Purification and properties of polygalacturonase associated with the infection process of Colletotrichum truncatum CP2 in chilli

In this study, polygalacturonase enzyme produced by Colletotrichum truncatum CP2 was partially purified by aqueous two-phase system and the properties of this enzyme was characterized. The highest yield (57.4%) and purification fold (5.1) was obtained using 22% PEG 6,000/15% sodium citrate comprising crude load of 16% (w/w) at pH 7.0 with addition of 1.0% (w/w) sodium chloride. The partially purified PG remained active over a wide range of pH (2.5-6.0) and the optimum activity was obtained at pH 5.0. Incubation of the partially purified PG at 40 and 50 °C for 30 min caused the activity of PG to decrease up to 20% and 40%, respectively. However, no significant changes in the activity when the enzymes were incubated up to 4 h at 40 and 50 °C. The results from this study suggested that ATPS comprising of PEG and sodium citrate could be potentially used as an alternative method for purification of PG

Bioconversion of rice straw into acetone-butanol-ethanol by Clostridium sporogenes A3

The inevitable depletion of the world’s energy supply and unstable oil market have renewed the interest of society in searching for alternative fuels. In addition to depletion of petroleum fuels, environmental issues like greenhouse effect, global warming and climate change, are also the issues to be resolved worldwide. Biobutanol has been considered as a suitable alternative to be a source of fuel. Abundant biomass from various agriculture sectors could be a source for biobutanol production. Rice straw is one of the most abundant lignocellulosic biomass that has a great potential as a cheap and affordable substrate for the production of reducing sugars and biofuel such as biobutanol. The feasibility of rice straw as a source of sugar production was evaluated in this study. The effectiveness of alkaline pretreatment on rice straw was assessed taking into consideration the yield of reducing sugars and changes in the morphological and chemical composition of rice straw. Pretreatment of rice straw by 2% (w/v) NaOH and KOH with autoclaving at 121°C, 15 psi (10 min) could be promising pretreatment methods for sugar production. Alkaline pretreatments with 2% (w/v) KOH and NaOH followed by thermal pretreatment at 121°C, 15 psi (20 min) resulted in 58.5 to 64.5% higher conversion rate of reducing sugars production than untreated rice straw showing that alkaline pretreatments were effective even when a higher temperature was used. FTIR and SEM investigations showed that alkaline pretreatments caused chemical and morphological changes in the rice straw. The peaks of the cellulose and lignin materials were decreased after alkaline pretreatment, indicating that some cellulose and lignin were degraded. The reducing sugars obtained were then converted to acetone-butanolethanol (ABE) by Clostridium sporogenes A3. The total acetone-butanol-ethanol (ABE) production by locally isolated C. sporogenes A3 using rice straw hydrolysate was 1.58±0.11 g/L in which 0.73±0.05 g/L was butanol after 120 h of fermentation. Higher ABE yield was obtained from rice straw hydrolysate when compared to using commercial glucose as a carbon source. Increasing concentration of sugars in the rice straw hydrolysate to 40 g/L did not showed any improvements to the total ABE obtained. A higher level of ABE was obtained (1.72±0.39 g/L) at initial culture pH 5.5 (37ºC), in which 0.93±0.22 g/L was butanol. In comparison, higher yield of ABE was obtained when using C. acetobutylicum ATCC824. The ABE yield obtained was 0.33 which corresponds to 64% of ABE increment compared to fermentation using C. sporogenes A3. These results suggested that the reducing sugars obtained from pretreated rice straw could be used as a substrate for ABE fermentation by C. sporogenes A3 and C. acetobutylicum ATCC824. This will reduce carbon emission and our dependency on fossil fuel, and at the same time makes butanol as one of our future energy for many applications

Characterization of Colletotrichum truncatum CP2 and its interaction with chillies (Capsicum annuum L.) during pathogenesis

Anthracnose caused by Colletotrichum species is the most destructive disease of chilli worldwide. It is responsible for worldwide yield losses and could be even more severe without a successful control that still relies on the use of fungicides. Due to the growing concern about environmental and health damages caused by this control, an understanding of the mechanisms leading to the fungal pathogenicity in a particular host is essential for the implementation of effective disease control. This study aimed to investigate the mechanism leading to pathogenesis of Colletotrichum species in chilli fruit as little is known about the pathogenicity factor involved in this interaction. Thirty five fungal isolates were isolated from chilli lesions of anthracnose from different geographic locations in Malaysia. The ability of fungal isolates to produce cell wall-degrading enzymes was screened and the best cell wall-degrading enzymes producer was selected for further study. Based on its morphological, biochemical and molecular identification, fungal isolate CP2 was identified as Colletotrichum truncatum. Successful inoculation of the C. truncatum CP2 on detached chilli fruits proved its pathogenicity and was confirmed to be a primary pathogen of chilli when it successfully infected the chilli fruits. In order to illustrate the infection strategy adopted by C. truncatum CP2, the infection process of this fungus in the chilli fruit was characterized using light, scanning and transmission microscope. C. truncatum CP2 exhibited a prolonged biotrophic phase of about 48 hour, before switched to necrotrophic phase at approximately 72 hour after inoculation. The first phase of necrotrophy in C. truncatum CP2 was characterized by formation of germ tube, appresorium and infectious hyphae. The destructive necrotrophic phase was characterized by formation of sunken lesions and production of numerous acervuli. The role of cell wall-degrading enzymes in facilitating the C. truncatum CP2 to colonize the host cell was investigated taking into consideration changes in the morphological and chemical compositions of the chilli fruits. The results of enzymatic activity experiment indicated that polygalacturonase (PG) was the first cell wall-degrading enzymes detected and the activities obtained were higher (0.24±0.10 U/mL) than other enzymes, which appeared later and in lower amount. Significant changes in the pectin (total uronide content increased up to 50.33% - 71.85%) and cellulose contents (decreased to 11.45% - 12.32%) in chilli treated with PG and combination of PG and cellulases showed the main role of these enzymes in facilitating the C. truncatum CP2 during pathogenesis in chilli fruits. According to Fourier transform infrared analysis, there were remarkable changes in the vibration side of cellulose (3290 cm-1 and 2924 cm-1) and ring and vibration side of pectin (1581, 1337 and 1029 cm-1) in the cell wall of chilli treated with PG and mixture of both enzymes. In order to understand the exact role of PG enzymes in pathogenesis, PG enzymes from C. truncatum CP2 was purified using aqueous two phase system. The optimum purification condition of PG was achieved using 22% (w/w) polyethylene glycol and 15% (w/w) sodium citrate comprising crude load of 16% (w/w) at pH 7.0 with addition of 1.0% (w/w) sodium chloride. The necrotizing activity of the crude and purified PG from C. truncatum CP2 was then tested on detached chilli fruits. The faster lesion formation on the chilli treated with purified PG had confirmed the involvement of this enzyme in anthracnose of chilli. In conclusion, C. truncatum CP2 possess all the features to be termed as a serious anthracnose pathogen with the presence of pathogenicity factors such as PG enzymes. The results from this study provide a better insight into the interaction of C. truncatum CP2 and chilli fruits and these findings may be used in the development of efficient disease management strategies in Malaysia

Effects of Chemical and Thermal Pretreatments on the Enzymatic Saccharification of Rice Straw for Sugars Production

The effects of alkaline pretreatment with NaOH, KOH, Ca(OH)2, and NaOCl at varying temperatures and concentrations on the production of sugars, changes in the morphological structure, and the chemical composition of rice straw were evaluated. Enzymatic saccharification of 2% (w/v) KOH-treated rice straw with autoclaving at 121 °C, 15 psi, 20 min, gave a maximum yield of 59.90 g/L of reducing sugars, which was slightly higher than that of NaOH (55.48 g/L) with the same conditions. Chemical composition analysis of the rice straw showed that the cellulose content was increased to 71% and 66% after pretreatments with NaOH and KOH, respectively. Fourier Transform Infrared (FTIR) spectroscopy revealed that solubilization and removal of the lignin component also took place. The scanning electron microscope (SEM) analysis showed a marked change in the morphological structure of the treated rice straw compared to the untreated rice straw. These results suggested that pretreatment of rice straw with either 2% (w/v) NaOH or KOH at high temperature could be a promising pretreatment method for sugars production