. BSP1 YwaD aminopeptidase in Pichia pastoris. Protein Expr Purif 122:23–30. https://doi.org/10.1016/j.pep.2016.02.009
Toelstede S, Hofmann T (2008) Sensomics mapping and identification of the key bitter metabolites in Gouda cheese. J Agric Food Chem 56(8):2795–2804. https://doi.org/10.1021/jf7036533
Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46(W1):W296-w303. https://doi.org/10.1093/nar/gky427
Winters P, Caldwell R, Enfield LEF (1996) The ampS-nprE (124°–-127°) region of the Bacillus subtilis 168 chromosome sequencing of a 27 kb segment and identification of several genes in the area free. Microbiology (Reading) 142(3033):3037
Wu Y, Hu X, Ge Y, Zheng D, Yuan Z (2012) Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis. FEMS Microbiol Lett 330(2):105–112. https://doi.org/10.1111/j.1574-6968.2012.02539.x
Xu K, Yuan Z, Rayner S, Hu X (2015) Genome comparison provides molecular insights into the phylogeny of the reassigned new genus Lysinibacillus. BMC Genomics 16:140. https://doi.org/10.1186/s12864-015-1359-x
Yang M, Zheng J, Jia H, Song M (2016) Functional characterization of X-prolyl aminopeptidase from Toxoplasma gondii. Parasitology 143(11):1443–1449. https://doi.org/10.1017/S0031182016000986
Yasbin RE, Wilson GA, Young FE (1975) Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. J Bacteriol 121(1):296–304. https://doi.org/10.1128/jb.121.1.296-304.1975
Zhang W, Yang Y, Liu X, Liu C, Bai Z (2019) Development of a secretory expression system with high compatibility between expression elements and an optimized host for endoxylanase production in Corynebacterium glutamicum. Microb Cell Fact 18(1):72. https://doi.org/10.1186/s12934-019-1116-y
Zhou C, Zheng Q, Peng Q, Du L, Shu C, Zhang J, Song F (2014) Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant. Appl Microbiol Biotechnol 98(18):7901–7909. https://doi.org/10.1007/s00253-014-5874-5
Acknowledgements
We thank Dr. Xiuxia Liu from Jiangnan University, China, and Dr. Puping Song from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, for kindly supplying C. glutamicum CGMCC1.15647, pXMJ19-5.7, and pHT315-8E21b.
Funding
This project was supported by the National Natural Science Foundation of China (NFSC) (32170008), Hubei Natural Science Foundation (2021CFB201), Hubei Province Innovation Major Program (2018ABA093), and “Fundamental Research Funds for the Central Universities” of South-Central University for Nationalities (CZZ20001). National Natural Science Foundation of China (NFSC),32170008,Xiaomin Hu,Hubei Natural Science Foundation,2021CFB201,Peiling Geng,Hubei Province Innovation Major Program,2018ABA093,Hairong Xiong,Fundamental Research Funds for Central Universities of South-Central University for Nationalities,CZZ20001,Xiaomin Hu
Author information
Authors and Affiliations
Contributions
XH and HX conceived and designed the research. PZ, MZ and XW conducted experiments. PZ and PG analyzed data. PZ and XH wrote the manuscript. All authors read and approved the manuscript.
Corresponding authors
Ethics declarations
Ethics approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhao, P., Zhang, M., Wan, X. et al. Characterization and heterologous expression of a novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from Lysinibacillus sphaericus. Appl Microbiol Biotechnol 106, 1139–1149 (2022). https://doi.org/10.1007/s00253-022-11767-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-022-11767-8