Amino Acid Regions of Family 45 Endoglucanases Involved in Cotton Defibrillation and in Resistance to Anionic Surfactants and Oxidizing Agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.     

Exploring amino acids responsible for the temperature profile of glycoside hydrolase family 45 endoglucanase EGL3 from Humicola grisea

EGL3 and RCE1 are glycoside hydrolase family 45 endoglucanases isolated from Humicola grisea and Rhizopus oryzae respectively. The amino acid sequences of the two endoglucanases are homologous; on the other hand, the optimum temperature of EGL3 is higher than that of RCE1. In this study, four chimeric endoglucanases, named ER1, ER2, ER3 and ER4, in which one of four sequential amino acid regions of the EGL3 catalytic domain (CAD) was replaced by the corresponding RCE1 amino acids, were constructed to explore the region responsible for the EGL3 temperature profile. Then their temperature profiles were compared with that of the recombinant EGL3. Replacement of the N-terminal region of EGL3 with that of RCE1 caused the EGL3 temperature profile to shift to a lower temperature. These results suggest that the N-terminal amino acids of the EGL3 are responsible for the EGL3 temperature profile.     

Cloning of the <Emphasis Type="Italic">Penicillium canescens</Emphasis> endo-1,4-β-glucanase gene <Emphasis Type="Italic">egl3</Emphasis> and the characterization of the recombinant enzyme

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-β-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for β-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54°C, respectively. The K _m and V _m values for CMC hydrolysis were determined; they amounted to 17.1 g/l and 0.31 μM/(mg s), respectively.     

Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae

The DNA (glaB) and a cDNA-encoding glucoamylase produced in solid-state culture of Aspergillus oryzae were cloned using oligodeoxyribonucleotide probes derived from internal amino acid sequences of the enzyme. Comparison of the nucleotide sequences of a genomic DNA fragment with its cDNA showed the glaB gene carried three exons interrupted by two introns and had an open reading frame encoding 493 aa residues. The 5′-flanking region had a TATA box at nt −87 from the start codon and two putative CAAT sequences at nt −276 and −288. The glaB gene shared 57% homology at the aa level with the glaA gene which was cloned previously from A. oryzae. Interestingly, the glucoamylase encoded by the glaB gene had no C-terminal domain such as that proposed to have starch binding activity in Aspergillus glucoamylases. Introduction of cDNA of the glaB gene to Saccharomyces cerevisiae caused the secretion of active glucoamylase to culture medium and introduction of the glaB gene to A. oryzae increased glucoamylase productivity in solid-state culture. Northern blot analysis showed the glaB gene was expressed in solid-state culture, but not in submerged culture.     

Isolation and characterization of the gene encoding a manganese peroxidase from <Emphasis Type="Italic">Lentinula edodes</Emphasis>

A genomic DNA sequence and cDNA encoding a putative manganese peroxidase were isolated from the white-rot basidiomycete Lentinula edodes. The gene, called lemnp1, consists of a 1985-bp open reading frame interrupted by 16 introns and was flanked by an upstream region having putative CAAT, TATA, and heat shock elements and by a downstream region having polyadenylation signals. The lemnp1 gene encodes a protein of 364 amino acids that shows high sequence homology to manganese peroxidases of other basidiomycetes. The deduced N-terminal amino acid sequence is different from the L. edodes manganese peroxidase reported previously.     

Properties of exgS,a gene for a major subunit of the Clostridium cellulovorans cellulosome

The nucleotide sequence of P70, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been determined. The gene designated as exgS (Genbank Accession No. U34793) consists of 2112 bp and encodes a protein containing 703 amino acids with a molecular mass of 77.7 kDa. ExgS has a putative signal peptide sequence of 32 amino acids. The N-terminal region is separated from the C-terminal region by a short Pro–Thr–Pro linker. The C-terminal region of ExgS contains a duplicated sequence (DS), each sequence consisting of 22 amino acids. exgS, located 67 bp downstream of cbpA in the chromosome, is immediately upstream of a gene encoding a family 9 type endoglucanase that we have designated as EngH. This gene cluster to date consists of regA–cbpA–exgS–engH. Recombinant ExgS (rExgS) containing no signal peptide was expressed in E. coli. The rExgS actively digested several forms of cellulose, including Avicel, Sigmacell101, crystalline cellulose, and xylan, but not carboxymethyl cellulose (CMC). Cellotetraose was the smallest oligosaccharide substrate for rExgS. The enzymatic studies indicated that ExgS was an exoglucanase and had some properties similar to that of CelS from C. thermocellum and CelF from C. cellulolyticum. An exoglucanase has now been found to be a component of the C. cellulovorans cellulosome as well as the previously reported endoglucanases.     

Drosophila melanogaster P1 genomic clone DS05563 contains the chaperonin-encoding gene Cctg

We report the sequence analysis of a Drosophila melanogaster (Dm) P1 genomic clone (DS05563) which contains the γ-chaperonin-encoding gene, Cctg. The (Hs) Cctg orthologue was found to share strong sequence identity with a 1603-bp region of DS05563, suggesting that Dm Cctg is located within this region. Detailed analysis has shown that Dm Cctg comprises four exons and is interrupted by three introns of 55, 85 and 66 bp. Dm Cctg encodes a predicted peptide of 545 amino acids (aa) (approx. 60 kDa). The predicted Dm CCTγ aa sequence shares a high degree of sequence identity with γ-orthologues from human (70%), mouse (70%), protozoa (60%) and yeast (60%), and also contains domains found in other chaperonins including bacterial GroEL, mitochondrial Hsp60 and plant Rubisco large subunit-binding protein. These data support the conclusion that the DS05563 clone contains the Dm Cctg gene.     

The promoter-proximal region of the Bacillus licheniformis penicillinase gene: Nucleotide sequence and predicted leader peptide sequence

Penicillinase (β-lactamase) is a major species of secreted protein produced by Bacillus licheniformis 749. From the pTB2 recombinant plasmid containing the cloned entire penicillinase (penP) gene, we have isolated and sequenced a 446-bp HpaII fragment carrying the beginning of penP. The 3′-end coding region of 216-bp on this DNA fragment codes for the first 72 amino acids of the prepenicillinase protein. The deduced structure of the leader peptide consists of a 34 amino acid signal sequence with a hydrophilic N-terminal region and a central hydrophobic core.     

Molecular Characterization of Laccase Genes from the Basidiomycete Coprinus cinereus and Heterologous Expression of the Laccase Lcc1

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K_m of 21 ± 2 μM and a catalytic constant of 200 ± 10 min⁻¹ for O₂ with 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.     

Amino acid regions of family 45 endoglucanases involved in cotton defibrillation and in resistance to anionic surfactants and oxidizing agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.     

Isolation and molecular characterisation of the gene encoding eburicol 14α-demethylase (CYP51) fromPenicillium italicum

TheCYP51 gene encoding eburicol 14α-demethylase (P450_14DM) was cloned from a genomic library of the filamentous fungal plant pathogenPenicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14α-demethylase from the yeastCandida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORF) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a characteristic haem-binding domain, HR2, common to all P450 genes. The deducedP. italicum P450_14DM protein and the P450_14DM proteins fromCandida albicans, C. tropicalis andSaccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of theCYP51 family. Multiple copies of a genomic DNA fragment ofP. italicum containing the cloned P450 gene were introduced intoAspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450_14DM activity, indicating that the cloned gene encodes a functional eburicol 14α-demethylase.     

Pigment and Virulence Deficiencies Associated with Mutations in the aroE Gene of Xanthomonas oryzae pv. oryzae

Xanthomonadins are yellow, membrane-bound pigments produced by members of the genus Xanthomonas. We identified an ethyl methanesulfonate-induced Xanthomonas oryzae pv. oryzae mutant (BXO65) that is deficient for xanthomonadin production and virulence on rice, as well as auxotrophic for aromatic amino acids (Pig⁻ Vir⁻ Aro⁻). Reversion analysis indicated that these multiple phenotypes are due to a single mutation. A genomic library of the wild-type strain was used to isolate a 7.0-kb clone that complements BXO65. By transposon mutagenesis, marker exchange, sequence analysis, and subcloning, the complementing activity was localized to a 849-bp open reading frame (ORF). This ORF is homologous to the aroE gene, which encodes shikimate dehydrogenase in various bacterial species. Shikimate dehydrogenase activity was present in the wild-type strain and the mutant with the complementing clone, whereas no activity was found in BXO65. This clone also complemented an Escherichia coli aroE mutant for prototrophy, indicating that aroE is functionally conserved in X. oryzae pv. oryzae and E. coli. The nucleotide sequence of the 2.9-kb region containing aroE revealed that a putative DNA helicase gene is located adjacent to aroE. Our results indicate that aroE is required for normal levels of virulence and xanthomonadin production in X. oryzae pv. oryzae.     

Pyret, a Ty3/Gypsy retrotransposon in Magnaporthe grisea contains an extra domain between the nucleocapsid and protease domains

A novel Ty3/Gypsy retrotransposon, named Pyret, was identified in the plant pathogenic fungus Magnaporthe grisea (anamorph Pyricularia oryzae). Pyret-related elements were distributed in a wide range of Pyricularia isolates from various gramineous plants. The Pyret element is 7250 bp in length with a 475 bp LTR and one conceptual ORF. The ORF contains seven nonsense mutations in the reading frame, indicating that the Pyret clone is lightly degenerate. Comparative domain analysis among retroelements revealed that Pyret exhibits an extra domain (WCCH domain) beyond the basic components of LTR retrotransposons. The WCCH domain consists of ~300 amino acids and is located downstream of the nucleocapsid domain. The WCCH domain is so named because it contains two repeats of a characteristic amino acid sequence, W-X₂-C-X₄-C-X₂-H-X₃-K. A WCCH motif-like sequence is found in the precoat protein of some geminiviruses, viral RNA-dependent RNA polymerase and also in an Arabidopsis protein of unknown function. Interestingly, detailed sequence analysis of the gag protein revealed that Pyret, as well as some other chromodomain-containing LTR retrotransposons, displays significant sequence homology with members of the gammaretroviruses (MLV-related retroviruses) in the capsid and nucleocapsid domains. This suggests that chromodomain-containing LTR retrotransposons and gammaretroviruses may share a common ancestor with the gag protein.     

Cloning and Characterization of the O-Methyltransferase I Gene (dmtA) from Aspergillus parasiticus Associated with the Conversions of Demethylsterigmatocystin to Sterigmatocystin and Dihydrodemethylsterigmatocystin to Dihydrosterigmatocystin in Aflatoxin Biosynthesis

O-Methyltransferase I catalyzes both the conversion of demethylsterigmatocystin to sterigmatocystin and the conversion of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis. In this study, both genomic cloning and cDNA cloning of the gene encoding O-methyltransferase I were accomplished by using PCR strategies, such as conventional PCR based on the N-terminal amino acid sequence of the purified enzyme, 5′ and 3′ rapid amplification of cDNA ends PCR, and thermal asymmetric interlaced PCR (TAIL-PCR), and genes were sequenced by using Aspergillus parasiticus NIAH-26. A comparison of the genomic sequences with the cDNA of the dmtA region revealed that the coding region is interrupted by three short introns. The cDNA of the dmtA gene is 1,373 bp long and encodes a 386-amino-acid protein with a deduced molecular weight of 43,023, which is consistent with the molecular weight of the protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal half of the deduced protein exhibits 76.3% identity with the coding region of the Aspergillus nidulans StcP protein, whereas the N-terminal half of dmtA exhibits 73.0% identity with the 5′ flanking region of the stcP gene, suggesting that translation of the stcP gene may start at a site upstream from methionine that is different from the site that has been suggested previously. Also, an examination of the 5′ and 3′ flanking regions of the dmtA gene in which TAIL-PCR was used demonstrated that the dmtA gene is located in the aflatoxin biosynthesis cluster between (and in the same orientation as) the omtA and ord-2 genes. Northern blotting revealed that expression of the dmtA gene is influenced by both medium composition and culture temperature and that the pattern correlates with the patterns observed for other genes in the aflatoxin gene cluster. Furthermore, Southern blotting and PCR analyses of the dmtA gene showed that a dmtA homolog is present in Aspergillus oryzae SYS-2.     

Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea

We report the cloning and characterisation of Pot2, a putative transposable element from Magnaporthe grisea. The element is 1857 by in size, has 43-bp perfect terminal inverted repeats (TIRs) and 16-bp direct repeats within the TIRs. A large open reading frame, potentially coding for a transposase-like protein, was identified. This putative protein coding region showed extensive identity to that of Fott, a transposable element from another phytopathogenic fungus, Fusarium oxysporum. Pot2, like the transposable elements Tc1 and Mariner of Caenorhabditis elegans and Drosophila, respectively, duplicates the dinucleotide TA at the target insertion site. Sequence analysis of DNA flanking 12 Pot2 elements revealed similarity to the consensus insertion sequence of Tct. Pot2 is present at a copy number of approximately 100 per haploid genome and represents one of the major repetitive DNAs shared by both rice and non-rice pathogens of M. grisea.     

Cloning and Structural Analysis of bglM Gene Coding for the Fungal Cell Wall-lytic β-1,3-Glucan-hydrolase BglM of Bacillus circulans IAM1165

Bacillus circulans IAM1165 produces isoforms of β-1,3-glucan-hydrolases. Of these enzymes, the 42-kDa enzyme BglM degrades Aspergillus oryzae cell walls the most actively. A gene coding for a BglM precursor consisting of 411 amino acid residues was cloned. The 27 N-terminal amino acid sequence of the precursor is a signal peptide. The 141 C-terminal amino acid sequence showed a motif of carbohydrate-binding module family 13. This domain bound to pachyman, lichenan, and A. oryzae cell walls. The central domain showed a bacterial β-1,3-glucan-hydrolase motif belonging to glycosyl hydrolase family 16. By removal of the C-terminal domain in the IAM1165 culture, mature BglM was processed to several 27-kDa fragments that hydrolyze a soluble β-1,3-glucan.     

Induction of a Metallothionein-like Protein in Tetrahymena pyriformis by Metal Ions

Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-β-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.