Enzyme mechanisms involved in cellulose hydrolysis by the rot fungus Sporotrichum pulverulentum

This article presents a review of the enzyme mechanisms involved in degradation of cellulose by the white-rot fungus Sporotrichum poulverulentum. The hydrolytic enzymes involved include: (1) five endo-1,4-β-glucanases; (2) one exo-1,4-β-glucanase, and (3) one or several 1,4-β-glucosidases. A recently discovered oxidative enzyme of importance in in vitro cellulose degradation seems to be a cellobiose oxidase. An oxidoreductase, cellobiose:quinone oxidoreductase, is of importance both in cellulose and in lignin degradation. Regulatory mechanisms of the extracellular enzyme activities, such as monosugar levels causing catabolite repression of the endoglucanases, have also been investigated. The enzymes used by S. pulverulentum in cellulose hydrolysis are compared to those used by Trichoderma viride. Very similar types of enzymes are used in both cases. However, no oxidative enzyme has so far been found to be involved in extracellular cellulose degradation in the case of T. viride. Recommendations for further research are given.     

Nutritional evaluation of the white-rot fungus Sporotrichum pulverulentum as a feedstuff to rats,pigs, and sheep

The production of single-cell protein (SCP) based on cheap carbon sources such as spent liquor from paper mills is of interest for different reasons. The White-rot fungus (Sporotrichum pulverulentum) has earlier been shown to degrade cellulose and lignin. The nutritive value of this fungus was investigated with rats, pigs, and sheep. The effect of different drying process was evaluated on rats. Experiments with piglets, growing pigs, and sheep were aimed at getting primary information on nutritive parameters with domestic animal species, Chemical analysis of S. pulverulentum showed that the sum of the amino acids corresponded to 70% and ammonia, GABA, and glucosamine to 20% of its crude protein content. Differences between drying treatments in their effect on protein digestibility were not noted. From a protein quality viewpoint, a tendency toward superiority was noted for two of the drying processes. The amino acid digestibility of S. pulverulentum was inferior to values for soybean oil meal given in textbooks. The piglet experiment confirmed the lower nutritive value of S. pulverulentum compared with soybean oil meal. in the piglet stage a content of metabolizable energy of S. pulverulentum was found which corresponded to 60% of that for soybean oil meal. With increasing age the ability of pigs to utilize the fungus increased. The limited nutritive value for monogastric animals is most certainly caused by the cell-wall structure of S. pulverulentum with poor digestibility of the carbohydrates. The experiment with sheep showed more satisfactory results than with monogastric species, with digestibility of crude protein of 82% and a content of metabolizable energy of 70% of soybean oil meal.     

The effect of acetylated xylan and sugar beet pulp on the expression and secretion of enzymes by <Emphasis Type="Italic">Penicillium purpurogenum</Emphasis>

Sugar beet pulp is a natural carbon source composed mainly of pectin and cellulose, which is utilized and degraded by the ascomycete Penicillium purpurogenum. The fungus also grows on and degrades acetylated xylan which lacks cellulose and pectin. Both carbon sources have been used in our laboratory to grow the fungus and to purify different enzymes secreted to the medium. The enzymes involved in the complex process of degradation of these carbon sources by the fungus have been explored previously under non-denaturing conditions; multienzyme complexes were separated and some subunits identified by Western blots and mass spectrometry. In this work, proteomic profiles show that the secretome is composed of numerous proteins varying in pI and molecular weight. Some enzymes are common to both growth conditions, while others are specific for each carbon source. The results show that the carbon sources utilized exert strong regulatory control over the proteins secreted. This is the first secretome study from a lignocellulolytic Penicillium.     

The importance of phenol oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum

The lignin degradation abilities of wildtype, a phenol oxidase-less mutant and a phenol oxidase-positive revertant of Sporotrichum pulverulentum were compared to determine if phenol oxidase activity is necessary for lignin degradation by white-rot fungi. The phenol oxidase-less mutant was unable to degrade kraft lignin or wood. The phenol oxidase-positive revertant, however, regained the ability of the wildtype to degrade kraft lignin and all of the major components of wood. It was found that kraft lignin and lignin-related phenols decreased cellulase and xylanase production by the phenol oxidase-less mutant. Addition of highly purified laccase increased the production of endo-1,4--glucanase in the phenol oxidase-less mutant in the presence of vanillic acid and kraft lignin. After addition of laccase to kraft lignin agar plates, the phenol oxidase-less mutant could degrade kraft lignin.It is proposed that phenol oxidase function in regulating the production of both lignin-and polysaccharide-degrading enzymes by oxidation of lignin and lignin-related phenols when S. pulverulentum is growing on wood.Abbreviation WT wildtype Sporotrichum pulverulentum Research supported by a grant from Stiftelsen Nils and Dorthi Troëdssons forskningsfond     

Zum Einfluß der physikalischen Struktur des Substrates auf die Hydrolyse von Cellulose durch verschiedene Enzymsysteme und Enzymkomponenten

Starting from cellulose samples prepared from cotton lintes and differing in lattice type, crystallinity and fibrillar morphology, enzymatic hydrolysis of fibre cellulose has been studied employing complete enzyme systems from Trichoderma, Sporotrichum, Gliocladium and Penicillium as well as isolated endo- and exo-1,4-β-glucanases from Trichoderma reesei and Sporotorichum pulverulentum. The effect of hydrolysis was characterized by content of reducing sugars (RS) and of glucose in the hydrolyzate as well as by DP and X-ray diffraction pattern of the residues. With all the complete enzyme systems investigated about the same order of degradability was found with a series of substrates differing in physical structure. The hydrolysis effect of cellulase from S. pulverulentum proved to be sensitive to the gas atmosphere above the system (N₂ or O₂), probably due to the interaction of an O₂-atmosphere with the activity of the cellubiose-oxydase existent in the system. Isolated endoglucanase from S. pulverulentum and T.reesei still led to a considerable formation of RS and glucose, a corrosion of the fibre surface and a significant descrease in DP. Influence of substrate physical structure was rather small with regard to RS, but still considerable with regard to residue-DP. The effect of isolated exoglucanases depends largely on the chemical structure of the cellobiohydrolase in question, as demonstrated with the two samples “CBH I” and “CBH II” from T. reesei. With CBH I, rather resembling endo-glucanase behaviour, a considerable formation of RS and a significant corrosion of the fibre surface has been observed. On the other hand, only negligibly small amounts of RS were formed by CBH II. Results are discussed with regard to the complex mechanism of cellulase action on fibrous cellulose and with regard to the relevance of different parameters of physical structure of cellulose in connection with enzymatic hydrolysis. A remarkable acceleration of the Cellulose III → Cellulose I lattice transition due to chain fragmentations in the presence of cellulase can be concluded the experiments.     

Russian Article pp. 191–195

Three cellulase components and one xylanase of Trichoderma sp. M-17 have been immobilzed on a soluble high molecular weight polymer (PVA), using carbodiimide. The immobilized enzymes retained about 80% of the cellulase, cellulose 1,4-β-cellobiosidase, β-glucosidase and 60% endo-1,4-β-xylanase activities. The bound enzymes catalyzed the hydrolysis of alkali-treated cornstalks with a higher efficiency than the free cellulase. The potential for reutilization of the immobilized enzymes was studied using membrane filters and the system was found to be active for three cycles.     

Crystal structure of basic 7S globulin, a xyloglucan-specific endo-β-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-β-glucanase

β-Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These β-linked glucans are constantly exposed to degradation by various endo-β-glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo-β-glucanases inhibitors, such as xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three-dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo-222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S.     

Regulation of Extracellular Chitinases and Proteases in the Entomopathogen and Acaricide <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS–PAGE. Received: 8 May 2002 / Accepted: 8 June 2002     

Bioconversion of kinnow-mandarin waste into single-cell protein

Summary Kinnow-mandarin waste (peel, pulp and seeds) was assessed for single-cell protein (SCP) production byChaetomium globosum andSporotrichum pulverulentum in shake-flask culture. The maximum protein enrichment (32% and 34%) of the substrate was achieved after 5 and 7 days of incubation by the two organisms, respectively. Of various nitrogen sources, NaNO₃ and NH₄Cl gave maximum protein enrichment of the substrate byC. globosum andS. pulverulentum, respectively.

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Bioconversion des résidus de la mandarine-kinnow en protéine uni-cellulaire

Résumé On a examiné la possibilité de produire des protéines uni-cellulaires (POU) à partir de résidus de la mandarine-kinnow (pelures, pulpe, et pépins) par la culture deChaetomium globosum et deSporotrichum pulverulentum en flacons agités. L'enrichissement maximum en protéines du substrat, soit 32 et 34% est obtenu après 5 et 7 jours d'incubation respectivement par les deux organismes. Parmi les diverses sources d'azote, NaNO₃ et NH₄Cl ont permis respectivement l'enrichissement maximum en protéines du substrat parC. globosum etS. pulverulentum.

     

Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost

In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.     

Label-free quantitative proteomics for the extremely thermophilic bacterium Caldicellulosiruptor obsidiansis reveal distinct abundance patterns upon growth on cellobiose, crystalline cellulose, and switchgrass

Mass spectrometric analysis of Caldicellulosiruptor obsidiansis cultures grown on four different carbon sources identified 65% of the cells' predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47_0549 together with the COB47_1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47_0096 and COB47_0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 β-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When C. obsidiansis was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation.     

Fungal pathogens secrete an inhibitor protein that distinguishes isoforms of plant pathogenesis-related endo-β-1,3-glucanases

Plant endo-β-1,3-glucanases and chitinases inhibit the growth of some fungi and generate elicitor-active oligosaccharides while depolymerizing polysaccharides of mycelial walls. Overexpression of the endo-β-1,3-glucanases and/ or chitinases in transgenic plants provides, in some cases, increased protection against fungal pathogens. However, most of the phytopathogenic fungi that have been tested in vitro are resistant to endo-β-1,3-glucanases and chitinases. Furthermore, some phytopathogenic fungi whose growth is inhibited by these enzymes are able to overcome the effect of these enzymes over a period of hours, indicating an ability of those fungi to adapt to the enzymes. Evidence is presented indicating that fungal pathogens secrete proteins that inhibit selective plant endo-β-1,3-glucanases.A glucanase inhibitor protein (GIP-1) has been purified to homogeneity from the culture fluid of the fungal pathogen of soybeans, Phytophthora sojae f. sp. glycines (Psg), and two basic pathogenesis-related endo-β-1,3-glucanases (EnGL_soy-A and EnGL_soy-B) have been purified from soybean seedlings. GIP-1 inhibits EnGL_soy-A but not EnGL_soy-B. Moreover, GIP-1 does not inhibit endo-β-1,3-glucanases secreted by Psg itself nor does GIP-1 inhibit PR-2c, a pathogenesis-related endo-β-1,3-glucanase of tobacco. Evidence is presented that Psg secretes other GIPs that inhibit other endo-β-1,3-glucanase(s) of soybean. Furthermore, GIP-1 does not exhibit proteolytic activity but does appear to physically bind to EnGL_soy-A. The results reported herein demonstrate specific interactions between gene products of the host and pathogen and establish the need to consider fungal proteins that inhibit plant endo-β-1,3-glucanases when attempting to use the genes encoding endo-β-1,3-glucanases to engineer resistance to fungi in transgenic plants.     

Application of solid waste from anaerobic digestion of poultry litter in Agrocybe aegerita cultivation: mushroom production, lignocellulolytic enzymes activity and substrate utilization

The degradation and utilization of solid waste (SW) from anaerobic digestion of poultry litter by Agrocybe aegerita was evaluated through mushroom production, loss of organic matter (LOM), lignocellulolytic enzymes activity, lignocellulose degradation and mushroom nutrients content. Among the substrate combinations (SCs) tested, substrates composed of 10–20% SW, 70–80% wheat straw and 10% millet was found to produce the highest mushroom yield (770.5 and 642.9 g per 1.5 kg of substrate). LOM in all SCs tested varied between 8.8 and 48.2%. A. aegerita appears to degrade macromolecule components (0.6–21.8% lignin, 33.1–55.2% cellulose and 14–53.9% hemicellulose) during cultivation on the different SCs. Among the seven extracellular enzymes monitored, laccase, peroxidase and CMCase activities were higher before fruiting; while xylanase showed higher activities after fruiting. A source of carbohydrates (e.g., millet) in the substrate is needed in order to obtain yield and biological efficiency comparable to other commercially cultivated exotic mushrooms.     

High expression of a neutral endo-β-glucanase gene from Humicola insolens in Trichoderma reesei

The neutral endo-β-glucanase gene cel5A from Humicola insolens was cloned and connected with the cellobiohydrolase 1 promoter from Trichoderma reesei to construct a recombinant plasmid pCB-hEG with the hygromycin B resistance marker. The plasmid was introduced into conidia of T. reesei using the Agrobacterium tumefaciens mediated transformation method. Eight transformants were obtained on screening plates with sodium carboxymethyl cellulose as the sole carbon source. Stable integration of the cel5A gene into the chromosomal DNA of T. reesei was confirmed by PCR. An obvious protein band (approximately 52 kDa) was detected by SDS-PAGE from fermentation broth, which showed that the cel5A gene in recombinant T. reesei successfully fulfilled efficient expression and extracellular secretion. After 96 h shaking-flask fermentation, the endo-β-glucanase activity at pH 6.5 from recombinant T. reesei reached 3,068 U/ml, which was 11 times higher than that of the host strain. In a 2 m³ fermenter, the endo-β-glucanase activity could be further increased to 8,012 U/ml after 96 h fermentation. The results showed a good prospect for application of neutral endo-β-glucanase in the textile industry.     

Regulation of protein synthesis in methylotrophic yeasts: Repression of methanol dissimilating enzymes by nitrogen limitation

The influence of nitrogen limitation on the regulation of the methanol oxidizing enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two methylotrophic yeastsHansenula polymorpha andKloeckera sp. 2201 was studied in continuous culture. When shifted from carbon-limited growth conditions (with a mixture of glucose and methanol as carbon sources) to a nitrogen-limited environment both cultures were found to go through a transition phase where neither enhanced residual concentrations of the nitrogen source nor of one of the two carbon sources could be detected in the supernatant. As soon as nitrogen became a limiting substrate an immediate reorganisation of the cell composition was initiated: protein content of the cells dropped to approximately 40% of its initial value, glycogen was synthesized and the enzyme composition of the cells was changed. The peroxisomal enzymes alcohol oxidase and catalase in both organisms and the two dehydrogenases for formaldehyde and formate in cells ofKloeckera sp. 2201 were subject to degradation (catabolite inactivation). The measured rates of inactivation indicated that in cells ofH. polymorpha this process might be limited to peroxisomes, whereas inKloeckera sp. 2201 the degradation was found to affect peroxisomal as well as cytoplasmic enzymes. In contrast to methanol dissimilating enzymes the net rate of synthesis of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was not affected by this process but those enzymes were synthesized with increased rates.     

Cellulases: Biosynthesis and applications

Strains of Trichoderma, particularly T. reesei and its mutants, are good sources of extracellular cellulase suitable for practical saccharification. They secrete a complete cellulase complex containing endo- and exo-glucanases plus β-glucosidase (cellobiase) which act syngergistically to degrade totally even highly resistant crystalline cellulose to soluble sugars. All strains investigated to date are inducible by cellulose, lactose, or sophorose, and all are repressible by glucose. Induction, synthesis and secretion of the β-glucanase enzymes appear to be closely associated. The composition and properties of the enzyme complex are similar regardless of the strain or inducing substrate although quantities of the enzyme secreted by the mutants are higher. Enzyme yields are proportional to initial cellulose concentration. Up to 15 filter paper cellulase units (20 mg of cellulase protein) per ml and productivities up to 80 cellulase units (130 mg cellulase protein) per litre per hour have been attained on 6% cellulose. The economics of glucose production are not yet competitive due to the low specific activity of cellulase (0.6 filter paper cellulase units/mg protein) and poor efficiency (about 15% of predicted sugar levels in 24 h hydrolyses of 10–25% substrate). As hydrolysis proceeds, the enzyme reaction slows due to increasing resistance of the residue, product inhibition, and enzyme inactivation. These problems are being attacked by use of pretreatments to increase the quantity of amorphous cellulose, addition of β-glucosidase to reduce cellobiose inhibition, and studies of means to overcome instability and increase efficiency of the cellulases. Indications are that carbon compounds derived from enzymatic hydrolysis of cellulose will be used as fermentation and chemical feedstocks as soon as the process economics are favourable for such application.     

Xylanases of anaerobic fungus <Emphasis Type="Italic">Anaeromyces mucronatus</Emphasis>

The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase, xylanase, α-xylosidase, β-xylosidase, α-glucosidase, β-glucosidase, β-galactosidase, mannosidase, cellobiohydrolase, amylase, laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but production into the medium was prevalent with the exception of intracellular β-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa.