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.     

Regulatory mutations affecting the synthesis of cellulase in Bacillus pumilus

A wild strain of Bacillus pumilus was investigated for cellulase production, and putative mutants of this strain were screened for catabolite repression insensitivity after chemical mutagenesis using ethyl methanesulphonate (EMS) as a mutagenic agent. Out of four classes of mutants studied and classified according to their cellulase induction rate and level of cellulase production in the presence of high concentrations of glucose (2.6%[w/v]), classes III and IV exhibited cellulase production up to 6.2 mg cellulase and 11.4 mg cellulase per gram of dry cell mass respectively. These mutants were referred to as catabolite repression-insensitive when compared to the wild strain which exhibited a total repression of cellulase synthesis under the same conditions. How EMS triggered the catabolite repression insensitivity in these mutants was not established. However this mutation brought out new strains of cellulase hyperproducers (mutants 6 and 11) in the presence of glucose when compared to other cellulase producers such as Aspergillus terreus, A. nidulans and Trichoderma reesei, which exhibited catabolite repression of cellulase synthesis. These mutants were selected as the most promising candidates for cellulase synthesis even at high glucose concentration.     

Some properties of carbohydrate and C4-dicarboxylic acid utilization negative mutants ofRhizobium leguminosarum biovarphaseoli strain P121

After NTG treatment of the very effective wild type strain P121 ofRhizobium leguminosarum biovarphaseoli, mutants defective in the utilization of sugars or organic acids were obtained. All the mutants nodulated the cultivar Goldie ofPhaseolus vulgaris. The arabinose, fructose, glucose and pyruvate utilization mutants formed nodules similar in shape and size to the nodules formed by the wild type strain. These mutants exhibited an acetylene reduction activity significantly lower than the activity observed with the wild type strain. All the C₄-dicarboxylic acid utilization mutatns, formed ineffective nodules that did not show a significant acetylene reduction activity. The C₄-dicarboxylic acids uptake system is apparently inducible in the free-living bacteria of strain P121. When P121 cells were grown on glucose in the presence of 2.5 mM malate, the rate of glucose-dependent O₂ consumption significantly decreased suggesting the presence of a catabolite repression-like phenomenon. Isolated bacteroids of strain P121, under the experimental conditions used, were able to oxidize succinate, fumarate or malate but did not oxidize pyruvate, glucose, fructose or sucrose.     

Purification and characterization of cellulolytic enzymes fromTrichoderma harzianum

Two forms of filter paper activity (filter paper activity; cellulose 1,4-β-cellobiosidase, EC 3.2.1.91) and single forms of CM-cellulase (carboxymethyl cellulase; endo-l,4-β-glucanase, EC 3.2.1.4) and β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) from the culture filtrate ofTrichoderma harzianum were separated and partially purified by (NH₄)₂SO₄ precipitation, ion-exchange chromatography and gel filtration. The final preparation was purified about 12-, 20- and 27-fold for FP-activity, CM-oellulase and β-glueosidase, respectively. The pH and temperature optima, stability, kinetic parameters, effeet of metal ions and molar mass of each was determined. A distinct type of synergistic action between cellulase components was observed for efficientin vitro saccharification of dewaxed cotton.     

Sophorose metabolism and cellulase induction in Trichoderma

The cellulase inducer sophorose was rapidly catabolized to CO₂ and H₂O by Trichoderma: only small amounts were used to induce the synthesis of cellulase. ³H-sophorose uptake began after a lag of 1 h and its half-life in the medium was less than 5 h. Cellulase activity in the medium did not increase till 6 h after the addition of sophorose and reached a half maximum value at 14 h. The presence of free sophorose in the medium was required for continuous cellulase production. Several small sophorose addition induced much more cellulase than an equivalent single dose. These results are attributed to two pathways of sophorose utilization, a catabolic pathway that has a high capacity but low affinity for sophorose and an inductive pathway having a lower capacity but higher affinity for sophorose.     

Increased endoplasmic reticulum content of a mutant of Trichoderma reesei (RUT-C30) in relation to cellulase synthesis

The structure and content of endoplasmic reticulum (ER) were studied in the shake cultures of two strains of Trichoderma reesei, one wild type (QM6a) and the other a cellulase (EC 3.2.1.4) hyper-producing and catabolite repression resistant mutant (RUT-C30). The results of quantitative electron microscopic and biochemical assays were correlated. At the stage of growth when cellulase secretion was high the ER in RUT-C30 cells was highly developed and more abundant than the ER in QM6a cells. A process regulating ER biogenesis may have been deactivated by mutation in RUT-C30 cells and thus the potential for extracellular enzyme synthesis and secretion has been enhanced.     

Induction of cellulase formation in Trichoderma reesei by cellobiono-1,5-lacton

Induction of synthesis of cellulolytic enzymes in Trichoderma reesei QM 9414 by cellobiono-1,5-lactone (CBL) has been investigated in a replacement system lacking additional carbon source. CBL induced cellulase secretion optimally at pH 5 and a concentration of 70 g/ml. Higher concentrations lead to lower induction. De novo induction of cellulases was proven by the inhibitory effect of cycloheximide addition. Induction by CBL was shown to act synergistically on induction by sophorose, as it decreased the concentration of sophorose required for maximal induction. Maximal endo--1,4-glucanase activities induced by either sophorose or CBL were comparable. The CBL-induced cellulase system contained all the major cellulolytic enzymes of T. reesei, i.e. cellobiohydrolase I and II, and endoglucanase I, as shown by SDS-PAGE, Western blotting and detection with specific mono- and polyclonal antibodies. No differences were seen in the types of individual enzymes formed upon induction by either sophorose or CBL. No other hydrolytic enzymes appear to be induced by CBL (i.e. amylase, laminarinase, xylanase).Abbreviations SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium-dodecylsulfate - CBL cellobiono-1,5-lacton - CBH cellobiohydrolase - EG endoglucanase - IgG immunoglobulin G     

Domain-specific determinants of catalysis/substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region (“NB loop”). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called “I-loop” region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1 mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high K_m with PPi (up to 33 mM), whereas Ncut deletions had greatly increased K_m with UDPG (up to 57 mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar K_m values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.     

Semiquantitative Plate Assay for Determination of Cellulase Production by Trichoderma viride

A plate clearing assay was devised to screen for high-producing cellulase mutants of Trichoderma viride. The method employs (i) the use of either rose bengal or oxgall to limit colony size and (ii) Phosfon D (tributyl-2, 4-dichloroben-zylphosphonium chloride) to enhance cellulase detection, in combination with acid-swollen cellulose on agar plates. The method was used to isolate constitutive cellulase mutants of T. viride and should prove useful for isolating high-producing mutants from a range of organisms. This technique has been also used to determine the concentration at which glucose and glycerol inhibit cellulase synthesis by catabolite repression in the wild-type strains.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.     

Fungus fruit body lytic enzyme from a myxomycete,Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).     

Cellulase induction by lactose in Trichoderma reesei PC-3-7

In an attempt to clarify the function of lactose in cellulase induction, experiments were carried out on cellulase formation by lactose along with other sugars in a resting cell system of Trichoderma reesei PC-3-7, a hypercellulase-producing mutant. Although lactose alone induces little cellulase under the conditions used, a synergistic effect on cellulase formation was observed following the respective addition of sophorose, cellobiose or galactose to lactose. The lactose consumption was more rapid when these sugars were added than in their absence. Furthermore, following lactose addition 10 h after the beginning of cultivation in the presence of cellobiose, cellulase formation was initiated with only a little lag, and lactose consumption started immediately, being complete in 14 h. \-Galactosidase induction experiments suggested that the rapid consumption of lactose is possibly not dependent on lactose degradation by the enzyme. From these results, it is suggested that lactose may function as an inducer for cellulase formation if it is taken up in the mycelium of T. reesei PC-3-7, and that sophorose, cellobiose or galactose may induce a putative lactose permease. *** DIRECT SUPPORT *** AG903066 00005     

Nitrogen fixation (acetylene reduction) inAquaspirillum magnetotacticum

Aquaspirillum magnetotacticum strain MS-1 and two nonmagnetic mutants derived from it reduced C₂H₂ microaerobically but not anaerobically even with NO₃ ^?. This organism apparently is not capable of NO₃ ^?-dependent nitrogen fixation. Cells ofA. magnetotacticum reduced C₂H₂ at rates comparable to those ofAzospirillum lipoferum grown under similar conditions, but much lower than that ofAzotobacter vinelandii grown aerobically. Cells ofA. magnetotacticum in anaerobic cultures lacking NO₃ ^? did not reduce C₂H₂ until O₂ was introduced. Optimum rates of C₂H₂ reduction byA. magnetotacticum were obtained at 200 Pa O₂. C₂H₂ reduction was inhibited by more than 1 kPa O₂ or 0.2 mM NO₃ ^? or NH₄ ⁺. These results suggest thatA. magnetotacticum fixes N₂ only under microaerobic, N-limited conditions.     

Oxydation of substrates in organic acids utilization negative mutants and the wild typeRhizobium meliloti strain S'14

Two mutants defective in succinate utilization were isolated by NTG mutagenesis of the effective wild typeRhizobium meliloti strain S₁₄. The mutants used carbon sources in a fashion similar to strain S₁₄, but they were not able to grow on succinate, fumarate or malate. The mutants nodulated alfalfa plants but did not exhibit any nitrogenase activity. The mutants oxidized glucose and fructose, but were not able to oxidize organic acids. Cultured free-living bacteria of strain S₁₄ appeared to have an inducible C₄-dicarboxylic acid uptake system and a constitutive glucose uptake system. When S₁₄ cells were grown on glucose in the presence of 5mM or more succinate or malate, the rate of glucose-dependent O₂ consumption significantly decreased suggesting the presence of a catabolite repression like phenomenom. Contribution no. 301, Station de Recherches, Agriculture Canada.     

Sophorolipids-induced cellulase production in cocultures of Hypocrea jecorina Rut C30 and Candida bombicola

Sophorose is a potent but expensive inducer for cellulase production. In this study the feasibility of using sophorolipids, natural lipids that contain sophorose, for cellulase induction was investigated. Enhanced cellulase production by Hypocrea jecorina Rut C30 grown on glycerol, a substrate without cellulase-inducing ability, was first confirmed by addition of the crude sophorolipids collected from Candida bombicola fermentation. Cocultures of H. jecorina Rut C30 and C. bombicola were then employed to evaluate the effects of coculture conditions: the cell concentration ratio between the two cultures, the concentration of vegetable oil (as lipid precursor for sophorolipid synthesis, in addition to glycerol as the primary carbon source), the presence of nitrogen source for growth, and the substitution of glucose for glycerol as the primary carbon source. Specific cellulase productivity of H. jecorina Rut C30 was significantly higher under the conditions that promoted sophorolipid production by C. bombicola. The ability of H. jecorina Rut C30 to degrade sophorolipids was also confirmed. The results of the study indicated that the sophorolipids produced by C. bombicola can be degraded by H. jecorina Rut C30 and the sophorose generated from the degradation can effectively induce the fungal cellulase synthesis.     

Indole alkaloids as systematic markers of the Apocynaceae

Indole alkaloids can be characterized by skeletal specialization (S), determined upon consideration of their relative position on a biogenetic map and the number of their naturally occurring substitutional derivatives, as well as by oxidation level (O). The mean (S) and (O) for contained alkaloids of a given plant taxon are taken to represent evolutionary advancement parameters, respectively EA_s and EA_o.A correlation of these EA_s/EA_o values for tribes of the Apocynaceae-Plumerioideae reveals a chemical gradient, given by gradually increasing EA_s and EA_o values, to link Carisseae-Alstonieae-Rauvolfieae-Tabernaemontaneae.     

Evaluation of β-1,3-glucanase and β-1,4-glucanase enzymes production in some Trichoderma species

Trichoderma species have become the important means of biological control for fungal diseases. This research was carried on to access the high β-1,3-glucanase and β-1,4-glucanase enzyme producer of Trichoderma species isolates using two different carbon sources for finding a method to obtain more concentrate culture filtrates. Therefore, 14 Trichoderma isolates belonging to species: Trichoderma ceramicum, T. virens, T. pseudokoningii, T. koningii, T. koningiosis, T. atroviridae, T. viridescens, T. asperellum, T. harzianum1, T. orientalis, T. harzianum2, T. brevicompactum, T. viride and T. spirale were cultured in Wiendling’s liquid medium plus 0.5% glycerol or 0.5% Phytophthora sojae-hyphe as the carbon source in shaking and non-shaking (stagnant) statuses. Enzyme activity rate and total protein were evaluated in raw, acetony and lyophilized concentrated culture filtrates and the specific enzyme activity of β-1,3-glucanase and β-1,4-glucanase were measured by milligramme glucose equivalent released per minute per milligramme total protein in culture filtrates. The results showed that using Phytophthora – hyphe in medium increased the enzyme activities as compared to glycerol at all Trichoderma species which suggested that these substrates can also act as inducer for synthesis of lytic enzymes, in addition the most enzymes activity was observed in the lyophilised concentrated culture filtrate. The most successful species in β-1,3-glucanase and β-1,4-glucanase enzymes activities were T. brevicompactum and T. virens and these species can be used for mass production of these enzymes which are supposed to be used in commercial formulation and also will be able to control P. sojae directly.     

Cellulase production by a thermophilic clostridium species

Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 μm) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C₁ cellulase (degrades native cellulose) and C_x cellulase (β-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl₂ precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C_x cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature.     

The cellulose-binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding

Cellulomonas fimi endo-β-1, 4-glucanase A (CenA) contains a discrete N-terminal cellulose-binding domain (CBD_cenA)- Related CBDs occur In at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDcenA- The adsorption of CBD_cenA to Crystalline cellulose was compared with that of two Trp mutants (W14A and W68A). The affinities of the mutant CBDs for cellulose were reduced by approximately 50- and 30-fold, respectively, relative to the wild type. Physical measurements indicated that the mutant CBDs fold normally. Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface.     

Intraspecific hybridisation of Trichoderma pseudokoningii by anastomosis and by protoplast fusion

Double auxotrophic and morphological mutants of Trichoderma pseudokoningii Rifai were fused by anastomosis and by protoplast fusion. The recovery of recombinants from heterokaryons on different selective media and from heterokaryotic colonies indicated the occurrence of parasexual events. Prototrophic colonies growing on minimal medium produced binucleate spores, green in colour, revealing a non-autonomous system for conidial pigmentation. Recombinants were obtained from these dikaryotic colonies suggesting the occurrence of a highly unstable diploid phase.