Cellulase

We describe the identification and structural characterization of a novel family of Arabidopsis genes related to ATL2 which encode a variant of the RING zinc finger domain, known as RING-H2. Analysis of genes selected by us and of sequences from Arabidopsis stored in databases permitted the prediction of several RING-H2 proteins that contain highly homologous RING domains. The ATL gene family is represented by fifteen sequences that contain, in addition to the RING, a transmembrane domain which is located in most of them towards the N-terminal end. Transgenic Arabidopsis seedlings carrying the ATL2 promoter fused to the GUS reporter gene revealed that the expression of ATL2 is rapidly induced after exposure to chitin or inactivated crude cellulase preparations. Rapid induction of transcript accumulation of another member of the ATL family was also observed under the same conditions. These results suggest that some ATLs may be involved in the early stages of the defense response triggered in plants in response to pathogen attack.

The Phasmatodea (stick insects) have multiple, endogenous, highly expressed copies of glycoside hy-drolase family 9 (GH9) genes. The purpose for retaining so many was unknown. We cloned and expressed the enzymes in transfected insect cell lines, and tested the individual proteins against different plant cell wall component poly-and oligosaccharides. Nearly all isolated enzymes were active against carboxymethylcellulose, however most could also degrade glucomannan, and some also either xylan or xyloglucan. The latter two enzyme groups were each monophyletic, suggesting the evolution of these novel substrate specificities in an early ancestor of the order. Such enzymes are highly unusual for Metazoa, for which no xyloglucanases had been reported. Phasmatodea gut extracts could degrade multiple plant cell wall components fully into sugar monomers, suggesting that enzymatic breakdown of plant cell walls by the entire Phasmatodea digestome may contribute to the Phasmatodea nutritional budget. The duplication and neofunctionalization of GH9s in the ancestral Phasmatodea may have enabled them to specialize as folivores and diverge from their omnivorous ancestors. The structural changes enabling these unprecedented activities in the cellulases require further study.

Fungal cellulases are well-studied enzymes and are used in various industrial processes. Much of the knowledge of enzymatic depolymerization of cellulosic material has come from Trichoderma cellulase system. Species of Trichoderma can produce substantial amounts of endoglucanase and exoglucanase but very low levels of β-glucosidase. This deficiency necessitates screening of fungi for cellulytic potential. A number of indigenously isolated fungi were screened for cellulytic potential. In the present study, the kinetics of cellulase production from an indigenous strain of Aspergillus niger MS82 is reported. Product formation parameters of endoglucanase and β-glucosidase (Qp + Yp/s) indicate that A. niger MS82 is capable of producing moderate to high levels of both endoglucanase and β-glucosidase when grown on different carbon containing natural substrates, for example, grass, corncob, bagasse along side purified celluloses. Furthermore, it was observed that the production of endoglucanase reaches its maximum during exponential phase of growth, while β-glucosidase during the Stationary phase. Enzyme production by solid-state fermentation was also investigated and found to be promising. Highest production of cellulase was noted at pH 4.0 at 35 °C under submerged conditions. Growth and enzyme production was affected by variations in temperature and pH.

The efficient cellulase producing bacteria was isolated from the soil of sacred groove and identified as P. aeruginosa SG21 by 16S rRNA sequencing. The objective of this study is to perform biochemical testing, antibiotic sensitivity testing and optimizing the growth conditions of the bacteria P. aeruginosa SG21. The culture conditions like pH, temperature, carbon sources, and nitrogen sources were optimized. The optimum conditions found for cellulase production are 35 and 40°C at pH 6-10 with CMC as carbon source and urea as nitrogen source.

Cassava starch production waste (cassava pulp) has been proposed as a high potential ethanolic fermentation substrate due to its high residual starch level and the small particle size of the lignocellulosic fibers. Saccharification of the residual starch from a 3% (w/v) dry weight basis (DS) of cassava pulp by α-amylase (100°C, 10 min) and glucoamylase (60°C, 2 h) resulted in a glucose yield of 22.6 g/l [67.8% (w/w) DS of cassava pulp] and in lignocellulosic fibers at 0.5 g/g DS cassava pulp. Pretreatment of the lignocellulosic fiber with dilute sulfuric acid and calcium hydroxide at 121°C, 15 lb/in2 for 30 min increased and decreased, respectively, its susceptibility to cellulase hydrolysis. Under the optimal conditions found, pretreatment of 6% (w/v) DS lignocellulosic fiber by 2% (w/v) H2SO4 for 30 min, followed by saccharification by cellulase (40°C, 9 h), yielded a glucose level of 26.6 g/l [79.8% (w/w) DS of the cassava pulp]. The starch and lignocellulosic fiber hydrolysates obtained from 30 g cassava pulp and 60 g H2SO4 pretreated lignocellulosic fiber were fermented by Saccharomyces cerevisiae, without the need for (NH4)2SO4 supplementation, to yield ethanol levels of 9.9 and 11.9 g/l, respectively, after 48 h.

Aspergillus niger strain HD-6, isolated from Anand, Gujarat
(India), was evaluated for endocellulase enzyme production
through solid state fermentation (SSF) using bajra straw as main
carbon source. Various cultural conditions such as temperature,
pH, moisture content, incubation period, nitrogen source, inoculum
age and size, etc. were standardized for optimum enzyme
production. Using 3 g bajra straw as sole carbon source maximum
endocellulase production from 96 hr old A. niger HD-6 was
achieved at pH 5.0, incubation temperature 28°C and moisture
content 65% after incubation for 4 days.

The application of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) as a tool for the characterization of complex carbohydrate structures was investigated for konjac glucomannan (KGM) oligosaccharide mixtures and the monitoring of their structural changes during 72 h of in vitro fermentation with human gut flora. Different types of KGM oligosaccharide mixtures were produced from a KGM polysaccharide using endo-beta-(1,4)-mannanase and endo-beta-(1,4)-glucanase. Distinction of structures emerging from different enzymatic KGM digests and detection of acetylated oligosaccharides were possible by both CE-LIF and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Using CE-LIF it could be shown that the endo-beta-(1,4)-glucanase digest exhibited a large degradability of the DP2, DP3, DP5, and DP6 components during in vitro fermentation, whereas the endo-beta-(1,4)-mannanase digest was digested only slightly, thereby highlighting the influence of structural characteristics on the fermentability by human gut flora.

Lignocellulosic biomass can be adequately utilized in the production of bio-ethanol, a promising alternative energy resource for non-renewable fossil fuels. The development of renewable energy resources involves mainly the hydrolysis of the cellulose component of waste biomass into glucose and the successive fermentation of the resulting sugars into ethanol. An investigation into the use of sawdust waste from twenty different tropical hardwoods for bio-ethanol production has been performed. Kraft-pretreated waste cellulose from the various trees have been saccharified with T. viride cellulase as well as sulphuric acid. The resulting sugars were converted into bio-ethanol by means of separate hydrolysis and fermentation (SHF) as well as simultaneous saccharification and fermentation (SSF). The highest alcohol concentration of 3.71 mg.ml-1 was obtained during the separate hydrolysis and fermentation process converting K. ivorensis wood sawdust whilst the lowest alcohol concentration of 2.54 mg.ml-1 was recorded with Ipomoea asarifolia cellulose. During SSF the highest alcohol production was calculated at a value of 3.89 mg.ml-1 , produced from K. ivorensis cellulose with the lowest concentration obtained at 2,73 mg.ml-1 obtained from cellulose extracted from Erythropleum suaveolens. Fermentation of sugars obtained after sulphuric acid catalyzed degradation of these cellulose materials produced the highest ethanol concentration of 3.32 mg.ml-1 with the lowest bio-ethanol concentration (1,86 mg.ml-1) obtained from Masonia altissima cellulose. The highest relative percentage yield of alcohol obtained after acid catalyzed saccharification was 40% calculated with cellulose from Kyaya ivorensis whilst a maximum extent of 65% alcohol production was evident during the fermentation of sugars produced from T. viride cellulase catalyzed saccharification of Masonia altissima cellulose.

ABSTRACT Study on the gelatinization kinetics of rice showed that gelatinization process was divided into two steps: swelling of the amorphous region and disruption of the crystalline region. Higher temperature storage (37 °C) resulted in an increase in the breaking point temperature suggesting that energy for the disorder of these two regions of starch in rice stored at 37 °C was higher than the rice stored at 4 °C. Storage-induced changes in rice led to significant increases in DSC peak temperature (p < 0.05) and significantly broadened peak width (p < 0.01) for rice stored at 37 °C compared to rice stored at 4 °C. As the peak temperature of rice stored at 37 °C was not influenced by the “annealing” treatment in contrast with the increased peak temperature of rice stored at 4 °C after the “annealing” treatment, the results indicate that the ageing process (37 °C storage) has already re-ordered the rice grain structure and that the annealing process under these conditions has no further effect on starch thermal properties. Because starches isolated from rice grain stored at 4 °C and 37 °C had similar thermal properties, this implies that the effects of storage on thermal properties are associated with the interactions between starch and non-starch components following storage. The gelatinization endotherm shifted to a lower temperature (p < 0.01) and a narrowed peak width was achieved after cellulase and protease treatments of stored rice, which indicates that the changes in cell wall remnants and proteins are responsible for the changes in rice thermal properties during storage. Scanning electron microscopy was applied to visualize the treatments of cellulase and protease on rice.