The brown-rot basidiomycete Fomitopsis palustris has the endo-glucanases capable of degrading microcrystalline cellulose

Two endoglucanases with processive cellulase activities, produced from Fomitopsis palustris grown on 2% microcrystalline cellulose (Avicel), were purified to homogeneity by anion-exchange and gel filtration column chromatography systems. SDS-PAGE analysis indicated that the molecular masses of the purified enzymes were 47 kDa and 35 kDa, respectively. The amino acid sequence analysis of the 47-kDa protein (EG47) showed a sequence similarity with fungal glycoside hydrolase family 5 endoglucanase from the white-rot fungus Phanerochaete chrysosporium. N-terminal and internal amino acid sequences of the 35-kDa protein (EG35), however, had no homology with any other glycosylhydrolases, although the enzyme had high specific activity against carboxymethyl cellulose, which is a typical substrate for endoglucanases. The initial rate of Avicel hydrolysis by EG35 was relatively fast for 48 h, and the amount of soluble reducing sugar released after 96 h was 100 microg/ml. Although EG47 also hydrolyzed Avicel, the hydrolysis rate was lower than that of EG35. Thin layer chromatography analysis of the hydrolysis products released from Avicel indicated that the main product was cellobiose, suggesting that the brown-rot fungus possesses processive EGs capable of degrading crystalline cellulose.     

Isolation and properties of a cellulosome-type multienzyme complex of the thermophilic Bacteroides sp. strain P-1

The extracellular form of cellulosome-type multienzyme complex of thermophilic Bacteroides sp. strain P-1 which was isolated from the anaerobic digester, is described. Multienzyme complex was isolated from the culture supernatant by an adsorption-desorption affinity chromatography on microcrystalline cellulose. The isolated multienzyme complex was found to form a complex that exhibited a high molecular weight (estimated at more than 1400 kDa) and was quite stable, requiring strong denaturing condition for dissociation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved multienzyme complex into at least 12 subunits with the molecular weight range of 49 to 209 kDa, respectively. The isolated multienzyme complex showed cellulose-binding ability, cellulase and xylanase activities and effected the hydrolysis of crystalline cellulose and lignocellulosic materials in the form of corncob, corn hull, rice straw, and sugarcane bagasse.     

The 1,4-beta-D-glucan glucanohydrolases from Phanerochaete chrysosporium. Re-assessment of their significance in cellulose degradation mechanisms.

A physico-chemical, functional and structural characterization, including partial sequence data, of three major 1,4-beta-D-glucan glucanohydrolases (EC. 3.2.1.4) isolated from the culture filtrate of the white-rot fungus Phanerochaete chrysosporium, shows that all three enzymes belong to a single family of cellulases. EG44, pI 4.3, (named after its apparent molecular mass in kDa), shows a clear homology with Schizopyllum commune Endoglucanase I (EGI); whereas EG38, pI 4.9, (named in the same manner) is related more closely to Trichoderma reesei (Trichoderma longibrachiatum) Endoglucanase III (EGIII). EG36, pI 5.6-5.7, is probably an EG38 protein lacking its cellulose binding domain. Strong synergistic action is induced by the enzymes acting in concert with cellobiohydrolases (CBHI and CBHII) from the same organism, indicating a highly effective enzymatic system for cellulose degradation. Controlled proteolysis with papain has allowed a so far unique cleavage of endoglucanases EG44 and EG38 into two domains: a core protein, which virtually lacks the capacity to absorb onto microcrystal-line cellulose but retains full catalytic activity against carboxymethyl cellulose and low molecular weight soluble substrates; and a peptide fragment corresponding to the cellulose binding domain. The latter appears to be of paramount significance in the mechanisms involved in the hydrolysis of microcrystalline cellulose.     

Purification of an extracellular cellulose-binding endoglucanase of Cellulomonas sp. ATCC 21399 by affinity chromatography on H3PO4-swollen cellulose

A cellulose-binding endoglucanase (endoglucanase A) of Cellulomonas sp. ATCC 21399 was purified to immunological homogeneity by affinity chromatography ob H(3)PO(4)-swollen cellulose. This method of purification turned out to be an easy and very gentle method for obtaining a high yield of cellulose-binding endoglucanase. The purified enzyme was immunologically homogeneous but appeared heterogeneous when analyzed by denaturing polyacrylamide gel electrophoresis. In addition to the cellulose-binding of endoglucanase A, the enzyme also had a strong affinity for Concanavaline A, indicating that the enzyme was glycosylated. Purified endoglucanase A showed an endo mode of action on carboxymethylcellulose. The enzyme could hydrolyze microcrystalline cellulose when acting alone, and the enzyme had a high specific activity on H(3)PO(4)-swollen cellulose.     

Exo-mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP-23. A unique type of cellulase among Bacillales.

Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose-binding module, and a putative fibronectin-III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45 degrees C and pH 6.0 on acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, pNP-glycosides or 4-methylumbeliferyl alpha-d-glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP-23 would contribute to widen up its range of action on natural cellulosic substrates.     

Visualization of the adsorption of a bacterial endo-β-1,4-glucanase and its isolated cellulose-binding domain to crystalline cellulose

Endo-β-1,4-glucanase A (CenA), a cellulase from the bacterium Cellulomonas fimi, is composed of two domains: a catalytic domain and a cellulose-binding domain. Adsorption of CenA and its isolated cellulose-binding domain (CBD·PT_CenA) to Valonia cellulose microcrystals was examined by transmission electron microscopy using an antibody sandwich technique (CenA/CBD·PT_CenA-CenA IgG-protein A-gold conjugate). Adsorption of both CenA and CBD·PT_CenA occurred along the lengths of the microcrystals, with an apparent preference for certain crystal faces or edges. CenA or CBD·PT_CenA, but not the isolated catalytic domain, were shown to prevent the flocculation of microcrystalline bacterial cellulose. The cellulose-binding domain may assist crystalline cellulose hydrolysis in vitro by promoting substrate dispersion.     

Cellobiose oxidase from Phanerochaete chrysosporium can be cleaved by papain into two domains

Cellobiose oxidase from the white rot fungus Phanerochaete chrysosporium has been purified to homogeneity by a new method. The enzyme has been cleaved by papain into two fragments: one containing the heme group and one containing the flavin group. The flavin fragment can oxidize cellobiose and is reoxidized by oxygen. Cellobiose oxidase binds to cellulose to approximately the same extent as cellobiohydrolase I. The cellulose-binding site is located on the flavin domain. The enzyme cannot be totally displaced from cellulose by cellobiose, and it is still active when adsorbed to cellulose. The possible role of the enzyme in lignocellulose degradation is discussed.     

一株嗜热毛壳菌β-葡萄糖苷酶的分离纯化及特性

研究了液体发酵嗜热毛壳菌Chaetomium thermophile产生的β-葡萄糖苷酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose 疏水层析、Sephacryl S-100分子筛层析等步骤后获得凝胶电泳均一的β-葡萄糖苷酶。经12.5%SDS-PAGE和凝胶过滤层析方法分别测得该酶的分子量大小约为78.4kDa和81kDa。该酶反应的最适温度和pH值分别为60℃和4.5-5.0。有较好的酸稳定性和热稳定性。金属离子对β-葡萄糖苷酶的活性影响较大, 其中Ca2+对酶有激活作用, 而Ag+、Cu2+ 、Hg2+对酶有显著的抑制作用。该酶对水杨苷具有很强的底物特异性。     

Presence of several cellulose-binding proteins in culture supernatant and cell lysate of Eubacterium cellulosolvens 5

Attempts were made to separate and characterize cellulose-binding proteins (CBPs) from both the culture supernatant and cell lysate of Eubacterium cellulosolvens 5. Once the CBPs were bound to Avicel cellulose, they were then effectively eluted with the solution containing 3.2 or 5% sodium dodecyl sulfate (SDS), but not eluted with the solution containing various kinds of carbohydrates and reagents. Namely, CBPs in both the culture supernatant and cell lysate of the bacterium bound tightly and strongly to cellulose. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted CBPs indicated that the CBPs contained the two major proteins having the molecular weights of approximately 160 and 84 kilodaltons (kDa) and one sub-major protein having a molecular weight of approximately 140 kDa. Zymogram analysis after the SDS-PAGE of the eluted CBPs showed that two proteins exhibited the highest levels of carboxymethyl cellulase (CMCase) activity corresponding to the molecular weights of approximately 160 and 90 kDa. A major protein having the molecular weight of approximately 160 kDa exhibited a distinct CMCase activity and was designated as CBPE1. Western immunoblot analysis indicated that the proteins prepared from 16 representative strains of rumen bacteria did not cross-react with rabbit antiserum raised against CBPE1. Thus, CBPE1 may be a unique CBP that plays an important role in the adhesion of the bacterium to cellulose.     

Enzymatic degradation of steam-pretreated Lespedeza stalk (Lespedeza crytobotrya) by cellulosic-substrate induced cellulases

The cellulase production by Trichoderma viride, cultivated on different substrates, namely steam-pretreated Lespedeza, filter paper, microcrystalline cellulose (MCC) or carboxymethyl cellulose (CMC), was studied. Different cellulase systems were secreted when cultivated on different substrates. The cellulolytic enzyme from steam-pretreated Lespedeza medium performed the highest filter paper activity, exoglucanase and endoglucanase activities, while the highest β-glucosidase activity was obtained from the enzyme produced on filter paper medium. The hydrolytic potential of the enzymes produced from different media was evaluated on steam-pretreated Lespedeza. The cellulase from steam-pretreated Lespedeza was found to have the most efficient hydrolysis capability to this specific substrate. The molecular weights of the cellulases produced on steam-pretreated Lespedeza, filter paper and MCC media were 33, 37 and 40 kDa, respectively, and the cellulase from CMC medium had molecular weights of 20 and 43 kDa. The degree of polymerization, crystallinity index and micro structure scanned by the scanning electron microscopy of degraded steam-pretreated Lespedeza residues were also studied.     

Production and Characterization of Recombinant Phanerochaete chrysosporium Cellobiose Dehydrogenase in the Methylotrophic Yeast Pichia pastoris

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.     

Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris.

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.     

Three Forms of α-Glucosidase from Suspension-cultured Rice Cells

Three forms of α-glucosidase (EC 3.2.1.20), designated as I, II, and III, have been isolated from suspension-cultured rice cells by a procedure including fractionation with ammonium sulfate, CM-cellulose column chromatography, and preparative disc gel electrophoresis. The three enzymes were homogeneous by Polyacrylamide disc gel electrophoresis. α-Glucosidase I was secreted in the culture medium during growth, α-glucosidase II was readily extracted from rice cells with the buffer alone, and α-glucosidase III required NaCl to be solubilized. The molecular weights of the three enzymes were 96,000 (I), 84,000 (II), and 58,000 (III). The three enzymes readily hydrolyzed maltose, maltotriose, maltotetraose, amylose, and soluble starch. α-Glucosidase I possessed strong isomaltose-hydrolyzing activity and hydrolyzed isomaltose about three times as rapidly as α-glucosidase III. The three enzymes produced panose as the main α-glucosyltransfer product from maltose. Half the maltose-hydrolyzing activities of the three enzymes were inhibited by 11.25 ng of castanospermine. The inhibition was competitive.     

Purification and Characterization of Kininogens from Sheep Plasma

High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) have been purified from sheep ( Avis Arias) plasma in three steps involving ammonium sulphate precipitation, column chromatography on Sephacryl-300HR and ion exchange chromatography on DEAE cellulose. HMWK gave a single band on native and SDS-PAGE with a molecular weight corresponding to 280 kDa. Under reducing conditions purified HMWK was again resolved to a single band with molecular weight corresponding to 140 kDa indicative of its dimeric nature. LMWK was resolved into two isoforms named as LMWK1 and LMWK2, with an apparent molecular weight of 68 kDa. The yield of HMWK, LMWK1 and 2 was about 8.1, 5.63 and 10.65 respectively. HMWK, LMWK1 and 2 strongly inhibited activities of ficin and papain but not of trypsin, chymotrypsin and bromelain. K_i values estimated for HMWK with papain and ficin was 0.8 and 0.6 nM respectively. K_i values estimated for LMWK1 and 2 with papain were 2.40 and 2.00 nM respectively. Binding of HMWK, LMWK1 and 2 to activated papain were accompanied by pronounced changes in secondary and tertiary structure that are compatible with perturbations of environment of aromatic residues.     

The role of the cellulolytic high molecular mass (HMM) complex of the anaerobic fungus Piromyces sp. strain E2 in the hydrolysis of microcrystalline cellulose

The anaerobic fungus Piromyces sp. strain E2 produces extracellular cellulolytic enzymes present both in a high molecular mass (HMM) complex or as individual proteins. Although the HMM complex was present in the culture fluid during all growth stages, the highest amounts of complex were obtained when cultures were harvested at the end of fungal growth. The complex obtained after gel-filtration chromatography on Sephacryl S-300 HR was found to be the major factor in hydrolysis of cellulose to glucose (sole product, up to 250 mM). The complex was very stable as demonstrated by identical hydrolysis patterns with fresh preparations or preparations stored at 4° C for 2 months. From inhibition experiments with gluconic acid lactone and glucose, it was concluded that the HMM complex must contain at least one glucohydrolase. SDS-PAGE analysis revealed that a partially purified HMM complex was composed of at least ten polypeptides and contained numerous endoglucanases and one β-glucosidase. Received: 10 October 1996 / Accepted: 11 December 1996     

Description of a cellulose-binding domain and a linker sequence from Aspergillus fungi

A family I cellulose-binding domain (CBD) and a serine- and threonine-rich linker peptide were cloned from the fungi Aspergillus japonicus and Aspergillus aculeatus. A glutathione S-transferase (GST) fusion protein comprising GST and a peptide linker with the CBD fused to its C-terminus, was expressed in Escherichia coli. The renatured GST-CBD recovered from inclusion bodies had a molecular mass of 36.5 kDa which agrees with the 29 kDa of the GST plus the calculated 7.5 kDa of the linker with the CBD. The isolated GST-CBD protein adsorbed to both bacterial microcrystalline cellulose and carboxymethyl cellulose. Deletion of the linker peptide caused a decrease in cellulose adsorbance and a higher sensitivity to protease digestion.     

Growth and Cellulase Formation by Cellvibrio fulvus

S ummary : The aerobic cellulolytic bacterium Cellvibrio fulvus grew on several sugars and polysaccharides, but not on highly substituted cellulose derivatives, organic acids and alcohols. Whereas no growth was obtained on long cotton fibres, it occurred on such fibres cut into small pieces, and on filter paper and chromatography powders derived from cotton. Lignin free wood pulp was rapidly degraded. The organism grew best at pH 7–8 and utilized nitrate, ammonium and some amino acids as nitrogen sources. The bacteria have cell-bound cellulase but enzyme was also found in the culture medium. Glucose repressed cellulase formation and the enzyme activity of cultures grown on cellulose was much higher than on sugars. Reducing sugar was not detected in cellulose cultures. The pH optimum for hydrolysis of carboxymethylcellulose (CMC) was 7 and the enzyme was inhibited by mercuric acetate but not by p -chloromercuribenzoate or EDTA. Fractionation of cellulase preparations from cultures grown on partially hydrolysed filter paper gave many components of different molecular weights. The activities of these components against carboxymethylcellulose and microcrystalline cellulose differed.     

The 1,4-beta-D-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei.

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.     

Purification and characterization of five cellulases and one xylanase from Penicillium brasilianum IBT 20888

The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l⁻¹ cellulose and 10 g l⁻¹ xylan for 111 h and the resulting culture filtrate was used for protein purification. From the cultivation broth, five cellulases and one xylanase were purified. Hydrolysis studies revealed that two of the cellulases were acting as cellobiohydrolases by being active on only microcrystalline cellulose (Avicel). Three of the cellulases were active on both Avicel and carboxymethyl cellulose indicating endoglucanase activity. Two of these showed furthermore mannanase activity by being able to hydrolyze galactomannan (locust bean gum). Adsorption studies revealed that the smaller of the two enzymes was not able to bind to cellulose. Similarity in molecular mass, pI and hydrolytic properties suggested that these two enzymes were identical, but the smaller one was lacking the cellulose-binding domain or an essential part of it. The basic xylanase (pI>9) was only active towards xylan. Two of the purified cellulases with endoglucanase activity were partly sequenced and based on sequence homology with known enzymes they were classified as belonging to families 5 and 12 of the glycosyl hydrolases.     

Dynamic interaction of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A and cellulose at equilibrium and during hydrolysis

The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two Trichoderma reesei cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.