Ciri inokulum bagi pengkulturan kulat oleaginus pencilan tempatan, Cunninghamella bainieri 2A1 dibangunkan dengan mengenal pasti kesan jenis, umur dan saiz inokulum terhadap pertumbuhan, penghasilan lipid dan GLA. Pengkulturan dijalankan pada suhu 30ºC dengan kadar goncangan 250 rpm dalam kelalang goncangan 500 mL yang mengandungi 200 mL medium terhad nitrogen. Inokulum spora didapati lebih sesuai berdasarkan produktiviti penghasilan lipid yang tinggi iaitu 0.71 (g/L/hari) berbanding penggunaan inokulum sel vegetatif vegetatif 24 jam dan 48 jam yang masingmasing memberikan produktiviti hanya 0.51 dan 0.45 (g/L/hari). Selain itu, penghasilan GLA (5.3 × 10-2 g/g biojisim tanpa lipid) dalam kultur yang dimulakan dengan inokulum spora (1 × 105 spora/mL) didapati lebih tinggi sebanyak 23% berbanding inokulum sel vegetatif. Kepekatan spora sebanyak 1 × 103 spora/mL menghasilkan morfologi pellet bersaiz 1.04 mm dan berkadaran dengan kandungan lipid dan GLA masing-masing sebanyak 40% (g/g biojisim) dan 8.34 × 10-2 (g/g biojisim tanpa lipid).
Cunninghamella bainieri 2A1 is an oleaginous fungus whose lipid accumulation profile is significantly influenced by metal ion concentrations in growth medium. Mg2+, Fe3+, Mn2+ and Cu2+ were found to be the important elements affecting lipid accumulation in this fungus. This study employs a statistical method (Response Surface Methodology – RSM) to study the combined effects of Mg2+, Fe3+, Mn2+ and Cu2+ on lipid accumulation of C. baineri 2A1. Cultivation was carried out in 250 mL Erlenmeyer flasks containing 100 mL nitrogen limited medium at 30oC and 250 rpm agitation for 120 h. A thirty-run central composite design experiment was employed to identify and optimize the significant factors. In addition to Mg2+ and Fe3+ which were shown to have significant effects on lipid accumulation, the interactions between Mg2+ and Cu2+, as well as the effect of Cu2+ in quadratic terms were also found to have significant effect on the process (p<0.05). The highest amount of lipid obtained in this study was 39% g/g biomass with optimal levels of Mg2+, Fe3+ and Cu2+ at 5.00, 0.017 and 0.0005 g/L, respectively, while Mn2+ was omitted. A 32% increment in lipid yield was recorded, where the lipid content increased to 38%, compared to initial yield of 29% g/g biomass prior to optimization. In conclusion, Mg2+ and Fe3+ have significant positive effect on the lipid accumulation of this fungus, whereas Mn2+ and Cu2+ exert negative effects in combination.
The effects of ammonium tartrate and glucose concentration on biomass, lipid and GLA accumulation in Cunninghamella sp. 2A1 were investigated using Response Surface Methodology (RSM). Cultivation was carried out in 250 mL shake flask containing 100 mL of nitrogen limiting medium (with various combinations of concentration of ammonium tartrate (1-3 g/L) and glucose (30-60 g/L) at 30°C and 250 rpm agitation for 120 h. The concentration of both compounds significantly affected the biomass, lipid and GLA yield (p<0.05), with the production of each of them being represented by quadratic models. Higher concentration of ammonium tartrate and glucose (2.99 and 59.33 g/L, respectively) was required for enhanced biomass production whereas low nitrogen content with excess glucose was otherwise favoured for lipid and GLA production. Ammonium tartrate and glucose concentration at 1 and 43 g/L, respectively were estimated by the model and proven to give the highest lipid production and GLA yield of 31.06 % (g/g biomass) and 4.15 ×10-2 (g/g lipid less biomass), respectively
We report here the isolation and sequence analysis of a cDNA fragment for an endoglucanase gene from Aspergillus terreus SUK-I. A pair of primers was designed based on conserved regions of endoglucanase gene. Amplification of A. terreus SUK-I cDNA using the primers produced a DNA band of 642 bp. The cDNA fragment was purified and cloned into pCR®ll-TOPO plasmid vector. The cDNA insert was designated as SA2 and sequenced. Analysis of the SA2 sequence showed a high degree of identity towards endoglucanase gene sequences from A. aculeatus, A kawachii and Talaromyces emersonii. While, the putative amino acid sequence of SA2 showed 74 % identity towards endoglucanase protein from Thermoascus aurantiacus and 69 % identity towards endoglucanase protein from A. kawachii, A. niger, A. aculeatus and A. nidulans. A 66 % identity between SA2 putative amino acid sequence and A. oryzae endoglucanase protein sequence was also observed. Therefore, we concluded that SA2 codes for a putative endoglucanase gene of A. terreus SUK-I.
[Kami melaporkan di sini pemencilan dan analisis satu fragmen cDNA untuk gen endoglukanase daripada Aspergillus terreus SUK-I. Sepasang pencetus telah direkabentuk berasaskan kepada kawasan terpelihara gen endoglukanase. Amplifikasi cDNA A. terreus SUK-I menggunakan pencetus berkenaan menghasilkan satu jalur DNA bersaiz 642 pb. Fragmen cDNA berkenaan telah ditulenkan dan diklonkan ke dalam vektor plasmid pCR®II-TOPO. cDNA selitan telah dilabelkan sebagai SA2 dan dijujukkan. Analisis terhadap jujukan SA2 menunjukkan sebahagian daripada jujukan SA2 mempunyai darjah identiti yang tinggi terhadap jujukan gen endoglukanase daripada A. aculeatus, A. kawachii dan Talaromyces emersonii. Sementara, jujukan asid amino putatif SA2 menunjukkan identiti sebanyak 74% terhadap protein endoglukanase daripada Thermoascus aurantiacus dan identiti sebanyak 69% terhadap protein endoglukanase daripada A. kawachii, A. niger, A. aculeatus dan A. nidulans. ldentiti sebanyak 66% juga diperhatikan di antara jujukan asid amino putatif SA2 dan jujukan protein endoglukanase A. oryzae. Oleh itu, kami membuat kesimpulan bahawa SA2 mengkodkan gen putative endoglukanase A. terreus SUK-I].