Papain-functionalized gold nanoparticles as heterogeneous biocatalyst for bioanalysis and biopharmaceuticals analysis.
Surface-modified gold nanoparticles (GNPs) were synthesized via layer-by-layer process with alternating cationic polyallylamine and anionic poly(acrylic acid) polyelectrolyte layers leading to a highly hydrophilic biocompatible shell supporting colloidal stability. Afterwards, papain was covalently immobilized on the modified GNPs via amide coupling between the amino groups on papain and the terminal carboxylic groups of the modified GNPs by using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium as coupling agents. The resultant papain-functionalized gold nanoparticles were characterized by surface plasmon resonance, dynamic light scattering and zeta potential measurements. The new technology resonant mass measurement was applied for determining the average number of papain molecules immobilized per GNP by measurement of the single nanoparticle buoyant mass in the range of femtograms. The activity of the immobilized enzyme was estimated by determination of the kinetic parameters (Km, Vmaxand kcat) with the standard chromogenic substrate Nα-benzoyl-dl-arginine-4-nitroanilide hydrochloride. It was found that Kmof immobilized and free enzyme are in the same order of magnitude. On contrary, turnover numbers kcatwere significantly higher for GNP-conjugated papain. Further, the gold nanobiocatalyst was applied for digestion of polyclonal human immunoglobulin G to yield protein fragments. The resultant fragment mixture was further analyzed by high-performance liquid chromatography-microelectrospray ionization-quadrupole-time-of-flight mass spectrometry, which demonstrated the applicability of the bioreactor based on papain functionalized GNPs. The immobilized papain not only has higher catalytic activity and better stability, but also can be easily isolated from the reaction medium by straightforward centrifugation steps for reuse.
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Siyao
S
Liu
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Markus
M
Höldrich
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Adrian
A
Sievers-Engler
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Jeannie
J
Horak
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany. Electronic address: Michael.laemmerhofer@uni-tuebingen.de.
Michael
M
Lämmerhofer
D055162:Biocatalysis; D015374:Biosensing Techniques / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006046:Gold / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000378:metabolism; D053768:Metal Nanoparticles / Q000737:chemistry*; D008958:Models, Molecular; D010206:Papain / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013499:Surface Properties
D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D006046:Gold; D010206:Papain
Biopharmaceuticals (biologicals) analytics; Gold nanoparticle; Immobilized enzyme reactor; Immobilized papain; Resonant mass measurement; Sample preparation