Sains Malaysiana 53(12)(2024): 3219-3227

http://doi.org/10.17576/jsm-2024-5312-07

 

Nanobunga Hibrid Lektin sebagai Pembawa Novel untuk Pemegunan Glikoenzim

(Lectin Hybrid Nanoflowers as Novel Carrier for Glycoenzyme Immobilisation)

 

WAN NURAZRA MARSYA WAN AHMAD¹, NOR NADIA SAAD¹, NUR NABILAH SHAHIDAN² & MUHAMMAD ASHRAF SHAHIDAN^1,*

 

¹Jabatan Sains Biologi dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
²Jabatan Teknologi Tenaga, Mineral dan Bahan Fakulti Biokejuruteraan dan Teknologi, Universiti Malaysia Kelantan, Kampus Jeli, 17600 Jeli, Kelantan, Malaysia

 

Received: 29 April 2024/Accepted: 19 August 2024

 

Abstrak

Interaksi khusus antara concanavalin A (Con A), sejenis lektin daripada tumbuhan jackbean, dengan teras manosa pada glikan-N membolehkannya digunakan untuk pemegunan glikoenzim. Dalam kajian ini, nanobunga hibrid Con A- CuSO₄ (CCHN) disediakan sebagai pembawa novel untuk pemegunan peroksidase lobak putih (HRP), glikoenzim yang digunakan secara meluas dalam aplikasi bioanalitikal. Morfologi CCHN yang disediakan dicerap menggunakan mikroskop elektron pengimbasan pancaran medan (FESEM) menunjukkan mikrostruktur persis-bunga dengan saiz purata diameter 51.5 μm serta saiz kelopak bunga yang luas sekitar 2 μm. Seterusnya, pengoptimuman pemegunan HRP pada CCHN dilakukan menggunakan format tiub Eppendorf dan plat mikro menunjukkan bacaan penyerapan A₄₅₀ tertinggi pada campuran 100 mg/mL HRP dan 10 mg/mL CCHN untuk kedua-dua format. Aktiviti HRP terpegun pada CCHN yang diukur menggunakan format tiub Eppendorf (A₄₅₀ = 1.58) adalah dua kali ganda lebih tinggi berbanding format plat mikro (A₄₅₀ = 0.76) dan dipilih untuk kajian seterusnya. Bacaan A₂₈₀ untuk setiap supernatan yang diperolehi selepas pengemparan dalam langkah pembasuhan yang diulang sebanyak empat kali pula didapati amat rendah menunjukkan HRP terpegun dengan kuat pada CCHN dan tiada kebocoran enzim pada larutan. Ujian penjanaan semula menunjukkan HRP terpegun boleh ditanggalkan daripada CCHN dengan sekali pembasuhan menggunakan gula perencat metil a-manosa pada kepekatan 0.1 M. Selain itu, ujian kebolehulangan turut dilakukan menggunakan tiga kelompok CCHN yang berbeza kerana tiada perbezaan signifikan pada bacaan A₄₅₀ menunjukkan kebolehulangan yang baik untuk pemegunan HRP. Keputusan kajian ini menunjukkan CCHN yang dihasilkan mempunyai potensi yang tinggi untuk digunakan sebagai pembawa untuk pemegunan glikoenzim secara mudah, tekal serta boleh diguna-semula.

 

Kata kunci: Con A; glikoenzim; lektin; nanobunga

 

Abstract

Specific interaction between concanavalin A (Con A), a plant lectin from jackbean, with mannose core in N-glycans can be exploited to immobilise glycoenzymes. In this study, we prepared Con A-copper sulphate hybrid nanoflower (CCHN) as a novel carrier to immobilise horseradish peroxidase (HRP), a widely used glycoenzyme in bioanalytical application. The morphology of the prepared CCHN was determined using field emission scanning electron microscopy (FESEM), which showed flower-like microstructures with an average diameter size of 51.5 μm with and wide petal size of an approximately 2 μm. Then, HRP immobilisation optimisation experiments were carried out in Eppendorf tubes and microplates, which both formats showed the highest A₄₅₀ measurements at 100 mg/mL HRP and 10 mg/mL CCHN mixture. However, twice HRP activities (A₄₅₀ =1.58) were recorded when using Eppendorf tubes format as compared to microplate format (A₄₅₀ = 0.76) and thus was used for further experiments. Furthermore, supernatant obtained after each washing step showed very low A₂₈₀ values up to four washing cycles, indicating HRP was strongly immobilised to the CCHN and no enzyme leakage into solution. Meanwhile, regeneration study showed that single washing step using inhibitory sugar, methyl a-mannose, at 0.1 M can effectively detach HRP from CCHN. Moreover, reproducibility test using CCHN prepared in three different batches showed consistent HRP activity readings indicating good reproducibility. This study highlighted the potential application of lectin hybrid nanoflower as a simple, reproducible and reusable carrier for glycoenzyme immobilisation.

 

Keywords: Con A; glycoenzyme; lectin; nanoflowers

 

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*Corresponding author; email: mashraf@ukm.edu.my