Open Access Original Research Article

Evaluation of Genetic Variability in Adansonia digitata L. by RAPD Analysis

Laxmi Gautam, Showkat Ahmad Ganie, Rajneesh Kumar Agnihotri, Rajendra Sharma

Biotechnology Journal International, Page 1-6
DOI: 10.9734/BBJ/2015/18727

Baobab (Adansonia digitata L.) belonging to Bombacaceae family, is one of the most widely used indigenous tree species in sub-Saharan Africa. The objective of the present study was to assess molecular variation among A. digitata and to determine the level of genetic similarity among them. The yield of DNA ranged from 15-40 µg/mg of leaf tissue and the purity was between 1.1- 2.9, indicating minimal levels of contaminating metabolites. The technique was ideal for isolation of DNA from A. digitata and the DNA isolated was used for randomly amplified polymorphic DNA (RAPD) analysis using the primer OPB07. The bands obtained ranged in size from 54-795 bp. Two clusters were observed, one group with 8 bands and the other with 11 bands. Present study could be important in domestication, conservation, management & improvement strategies of A. digitata.

Open Access Original Research Article

New Assay Method for Allene Oxide Cyclase, an Important Enzyme in Jasmonic Acid Biosynthesis

Keimei Oh, Yoichiro Shimura

Biotechnology Journal International, Page 1-7
DOI: 10.9734/BBJ/2015/20089

Aims: Allene oxide cyclase (AOC) (EC 5.3.99.6) is an important enzyme of jasmonates (JAs) biosynthesis. JAs are important signals that play a pivotal role in defense response of plants to environmental cues. Regulation JA biosynthesis is believed useful for elucidating the mechanism of plant defense system. Despite the high potential of AOC as a target for JA biosynthesis inhibitors, an efficient assay method suitable for screening AOC inhibitors is still not available. The aim of this work is to develop an efficient AOC assay method.

Study Design: Using excess amount of purified recombinant allene oxide synthase (AOS) combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-HPOT), we established an efficient method to generate (12,13S)-epoxyoctadecatrienoic acid (EOT), the substrate of AOC. The AOS produced EOT was subsequently converted to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid (OPDA) by using purified recombinant AOC in a real time manner and the amount of OPDA was determined by HPLC.

Place and Duration of Study: All the experiments were conducted from October 2009 to March 2013 at Akita Prefectural University, Japan.

Methodology: The recombinant AOS and AOC were expressed in E. coli. The target proteins were purified using affinity chromatography, respectively. The unstable EOT was generated by using excess AOS combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The AOC synthesized OPDA was characterized by the comparison of HPLC retention time with the OPDA standard. AOC activity was calculated by determine the amount of OPDA in the assay system.

Results: We found in the presence of 50 nmol of purified AOS together with 20 mM 13-HPOT, the synthesis of OPDA was saturated when using 5 nmol of purified AOC in the enzyme reaction for 30 min. Our results indicated that the AOC activity can be determined by dual enzyme system.

Conclusion: We established an efficient assay method for AOC which may be applied for screening of AOC inhibitors.

Open Access Original Research Article

Cellulase Producing Potential of Aspergillus terreus Uv2 on Cellulosic Wastes Pretreated with Acid and Alkali

D. Damisa, F. A. Kuta, O. P. Abioye, J. D. Bala, N. E. Egbe

Biotechnology Journal International, Page 1-10
DOI: 10.9734/BBJ/2015/18773

Aims: Cellulases offer very wide applications in biotechnology and enzymes from microbial origins present inexpensive source. Production of value added chemicals from wastes will be an exciting translation from waste to wealth and an eco-friendly initiative instead of the incineration option often given to cellulosic wastes.

Study Design: Sulphuric acid and Sodium hydroxide solutions were prepared at 0.5 M and 2 M concentrations to pretreat three cellulosic wastes that had been made neutral prior to fermentation with a known cellulase producing mold

Place and Duration of Study: All experiments were conducted in the laboratory of the Department of Microbiology, Federal University of Technology, Minna, Nigeria for a period of six weeks.

Methodology: Hypercellulase producing Aspergillus terreus UV2 strain was used to ferment pretreated cellulosic wastes: Corn cob, corn straw and bagasse, using submerged fermentation in Mandel basal medium. The crystalline lignocelluloses were milled and fractionated into 850 μ particle size and pretreated in two concentrations (0.5 M and 2 M) of both acid (sulphuric acid) and alkali (sodium hydroxide) independently and were left for varying residence time of one hour or three hours in the digester at ambient temperature, Optimum spore concentration of 1.0 x 106 spores/ml and pH of 4.8. Supernatants of crude enzyme were taken and assayed at 24 hours interval.

Results: Cellulase activity peaked at 96 hours. Enzyme secretion in the cellulosic wastes was highest in sugarcane bagasse, followed by the corn cob and then the corn straw corresponding to 51%, 40% and 16% respectively. Alkali pretreated cellulosics gave higher yield of cellulase than its counterpart acid. Non-pretreated residues gave only low enzyme titers. Bagasse produced optimum cellulase yield of 0.068 IU/ml/min within 120 hours when subjected to 2 M NaOH digestion for one hour before fermentation. This translated to 39% increase in enzyme expression when compared with non-treated bagasse of 0.049 IU/ml/min.

Conclusion: Sugarcane bagasse therefore when digested with mild alkali (2 M NaOH) for a pretreatment period of one hour holds a great possibility for cellulase production using a mutant mold, Aspergillus terreus UV2. Production of value added chemicals from cellulosic wastes will be an exciting translation from waste to wealth.

Open Access Original Research Article

Catalyzed Mediator-Based Decolorization of Five Synthetic Dyes by Pleurotus ostreatus ARC280 Laccase

Ali M. Elshafei, Abdelmageed M. Othman, Mohamed M. Hassan, Bakry M. Haroun, Maysa A. Elsayed, Ayman A. Farrag

Biotechnology Journal International, Page 1-15
DOI: 10.9734/BBJ/2015/19505

Aim: The aim of the present study was to evaluate qualitatively the decolorization of five dyes by Pleurotus ostreatus (P. ostreatus) ARC280 using solid medium. The laccase produced by the fungus was used in terms of its concentration and thermal stability for enzymatic decolorization and also in combination with Hydroxybenzotriazole (HBT) as a redox mediator.

Study Design: Qualitative evaluation of decolorization of dyes and determining the best conditions required for decolorization in the presence and absence of HBT.

Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between January 2013 and February 2014.

Methodology: P. ostreatus ARC280 fungal ability for dyes decolorization was qualitatively evaluated on solid medium containing (g/L): dye, 0.1; glucose, 10; agar, 30; 100 mL mineral solution and 100 mL wheat bran washing water obtained by boiling 50 g of wheat bran in 1000 mL of distilled water. The efficiency of decolorization was expressed in terms of decolorization percentage as follows:

Decolorization (%)

=

100

×

Absorbance t0 - Absorbance tf

 

Absorbance t0

Where Absorbance t0 is the absorbance at the optimum wavelength of the reaction mixture before incubation with the enzyme and Absorbance tf is the absorbance at the optimum wavelength after incubation time.

Results: The enzyme was efficient in decolorizing Acid Blue C.I. 220 (100%), Dichlorophenol indophenol sodium salt D 5110 (92.6%) and Brilliant Green C.I. 42040 (78.6%) after 6 h of incubation at 30ºC. In the presence of HBT (1 mM), Lanasol Red 6G was greatly affected by HBT as a laccase mediator system with decolorization percentage of 53.85% instead of 10.90 in case of laccase alone, however the enzyme could not efficiently decolorize Foron Yellow Brown S 2RFLI dye even in presence of HBT. The decolorization efficiency of all dyes was decreased by increasing reaction temperature from 30 to 50ºC. The absorbance reduction at the maximum wavelength was recorded with all the tested dyes.

Conclusion: The results obtained clearly confirmed the role of P. ostreatus ARC280 laccase and its mediated system in the decolorization of structurally different dyes.

Open Access Original Research Article

Application of Box-Behnken Design for the Optimized Production of Lactic Acid by Newly Isolated Lactobacillus plantarum JX183220 Using Cassava (Manihot esculenta Crantz) Flour

V. Sridevi, M. Padmaja, A. Sahitya, N. Harsha Vardhan, G. H. Rao

Biotechnology Journal International, Page 1-9
DOI: 10.9734/BBJ/2015/20236

Aim: The present study aimed at optimization of Lactic acid production using new isolate, Lactobacillus plantarum JX183220 with cassava flour (Manihot esculenta Crantz) in semi-solid fermentation by Response Surface Methodology.

Study Design: Box-Behnken design of Response Surface Methodology was used.

Place: Department of Chemical Engineering and Biotechnology, ANITS, Visakhapatnam.

Materials and Methods: Lactobacillus plantarum JX183220 isolated from goat milk was used for the production of Lactic acid using cassava flour (CF) in semi-solid fermentation. Different fermentation parameters such as incubation time, inoculum volume, pH, temperature, substrate concentration (cassava flour), and Calcium carbonate concentration were initially optimized in preliminary studies. The substrate concentration, temperature and pH were chosen as potential variables and further optimized using Box-Behnken design of Response Surface Methodology. A second order polynomial regression model was fitted and was found adequate with a high coefficient of determination, R2 (0.9913). Validation experiment was carried out at optimum conditions of the parameters as determined from the model.

Results: The preliminary experiments revealed that maximum production of lactic acid by Lactobacillus plantarum JX183220 was observed on 4th day of incubation with 2% inoculum and 0.3% Calcium carbonate. Optimization using Box -Behnken design of RSM resulted in maximum Lactic acid production of 18.3679 g/100 g of cassava at optimum conditions of substrate concentration, 1.225%; Temperature, 36.39°C and pH 6.43. These results were confirmed by validation experiment.

Conclusion: Optimum parameters for the direct conversion of cassava flour starch to Lactic acid by new isolate, Lactobacillus plantarum JX183220 were determined. Box Behnken design of RSM was found to be convenient tool with 15 runs for optimizing lactic acid production. The lactic acid production could be further enhanced by saccharification and fermentation in future studies.