Structure and characteristics of an endo-beta-1,4-glucanase, isolated from Trametes hirsuta, with high degradation to crystalline cellulose

Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-beta-1,4-glucanase (ThEG) was purified from the culture filtrate, and the gene and the cDNA were isolated. The gene consisted of an open reading frame encoding 384 amino acids, interrupted by 11 introns. The whole sequence showed high homology with that of family 5 glycoside hydrolase. The properties of the recombinant enzyme (rEG) in Aspergillus oryzae were compared with those of the En-1 from Irpex lacteus, which showed the highest homology among all the endoglucanases reported. The rEG activity against Avicel was about 8 times higher than that of En-1 when based on CMC degradation. A remarkable structural difference between the two enzymes was the length of the linker connecting the cellulose-binding domain to the catalytic domain.     

Gene Cloning of Cellobiohydrolase II from the White Rot Fungus Irpex lacteus MC-2 and Its Expression in Pichia pastoris

A gene (cel4) coding for a cellobiohydrolase II (Ex-4) was isolated from the white rot basidiomycete, Irpex lacteus strain MC-2. The cel4 ORF was composed of 452 amino acid residues and was interrupted by eight introns. Its deduced amino acid sequence revealed a multi domain structure composed of a cellulose-binding domain, a linker, and a catalytic domain belonging to family 6 of glycosyl hydrolases, from the N-terminus. cel4 cDNA was successfully expressed in the yeast Pichia pastoris. Recombinant Ex-4 showed endo-processive degrading activity towards cellulosic substrates, and a synergistic effect in the degradation of Avicel was observed when the enzyme acted together with either cellobiohydrolase I (Ex-1) or endoglucanase (En-1) produced by I. lacteus MC-2.     

Gene cloning of an endoglucanase from the basidiomycete Irpex lacteus and its cDNA expression in Saccharomyces cerevisiae

A gene (cen1) coding for an endoglucanase I (En-1) was isolated from white rot fungus Irpex lacteus strain MC-2. The cen1 ORF was comprised of 399 amino acid residues and interrupted by 14 introns. The deduced amino acid sequence of the cen1 ORF revealed a multi-domain structure composed of a cellulose-binding domain, a Ser-/Thr-rich linker, and a catalytic domain from the N-terminus. It showed a significant similarity to those of other endoglucanases that belong to family 5 of glycosyl hydrolases. cen1 cDNA was inserted into a yeast expression vector, YEpFLAG-1, and introduced into Saccharomyces cerevesiae. The resulting S. cerevisiae transformant secreted a recombinant En-1 that had enzymatic properties similar to the original En-1. A strong synergistic effect for a degradation of Avicel and phosphoric acid swollen cellulose was observed when recombinant En-1 was used together with a major exo-type cellobiohydrolase I of I. lacteus MC-2.     

Molecular cloning of the gene encoding β-1,3(4)-glucanase A from a marine bacterium, Pseudomonas sp. PE2, an essential enzyme for the degradation of Pythium porphyrae cell walls

Abstrfsact The β-1,3(4)-glucanase A (GluA)-encoding gene named gluA was cloned from the genomic library of a marine bacterium Pseudomonas sp. PE2 by expression in Escherichia coli, and the complete DNA sequence was determined. The recombinant enzyme from Pseudomonas sp. PE2 was examined to determine the essential enzymes for degrading Pythium porphyrae cell walls, comparatively using other two recombinant enzymes, chitinase A and β-1,3-glucanase B from the same bacterial strain. GluA most degraded the cell walls among these three enzymes, suggesting that GluA seems to be most important to P. porphyrae cell-wall-degrading activity. The deduced GluA is a modular enzyme composed of an N-terminal signal peptide, the tandem-duplicated carbohydrate-binding module family 6 (CBM_GluA-1 and CBM_GluA-2), and a glycoside hydrolase family 16 catalytic domain. Deletion analysis clearly indicated that GluA lacking CBM_GluA-1 and CBM_GluA-2 does not bind to Avicel and xylan. These results suggest that the tandem-repeated CBM of GluA may play a key role in the binding of Avicel and xylan as well as β-1,3- and β-1,3;1,4-glucans and is very important to bind to insoluble polysaccharides.     

Successful PCR-based reverse genetic screens using an En-1-mutagenised Arabidopsis thaliana population generated via single-seed descent

 The development of an Arabidopsis population via single-seed descent is described which includes 3,000 lines that carry approximately 15,000 independent insertions of the autonomous maize element En-1. A PCR strategy is outlined which allows the recovery of En-1-insertion mutants among this population in any random gene sequence of Arabidopsis thaliana. The method employs PCR reactions on pooled DNA. Positive amplification using a target-specific primer and an En-1-specific primer on row, column and single-tray pools identifies the putative insertion mutant. In a control experiment two insertion mutants of the PIN gene were successfully identified. In addition, a new independent insertion in the PIN gene was detected which was transmitted to the next generation and showed co-segregation with the pin phenotype. These examples demonstrate that the inheritance of inserts of the autonomous element En-1 is stable enough to make a proper genetic analysis feasible in a genomic background with multiple En-1 inserts. Received: 20 March 1998 / Accepted: 31 March 1998     

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus<Emphasis Type="Italic"> Cercospora nicotianae</Emphasis>

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.Communicated by E. Cerdá-Olmedo     

Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw

The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg²⁺ and Ca²⁺ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by CelA. Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998     

Improvement of the enzymatic activity of the hyperthermophilic cellulase from <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>

A hyperthermophilic β-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).     

Expression and characterization of full-length Ampullaria crossean endoglucanase EG65s and their two functional modules

Three endoglucanase cDNAs, eg65a, eg65b, and eg65c, were cloned from the mollusk Ampullaria crossean in previous work. To characterize the full-length enzymes as well as their individual functional modules via heterologous expression analysis, the three full-length putative endoglucanases (rEG65a, rEG65b, and rEG65c) and the corresponding catalytic modules (EG65a-CM, EG65b-CM, and EG65c-CM) were expressed in Pichia pastoris GS115, and the three corresponding carbohydrate-binding modules (EG65a-CBM, EG65b-CBM, and EG65c-CBM) were expressed in Escherichia coli BL21 (DE3). The properties of recombinant rEG65b, EG65a-CM, EG65b-CM, and EG65c-CM were characterized. Binding assays of CBMs with insoluble polysaccharides indicated that both EG65b-CBM and EG65c-CBM bound to phosphoric-acid swollen cellulose (PASC), Avicel, and oat-spelt xylan, while EG65a-CBM did not. The relative equilibrium constants (K(r)) of EG65b-CBM and EG65c-CBM were determined by absorption isotherm measurements. In this study, the CBMs of animal cellulases were expressed and characterized for the first time.     

Cloning and Sequence Analysis of Endoglucanase Genes from an Industrial Fungus,Aspergillus kawachii

Three endoglucanase genes (cel5A, cel5B, and cel61A) were cloned from an industrial fungus, Aspergillus kawachii. Yeasts transformed with these cDNAs showed endoglucanase activity in medium. Cel5A and Cel61A contained a type 1 cellulose-binding domain (CBD1) at the C-terminus of the enzyme. The putative catalytic regions of Cel5A and Cel5B showed homology with various endoglucanases belonging glycosyl hydrolase family 5 (GH5). Cel5B showed high homology with Cel5A in catalytic region, but it lacked CBD1 and linker. The cel5A contained four introns, whereas cel5B contained five introns. The putative catalytic region of Cel61A showed homology with enzymes belonging to GH61. The cel61A contained no introns.     

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.     

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the V_max without significant K _m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.     

Domain engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> exoglucanases

To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg ^–1 respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.     

新型甘露聚糖酶-乙酰酯酶双功能酶的功能和结构域协同研究

[目的] 分析鉴定高效木质纤维素降解菌群EMSD5来源的新型甘露聚糖酶-乙酰酯酶双功能酶44884,解析催化域间的协同关系,以及碳水化合物结合模块（CBM）对催化域特性的影响,拓展对该类双功能酶的认识,为甘露聚糖酶的升级改造和应用提供依据。[方法] 通过大肠杆菌异源表达甘露聚糖酶-乙酰酯酶双功能酶44884,并构建截短和定点突变的突变体,利用TLC和DNS法比较野生型和突变体的酶学性质。[结果] 成功对44884全长和突变蛋白进行克隆表达,并发现44884中2个催化域能够彼此促进各自产物的释放,而且以双功能酶形式存在时,这种促进效果更为明显。44884中的2个CBM65均有甘露聚糖和结晶纤维素结合活性,且CBM65的存在并不改变甘露聚糖酶和乙酰酯酶的最适反应条件和水解模式。虽然CBM65显著降低了2个催化域的热稳定性,但水解天然底物时,2个CBM65对各自临近催化域的水解具有明显的促进效果。[结论] 本研究首次发现并探究了新型甘露聚糖酶和乙酰酯酶形成的双功能酶44884的功能,解析了催化域之间高效的协同效应,以及新型甘露聚糖结合模块CBM65对双功能酶水解的促进作用。     

Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining

The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known β-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca²⁺ and showed its maximum at 53 °C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect. Received: 25 February 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Analysis of functional domains of endoglucanases from Clostridium cellulovorans by gene cloning, nucleotide sequencing and chimeric protein construction.

Summary The nucleotide sequence of engD, an endo--1,4-glucanase gene from Clostridium cellulovorans was determined (Genbank Accession No. M37434). The COON-terminal part of the gene product, EngD, contained a Thr-Thr-Pro repeated sequence followed by a region that has homology to the exoglucanase of Cellulomonas fimi. EngD and EngB, another C. cellulovorans endoglucanase, show 75% amino acid sequence homology at their NH₂-termini, in contrast to their carboxyterminal domains which show no homology. EngD had endoglucanase activity on carboxymethylcellulose (CMC), cellobiosidase activity on p-nitrophenyl-cellobioside (p-NPC), and partial hydrolytic activity on crystalline cellulose (Avicel), while EngB showed hydrolytic activity against only CMC. Chimeric proteins between EngB and EngD were constructed by exchanging the non-homologous COOH-terminal regions. Chimeric proteins that contained the NH₂-terminus of EngD retained cellobiosidase activity but chimeras with the EngB NH₂-terminus showed no cellobiosidase activity. Hydrolysis of crystalline cellulose (Avicelase activity) was observed only with the enzyme containing the EngD NH₂-terminus and EngD COOH-terminus.     

Identification and functional analysis of a gene encoding β-glucosidase from the brown-rot basidiomycete <Emphasis Type="Italic">Fomitopsis palustris</Emphasis>

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and β-glucosidases. A novel β-glucosidase designated as Cel3A was identified from F. palustris grown at the expense of Avicel. The deduced amino acid sequence of Cel3A showed high homology with those of other fungal β-glucosidases that belong to glycosyl hydrolase (GH) family 3. The sequence analysis also indicated that Cel3A contains the N- and C-terminal domains of GH family 3 and Asp-209 was conserved as a catalytic nucleophile. The cloned gene was successfully expressed in the yeast Pichia pastoris and the recombinant protein exhibited β-glucosidase activity with cellobiose and some degree of thermostability. Considering the size and sequence of the protein, the β-glucosidase identified in this study is different from the protein purified directly from F. palustris in the previous study. Our results suggest that the fungus possesses at least two β-glucosidase genes.     

Family 19 Chitinases from Streptomyces thermoviolaceus OPC-520: Molecular Cloning and Characterization

Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70°C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.