Analysis of free oligosaccharides (fOS) from wild-type Saccharomyces cerevisiae (Baker’s yeast) using two different extraction methods

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

Kadar pelepasan bromokarbon jangka hayat pendek oleh rumpai laut tropika menggunakan simulasi laut tropika

Bagi negara tropika yang mempunyai keberhasilan marin yang tinggi seperti Malaysia, makroalga (rumpai laut) telah menjadi penyumbang utama kepada pelepasan bromokarbon jangka hayat pendek (VSL) ke dalam atmosfera. Faktor abiotik seperti keamatan cahaya dan kepekatan klorofil a telah diketahui mempengaruhi pengeluaran bromokarbon oleh rumpai laut, namun begitu masih lagi kurang kajian yang mengukur secara sistematik pengaruh rumpai laut terhadap kadar pelepasan bromokarbon VSL dijalankan. Oleh itu, sistem pengkulturan rumpai laut yang diselaraskan dengan keadaan persekitaran semula jadi disediakan bagi mengkaji kadar pelepasan bromokarbon VSL (CH2Br2, CHBr3 dan CHBr2Cl) bagi tujuh rumpai laut merah, perang dan hijau iaitu Gracilaria changii, Ulva reticulata, Caulerpa racemosa var. macrophysa, Kappaphycus alvarezii, Sargassum binderi, Sargassum siliquosum dan Padina australis. Penghasilan bromokarbon VSL menunjukkan kitaran diurnal dengan kepekatan halokarbon meningkat kepada tahap maksimum pada waktu tengahari (1738 pmolL-1) dan menurun apabila keamatan cahaya dan suhu permukaan laut (SST) berkurang. Penghasilan bromokarbon VSL rumpai laut yang diletakkan di bawah cahaya matahari adalah lima kali ganda lebih tinggi daripada penghasilan tangki akuakultur yang diletakkan dalam persekitaran gelap yang menunjukkan berlakunya penghasilan fotokimia. Purata kadar penghasilan fotokimia untuk bromokarbon VSL daripada uji kaji tangki akuakultur berjulat antara 1 dan 137 pmol per g-1 FW-1 h-1. Ini menjadikan rumpai laut merah (Gracilaria changii) sebagai pengeluar tertinggi. Begitu juga, bromoperoksida (BPO) yang diekstrak daripada kesemua rumpai laut juga menunjukkan aktiviti tertinggi dalam rumpai laut merah diikuti oleh rumpai laut perang dan hijau

Functional characterisation and product specificity of Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1

Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides