OBJECTIVE: The main objective of this study was to explore oleaginous yeast, Yarrowia lipolytica isolated from soil and optimization of culture conditions and medium components to obtained better quality microbial oil for biodiesel production.
METHODS: Fifty yeast strains were isolated from soil from different regions of Lahore and eleven of them were selected for oil production. The isolated yeast colonies were screened to further check their lipid producing capabilities by the qualitative analysis. Five yeast strains were designated as oleaginous because they produced more than 16% of oil based on their biomass. To estimate the total lipid content of yeast cells, the extraction of lipids was done by performing the procedure proposed by Bligh and Dyer. The transesterification of yeast oils was performed by using different methods. There were three different strategies customized to transesterifying microbial oil using base catalyzed transesterification, acid catalyzed transesterification and enzyme-based transesterification. After completion of transesterification, sample was used for fatty acid methyl esters (FAMEs) were analyzed by gas-chromatograph with ionization detector type MS.
RESULTS: The isolate IIB-10 identified as Yarrowia lipolytica produced maximum amount of lipids i.e. 22.8%. More amount of biomass was obtained when cane molasses was utilized as carbon source where it produced 29.4 g/L of biomass while sucrose and lactose were not utilized by IIB-10 and no biomass was obtained. Similarly, meat extracts showed best results when it was used as nitrogen source because it resulted in 35.8 g/L biomass of Yarrowia lipolytica IIB-10. The culturing conditions like size of inoculum, effect of pH and time of incubation were also studied. The 10% of inoculum size produced 25.4 g/L biomass at 120 h incubation time, while the pH 7 was the optimum pH at which 24.8 g/L biomass was produced by Yarrowia lipolytica IIB-10. GC-MS analysis showed that biodiesel produced by transesterification contained similar fatty acids as found in vegetable oil for this reason it is widely accepted feedstock for biodiesel production.
CONCLUSION: The analysis of fatty acids methyl esters showed the similar composition of microbial oil as in vegetable oils and high amount of methyl esters were obtained after transesterification. Therefore, potentially oleaginous yeast could be used to generate a large amount of lipids for biodiesel production that will be the better substitute of petroleum-based diesel and will also control the environmental pollution.
METHODS: In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic(®) CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment.
RESULTS: Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel(®) and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 °C. Although CBHB on its own was unable to digest crystalline substrates, supplementation of CBHB (0.37%) with Cellic(®) CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CBHB supplementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples.
DISCUSSION: Since CBHB alone was inactive against crystalline cellulose, these data suggested that it might work synergistically with other components of Cellic(®) CTec2. CBHB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic(®) CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CBHB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.
RESULTS: Yeast two-hybrid (Y2H) experiment was used to identify the binding partners of surface antigens of T. gondii by using SAG2 as bait. Colony PCR was performed and positive clones were sent for sequencing to confirm their identity. The yeast plasmids for true positive clones were rescued by transformation into E. coli TOP 10F' cells. The interplay between bait and prey was confirmed by β-galactosidase assay and co-immunoprecipitation experiment. We detected 20 clones interacting with SAG2 based on a series of the selection procedures. Following the autoactivation and toxicity tests, SAG2 was proven to be a suitable candidate as a bait. Thirteen clones were further examined by small scale Y2H experiment. The results indicated that a strong interaction existed between Homo sapiens zinc finger protein and SAG2, which could activate the expressions of the reporter genes in diploid yeast. Co-immunoprecipitation experiment result indicated the binding between this prey and SAG2 protein was significant (Mann-Whitney U-test: Z = -1.964, P = 0.05).
CONCLUSIONS: Homo sapiens zinc finger protein was found to interact with SAG2. To improve the understanding of this prey protein's function, advanced investigations need to be carried out.