NORMAL GENOMIC

• Chen YC, Chen WT, Liu JC, Tsai LC, Cheng HL
• A highly active beta-glucanase from a new strain of rumen fungus Orpinomyces sp.Y102 exhibits cellobiohydrolase and cellotriohydrolase activities.
• Bioresour Technol. 2014; 170: 513-21
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• A new strain of rumen fungus was isolated from Bos taurus, identified and designated Orpinomyces sp.Y102. A clone, celC7, isolated from the cDNA library of Orpinomyces sp.Y102, was predicted to encode a protein containing a signal peptide (Residues 1-17), an N-terminal dockerin-containing domain, and a C-terminal cellobiohydrolase catalytic domain of glycoside hydrolase family 6. CelC7 was insoluble when expressed in Escherichia coli. Deletion of 17 or 105 residues from the N-terminus significantly improved its solubility. The resulting enzymes, CelC7(-17) and CelC7(-105), were highly active to beta-glucan substrates and were stable between pH 5.0 and 11.0. CelC7(-105) worked as an exocellulase releasing cellobiose and cellotriose from acid-swollen Avicel and cellooligosaccharides, and displayed a Vmax of 6321.64mumole/min/mg and a Km of 2.18mg/ml to barley beta-glucan. Further, the crude extract of CelC7(-105) facilitated ethanol fermentation from cellulose. Thus, CelC7(-105) is a good candidate for industrial applications such as biofuel production.

• Wang TY et al.
• Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses.
• Biotechnol Biofuels. 2011; 4: 24-24
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• BACKGROUND: Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus. RESULTS: We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to N. patriciarum to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative GH contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: beta-glucosidase, endo-1,4-beta-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks. CONCLUSIONS: These GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One beta-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.

• Peer A, Smith SP, Bayer EA, Lamed R, Borovok I
• Noncellulosomal cohesin- and dockerin-like modules in the three domains of life.
• FEMS Microbiol Lett. 2009; 291: 1-16
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• The high-affinity cohesin-dockerin interaction was originally discovered as modular components, which mediate the assembly of the various subunits of the multienzyme cellulosome complex that characterizes some cellulolytic bacteria. Until recently, the presence of cohesins and dockerins within a bacterial proteome was considered a definitive signature of a cellulosome-producing bacterium. Widespread genome sequencing has since revealed a wealth of putative cohesin- and dockerin-containing proteins in Bacteria, Archaea, and in primitive eukaryotes. The newly identified modules appear to serve diverse functions that are clearly distinct from the classical cellulosome archetype, and the vast majority of parent proteins are not predicted glycoside hydrolases. In most cases, only a few such genes have been identified in a given microorganism, which encode proteins containing but a single cohesin and/or dockerin. In some cases, one or the other module appears to be missing from a given species, and in other cases both modules occur within the same protein. This review provides a bioinformatics-based survey of the current status of cohesin- and dockerin-like sequences in species from the Bacteria, Archaea, and Eukarya. Surprisingly, many identified modules and their parent proteins are clearly unrelated to cellulosomes. The cellulosome paradigm may thus be the exception rather than the rule for bacterial, archaeal, and eukaryotic employment of cohesin and dockerin modules.

• Steenbakkers PJ et al.
• A serpin in the cellulosome of the anaerobic fungus Piromyces sp. strain E2.
• Mycol Res. 2008; 112: 999-1006
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• A gene encoding a novel component of the cellulolytic complex (cellulosome) of the anaerobic fungus Piromyces sp. strain E2 was identified. The encoded 538 amino acid protein, named celpin, consists of a signal peptide, a positively charged domain of unknown function followed by two fungal dockerins, typical for components of the extracellular fungal cellulosome. The C-terminal end consists of a 380 amino acid serine proteinase inhibitor (or serpin) domain homologue, sharing 30% identity and 50% similarity to vertebrate and bacterial serpins. Detailed protein sequence analysis of the serpin domain revealed that it contained all features of a functional serpin. It possesses the conserved amino acids present in more than 70% of known serpins, and it contained the consensus of inhibiting serpins. Because of the confined space of the fungal cellulosome inside plant tissue and the auto-proteolysis of plant material in the rumen, the fungal serpin is presumably involved in protection of the cellulosome against plant proteinases. The celpin protein of Piromyces sp. strain E2 is the first non-structural, non-hydrolytic fungal cellulosome component. Furthermore, the celpin protein of Piromyces sp. strain E2 is the first representative of a serine proteinase inhibitor of the fungal kingdom.

• Chen H, Li XL, Xu H, Ljungdahl LG, Cerniglia CE
• High level expression and characterization of the cyclophilin B gene from the anaerobic fungus Orpinomyces sp. strain PC-2.
• Protein Pept Lett. 2006; 13: 727-32
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• Cyclophilins are an evolutionarily conserved family of peptidyl-prolyl cis-trans isomerases (PPIases). A cyclophilin B (cypB) gene from the anaerobic fungus Orpinomyces sp. strain PC-2 was cloned and overexpressed in Escherichia coli. It was expressed as an amino-terminal 6 x His-tagged recombinant protein to facilitate purification. Highly purified protein (26.5 kDa) was isolated by two chromatographic steps involving affinity and gel filtration for biochemical studies of the enzyme. The recombinant CypB displayed PPIase activity with a k(cat)/K(m) of 8.9 x 10(6) M(-1) s(-1) at 10 degrees C and pH 7.8. It was inhibited by cyclosporin A (CsA) with an IC(50) of 23.5 nM, similar to those of the native protein and other cyclophilin B enzymes from animals. Genomic DNA analysis of cypB revealed that it was present as a single copy in Orpinomyces PC-2 and contained two introns, indicating it has a eukaryotic origin. It is one of the most heavily interrupted genes with intron sequences found in anaerobic fungi. The three-dimensional model of Orpinomyces PC-2 CypB was predicted with a homology modeling approach using the Swiss-Model Protein Modeling Server and three dimensional structure of human CypB as a template. The overall architecture of the CypB molecule is very similar to that of human CypB.

• Harhangi HR et al.
• Cel6A, a major exoglucanase from the cellulosome of the anaerobic fungi Piromyces sp. E2 and Piromyces equi.
• Biochim Biophys Acta. 2003; 1628: 30-9
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• Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.

• McCabe BK, Kuek C, Gordon GL, Phillips MW
• Production of beta-glucosidase using immobilised Piromyces sp. KSX1 and Orpinomyces sp. 478P1 in repeat-batch culture.
• J Ind Microbiol Biotechnol. 2003; 30: 205-9
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• Two anaerobic fungi, one a monocentric strain ( Piromyces sp. KSX1) and the other a polycentric strain ( Orpinomyces sp. 478P1), were immobilised in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. beta-Glucosidase production with KSX1 was maintained for 45 days over six repeated batch cultures yielding a maximum level of 107 mIU/ml. For 478P1, beta-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/ml. Although repeat-batch cultures of KSX1 produced more beta-glucosidase than strain 478P1, the maximum specific beta-glucosidase produced from these immobilised cultures was similar. The immobilised polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any growth in the culture liquor.

• Doi RH, Kosugi A, Murashima K, Tamaru Y, Han SO
• Cellulosomes from mesophilic bacteria.
• J Bacteriol. 2003; 185: 5907-14

• Steenbakkers PJ et al.
• The major component of the cellulosomes of anaerobic fungi from the genus Piromyces is a family 48 glycoside hydrolase.
• DNA Seq. 2002; 13: 313-20
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• Sequencing of two cDNAs from the anaerobic fungi Piromyces equi and Piromyces sp. strain E2 revealed that they both encode a glycoside hydrolase (GH) family 48 cellulase, containing two C-terminal fungal dockerin domains. N-terminal sequencing of the major component of the Piromyces multi-enzyme cellulase/hemicellulase complex, termed the cellulosome, showed that these 80 kDa proteins corresponded to the GH family 48 enzyme. These data show for the first time that GH family 48 cellulases are not confined to bacteria, and that bacterial and fungal cellulosomes share the same pivotal component.

• Steenbakkers PJ et al.
• An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2.
• Biochem J. 2002; 365: 193-204
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• The cellulosome produced by Piromyces sp. strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water. Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90 kDa. Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested. Incubation with 0.5% (w/v) SDS or 8 M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170 kDa components. To investigate the major 90 kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing. Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi. The 2800 bp cDNA clone contained an open reading frame of 2334 bp encoding a 757-residue extracellular protein. Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family. Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase. The catalytic domain is succeeded by a putative beta-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains. Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges. The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.

• Schaeffer F, Matuschek M, Guglielmi G, Miras I, Alzari PM, Beguin P
• Duplicated dockerin subdomains of Clostridium thermocellum endoglucanase CelD bind to a cohesin domain of the scaffolding protein CipA with distinct thermodynamic parameters and a negative cooperativity.
• Biochemistry. 2002; 41: 2106-14
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• Mutagenized dockerin domains of endoglucanase CelD (type I) and of the cellulosome-integrating protein CipA (type II) were constructed by swapping residues 10 and 11 of the first or the second duplicated segment between the two polypeptides. These residues have been proposed to determine the specificity of cohesin-dockerin interactions. The dockerin domain of CelD still bound to the seventh cohesin domain of CipA (CohCip7), provided that mutagenesis occurred in one segment only. Binding was no longer detected by nondenaturing gel electrophoresis when both segments were mutagenized. The dockerin domain of CipA bound to the cohesin domain of SdbA as long as the second segment was intact. None of the mutated dockerins displayed detectable binding to the noncognate cohesin domain. Isothermal titration calorimetry showed that binding of the CelD dockerin to CohCip7 occurred with a high affinity [K(a) = (2.6 +/- 0.5) x 10(9) M(-1)] and a 1:1 stoichiometry. The reaction was weakly exothermic (DeltaHdegrees = -2.22 +/- 0.2 kcal x mol(-1)) and largely entropy driven (TDeltaSdegrees = 10.70 +/- 0.5 kcal x mol(-1)). The heat capacity change on complexation was negative (DeltaC(p) = -305 +/- 15 cal x mol(-1) x K(-1)). These values show that cohesin-dockerin binding is mainly hydrophobic. Mutations in the first or the second dockerin segment reduced or enhanced, respectively, the hydrophobic character of the interaction. Due to partial enthalpy-entropy compensation, these mutations induced only small changes in binding affinity. However, the binding affinity was strongly decreased when both segments were mutated, indicating strong negative cooperativity between the two mutated sites.

• Fierobe HP et al.
• Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes.
• J Biol Chem. 2001; 276: 21257-61
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• Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.

• Freelove AC, Bolam DN, White P, Hazlewood GP, Gilbert HJ
• A novel carbohydrate-binding protein is a component of the plant cell wall-degrading complex of Piromyces equi.
• J Biol Chem. 2001; 276: 43010-7
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• The recycling of photosynthetically fixed carbon by the action of microbial plant cell wall hydrolases is a fundamental biological process that is integral to one of the major geochemical cycles and, in addition, has considerable industrial potential. Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Anaerobic fungi that colonize herbivores are the most efficient plant cell wall degraders known, and this activity is vested in a high molecular weight complex that binds tightly to the plant cell wall. To investigate whether plant cell wall attachment is mediated by noncatalytic proteins, a cDNA library of the anaerobic fungus Piromyces equi was screened for sequences that encode noncatalytic proteins that are components of the cellulase-hemicellulase complex. A 1.6-kilobase cDNA was isolated encoding a protein of 479 amino acids with a M(r) of 52548 designated NCP1. The mature protein had a modular architecture comprising three copies of the noncatalytic dockerin module that targets anaerobic fungal proteins to the cellulase-hemicellulase complex. The two C-terminal modules of NCP1, CBM29-1 and CBM29-2, respectively, exhibit 33% sequence identity with each other but have no homologues in protein data bases. A truncated form of NCP1 comprising CBM29-1 and CBM29-2 (CBM29-1-2) and each of the two individual copies of CBM29 bind primarily to mannan, cellulose, and glucomannan, displaying the highest affinity for the latter polysaccharide. CBM29-1-2 exhibits 4-45-fold higher affinity than either CBM29-1 or CBM29-2 for the various ligands, indicating that the two modules, when covalently linked, act in synergy to bind to an array of different polysaccharides. This paper provides the first report of a CBM-containing protein from an anaerobic fungal cellulase-hemicellulase complex. The two CBMs constitute a novel CBM family designated CBM29 whose members exhibit unusually wide ligand specificity. We propose, therefore, that NCP1 plays a role in sequestering the fungal enzyme complex onto the plant cell wall.

• Novo C, Simoes F, Mendonca D, Matos J, Clemente A
• Primary structure deduction and molecular modelling from a cDNA of a cellobiohydrolase-like protein from the white-rot fungus Coriolus versicolor.
• Int J Biol Macromol. 2001; 28: 285-92
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• Molecular cloning and cDNA sequencing analysis were used to elucidate the primary structure of a cellulase-like structure from the white-rot fungus Coriolus versicolor. The cDNA of interest was isolated from a cDNA library obtained from C. versicolor mycelia grown on cellulase inducer medium. A pattern search showed that this cellulase belongs to the glycosyl hydrolases family 6. From the deduced amino acid sequence, models of the binding and catalytic domains were built by homology modelling. The constructed models present a typical cellulose-binding domain at the N-terminal region, a rich Pro, Ser, Thr linker peptide, and a catalytic domain at the C-terminus region.

• Ponpium P, Ratanakhanokchai K, Kyu KL
• Isolation and properties of a cellulosome-type multienzyme complex of the thermophilic Bacteroides sp. strain P-1.
• Enzyme Microb Technol. 2000; 26: 459-465
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• 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.

• Shoham Y, Lamed R, Bayer EA
• The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides.
• Trends Microbiol. 1999; 7: 275-81
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• The cellulosome is an extracellular supramolecular machine that can efficiently degrade crystalline cellulosic substrates and associated plant cell wall polysaccharides. The cellulosome arrangement can also promote adhesion to the insoluble substrate, thus providing individual microbial cells with a direct competitive advantage in the utilization of the soluble hydrolysis products.

• Lytle B, Wu JH
• Involvement of both dockerin subdomains in assembly of the Clostridium thermocellum cellulosome.
• J Bacteriol. 1998; 180: 6581-5
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• Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.

• Chen H, Li XL, Blum DL, Ljungdahl LG
• Two genes of the anaerobic fungus Orpinomyces sp. strain PC-2 encoding cellulases with endoglucanase activities may have arisen by gene duplication.
• FEMS Microbiol Lett. 1998; 159: 63-8
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• A cDNA designated celE cloned from Orpinomyces PC-2 consisted of an open reading frame encoding a polypeptide (CelE) of 477 amino acids. CelE was highly homologous to CelBs of Orpinomyces (72.3% identity) and neocallimastix (67.9% identity) and like them it had a non-catalytic repeated peptide domain (NCRPD) at the C-terminal end. The catalytic domain of CelE was homologous to glycosyl hydrolases of Family 5, found in several anaerobic bacteria. The gene of celE was devoid of introns. The recombinant proteins CelE and CelB of Orpinomyces PC-2 randomly hydrolyzed carboxymethylcellulose and cello-oligosaccharides in the pattern of endoglucanases. The results indicated that a gene of bacterial origin was duplicated to form celE and celB of Orpinomyces PC-2.

• Birch PR
• Targeted differential display of abundantly expressed sequences from the basidiomycete Phanerochaete chrysosporium which contain regions coding for fungal cellulose-binding domains.
• Curr Genet. 1998; 33: 70-6
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• Cellulose-binding domains (CBDs) are present in the majority of fungal cellulases studied to-date. This work describes the use of targeted differential display, employing degenerate primers designed to anneal to variants of a region conserved in fungal CBDs, each in combination with an oligo-dT primer, to PCR-amplify cDNA sequences containing regions coding for such domains from Phanerochaete chrysosporium. After growth on either Avicel or carboxymethyl cellulose (CMC), five distinct, abundantly expressed cDNA sequences were obtained. Two of these originated from transcripts of the previously characterised cbhI.1 and cbhI.2 genes, whereas three were from novel genes. One of the latter was isolated only after growth on CMC. No such sequences were obtained after growth on xylan, suggesting that the expression of sequences containing such regions is down-regulated on this substrate. The use of targeted differential display both for isolating novel sequences and for studying the expression of known genes within a family is discussed.

• Fanutti C, Ponyi T, Black GW, Hazlewood GP, Gilbert HJ
• The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain.
• J Biol Chem. 1995; 270: 29314-22
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• Two cDNAs, designated xynA and manA, encoding xylanase A (XYLA) and mannanase A (MANA), respectively, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Piromyces. XYLA and MANA displayed properties typical of endo-beta 1,4-xylanases and mannanases, respectively. Neither enzyme hydrolyzed cellulosic substrates. The nucleotide sequences of xynA and manA revealed open reading frames of 1875 and 1818 base pairs, respectively, coding for proteins of M(r) 68,049 (XYLA) and 68,055 (MANA). The deduced primary structure of MANA revealed a 458-amino acid sequence that exhibited identity with Bacillus and Pseudomonas fluorescens subsp. cellulosa mannanases belonging to glycosyl hydrolase Family 26. A 40-residue reiterated sequence, which was homologous to duplicated noncatalytic domains previously observed in Neocallimastix patriciarum xylanase A and endoglucanase B, was located at the C terminus of MANA. XYLA contained two regions that exhibited sequence identity with the catalytic domains of glycosyl hydrolase Family 11 xylanases and were separated by a duplicated 40-residue sequence that exhibited strong homology to the C terminus of MANA. Analysis of truncated derivatives of MANA confirmed that the N-terminal 458-residue sequence constituted the catalytic domain, while the C-terminal domain was not essential for the retention of catalytic activity. Similar deletion analysis of XYLA showed that the C-terminal catalytic domain homologue exhibited catalytic activity, but the corresponding putative N-terminal catalytic domain did not function as a xylanase. Fusion of the reiterated noncatalytic 40-residue sequence conserved in XYLA and MANA to glutathione S-transferase, generated a hybrid protein that did not associate with cellulose, but bound to 97- and 116-kDa polypeptides that are components of the multienzyme cellulase-hemicellulase complexes of Piromyces and Neocallimastix patriciarum, respectively. The role of this domain in the assembly of the enzyme complex is discussed.

• Han SJ, Yoo YJ, Kang HS
• Characterization of a bifunctional cellulase and its structural gene. The cell gene of Bacillus sp. D04 has exo- and endoglucanase activity.
• J Biol Chem. 1995; 270: 26012-9
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• Bacillus sp. D04 secreted a bifunctional cellulase that had a molecular weight of 35,000. This cellulase degraded Cm-cellulose, cellotetraose, cellopentaose, p-nitrophenyl-beta-D-cellobioside, and avicel PH101. Based on the high performance liquid chromatography analysis of the degradation products, this cellulase randomly cleaved internal beta-1, 4-glycosidic bonds in cellotetraose and cellopentaose as an endoglucanase. It also hydrolyzed the aglycosidic bond in p-nitrophenyl-beta-D-cellobioside and cleaved avicel to cellobiose as an exoglucanase. Cellobiose competitively inhibited the p-nitrophenyl-beta-D-cellobioside degrading activity but not Cm-cellulose degrading activity. Ten mM p-chloromercuribenzoate inhibited p-nitrophenyl-beta-D-cellobioside degrading activity completely, but Cm-cellulose degrading activity incompletely. Cm-cellulose increased p-nitrophenyl-beta-D-cellobioside degrading activity, and vice versa, whereas methylumbelliferyl-beta-D-cellobiose strongly inhibited p-nitrophenyl-beta-D-cellobioside degrading activity. The cellulase gene (cel gene), 1461 base pairs, of Bacillus sp. D04 was cloned. The nucleotide sequence of the cel gene was highly homologous to those of Bacillus subtilis DLG and B. subtilis BSE616. The cel gene was overexpressed in Escherichia coli, and its product was purified. The substrate specificity and substrate competition pattern of the purified recombinant cellulase were the same as those of the purified cellulase from Bacillus sp. D04. These results suggest that a single polypeptide cellulase had both endo- and exoglucanase activities and each activity exists in a separate site.

• Ali BR et al.
• Cellulases and hemicellulases of the anaerobic fungus Piromyces constitute a multiprotein cellulose-binding complex and are encoded by multigene families.
• FEMS Microbiol Lett. 1995; 125: 15-21
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• More than 80% of the extracellular Avicelase, endoglucanase, xylanase and mannanase activities of the anaerobic fungus Piromyces were associated with a cellulose-binding complex. The complex was composed of at least 10 polypeptides ranging in size from 190 kDa to 50 kDa, and contained numerous endoglucanases, xylanases and mannanases. Multiple genes encoding each of these activities were isolated from an expressing cDNA library.

• Herscovics A, Schneikert J, Athanassiadis A, Moremen KW
• Isolation of a mouse Golgi mannosidase cDNA, a member of a gene family conserved from yeast to mammals.
• J Biol Chem. 1994; 269: 9864-71
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• The amino acid sequence of the specific alpha-mannosidase involved in N-oligosaccharide processing in Saccharomyces cerevisiae was found to have a high degree of similarity to the deduced amino acid sequence of a rabbit liver alpha-mannosidase partial cDNA, demonstrating that processing mannosidases have been conserved through eukaryotic evolution. Regions of sequence identity were chosen to design degenerate oligonucleotide primers that can be used to prepare probes using the polymerase chain reaction (PCR) for cloning processing mannosidases from other eukaryotes. Using these primers for PCR with mouse liver cDNA as template, two related but distinct PCR products were obtained. The amino acid sequences of PCR1 and PCR2 were 88 and 65% identical with the corresponding sequence of the rabbit enzyme, respectively. Southern blot analysis of mouse genomic DNA using PCR1 and PCR2 as probes revealed that they are derived from two different genes, indicating the existence of a mammalian mannosidase gene family with at least two members. Using PCR2 as a probe, a novel mouse cDNA was isolated from a 3T3 cDNA library. It contains an open reading frame which encodes a type II membrane protein of 73 kDa with a cytoplasmic region of about 35 amino acids, a Ca2+ binding consensus sequence, and a single N-glycosylation site. Northern blot analysis of mouse tissues and L cells revealed tissue-specific expression of multiple transcripts, ranging in size from 4.2 to 8.5 kilobases, that suggests a complex pattern of gene regulation. Transient expression of the influenza hemagglutinin epitope-tagged cDNA in COS cells followed by indirect immunofluorescence with monoclonal antibody 12CA5 showed that the cloned mannosidase is primarily localized in a juxtanuclear position corresponding to the Golgi. The C-terminal domain lacking the putative transmembrane region was shown to have alpha-mannosidase activity when expressed in COS cells as a secreted Protein A fusion product.

• Gibbs MD, Saul DJ, Luthi E, Bergquist PL
• The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme.
• Appl Environ Microbiol. 1992; 58: 3864-7
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• The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues. The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase. Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.

• Camirand A, Heysen A, Grondin B, Herscovics A
• Glycoprotein biosynthesis in Saccharomyces cerevisiae. Isolation and characterization of the gene encoding a specific processing alpha-mannosidase.
• J Biol Chem. 1991; 266: 15120-7
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• We have isolated the gene from Saccharomyces cerevisiae encoding an alpha-mannosidase of unique specificity which catalyzes the removal of one mannose residue from Man9GlcNAc to produce a single isomer of Man8GlcNAc (Jelinek-Kelly, S., and Herscovics, A. (1988) J. Biol. Chem. 263, 14757-14763). Amino acid sequence information was obtained and corresponding degenerate oligonucleotide primers were synthesized for polymerase chain reactions on yeast genomic DNA. The labeled polymerase chain reaction products were used to screen a S. cerevisiae genomic library in YEp24, and positive clones of different lengths with similar restriction maps were isolated. A 4.6-kilobase fragment which hybridized with the probes was sequenced. It contained a 1650-base pair open reading frame encoding peptide sequences corresponding to the amino acid sequences of the purified alpha-mannosidase. The gene, designated MNS1, encodes a 549-amino acid polypeptide of calculated molecular size 63,017 Da produced by an mRNA species of approximately 1.7 kilobases. The protein possesses a putative noncleavable signal sequence near its N-terminal region which probably acts as a transmembrane domain. It has three potential N-glycosylation sites and a calcium-binding consensus sequence. Its amino acid sequence is homologous to the recently isolated cDNA from rabbit liver alpha-1,2 mannosidase which can transform Man9GlcNAc to Man5GlcNAc (Moremen, K. W., Schutzbach, J. S., Forsee, W. T., Neame, P., Bishoff, J., Lodish, H. F., and Robbins, P. W. (1990) Glycoconjugate J. 7, 401). Overexpression of the MNS1 gene caused an 8-10-fold increase in specific alpha-mannosidase activity. Disruption of the MNS1 gene resulted in undetectable specific alpha-mannosidase activity but no apparent effect on growth. These results demonstrate that MNS1 is the structural gene for the specific alpha-mannosidase and that its activity is not essential for viability.