Expression of Recombinant Alcohol Dehydrogenase in Escherichia coli Strain BL21 (DE3) and In Planta Agrobacterium Transformation of Tomato Seeds

Alcohol dehydrogenase is an enzyme that is involved in various roles in plant such as in plant development, growth and plant responses to abiotic and biotic stresses. A recombinant alcohol dehydrogenase 1 (Adh1) cDNA (r-msAdh1) from Metroxylon sagu has been previously isolated, containing 20 nucleotides derived from Elaeis guineensis at the 5’-end, with a molecular weight of 1.14 kb. The objective of this study is to determine the function of r-msAdh1 via analyses in prokaryotic and eukaryotic hosts. For expression in prokaryotic system, pET-41a(+) with a 8x His tag at the C terminal was used for r-msAdh1 protein purification and expression was achieved using IPTG for four to six hours in Escherichia coli strain BL21 (DE3) incubated at low temperature. The induced BL21 strain produced a small amount of soluble r-msAdh1 protein while large amount was present as insoluble aggregates. Subsequently, the r-msAdh1 cDNA was transformed into tomato seeds (Solanum lycopersicum cv. MT1) via Agrobacterium-mediated in planta transformation. The integration of r-msAdh1 cDNA and the selectable marker were detected in transformed seedlings, T0, using polymerase chain reaction technique. The transformation efficiency was determined to be 33% for r-msAdh1 cDNA and 46% for the selectable marker. For stability analysis of the transgene, eleven T1 generation randomly selected from the transgenic T0 were analyzed for the presence of the cDNA, and all seedlings were found to contain the full length of r-msAdh1 cDNA. However, out of eleven T1 transgenic lines produced, only four seedlings were used for expression analysis using the reverse transcriptase PCR (RT-PCR). Two transgenic lines, T19 and T111, were determined to contain r-msAdh1 cDNA and verified by nucleotide sequencing. Although only a small number of T1 transgenic seedlings was obtained, this study shows that tomato seeds could be used as a target tissue for Agrobacterium-mediated in planta transformation primarily because the protocol is easy, rapid and cheaper compared to tissue culture-based method

Optimisation of Remazol Brilliant Blue R dye decolourisation and laccase enzyme production by Marasmius cladophyllus using response surface methodology

The decolourisation of Remazol Brilliant Blue R dye and laccase activity was investigated using pure culture of an endophytic fungus, Marasmius cladophyllus. The fungus is found capable of decolourising 99% of the dye after 12 days of incubation in Glucose Minimal (GM) liquid media (pH 5.5) and laccase activity of 285 U/L was recorded. Response surface methodology (RSM) was used to determine and optimise the significant variable(s) in order to obtain the optimum dye decolourisation conditions and laccase production. It was also used to study the interaction effect of the variables on both responses. Box-Behnken Design was used to identify the significant variable(s) whereas the optimisation process was done by using Central Composite Design. It was found that initial dye concentration of 100-300 mg/L, incubation period of 4-20 days and pH of liquid medium of 4-8 significantly influenced the decolourisation of dye and laccase activity. However, only the relationship of the incubation period and pH is significantly affected both the responses. Maximum dye decolourisation of 100% was successfully achieved and the highest laccase activity of 504.53 U/L was recorded after 16 days of incubation period at pH 7 with 259.46 mg/L initial dye concentration

Lignocellulolytic enzymes produced by tropical white rot fungi during biopulping of Acacia mangium wood chips

Pycnoporus coccineus and Coriolus versicolor are among the tropical white rot basidiomycetes that degrade lignin selectively. In this research work, crude enzyme produced during biopulping using both fungi were extracted, filtered and assayed using specific substrates. Both the peroxidases enzyme activities and residual lignin content were measured for the incubation period of 20, 40 and 60 days of biopulping. For both fungi, manganese peroxidase is predominant and highly expressed thus shows the highest rate compared to other ligninolytic enzymes activities in all of extract preparations. After 60 days of inoculation, manganese peroxidase activities are recorded as 270.51 U/mL and 274.36 U/mL for C. versicolor and P. coccineus, respectively. On the evaluation of the lignin content after biopulping, the lignin content showed significant decreased. Wood chip biotreated with C. versicolor showed higher percentage in lignin loss (9.42%) compared to with P. coccineus (8.10%)