New chromogenic substrates for the assay of alpha-amylase and (1→4)-β-D-glucanase.
McCleary, B. V. (1980). Carbohydrate Research, 86(1), 97-104.
New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1→4)-β-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1→4)-β-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.
Evaluation of substrate composition for lignocellulolytic enzymes production by solid state fermentation from wastes of olive oil and wine industries.
Salgado, J. M., Moreira, C., Abrunhosa, L., Venâncio, A., Domínguez, J. M. & Belo, I. (2012). American programme for Science, Technology and Development, 95-101.
Wastes from olive oil and wine industries (as exhausted grape mark, vineshoot trimmings, two-phase olive mill waste, vinasses and olive mill wastewater were evaluated for lignocellulolytic enzymes production (as cellulases, xylanases and feruloyl esterases) by solid state fermentation with Aspergillus niger, Aspergillus ibericus and Aspergillus japonicus. To study the effect of different substrates in enzymes production a Plackett-Burman experimental design was presented. The variables that had a higher positive effect in lignocellulolytic enzymes were urea, time and exhausted grape mark. The mixture of two-phase olive mill waste with exhausted grape mark and vineshoot trimmings had maxima activity of cellulases, xylanases and feruloyl esterases.
Cellulolytic potential of thermophilic species from four fungal orders.
Busk , P.K. & Lange. L. (2013). AMB Express, 3(1), 47.
Elucidation of fungal biomass degradation is important for understanding the turnover of biological materials in nature and has important implications for industrial biomass conversion. In recent years there has been an increasing interest in elucidating the biological role of thermophilic fungi and in characterization of their industrially useful enzymes. In the present study we investigated the cellulolytic potential of 16 thermophilic fungi from the three ascomycete orders Sordariales, Eurotiales and Onygenales and from the zygomycete order Mucorales thus covering all fungal orders that include thermophiles. Thermophilic fungi are the only described eukaryotes that can grow at temperatures above 45°C. All 16 fungi were able to grow on crystalline cellulose but their secreted enzymes showed widely different cellulolytic activities, pH optima and thermostabilities. Interestingly, in contrast to previous reports, we found that some fungi such as Melanocarpus albomyces readily grew on crystalline cellulose and produced cellulases. These results indicate that there are large differences in the cellulolytic potential of different isolates of the same species. Furthermore, all the selected species were able to degrade cellulose but the differences in cellulolytic potential and thermostability of the secretome did not correlate to the taxonomic position. PCR amplification and sequencing of 22 cellulase genes from the fungi showed that the level of thermostability of the cellulose-degrading activity could not be inferred from the phylogenetic relationship of the cellulases.
Transcriptional comparison of the filamentous fungus Neurospora crassa growing on three major monosaccharides D-glucose, D-xylose and and L-arabinose.
Li, J., Lin, L., Li. H., Tian, C. & Ma, Y. (2014). Biotechnology for Biofuels, 7(1), 31.
Background: D-glucose, D-xylose and L-arabinose are the three major monosaccharides in plant cell walls. Complete utilization of all three sugars is still a bottleneck for second-generation cellulolytic bioethanol production, especially for L-arabinose. However, little is known about gene expression profiles during L-arabinose utilization in fungi and a comparison of the genome-wide fungal response to these three major monosaccharides has not yet been reported. Results: Using next-generation sequencing technology, we have analyzed the transcriptome of N. crassa grown on L-arabinose versus D-xylose, with D-glucose as the reference. We found that the gene expression profiles on L-arabinose were dramatically different from those on D-xylose. It appears that L-arabinose can rewire the fungal cell metabolic pathway widely and provoke the expression of many kinds of sugar transporters, hemicellulase genes and transcription factors. In contrast, many fewer genes, mainly related to the pentose metabolic pathway, were upregulated on D-xylose. The rewired metabolic response to L-arabinose was significantly different and wider than that under no carbon conditions, although the carbon starvation response was initiated on L-arabinose. Three novel sugar transporters were identified and characterized for their substrates here, including one glucose transporter GLT-1 (NCU01633) and two novel pentose transporters, XAT-1 (NCU01132), XYT-1 (NCU05627). One transcription factor associated with the regulation of hemicellulase genes, HCR-1 (NCU05064) was also characterized in the present study. Conclusions: We conducted the first transcriptome analysis of Neurospora crassa grown on L-arabinose and performed a comparative analysis with cells grown on D-xylose and D-glucose, which deepens the understanding of the utilization of L-arabinose and D-xylose in filamentous fungi. The dataset generated by this research will be useful for mining target genes for D-xylose and L-arabinose utilization engineering and the novel sugar transportes identified are good targets for pentose untilization and biofuels production. Moreover, hemicellulase production by fungi could be improved by modifying the hemicellulase regulator discovered here.
Relevance of the light signaling machinery for cellulase expression in trichoderma reesei (hypocrea jecorina).
Gyalai-Korpos, M., Nagy, G., Mareczky, Z., Schuster, A., Réczey, K. & Schmoll, M. (2010). BMC Research Notes, 3, 330.
Background: In nature, light is one of the most important environmental cues that fungi perceive and interpret. It is known not only to influence growth and conidiation, but also cellulase gene expression. We therefore studied the relevance of the main components of the light perception machinery of Trichoderma reesei (Hypocrea jecorina), ENV1, BLR1 and BLR2, for production of plant cell wall degrading enzymes in fermentations aimed at efficient biosynthesis of enzyme mixtures for biofuel production. Findings: Our results indicate that despite cultivation in mostly dark conditions, all three components show an influence on cellulase expression. While we found the performance of the enzyme mixture secreted by a deletion mutant in env1 to be enhanced, the higher cellulolytic activity observed for Δblr2 is mainly due to an increased secretion capacity of this strain. Δblr1 showed enhanced biomass accumulation, but due to its obviously lower secretion capacity still was the least efficient strain in this study. Conclusions: We conclude that with respect to regulation of plant cell wall degrading enzymes, the blue light regulator proteins are unlikely to act as a complex. Their regulatory influence on cellulase biosynthesis involves an alteration of protein secretion, which may be due to adjustment of transcription or posttranscriptional regulation of upstream factors. In contrast, the regulatory function of ENV1 seems to involve adjustment of enzyme proportions to environmental conditions.
Dehydrogenase GRD1 Represents a Novel Component of the Cellulase Regulon in Trichoderma reesei (Hypocrea jecorina).
Schuster, A., Kubicek, C. P. & Schmoll, M. (2011). Applied and Environmental Microbiology, 77(13), 4553-4563.
Trichoderma reesei (Hypocrea jecorina) is nowadays the most important industrial producer of cellulase and hemicellulase enzymes, which are used for pretreatment of cellulosic biomass for biofuel production. In this study, we introduce a novel component, GRD1 (glucose-ribitol dehydrogenase 1), which shows enzymatic activity on cellobiose and positively influences cellulase gene transcription, expression, and extracellular endo-1,4-β-D-glucanase activity. grd1 is differentially transcribed upon growth on cellulose and the induction of cellulase gene expression by sophorose. The transcription of grd1 is coregulated with that of cel7a (cbh1) under inducing conditions. GRD1 is further involved in carbon source utilization on several carbon sources, such as those involved in lactose and D-galactose catabolism, in several cases in a light-dependent manner. We conclude that GRD1 represents a novel enhancer of cellulase gene expression, which by coregulation with the major cellulase may act via optimization of inducing mechanisms.
Influence of the carbon source on production of cellulases, hemicellulases and pectinases by Trichoderma reesei Rut C-30.
Olsson, L., Christensen, T. M. I. E., Hansen, K. P. & Palmqvist, E. A. (2003). Enzyme and Microbial Technology, 33(5), 612-619.
The growth and enzyme production by Trichoderma reesei Rut C-30 using different lignocellulosic materials as carbon source were investigated. Cellulose, sugar beet pulp and alkaline extracted sugar beet pulp (resulting in partial removal of hemicellulose, lignin and pectin) or mixtures thereof were used as carbon sources. It was found that endoglucanase and endoxylanse activities were produced throughout the cultivations, whereas α-arabinosidase was induced late during the cultivation. The highest amounts of endoglucanse, could be measured when T. reesei Rut C-30 was grown on cellulose or cellulose containing mixtures. Endoxylanase was produced on all substrates, but the presence of cellulose was favourable for the production. Polygalacturonase activity could be measured at high varying levels throughout the cultivations, except during growth on cellulose. The varying levels might originate from the production of different isoenzymes of polygalacturonase.
Jeongeupia naejangsanensis gen. nov., sp. nov., a cellulose-degrading bacterium isolated from forest soil from Naejang Mountain in Korea.
Yoon, J. H., Choi, J. H., Kang, S. J., Choi, N. S., Lee, J. S. & Song, J. J. (2010). International Journal of Systematic and Evolutionary Microbiology, 60(3), 615-619.
A Gram-stain-negative, motile, rod-shaped, cellulose-degrading bacterial strain, BIO-TAS4-2T, which belongs to the Betaproteobacteria, was isolated from forest soil from Naejang Mountain, Korea, and its taxonomic position was investigated by using a polyphasic study. Strain BIO-TAS4-2T grew optimally at pH 7.0–8.0, at 30°C and in the presence of 0–1.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences showed that strain BIO-TAS4-2T clustered with members of the genera Andreprevotia, Silvimonas and Deefgea of the family Neisseriaceae, with which it exhibited 16S rRNA gene sequence similarities of 93.5–94.2 %. Strain BIO-TAS4-2T contained Q-8 as the predominant ubiquinone and summed feature 3 (C16:1ϖ7c and/or iso-C15:0 2-OH) and C16:0 as the major fatty acids. The DNA G+C content was 63.8 mol%. Strain BIO-TAS4-2T could be differentiated from members of phylogenetically related genera by differences in fatty acid composition, DNA G+C content and some phenotypic properties. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BIO-TAS4-2T is considered to represent a novel species in a new genus, for which the name Jeongeupia naejangsanensis gen. nov., sp. nov. is proposed, with BIO-TAS4-2T(=KCTC 22633T=CCUG 57610T) as the type strain.
Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan-and mannan-based polysaccharides.
Chhabra, S. R., Shockley, K. R., Ward, D. E. & Kelly, R. M. (2002). Applied and Environmental Microbiology, 68(2), 545-554.
The genome sequence of the hyperthermophilic bacterium Thermotoga maritima encodes a number of glycosyl hydrolases. Many of these enzymes have been shown in vitro to degrade specific glycosides that presumably serve as carbon and energy sources for the organism. However, because of the broad substrate specificity of many glycosyl hydrolases, it is difficult to determine the physiological substrate preferences for specific enzymes from biochemical information. In this study, T. maritima was grown on a range of polysaccharides, including barley β-glucan, carboxymethyl cellulose, carob galactomannan, konjac glucomannan, and potato starch. In all cases, significant growth was observed, and cell densities reached 109 cells/ml. Northern blot analyses revealed different substrate-dependent expression patterns for genes encoding the various endo-acting β-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example, cel74 (TM0305), a gene encoding a putative β-specific endoglucananse, was strongly expressed on all substrates tested, including starch, while no evidence of expression was observed on any substrate for lam16 (TM0024), xyl10A (TM0061), xyl10B (TM0070), and cel12A (TM1524), which are genes that encode a laminarinase, two xylanases, and an endoglucanase, respectively. The cel12B (TM1525) gene, which encodes an endoglucanase, was expressed only on carboxymethyl cellulose. An extracellular mannanase encoded by man5 (TM1227) was expressed on carob galactomannan and konjac glucomannan and to a lesser extent on carboxymethyl cellulose. An unexpected result was the finding that the cel5A (TM1751) and cel5B (TM1752) genes, which encode putative intracellular, β-specific endoglucanases, were induced only when T. maritima was grown on konjac glucomannan. To investigate the biochemical basis of this finding, the recombinant forms of Man5 (Mr, 76,900) and Cel5A (Mr, 37,400) were expressed in Escherichia coli and characterized. Man5, a T. maritima extracellular enzyme, had a melting temperature of 99°C and an optimun temperature of 90°C, compared to 90 and 80°C, respectively, for the intracellular enzyme Cel5A. While Man5 hydrolyzed both galactomannan and glucomannan, no activity was detected on glucans or xylans. Cel5A, however, not only hydrolyzed barley β-glucan, carboxymethyl cellulose, xyloglucan, and lichenin but also had activity comparable to that of Man5 on galactomannan and higher activity than Man5 on glucomannan. The biochemical characteristics of Cel5A, the fact that Cel5A was induced only when T. maritima was grown on glucomannan, and the intracellular localization of Cel5A suggest that the physiological role of this enzyme includes hydrolysis of glucomannan oligosaccharides that are transported following initial hydrolysis by extracellular glycosidases, such as Man5.
Cellulose degradation by Sulfolobus solfataricus requires a cell-anchored endo-β-1-4-glucanase.
Girfoglio, M., Rossi, M. & Cannio, R. (2012). Journal of Bacteriology, 194(18), 5091–5100.
A sequence encoding a putative extracellular endoglucanase (sso1354) was identified in the complete genome sequence of Sulfolobus solfataricus. The encoded protein shares signature motifs with members of glycoside hydrolases family 12. After an unsuccessful first attempt at cloning the full-length coding sequences in Escherichia coli, an active but unstable recombinant enzyme lacking a 27-residue N-terminal sequence was generated. This 27-amino-acid sequence shows significant similarity with corresponding regions in the sugar binding proteins AraS, GlcS, and TreS of S. solfataricus that are responsible for anchoring them to the plasma membrane. A strategy based on an effective vector/host genetic system for Sulfolobus and on expression control by the promoter of the S. solfataricus gene which encodes the glucose binding protein allowed production of the enzyme in sufficient quantities for study. In fact, the enzyme expressed in S. solfataricus was stable and highly thermoresistant and showed optimal activity at low pH and high temperature. The protein was detected mainly in the plasma membrane fraction, confirming the structural similarity to the sugar binding proteins. The results of the protein expression in the two different hosts showed that the SSO1354 enzyme is endowed with an endo-β-1-4-glucanase activity and specifically hydrolyzes cellulose. Moreover, it also shows significant but distinguishable specificity toward several other sugar polymers, such as lichenan, xylan, debranched arabinan, pachyman, and curdlan.
Unravelling the molecular basis for light modulated cellulase gene expression-the role of photoreceptors in Neurospora crassa.
Schmoll, M., Tian, C., Sun, J., Tisch, D. & Glass, N. L. (2012). BMC genomics, 13(1), 127.
Background: Light represents an important environmental cue, which exerts considerable influence on the metabolism of fungi. Studies with the biotechnological fungal workhorse Trichoderma reesei (Hypocrea jecorina) have revealed an interconnection between transcriptional regulation of cellulolytic enzymes and the light response. Neurospora crassa has been used as a model organism to study light and circadian rhythm biology. We therefore investigated whether light also regulates transcriptional regulation of cellulolytic enzymes in N. crassa. Results: We show that the N. crassa photoreceptor genes wc-1, wc-2 and vvd are involved in regulation of cellulase gene expression, indicating that this phenomenon is conserved among filamentous fungi. The negative effect of VVD on production of cellulolytic enzymes is thereby accomplished by its role in photoadaptation and hence its function in White collar complex (WCC) formation. In contrast, the induction of vvd expression by the WCC does not seem to be crucial in this process. Additionally, we found that WC-1 and WC-2 not only act as a complex, but also have individual functions upon growth on cellulose. Conclusions: Genome wide transcriptome analysis of photoreceptor mutants and evaluation of results by analysis of mutant strains identified several candidate genes likely to play a role in light modulated cellulase gene expression. Genes with functions in amino acid metabolism, glycogen metabolism, energy supply and protein folding are enriched among genes with decreased expression levels in the wc-1 and wc-2 mutants. The ability to properly respond to amino acid starvation, i. e. up-regulation of the cross pathway control protein cpc-1, was found to be beneficial for cellulase gene expression. Our results further suggest a contribution of oxidative depolymerization of cellulose to plant cell wall degradation in N. crassa.
Characterisation of cellulase activity in the digestive system of the redclaw crayfish (Cherax quadricarinatus).
Xue, X. M., Anderson, A. J., Richardson, N. A., Anderson, A. J., Xue, G. P. & Mather, P. B. (1999). Aquaculture, 180(3), 373-386.
Endogenous cellulase activity was identified in the gastric fluid and digestive gland of the redclaw crayfish. Cellulase showed maximal activity from pH 4 to 5 and was stable for up to 2 h at 40°C. Cellulase activity in the digestive gland was unaffected by antibiotic treatment. Taken together these findings suggest a significant endogenous component for redclaw cellulase activity. Partial purification of cellulase activity was performed using anion exchange and gel filtration chromatography. One major and one minor band of activity were identified subsequently by SDS-PAGE and zymography. The molecular weight of the major band was estimated at 40 kDa while the minor band was estimated at 30 kDa. Redclaw cellulase enzymes demonstrated broad substrate specificity, hydrolysing polysaccharides containing β-1,4 and mixed β-1,4 and β-1,3 glycosidic bonds but showed a preference for soluble substrates. Hydrolysis products of cellodextrins of various lengths also showed that the enzymes liberated free glucose. Exposure of redclaw to antibiotics resulted in a dramatic decline in bacterial populations in the gastric contents (>90%) but only a 40% decline in cellulase activity.
Identification of thermostable β-xylosidase activities produced by Aspergillus brasiliensis and Aspergillus niger.
Pedersen, M., Lauritzen, H. K., Frisvad, J. C. & Meyer, A. S. (2007). Biotechnology Letters, 29(5), 743-748.
Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicas produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable β-xylosidases. The β-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75°C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60°C. At 75°C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable β-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.
The effect of Pleurotus ostreatus arabinofuranosidase and its evolved variant in lignocellulosic biomasses conversion.
Marcolongo, L., Ionata, E., Cara, F. L., Amore, A., Giacobbe, S., Pepe, O. & Faraco, V. (2014). Fungal Genetics and Biology, 72, 162-167.
The fungal arabinofuranosidase from Pleurotus ostreatus PoAbf recombinantly expressed in Pichia pastoris rPoAbf and its evolved variant rPoAbf F435Y/Y446F were tested for their effectiveness to enhance the enzymatic saccharification of three lignocellulosic biomasses, namely Arundo donax, corn cobs and brewer’s spent grains (BSG), after chemical or chemical–physical pretreatment. All the raw materials were subjected to an alkaline pretreatment by soaking in aqueous ammonia solution whilst the biomass from A. donax was also pretreated by steam explosion. The capability of the wild-type and mutant rPoAbf to increase the fermentable sugars recovery was assessed by using these enzymes in combination with different (hemi)cellulolytic activities. These enzymatic mixtures were either entirely of commercial origin or contained the cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli in substitution to the commercial counterparts. The addition of the arabinofuranosidases from P. ostreatus improved the hydrolytic efficiency of the commercial enzymatic cocktails on all the pretreated biomasses. The best results were obtained using the rPoAbf evolved variant and are represented by increases of the xylose recovery up to 56.4%. These data clearly highlight the important role of the accessory hemicellulolytic activities to optimize the xylan bioconversion yields.
Growth and Enzyme Production in Blue Crabs (Callinectes sapidus) Fed Cellulose and Chitin Supplemented Diets.
Allman, A. L., Williams, E. P. & Place, A. R. (2017). Journal of Shellfish Research, 36(1), 283-291.
The blue crab [Callinectes sapidus (Rathbun, 1896)] is a benthic decapod with a varied diet. The diet includes invertebrates and detrital material that can have relatively large amounts of chitin and cellulose, both of which can be difficult to digest for many organisms and often require the aid of specific bacteria in the gut microbiome. In this study, juvenile blue crabs were fed an optimized defined pelleted diet with a 20% replacement of wheat flour filler with either chitin, cellulose, or a 14%/6% mix of both, followed by a diet switch to the opposing ingredient. Crabs had increasing growth performance with increasing amounts of cellulose in the diet versus chitin and had an additional molt in most cases. This occurred during the initial phase and following the switch, indicating that performance can be recovered. Subsequently, cellulose and chitin digestion assays were used to show that the foregut, midgut, and hindgut were all able to significantly digest more cellulose than chitin with the majority of activity in the foregut and midgut. Implications for rearing and diet formulations as well as the role of cellulose and chitin digestion in the natural diet are discussed.
Conferring cellulose-degrading ability to Yarrowia lipolytica to facilitate a consolidated bioprocessing approach.
Guo, Z. P., Duquesne, S., Bozonnet, S., Cioci, G., Nicaud, J. M., Marty, A. & O’Donohue, M. J. (2017). Biotechnology for Biofuels, 10(1), 132.
Background: Yarrowia lipolytica, one of the most widely studied “nonconventional” oleaginous yeast species, is unable to grow on cellulose. Recently, we identified and overexpressed two endogenous β-glucosidases in Y. lipolytica, thus enabling this yeast to use cello-oligosaccharides as a carbon source for growth. Using this engineered yeast platform, we have now gone further toward building a fully cellulolytic Y. lipolytica for use in consolidated bioprocessing of cellulose. Results: Initially, different essential enzyme components of a cellulase cocktail (i.e,. cellobiohydrolases and endoglucanases) were individually expressed in Y. lipolytica in order to ascertain the viability of the strategy. Accordingly, the Trichoderma reesei endoglucanase I (TrEG I) and II (TrEG II) were secreted as active proteins in Y. lipolytica, with the secretion yield of EG II being twice that of EG I. Characterization of the purified His-tagged recombinant EG proteins (rhTrEGs) revealed that rhTrEG I displayed higher specific activity than rhTrEG II on both cellotriose and insoluble cellulosic substrates, such as Avicel, β-1, 3 glucan, β-1, 4 glucan, and PASC. Similarly, cellobiohydrolases, such as T. reesei CBH I and II (TrCBH I and II), and the CBH I from Neurospora crassa (NcCBH I) were successfully expressed in Y. lipolytica. However, the yield of the expressed TrCBH I was low, so work on this was not pursued. Contrastingly, rhNcCBH I was not only well expressed, but also highly active on PASC and more active on Avicel (0.11 U/mg) than wild-type TrCBH I (0.065 U/mg). Therefore, work was pursued using a combination of NcCBH I and TrCBH II. The quantification of enzyme levels in culture supernatants revealed that the use of a hybrid promoter instead of the primarily used TEF promoter procured four and eight times more NcCBH I and TrCBH II expressions, respectively. Finally, the coexpression of the previously described Y. lipolytica β-glucosidases, the CBH II, and EG I and II from T. reesei, and the N. crassa CBH I procured an engineered Y. lipolytica strain that was able to grow both on model cellulose substrates, such as highly crystalline Avicel, and on industrial cellulose pulp, such as that obtained using an organosolv process. Conclusions: A Y. lipolytica strain coexpressing six cellulolytic enzyme components has been successfully developed. In addition, the results presented show how the recombinant strain can be optimized, for example, using artificial promoters to tailor expression levels. Most significantly, this study has provided a demonstration of how the strain can grow on a sample of industrial cellulose as sole carbon source, thus revealing the feasibility of Yarrowia-based consolidated bioprocess for the production of fuel and chemical precursors. Further, enzyme and strain optimization, coupled to appropriate process design, will undoubtedly lead to much better performances in the future.