Abstract
Polyurethane (PUR) is a soil and aquatic contaminant throughout the world. Towards bioremediation, in a previous study, a soil bacterium, Pseudomonas sp. AKS31, capable of efficiently degrading PUR was isolated. Polyurethanase (PURase) enzyme is capable of cleaving the ester bond of PUR and is considered as a key regulator of PUR biodegradation. Hence, for a high yield, easy purification, and further characterization, the aim of this study was to clone and overexpress the PURase gene of this isolate. The current study also investigated structural aspects of this enzyme through predictive bioinformatics analyses. In this context, the PURase gene of the isolate was cloned and expressed in E. coli using pET28(a)+ vector. The obtained recombinant protein was found insoluble. Therefore, first, the protein was made soluble with urea and purified using nickel-NTA beads. The purified enzyme exhibited substantial activities when tested on the LA-PUR plate. Bioinformatics-based analysis of the protein revealed the presence of a lipase serine active site and indicated that this PURase belongs to the Family 1.3 lipase. Hence, the present study shows that active PURase can be produced in large quantities using a prokaryotic expression system and thus, provides an effective strategy for in-vitro PUR-degradation.
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References
Aldila F, Susilowati A, Setyaningsih R (2019) Polyurethane degrading bacteria isolated from decayed teak wood (Tectonagrandis Linn. F.). J Biodjati 4(2):225–235. https://doi.org/10.1557/biodjati.v4i2.4525
Ali SS, Elsamahy T, Koutra E, Kornaros M, El-Sheekh M, Abdelkarim EA, Zhu D, Sun J (2021) Degradation of conventional plastic wastes in the environment: a review on current status of knowledge and future perspectives of disposal. Sci Total Environ 771:144719. https://doi.org/10.1016/j.scitotenv.2020.144719
Angkawidjaja C, You DJ, Matsumura H, Kuwahara K, Koga Y, Takano K, Kanaya S (2007) Crystal structure of a family I3 lipase from Pseudomonas sp. MIS38 in a closed conformation. FEBS Lett 581(26):5060–5064. https://doi.org/10.1016/j.febslet.2007.09.048
Baeshen MN, Al-Hejin AM, Bora RS, Ahmed MM, Ramadan HA, Saini KS, Baeshen NA, Redwan EM (2015) Production of biopharmaceuticals in E. coli: current scenario and future perspectives. J Microbiol Biotechnol 25(7):953–962. https://doi.org/10.4014/jmb.1412.12079
Baumann U (2019) Structure-function relationships of the repeat domains of RTX toxins. Toxins 11(11):657. https://doi.org/10.3390/toxins11110657
Blow D (1990) More of the catalytic triad. Nature 343:694–695. https://doi.org/10.1038/343694a0
Borchert E, Hammerschmidt K, Hentschel U, Deines P (2021) Enhancing microbial pollutant degradation by integrating eco-evolutionary principles with environmental biotechnology. Trends Microbiol 29(10):908–918. https://doi.org/10.1016/j.tim.2021.03.002
Bracco P, Van Midden N, Arango E, Torrelo G, Ferrario V, Gardossi L, Hanefeld U (2020) Bacillus subtilis lipase a—lipase or esterase? Catalysts 10(3):308. https://doi.org/10.3390/catal10030308
Cárdenas Espinosa MJ, Colina Blanco A, Heipieper HJ, Eberlein C (2021) Screening and cultivating microbial strains able to grow on building blocks of polyurethane. Meth Enzymol 648:423–434. https://doi.org/10.1016/bs.mie.2020.12.008
Esmaili I, Mohammad Sadeghi HM, Akbari V (2018) Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies. Res Phar Sci 13(5):413–421. https://doi.org/10.4103/1735-5362.236834
Foschi E, D’Addato F, Bonoli A (2021) Plastic waste management: a comprehensive analysis of the current status to set up an after-use plastic strategy in Emilia-Romagna Region (Italy). Environ Sci Pollut Res 28(19):24328–24341. https://doi.org/10.1007/s11356-020-08155-y
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch (2005) Protein identification and analysis tools on the ExPASy SERVER. In: Walker JM (eds) The proteomics protocols handbook. Totowa: Humana Press, pp 571–607. https://archiveouverte.unige.ch/unige:37793. Accessed 9 Mar 2005
Ghosal D, Ghosh S, Dutta TK, Ahn Y (2016) Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons (PAHs): a review. Front Microbiol 7:1369. https://doi.org/10.3389/fmicb.2016.01369
Gurung N, Ray S, Bose S, Rai V (2013) A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. BioMed Res Int. https://doi.org/10.1155/2013/329121
Haimov B, Srebnik S (2016) A closer look into the α-helix basin. Sci Rep 6:38341. https://doi.org/10.1038/srep3834
Howard GT (2002) Biodegradation of polyurethane: a review. Int Biodeterior Biodegr 49(4):245–252. https://doi.org/10.1016/s0964-8305(02)00051-3
Jaiswal S, Shukla P (2020) Alternative strategies for microbial remediation of pollutants via synthetic biology. Front Microbiol 11:808. https://doi.org/10.3389/fmicb.2020.00808
Kelley L, Mezulis S, Yates C et al (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858. https://doi.org/10.1038/nprot.2015.053
Liu J, He J, Xue R, Xu B, Qian X, Xin F, Blank LM, Zhou J, Wei R, Dong W, Jiang M (2021) Biodegradation and up-cycling of polyurethanes: progress, challenges, and prospects. Biotechnol Adv 48:107730. https://doi.org/10.1016/j.biotechadv.2021.107730
Maruthamuthu M, van Elsas JD (2017) Molecular cloning, expression, and characterization of four novel thermo-alkaliphilic enzymes retrieved from a metagenomic library. Biotechnol Biofuels 10:142. https://doi.org/10.1186/s13068-017-0808-y
Millet H, Vangheluwe P, Block C, Sevenster A, Garcia L, Antonopoulos R (2018) The nature of plastics and their societal usage. In: Hester RE, Harrison RM (eds) Plastics and the environment, Royal Society of Chemistry: Cambridge, UK, pp 1–20. https://doi.org/10.1039/9781788013314-00001
Okunola AA, Kehinde IO, Oluwaseun A, Olufiropo EA (2019) Public and environmental health effects of plastic wastes disposal: a review. J Toxicol Risk Assess. https://doi.org/10.23937/2572-4061.1510021
Omori K, Idei A, Akatsuka H (2001) Serratia ATP-binding cassette protein exporter, Lip, recognizes a protein region upstream of the C terminus for specific secretion. J Biol Chem 276(29):27111–27119. https://doi.org/10.1074/jbc.M101410200
Pathak VM, Navneet (2017) Review on the current status of polymer degradation: a microbial approach. Bioresour Bioprocess. https://doi.org/10.1186/s40643-017-0145-9
Ramachandran GN, Ramakrishnan C, Sasisekharan V (1963) Stereochemistry of polypeptide chain configurations. J Mol Biol 7:95–99. https://doi.org/10.1016/s0022-2836(63)80023-6
Ramos CR, Abreu PA, Nascimento AL, Ho PL (2004) A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide. Braz J Med Biol 37(8):1103–1109. https://doi.org/10.1590/s0100-879x2004000800001
Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. https://doi.org/10.3389/fmicb.2014.00172
Rosano GL, Morales ES, Ceccarelli EA (2019) New tools for recombinant protein production in Escherichia coli: a 5-year update. Protein Sci 28:1412–1422. https://doi.org/10.1002/pro.3668
Roy R, Mukherjee G, Das Gupta A, Tribedi P, Sil AK (2021) Isolation of a soil bacterium for remediation of polyurethane and low-density polyethylene: a promising tool towards sustainable cleanup of the environment. 3 Biotech 11(1):29. https://doi.org/10.1007/s13205-020-02592-9
Sarkar A, Chatterjee A, Ansari S, Chakraborty N (2016) Characterization of molecular mimicry between UL18 glycoprotein of human Cytomegalovirus [HCMV] and class-I MHC molecule through pattern-based analysis: an In-silico approach. J Health Med Informat 7:230. https://doi.org/10.4172/2157-7420.1000230
Siracusa V (2019) Microbial degradation of synthetic biopolymers waste. Polymers 11(6):1066. https://doi.org/10.3390/polym11061066
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. https://doi.org/10.1093/molbev/msr121
Tribedi P, Gupta AD, Sil AK (2015) Adaptation of Pseudomonas sp. AKS2 in biofilm on low-density polyethylene surface: an effective strategy for efficient survival and polymer degradation. Bioresour Bioprocess. https://doi.org/10.1186/s40643-015-0044-x
Young CL, Britton ZT, Robinson AS (2012) Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 7(5):620–634. https://doi.org/10.1002/biot.201100155
Acknowledgements
Authors would like to thank Dr. Soumalee Basu and Dr. Biraj Sarkar for their help in the analysis of Bioinformatics data and critical reading. Authors would also like to thank Department of Microbiology, University of Calcutta for providing laboratory facilities, high-speed internet and computational laboratory facilities. The authors would also like to thank DBT-IPLS program at the University of Calcutta for various instrumental support. SPD is supported by DST, Government of India. GM is supported by CSIR, Government of India. DM like to thank DST, Govt. of India.
Funding
Dr. Mousumi Saha acknowledges the financial support from National Post Doctoral Fellowship (NPDF) scheme by Science and Engineering Research Board, Department of Science and Technology (SERB-DST), Government of India (PDF/2017/000854). The contingency grant was used for the purchase of chemicals, used in the different experiments. It was also partly supported by DST, Govt. of West Bengal [701(SANC)/ST/P/S&T/2G-3/2010, dt- 03/12/2015]. No additional grants were available to support the article processing charges.
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MS, SPD, GM, AB, DG perform the experiments and analysed the results. MS and AKS conceived the idea, designed the experiments, analysed the results and wrote the manuscript.
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Saha, M., Dutta, S.P., Mukherjee, G. et al. Cloning, expression and characterization of PURase gene from Pseudomonas sp. AKS31. Arch Microbiol 204, 498 (2022). https://doi.org/10.1007/s00203-022-03110-6
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DOI: https://doi.org/10.1007/s00203-022-03110-6