Malaysian Journal of Analytical Sciences Vol 22 No 5 (2018): 857 - 866

IMMOBILIZED METAL AFFINITY CHROMATOGRAPHIC MEMBRANE FOR TRYPSIN SEPARATION

(Membran Kromatografi Afiniti Logam Dipegun untuk Pemisahan Tripsin)

Sofiah Hamzah¹*, Nurul Hidayati Mat Alim¹, Nurul Fakhriah Ismail¹, Norhafiza Ilyana Yatim², Nur A’lya Mohd Sani¹

¹School of Ocean Engineering

²School of Marine and Environmental Sciences

Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

*Corresponding author: sofiah@umt.edu.my

Received: 16 April 2017; Accepted: 7 March 2018

Abstract

As a new technology, Immobilized Metal Affinity Chromatographic Membrane (IMAC) has proven its efficiency for protein purification. It is also a separation technique that use covalently bound chelating compounds on the membrane supports to immobilize metal ions, which serve as affinity ligands for various proteins. This study aims to prepare and characterize highly specific IMAC for trypsin separation. Chitosan and polyethylene glycol were used as modification solutions to impart the membrane porosity and flux recovery ratio (FRR) of IMAC matrix. The modified PSf with chitosan improved its FRR up to 82.11% which indicated that PSf/Chitosan was a suitable matrix for the affinity membrane. Glutaraldehyde and Cu²⁺ served as crosslinker agents and affinity ligands respectively. Maximum immobilization capacity of Cu²⁺ occurred at 120 ppm within 60 minutes’ incubation time. The optimum capacity of trypsin adsorption (12.67 mg/cm²) onto IMAC membrane occurred when 0.3 M ionic strength of trypsin solution was used. Desorption of the enzyme by displacing salt of potassium chloride showed the highest trypsin recovery at 72.3%.

Keywords: affinity, chitosan, membrane, trypsin, ultrafiltration

Abstrak

Sebagai teknologi baru, Membran Kromatografi Afiniti Logam Dipegun (IMAC) telah membuktikan kecekapannya untuk penulenan protein. Ia juga merupakan teknik pemisahan yang menggunakan sebatian pelekat yang terikat secara kovalen pada membran sokongan untuk pegunkan ion logam, yang berfungsi sebagai ligan afiniti untuk pelbagai protein. Kajian ini bertujuan untuk menyediakan dan mencirikan IMAC yang sangat spesifik untuk pemisahan trypsin. Kitosan dan polietilen glikol digunakan sebagai larutan pengubahsuai bagi meningkatkan keliangan membran dan nisbah pemulihan fluks (FRR) matriks IMAC. PSf yang telah diubah suai dengan kitosan telah meningkatkan FRR sehingga 82.11 % yang menunjukkan bahawa PSf/kitosan adalah matrik yang sesuai untuk membran afiniti. Glutaraldehid dan Cu²⁺ masing-masing berfungsi sebagai agen silangan dan ligan afiniti. Kapasiti imobilisasi maksimum Cu²⁺ berlaku pada 120 ppm dalam masa inkubasi selama 60 minit. Kapasiti jerapan trypsin paling optimum (12.67 mg / cm²) ke atas membran IMAC berlaku apabila 0.3M larutan tripsin digunakan. Penyerapan enzim dengan menggunakan garam gantian kalium klorida menunjukkan dapatan tripsin paling tinggi iaitu kira-kira 72.3%.

Kata kunci: afiniti, kitosan, membran, tripsin, penurasan-ultra

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