Background Major cost of bioethanol is attributed to enzymes employed in biomass hydrolysis. Biomass hydrolyzing enzymes are predominantly produced from the hyper-cellulolytic mutant filamentous fungus Trichoderma reesei RUT-C30. Several decades of research have failed to provide an industrial grade organism other than T. reesei, capable of producing higher titers of an effective synergistic biomass hydrolyzing enzyme cocktail. Penicillium janthinellum NCIM1366 was reported as a cellulase hyper producer and a potential alternative to T. reesei, but a comparison of their hydrolytic performance was seldom attempted. Results Hydrolysis of acid or alkali-pretreated rice straw using cellulase enzyme preparations from P. janthinellum and T. reesei indicated 37 and 43% higher glucose release, respectively, with P. janthinellum enzymes. A comparison of these fungi with respect to their secreted enzymes indicated that the crude enzyme preparation from P. janthinellum showed 28% higher overall cellulase activity. It also had an exceptional tenfold higher beta-glucosidase activity compared to that of T. reesei, leading to a lower cellobiose accumulation and thus alleviating the feedback inhibition. P. janthinellum secreted more number of proteins to the extracellular medium whose total concentration was 1.8-fold higher than T. reesei. Secretome analyses of the two fungi revealed higher number of CAZymes and a higher relative abundance of cellulases upon cellulose induction in the fungus. Conclusions The results revealed the ability of P. janthinellum for efficient biomass degradation through hyper cellulase production, and it outperformed the established industrial cellulase producer T. reesei in the hydrolysis experiments. A higher level of induction, larger number of secreted CAZymes and a high relative proportion of BGL to cellulases indicate the possible reasons for its performance advantage in biomass hydrolysis.
BackgroundMajor cost of bioethanol is attributed to enzymes employed in biomass hydrolysis. Lignocellulolytic enzymes are predominantly produced from the hyper cellulolytic mutant filamentous fungus Trichoderma reesei RUT-C30. Several decades of research have failed to provide an industrial grade organism producing higher titers of an effective synergistic biomass hydrolyzing enzyme cocktail. Penicillium janthinellum NCIM1366 was reported as a cellulase hyper producer and a potential alternative to T. reesei, but a comparison of their hydrolytic performance was seldom attempted. ResultsHydrolysis of acid or alkali pretreated rice straw using cellulase enzyme preparations from P. janthinellum and T. reesei indicated 37 and 43 % higher glucose release respectively with P. janthinellum enzymes. A comparison of these fungi with respect to their secreted enzymes indicated that the crude enzyme preparation from P. janthinellum showed 28 % higher overall cellulase activity. It also had an exceptional 10-fold higher beta-glucosidase activity compared to that of T. reesei, leading to a lower cellobiose accumulation and thus alleviating the feedback inhibition. P. janthinellum secreted more number of proteins to the extracellular medium whose total concentration was 1.8 fold higher than T. reesei. Secretome analyses of the two fungi revealed more number of CAZymes and a higher relative abundance of cellulases upon cellulose induction in the fungus.ConclusionsThe results revealed the ability P. janthinellum for efficient biomass degradation through hyper cellulase production, and it outperformed the established industrial cellulase producer T. reesei in the hydrolysis experiments. A higher level of induction, larger number of secreted CAZymes and a high relative proportion of BGL to cellulases could be the possible reasons for its performance advantage in biomass hydrolysis.
The cellulase production by P. janthinellum mutants on lignocellulosic material such as cellulose or steam exploded bagasse (SEB) in combination with wheat bran was studied in solid state fermentation (SSF). One of the mutants, EU2D21, produced the highest levels of endoglucanase (3710 IU g-1 carbon source) and β-glucosidase (155 IU g-1 carbon source). Ionic liquids are so-called green solvents that have become attractive for biocatalysis. Stability of mutant cellulases was tested in 10-50% of the ionic liquid 1-butyl-3-methylimidazolium chloride ([bmim]Cl). FPA and CMCase were significantly stable in 10% ionic liquid after 5h. β-glucosidase showed 85% of its original activity after 5 h incubation in 30% ionic liquid and retained 55% of its activity after 24 h. This enzyme preparation hydrolyzed ionic-liquid-treated SEB completely in 15 h in the presence of 20% ionic liquid. These studies revealed that there is no need of regenerating cellulose after ionic liquid treatment, since cellulase of mutant strain was found to be significantly stable in the ionic liquid.
Aspergillus niger NCIM 1207 produced significantly high levels of β-glucosidase and β-xylosidase activities in submerged fermentation. Cellulose induced only β-glucosidase, while xylan induced both β-glucosidase and β-xylosidase activities. Both the enzymes of this strain were found to undergo catabolite repression in the presence of high concentrations of glucose and glycerol. The sudden drop in pH of the fermentation medium below 3.5 caused the inactivation of enzymes when the fungus was grown in glycerol-containing media at lower temperatures. The growth of the organism at 36 oC led to an increase in pH of the fermentation above 6.0 that affected β-xylosidase activity significantly. Highest levels of β-glucosidase ((19 IU mL-1 or 633 IU g-1 of substrate) and β-xylosidase (18.7 IU/mL-1 or 620 IU g-1 of substrate) activities were detected when A. niger was grown at 30 oC for first five days followed by further incubation at 36 oC. Such a process of growing the organism at lower temperatures (growth phase) followed by producing the enzymes at higher temperatures (production phase) in case of fungal systems has not been reported so far. The zymogram staining of the β-glucosidase demonstrated that A. niger produced only single species of β-glucosidase. We feel that A. niger NCIM 1207 is a potential candidate to produce both β-glucosidase and β-xylosidase in high amounts that can be used to supplement commercial cellulase preparation.
The genus pseudozyma The species of the genus Pseudozyma belong to ustilaginales based on the morphological studies 1 and molecular characterization. 2,3 There are 11 species reported so far from this genus that are distinguished by analysing the sequences of combined ITS and D1/D2 regions. Boekhout & Fell 4 reported seven species and Sugita et al. 5 described two species isolated from blood of the patients. Two more species of Pseudozyma were isolated from wilting leaves of different plants that are named as Pseudozyma hubeiensis and Psedozyma shanxiensis. 6 These species are distinguished from other reported species by morphological studies and physiological characterization. The novelty of these two species was also confirmed by molecular taxonomic analysis based on sequencing of 26S rRNA gene, D1/ D2 domain and internal transcribed spacer (ITS) regions (6). All 11 species of Pseudozyma were further studied for assimilation reactions which differentiated all the species of the genus Pseudozyma from one another. The ability to grow at 40°C and no assimilation of erythritol are the characteristics that differentiate P. shanxiensis from all other species of Pseudozyma. However, P. hubeiensis does not assimilate inositol and this characteristic differentiates P. hubeiensis from all other species. 6 P. hubeiensis was first isolated from decaying sandal wood in our laboratory 7 and then it was sent for identification to National Collection of Yeast Cultures (NCYC) in 2008. The sequencing of 26 rDNA D1/D2 domain and standard taxonomic tests confirmed that it is a novel strain of P. hubeiensis. It was then deposited in NCIM Resource Center, CSIR-National Chemical Laboratory, Pune, with an accession number NCIM 3574. Among all species of Pseudozyma, Pseudzyma antarctica (Formally known as Candida antarctica) has been a most studied. It is well known producer of glycolipid, manosylerythritol (MEL) from vegetable oil including soybean oil, alkanes, glycerol, glucose and xylose 8 and also from cellulosic materials. 9 MELs show excellent surface active properties in addition to versatile biochemical actions. In addition to its bio-surfactant property, MEL possesses antitumor and cell differentiation induction activities. 10 The other species such as P. aphidis, P. rugulosa, P. fusiformata are also known to produce MEL. 11 Extracellular esterases 12 and biodegradable plastic degrading enzymes 13 have been reported from P. antarctica. The plastic degrading enzyme named PaE produced by P. antarctica degrades biodegradable plastic films composed of poly (butylele succinate) (PBS), poly (butylene succinate-co-adipate) (PBSA) and poly (lactic acid) (PLA). Xylose induced xylanases were reported from P. antarctica and the 33 kDa purified protein was found to produce xylose from xylan indicating that they are endo-xylanases. 14 Genome and transcriptome analysis of P. antarctica was recently reported. 15 The another novel yeast species, P. brasiliensis produced the xylan induced secretome containing endo-xylanase and β-xylosidase. 16
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