Cloning of the cel8Y gene from Pectobacterium chrysanthemi PY35 and its comparison to cel genes of soft-rot Pectobacterium

The phytopathogenic Pectobacterium chrysanthemi (Pch) PY35 secretes multiple isozymes of plant cell wall degrading enzyme cellulases. We cloned a second cel gene that encodes cellulase in Pch PY35. The inserted 2 kb fragment was subcloned in order to geneate pPY710 (cel8Y). The structural organization of the cel8Y gene consists of an open reading frame (ORF) of 999 bp that encodes 332 amino acid residues with a signal peptide of 23 amino acids. The predicted amino acid sequence of Cel8Y was very similar to that of Cellulomonas uda, but completely different from that of the Cel5Z of Pch PY35. It belonged to the glycoside hydrolase family 8, based on amino acid sequence similarities in contrast to Cel5Z of Pch PY35, which was confirmed as family 5. Cel8Y was not closely related to the known cellulases of Pectobacterium. It had the conserved region of the glycoside hydrolase family 8, ASDGDVLIAWALLKAGNKW. The apparent molecular mass of the Cel8Y protein was calculated to be approximately 34 kDa by a carboxymethylcellulosesodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). The Cel8Y had a calculated pl of 6.49. It was optimally active at pH 7 with an approximate optimal temperature around 40 degrees C. The cellulase activity of Cel8Y was lower than that of Cel5Z.     

Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM)

The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C. The cel74 gene was previously found to be induced during T. maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.     

Identification of a genetic element (psr) which negatively controls expression of Enterococcus hirae penicillin-binding protein 5.

Enterococcus hirae ATCC 9790 produces a penicillin-binding protein (PBP5) of low penicillin affinity which under certain conditions can take over the functions of all the other PBPs. The 7.1-kb EcoRI fragment containing the pbp5 gene of this strain and of two mutants, of which one (E. hirae R40) overproduces PBP5 and the other (E. hirae Rev14) does not produce PBP5, was cloned in pUC18 and sequenced. In the 7.1-kb EcoRI fragment cloned from strain ATCC 9790, an open reading frame (psr) potentially encoding a 19-kDa protein was identified 1 kb upstream of the pbp5 gene. An 87-bp deletion in this element was found in the 7.1-kb EcoRI fragment cloned from strains R40 and Rev14. In addition, several base substitutions were found in the pbp5 genes of strains R40 and Rev14. One of these converted the 42nd codon, TCA, to the stop codon, TAA, in the pbp5 gene of Rev14. Escherichia coli strains were transformed with plasmids carrying the 7.1-kb EcoRI insert or a 2.6-kb HincII insert containing only the pbp5 gene of the three strains. Immunoblotting analysis of proteins expressed by these transformants showed that the 87-bp deletion in psr was associated with the PBP5 overproducer phenotype of strain R40 and the conversion of the TCA codon to the stop codon was associated with the PBP5 nonproducer phenotype of strain Rev14. None of the other nucleotide substitutions had any apparent effect on the level of PBP5 synthesized.     

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.     

A comparative analysis of two cDNA clones of the cellulase gene family from anaerobic fungus Piromyces rhizinflata

Cellulase family and some other glycosyl hydrolases of anaerobic fungi inhabiting the digestive tract of ruminants are believed to form an enzyme complex called cellulosome. Study of the individual component of cellulosome may shed light on understanding the organization of this complex and its functional mechanism. We have analysed the primary sequences of two cellulase clones, cel5B and cel6A, isolated from the cDNA library of ruminal fungus, Piromyces rhizinflata strain 2301. The deduced amino acid sequences of the catalytic domain of Cel5B, encoded by cel5B, showed homology with the subfamily 4 of the family 5 (subfamily 5(4)) of glycosyl hydrolases, while cel6A encoded Cel6A belonged to family 6 of glycosyl hydrolases. Phylogenetic tree analysis suggested that the genes of subfamily 5(4) glycosyl hydrolases of P. rhizinflata might have been acquired from rumen bacteria. Cel5B and Cel6A were modular enzymes consisting of a catalytic domain and dockerin domain(s), but not a cellulose binding domain. The occurrence of dockerin domains indicated that both enzymes were cellulosome components. The catalytic domain of the Cel5B (Cel5B') and Cel6A (Cel6A') recombinant proteins were purified. The optimal activity conditions with carboxymethyl cellulose (CMC) as the substrate were pH 6.0 and 50 degrees C for Cel5B', and pH 6.0 and 37-45 degrees C for Cel6A'. Both Cel5B' and Cel6A' exhibited activity against CMC, barley beta-glucan, Lichenan, and oat spelt xylan. Cel5B' could also hydrolyse p-nitrophenyl-beta-d-cellobioside, Avicel and filter paper while Cel6A' did not show any activity on these substrates. It is apparent that Cel6A' acted as an endoglucanase and Cel5B' possessed both endoglucanase and exoglucanase activities. No synergic effect was observed for these recombinant enzymes in vitro on Avicel and CMC.     

Loss of the gene for the alpha subunit of ATP synthase (ATP5A1) from the W chromosome in the African grey parrot (Psittacus erithacus)

This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceeded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study. Received: 6 September 2000 / Accepted: 7 March 2001     

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.     

A plant serpin gene. Structure, organization and expression of the gene encoding barley protein Z4

A 3133-bp nucleotide sequence of the gene Paz1 on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5' upstream and 523 bp 3' downstream of the coding region. The 1079-bp 5' upstream region of the gene shows little similarity to 5' regions of other sequences genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334-bp-long intron (bases 1497-1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43,128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25-30% and pronounced in the carboxy-terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane-bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36-56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants. The 'high-lysine' allees, lys1 (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4 gene. Also, 1554 bp of another 8-kbp fragment of the barley genome Paz psi, similar to the protein-Z4-coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene.     

Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake

Alkaline cellulase-producing actinomycete strains were isolated from mud samples collected from East African soda lakes. The strains were identified as novel Streptomyces spp. by 16S rDNA sequence analysis. A cellulase gene (cel12A) from Streptomyces sp. strain 11AG8 was cloned by expression screening of a genomic DNA library in Escherichia coli. From the nucleotide sequence of a 1.5-kb DNA fragment, an open reading frame of 1,113 nucleotides was identified encoding a protein of 371 amino acids. From computer analysis of the sequence, it was deduced that the Cel12A mature enzyme is a protein of 340 amino acids. The protein contained a catalytic domain, a glycine-rich linker region, and a cellulose-binding domain of 221, 12, and 107 amino acids, respectively. FASTA analysis of the catalytic domain of Cel12A classified the enzyme as a family 12 endoglucanase and the cellulose-binding domain as a family IIa CBD. Streptomyces rochei EglS was determined as nearest neighbor with a similarity of 75.2% and 61.0% to the catalytic domain and the cellulose-binding domain, respectively. The cell2A gene was subcloned in a Bacillus high-expression vector carrying the Bacillus amyloliquefaciens amylase regulatory sequences, and the construct was transformed to a Bacillus subtilis host strain. Crude enzyme preparations were obtained by ultrafiltration of cultures of the Bacillus subtilis recombinant strain containing the 11AG8 cell2A gene. The enzyme showed carboxymethylcellulase (CMCase) activities over a broad pH range (5-10) with an optimum activity at pH 8 and 50 degrees C. The enzyme retained more than 95% of its activity after incubation for 30 min under these conditions.     

High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions

The Thermobifida fusca cel6A gene encoding an endoglucanase was fused to three different downstream box (DB) regions to generate cel6A genes with 14 amino acid fusions. The DB-Cel6A fusions were inserted into the tobacco (Nicotiana tabacum cv. Samsun) chloroplast genome for protein expression. Accumulation of Cel6A protein in transformed tobacco leaves varied over approximately two orders of magnitude, dependent on the identity of the DB region fused to the cel6A open reading frame (ORF). Additionally, the DB region fused to the cel6A ORF affected the accumulation of Cel6A protein in aging leaves, with the most effective DB regions allowing for high level accumulation of Cel6A protein in young, mature, and old leaves, while Cel6A protein accumulation decreased with leaf age when less effective DB regions were fused to the cel6A ORF. In the most highly expressed DB-Cel6A construct, enzymatically active Cel6A protein accumulated at up to 10.7% of total soluble leaf protein (%TSP). The strategy used for high-level endoglucanase expression may be useful for expression of other cellulolytic enzymes in chloroplasts, ultimately leading to cost-effective heterologous enzyme production for cellulosic ethanol using transplastomic plants.     

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.     

Cloning of the Ruminococcus albus cel5D and cel9A genes encoding dockerin module-containing endoglucanases and expression of cel5D in Escherichia coli

An EcoRI chromosomal DNA fragment of Ruminococcus albus F-40 that conferred endoglucanase activity on Escherichia coli was cloned. An open reading frame (ORF1) and another incomplete reading frame (ORF2) were found in the EcoRI fragment. The ORF2 was completed using inverse PCR genome walking technique. ORF1 and ORF2, which confront each other, encoded cellulases belonging to families 5 and 9 of the glycoside hydrolases and were designated cel5D and cel9A respectively. The cel5D gene encodes 753 amino acids with a deduced molecular weight of 83,409. Cel5D consists of a signal peptide of 24 amino acids, a family-5 catalytic module, a dockerin module, and two family-4 carbohydrate-binding modules (CBMs). The cel9A gene encodes 936 amino acids with a deduced molecular weight of 104,174, consisting of a signal peptide, a family-9 catalytic module, a family-3 CBM, and a dockerin module. The catalytic module polypeptide (rCel5DCat) derived from Cel5D was constructed, expressed, and purified from a recombinant E. coli. The truncated enzyme hydrolyzed cellohexaose, cellopentaose, and cellotetraose to yield mainly cellotriose and cellobiose with glucose as a minor product, but the enzyme was less active toward cellotriose and not active toward cellobiose, suggesting that this enzyme is a typical endoglucanase. rCel5DCat had a Km of 3.9 mg/ml and a Vmax of 37.2 micromol/min/mg for carboxymethycellulose.     

Cloning and characterization of thermostable endoglucanase (Cel8Y) from the hyperthermophilic Aquifex aeolicus VF5

Aquifex aeolicus is the hyperthermophilic bacterium known, with growth-temperature maxima near 95 degrees C. The cel8Y gene, encoding a thermostable endoglucanase (Cel8Y) from Aquifex aeolicus VF5, was cloned into a vector for expression and expressed in Escherichia coli XL1-Blue. A clone of 1.7 kb fragment containing endoglucanase activity, designated pKYCY100, was sequenced and found to contain an ORF of 978 bp encoding a protein of 325 amino acid residues, with a calculated molecular mass of 38,831 Da. This endoglucanase was designated cel8Y gene. The endoglucanase has an 18-amino-acid signal peptide but not cellulose-binding domain. The endoglucanase of A. aeolicus VF5 had significant amino acid sequence similarities with endoglucanases from glycosyl hydrolase family 8. The predicted amino acid sequence of the Cel8Y protein was similar to that of CMCase of Cellulomonas uda, BcsC of Escherichia coli, CelY of Erwinia chrysanthemi, and CMCase of Acetobacter xylinum. The molecular mass of Cel8Y was calculated to be 36,750 Da, which is consistent with the value obtained from result of CMC-SDS-PAGE of the purified enzyme. Cel8Y was thermostable, exhibiting maximal activity at 80 degrees C and pH optima of 7.0 and with half-lives of 2 h at 100 degrees C, 4 h at 90 degrees C.