Abstract
The objective of this study was to use for the first time depth filters, which are usually intended for clarification of cell culture broth, as a direct immobilization support/matrix for industrially relevant enzymes. With this method, it is not only possible to immobilize pure enzymes; it can be also used for capturing recombinant enzymes directly out of culture supernatant. Therefore, the depth filters were coated with different anionic and cationic polymer layers by Layer-by-Layer (LbL) technology. The immobilization behavior of the model enzyme Candida antarctica lipase B (CalB) was examined. Optimal conditions for lipase immobilization were found for anionic surfaces with Poly (allylamin hydrochlorid) (PAH)/Poly (sodium-4-styrene sulfonate) (PSS) coating in 20 mM acetate buffer pH 4. Stability studies showed that immobilized CalB is 1.7-fold more stable when storage is carried out in buffer at 4 °C, compared to storage in buffer at room temperature or storage after drying at 30 °C for 24 h. The calculated half-life period is 108 days until half of the activity was lost. Furthermore, the possibility of direct capture of the CalB either from sonicated culture broth (Escherichia coli) or from cell-free supernatant was tested. Filter blocking prevented the immobilization of lipase from sonicated culture broth, but immobilization from cell-free supernatant could be performed successfully at moderate biomass content (OD600 = 7.0).
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References
Anderson EM, Larsson KM, Kirk O (1998) One biocatalyst—many applications: the use of Candida antarctica B-lipase in organic synthesis. Biocatal Biotransfor 16:181–204
Arroyo M, Sánchez-Montero JM, Sinisterra JV (1999) Thermal stabilization of immobilized lipase B from Candida antarctica on different supports: Effect of water activity on enzymatic activity in organic media. Enzyme Microb Tech 24:3–12
Bastida A, Sabuquillo P, Armisen P, Fernandez-Lafuente R, Huguet J, Guisan JM (1998) A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnol Bioeng 58:486–493
Caruso F, Niikura K, Furlong DN, Okahata Y (1997) 2. Assembly of alternating polyelectrolyte and protein multilayer films for immunosensing. Langmuir 13:3427–3433
Cesarini S, Infanzón B, Pastor FIJ, Diaz P (2014) Fast and economic immobilization methods described for non-commercial Pseudomonas lipases. BMC Biotechnol 14:27
Decher G (1997) Fuzzy nanoassemblies: toward layered polymeric multicomposites. Science 277:1232–1237
Fernandez-Lafuente R, Armisén P, Sabuquillo P, Fernández-Lorente G, M. Guisán J (1998) Immobilization of lipases by selective adsorption on hydrophobic supports. Chem Phys Lipids 93:185–197
Ghostine RA, Markarian MZ, Schlenoff JB (2013) Asymmetric growth in polyelectrolyte multilayers. J Am Chem Soc 135:7636–7646
Gotor-Fernández V, Busto E, Gotor V (2006) Candida antarctica lipase B: an ideal biocatalyst for the preparation of nitrogenated organic compounds. Adv Synth Catal 348:797–812
Gumí T, Paolucci-Jeanjean D, Belleville M-P, Rios GM (2007) Enzymatic membrane reactor involving a hybrid membrane in supercritical carbon dioxide. J Membrane Sci 297:98–103
Gupta S, Bhattacharya A, Murthy CN (2013) Tune to immobilize lipases on polymer membranes: techniques, factors and prospects. Biocatal Agric Biotechnol 2:171–190
Ji X, Su Z, Wang P, Ma G, Zhang S (2014) Polyelectrolyte doped hollow nanofibers for positional assembly of bienzyme system for cascade reaction at O/W interface. ACS Catal 4:4548–4559
Ladam G, Schaaf P, Frédéric JGC, Decher G, Voegel J-C (2001) Protein adsorption onto auto-assembled polyelectrolyte films. Langmuir 17:878–882
Larsen MW, Bornscheuer UT, Hult K (2008) Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems. Protein Expres Purif 62:90–97
Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Tech 40:1451–1463
Minow B, Egner F, Jonas F, Lagrange B (2014) High-cell-density clarification by single-use diatomaceous Earth filtration. BioProcess Int 12:2–9
Nicoletti G, Cipolatti EP, Valério A, Carbonera NG, Soares NS, Theilacker E, Ninow JL, de Oliveira D (2015) Evaluation of different methods for immobilization of Candida antarctica lipase B (CalB lipase) in polyurethane foam and its application in the production of geranyl propionate. Bioprocess Biosyst Eng 38:1739–1748
Ohlrogge K, Ebert K (2006) Membranen. Grundlagen, Verfahren und industrielle Anwendungen. VCH Verlag GmbH & Co. KGaA, Weinheim, Wiley
Ong AL, Kamaruddin AH, Bhatia S, Aboul-Enein HY (2008) Enantioseparation of (RS)-ketoprofen using Candida antarctica lipase B in an enzymatic membrane reactor. J Sep Sci 31:2476–2485
Peyratout CS, Dähne L (2004) Tailor-made polyelectrolyte microcapsules: from multilayers to smart containers. Angew Chem Int Ed 43:3762–3783
Prashad M, Tarrach K (2006) Depth filtration: cell clarification of bioreactor offloads. Filtr Separat 43:28–30
Rios GM, Belleville MP, Paolucci D, Sanchez J (2004) Progress in enzymatic membrane reactors—a review. J Membrane Sci 242:189–196
Sharma R, Chisti Y, Banerjee UC (2001) Production, purification, characterization, and applications of lipases. Biotechnol Adv 19:627–662
Trodler P, Nieveler J, Rusnak M, Schmid RD, Pleiss J (2008) Rational design of a new one-step purification strategy for Candida antarctica lipase B by ion-exchange chromatography. J Chrom A 1179:161–167
Trusek-Holownia A, Noworyta A (2002) Catalytic membrane preparation for enzymatic hydrolysis reactions carried out in the membrane phase contactor. Desalination 144:427–432
Uppenberg J, Hansen MT, Patkar S, Jones TA (1994) The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure 15:293–308
Venkiteshwaran A, Fogle J, Patnaik P, Kowle R, Chen D (2015) Mechanistic evaluation of virus clearance by depth filtration. Biotechnol Progr 31:431–437
Yigzaw Y, Piper R, Tran M, Shukla AA (2006) Exploitation of the adsorptive properties of depth filters for host cell protein removal during monoclonal antibody purification. Biotechnol Progr 22:288–296
Acknowledgments
This project is in cooperation between the Institute of Technical Chemistry, Sartorius Stedim Biotech GmbH, and Surflay Nanotec GmbH. It has been carried out as an integral part of the Biocatalysis 2021 Cluster, which is financially supported by the BMBF (Bundesministerium für Bildung und Forschung). Special thanks also to Uwe Bornscheuer and his group from the Institute of Biochemistry, Greifswald University, Germany for providing the expression vector. Funmilola Heinen is gratefully acknowledged for assistance in the laboratory work.
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Schreiber, S., Thiefes, A., Schuldt, U. et al. New application of depth filters for the immobilization of Candida antarctica lipase B. Appl Microbiol Biotechnol 101, 599–607 (2017). https://doi.org/10.1007/s00253-016-7764-5
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DOI: https://doi.org/10.1007/s00253-016-7764-5