Sains Malaysiana 49(1)(2020): 85-92

http://dx.doi.org/10.17576/jsm-2020-4901-10

Analysis of Free Oligosaccharides (fOS) from Wild-Type Saccharomyces cerevisiae (Baker's Yeast) using Two Different Extraction Methods

(Analisis Oligosakarida Bebas (fOS) daripada Saccharomyces cerevisiae (Yis Baker) Jenis Liar menggunakan Dua Kaedah Pengekstrakan Berbeza)

IQBAL JALALUDIN¹, AMIRUL HUSNA SUDIN¹, DHARSHINI ELANGOVAN¹, HUSSEIN M. AL-BAJALAN¹, NUR MAISARAH SARIZAN², NOOR LIANA MAT YAJIT³, KAMALRUL AZLAN AZIZAN², ABDUL MUNIR ABDUL MURAD³, FARAH DIBA ABU BAKAR3, DOMINIC S. ALONZI⁴ & MUKRAM MOHAMAD MACKEEN^1,2*

¹Department of Chemical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

²Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

³Department of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

⁴Oxford Glycobiology Institute, University of Oxford, Oxford OX1 3QU, United Kingdom

Received: 1 October 2019/Accepted: 22 October 2019

ABSTRACT

The glycomic profiles of free oligosaccharides (fOS) derived from misfolded N- and O-linked glycoproteins and lipid-linked oligosaccharides are important molecular signatures in various biological processes and serve as a readout of functional properties such as glycosidase inhibition. Several glycan extraction methods are available based on different sorbent chemistries that may influence the analytical profiles obtained. However, there is limited availability of studies comparing the effects of sorbent chemistries on glycan profiles.  Therefore, in our study, the fOS profiles from wild-type Saccharomyces cerevisiae (Baker's yeast) extracted using two common methods namely mixed-bed ion-exchange (MBIE) [AG50W-X12 (H⁺) and AG2-X8 (Cl^-)] and reversed-phase (C18) sorbents were compared using total carbohydrate (phenol sulfuric acid) and total protein (bicinchoninic acid, BCA) assays, thin-layer chromatography (TLC) and high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) analyses. MBIE extraction contained higher oligosaccharide and protein (0.26 mg/mL and 1.8 mg/mL) content than C18 extraction (0.11 mg/mL and 0.2 mg/mL). TLC analysis (butanol: ethanol: water = 6:3:1 and 5:4:1) showed the presence of fOS in both the MBIE and C18 extracts based on the detection of orcinol active (UV-inactive) spots. Similar peaks were present in the HPLC-ELSD chromatograms for both extractions methods with MBIE showing higher abundance.  Glycan unit (GU) analysis of the dextran standard using HPLC-ELSD showed that the largest possible oligosaccharide structures detected were only di/trisaccharides. Based on all these results, MBIE extraction is a more suitable carbohydrate extraction technique compared to C18 extraction for subsequent profiling and functional studies of fOS.

Keywords: Free oligosaccharides (fOS); glycans; HPLC-ELSD; Saccharomyces cerevisiae; TLC

ABSTRAK

Profil glikomik oligosakarida bebas (fOS) yang diterbit daripada glikoprotein N- dan O- yang tersalah lipat serta oligosakarida terpaut-lipid (LLO) adalah penanda molekul penting dalam pelbagai proses biologi dan berfungsi sebagai bacaan terhadap sifat berfungsi seperti perencatan glikosidase. Beberapa kaedah pengekstrakan glikan boleh didapati berdasarkan pelbagai kimia bahan erap yang berbeza yang mungkin mempengaruhi profil analitik. Walau bagaimanapun, kajian terhadap perbandingan kesan kimia bahan erap terhadap profil glikan adalah terhad. Oleh itu, dalam kajian ini profil fOS daripada Saccharomyces cerevisiae (yis Baker) jenis liar diekstrak menggunakan dua kaedah yang digunakan secara meluas iaitu bahan erap dan pertukaran ion lapisan-campuran (MBIE) AG50W-X12 (H⁺) dan AG2-X8 (Cl^-)] fasa berbalik (C18) dibandingkan dengan jumlah karbohidrat (asid fenol sulfurik) dan jumlah protein (asid bisikoninik, BCA), kromatografi lapisan nipis (TLC) dan kromatografi cecair berprestasi tinggi-penyejatan penyerakan cahaya (HPLC-ELSD). Pengekstrakan MBIE mengandungi kandungan oligosakarida dan protein yang lebih tinggi iaitu (0.26 mg/mL dan 1.8 mg/mL) berbanding dengan pengekstrakan C18 (0.11 mg/mL dan 0.2 mg/mL). Analisis TLC (butanol: etanol: air = 6: 3: 1 dan 5: 4: 1) menunjukkan kehadiran fOS dalam kedua-dua ekstrak MBIE dan C18 berdasarkan tompok aktif orsinol (tidak aktif-UV). Puncak yang sama hadir dalam kromatogram HPLC-ELSD untuk kedua-dua kaedah pengekstrakan dengan MBIE menunjukkan kelimpahan yang lebih tinggi. Analisis unit glikan (GU) terhadap data HPLC-ELSD piawai dekstran menunjukkan struktur oligosakarida yang paling besar dikesan hanya di/trisakarida. Berdasarkan keputusan yang diperoleh, pengekstrakan MBIE merupakan teknik pengekstrakan karbohidrat yang lebih sesuai berbanding dengan pengekstrakan C18 untuk kajian profil dan fungsi fOS.

Kata kunci: Glikan; HPLC-ELSD; Oligosakarida bebas (fOS); Saccharomyces cerevisiae; TLC

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*Corresponding author; email: mukram.mackeen@ukm.edu.my