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.     

Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii

The glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii (AkCel61) is a modular enzyme that consists of a catalytic domain and a carbohydrate-binding module belonging to family 1 (CBM1) that are connected by a Ser-Thr linker region longer than 100 amino acids. We expressed the recombinant AkCel61, wild-type enzyme (rAkCel61), and a truncated enzyme consisting of the catalytic domain (rAkCel61ΔCBM) in Pichia pastoris and analyzed their biochemical properties. Purified rAkCel61 and rAkCel61ΔCBM migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were demonstrated to have apparent molecular masses of 81,000 and 34,000 Da, respectively. After treatment with endoglycosidase H, both proteins showed an increase in mobility, thus, demonstrating estimated molecular masses of 78,000 and 28,000 Da, respectively. Mass spectrometry analysis revealed that rAkCel61 and rAkCel61ΔCBM expressed in P. pastoris are heterogeneous due to protein glycosylation. The rAkCel61 protein bound to crystalline cellulose but not to arabinoxylan. The rAkCel61 and rAkCel61ΔCBM proteins produced small amounts of oligosaccharides from soluble carboxymethylcellulose. They also exhibited a slight hydrolytic activity toward laminarin. However, they showed no detectable activity toward microcrystalline cellulose, arabinoxylan, and pectin. Both recombinant enzymes also showed no detectable activity toward p-nitrophenyl β-d-glucoside, p-nitrophenyl β-d-cellobioside, and p-nitrophenyl β-d-cellotrioside. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of an alpha-L-rhamnosidase from Aspergillus kawachii and its gene

An α-l-rhamnosidase was purified by fractionating a culture filtrate of Aspergillus kawachii grown on l-rhamnose as the sole carbon source. The α-l-rhamnosidase had a molecular mass of 90 kDa and a high degree of N-glycosylation of approximately 22%. The enzyme exhibited optimal activity at pH 4.0 and temperature of 50 °C. Further, it was observed to be thermostable, and it retained more than 80% of its original activity following incubation at 60 °C for 1 h. Its T ₅₀ value was determined to be 72 °C. The enzyme was able to hydrolyze α-1,2- and α-1,6-glycosidic bonds. The specific activity of the enzyme was higher toward naringin than toward hesperidin. The A. kawachii α-l-rhamnosidase-encoding gene (Ak-rhaA) codes for a 655-amino-acid protein. Based on the amino acid sequence deduced from the cDNA, the protein possessed 13 potential N-glycosylation recognition sites and exhibited a high degree of sequence identity (up to 75%) with the α-l-rhamnosidases belonging to the glycoside hydrolase family 78 from Aspergillus aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and partial characterization of an acidic polygalacturonase from Aspergillus kawachii

An endo-polygalacturonase, named PGI, was purified to homogeneity from the culture filtrate of Aspergillus kawachii IFO 4033 grown in a glucose-tryptone medium. The molecular mass of PGI was estimated to be 60 kDa by SDS-PAGE and 40 kDa by gel filtration on Sephacryl S-100. The isoelectric point was 3.55 as determined by isoelectic focusing. PGI exhibited binding properties to ConA-Sepharose suggesting that the protein is glycosylated. The N-terminal amino acid sequence was also determined as S-T-C-T-F-T-D-A-A-T-A-S-E-S-K. The remarkable property of PGI was its high activity in the pH range 2.0-3.0 towards soluble and insoluble substrates, while being inactive at pH 5.0. Enzyme stability at low pHs was markedly enhanced by different compounds, such as proteins, polysaccharides, simple sugars and the substrate pectin. PGI was very efficient to extract pectin from lemmon protopectin and to macerate carrot tissues at pH 2.0. These properties make PGI an interesting biocatalyst for industrial applications under highly acidic conditions.     

Cloning of the xynNB gene encoding xylanase B from Aspergillus niger and its expression in Aspergillus kawachii

Aspergillus niger IFO 4066 produced two xylanases, xylanase A (XynNA) and xylanase B (XynNB), in culture medium, and these enzymes were purified. Acidophilic xylanase such as xylanase C (XynC) of white koji mold (Aspergillus kawachii IFO 4308) was not detected in A. niger cultures. However, results of Southern analysis using xynC cDNA of A. kawachii as a probe suggested that A. niger contained a gene homologous to xynC of A. kawachii. Therefore, we cloned this xylanase gene from A. niger. The predicted amino acid sequence of the cloned xylanase showed a homology to that of xynC of A. kawachii. However, a large number of amino acid substitutions were detected, especially in the N-terminal region. Both this cloned gene and xynC gene of A. kawachii had an intron at the same position in the coding region. The cloned gene was expressed in A. kawachii and a large quantity of xylanase was produced. The elution profile on an anion exchange chromatogram and the N-terminal amino acid sequence of the xylanase purified from the transformant were the same as those of XynNB. This confirmed that the cloned gene encoded XynNB.     

Cloning and Sequencing of the xynC Gene Encoding Acid Xylanase of Aspergillus kawachii

The molecular conformation of elsinan, consisting of (1 → 3)-α-linked maltotriose and α-maltotetraose units, was studied by X-ray diffraction coupled with conformational analysis. The quality of the X-ray fiber pattern obtained from elsinan was very poor, but the layer line spacing (45 Å), the probable presence of (005) reflection and a similar pattern with the powder pattern of a low molecular weight poly[(1 →3)-α-maltotriose] segment (DP about 35) suggested that the poly[(1 →3)-linked-α-maltotriose] segment (MTR part) of elsinan chain took a five-fold helical structure with an asymmetric unit of maltotriose. Conformational analysis for the five-fold helix of the MTR part pointed out that two left handed helices, - 5/1 and - 5/2, were energetically probable.     

分离纯化内切β—葡聚糖苷酶和部分N—末端序列分析

使用硫酸铵分级沉淀、DEAE－TOYOPEARL650M和POROS20PI弱阴离子交换色谱等分离技术,从黑曲霉（Aspergillus niger）发酵酶粉中分离提纯出一种新内切β－葡聚糖苷酶,在非还原条件下分子量为36kD,还原条件为38kD,该酶最适pH3．5,最适温度55℃,N－末端部分氨基酸序列为XXXFKCVGSMEDGAES。     

Cloning and sequencing of the xynA gene encoding xylanase A of Aspergillus kawachii.

We have cloned the xynA gene coding for xylanase A, a major component of the xylanase family, from Aspergillus kawachii. The cDNA was isolated from an A. kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein. Nucleotide sequence analysis of the cDNA showed a 981-bp open reading frame that encoded a protein of 327 amino acid residues. The signal peptide was composed of 25 amino acid residues and the N-terminus of the mature protein was pyroglutamic acid. The transformed yeast with a cloned cDNA produced xylanase. The genomic DNA was arranged as ten exons and nine introns.     

Aspergillus terreus as an industrial filamentous fungus for pharmaceutical biotechnology

1. Download : Download high-res image (112KB)
2. Download : Download full-size image

     

Cloning and Sequencing of the xynA Gene Encoding Xylanase A of Aspergillus kawachii

We have cloned the xynA gene coding for xylanase A, a major component of the xylanase family, from Aspergillus kawachii. The cDNA was isolated from an A. kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein. Nucleotide sequence analysis of the cDNA showed a 981-bp open reading frame that encoded a protein of 327 amino acid residues. The signal peptide was composed of 25 amino acid residues and the N-terminus of the mature protein was pyroglutamic acid. The transformed yeast with a cloned cDNA produced xylanase. The genomic DNA was arranged as ten exons and nine introns.     

Cloning and sequence analysis of a novel xylanase gene, Auxyn10A, from Aspergillus usamii

A full-length cDNA sequence, encoding a novel endo-1,4-β-d-xylanase (AuXyn10A) of Aspergillus usamii, was obtained by using rapid amplification of cDNA ends (RACE) methods and cloned into the pUCm-T vector, followed by DNA sequencing. The cDNA gene, designated as Auxyn10A, is 1,235 bp in length harboring 5′- and 3′-non-encoding regions, as well as an ORF of 984 bp that encodes a 19-aa signal peptide, a 6-aa propeptide and a 302-aa mature peptide with a calculated MW of 32,756 Da. The AuXyn10A displays high similarity to the xylanases of Aspergillus niger, Aspergillus kawachii and Aspergillus niger, members of the glycoside hydrolase family 10. Its three-dimensional structure was predicted using programs based on the crystal structure of Penicillium simplicissimum xylanase (1B30_A) from the family 10. The complete DNA gene was cloned from the genomic DNA of A. usamii using conventional PCR and hairpin structure-mediated PCR techniques. The DNA gene is 2,255 bp in length, containing a 510 bp of 5′-flanking promoter region and a 1,745 bp of downstream fragment that consists of ten exons and nine short introns ranging from 52 to 62 bp.     

Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris

Using 3′ and 5′ rapid amplification of cDNA ends methods, the full-length cDNA sequence encoding an endo-1,4-β-glucanase of Aspergillus usamii E001 (abbreviated as AuCel12A) was amplified from the total RNA. The clone cDNA sequence of the gene encoding the AuCel12A, named as Aucel12A, is 1,027 bp in length harboring 5′ and 3′ non-coding regions, as well as a 720 bp of open reading frame that encodes a 16-aa signal peptide, and a 223-aa mature AuCel12A with a theoretical M.W. of 24,294 Da, a calculated pI of 4.15, and one putative N-glycosylation site. The complete DNA sequence of the gene Aucel12A was amplified from the genomic DNA of A. usamii E001 by using the conventional PCR and pUCm-T vector-mediated PCR initially developed in our lab. The clone DNA sequence is 1,576 bp in length, consisting of a 5′ flanking regulatory region, three exons, and two introns with sizes of 50 and 66 bp. The cDNA fragment encoding the mature AuCel12A was expressed in a fully active form in Pichia pastoris. One P. pastoris transformant expressing the highest recombinant AuCel12A (rAuCel12A) activity, labeled as P. pastoris GSCel2-1, was chosen for subsequent studies. Integration of the Aucel12A into P. pastoris genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS-PAGE and enzyme activity assays demonstrated that the rAuCel12A, a glycosylated protein with an apparent M.W. of 27.0 kDa and a carbohydrate content of 4.82%, was secreted into the culture medium. The purified rAuCel12A displayed the highest activity at pH 5.0 and 60°C. It was highly stable at a pH range of 3.5–7.0, and at a temperature of 55°C or below. Its activity was not significantly affected by an array of metal ions and EDTA, but inhibited by Ag⁺, Hg²⁺ and Fe²⁺. The K _m and V _max of the rAuCel12A, towards carboxymethylcellulose-Na (CMC-Na) at pH 5.0 and 50°C were 4.85 mg/ml and 160.5 U/mg, respectively.     

Cloning of three endoglucanase genes from Thermomonospora curvata into Escherichia coli.

A BamHI genomic library from Thermomonospora curvata was constructed in E. coli using cosmid vector pHC79. Four clones able to hydrolyze CMC were isolated. Restriction digests and Southern gel analysis revealed the presence of three different endoglucanase genes. DNA fragments contained in all of the endoglucanase cosmids hybridized to T. curvata chromosomal DNA. The cellulase genes were expressed in E. coli, but at rather low levels.     

Cloning and DNA sequence analysis of a polygalacturonase cDNA from Aspergillus niger RH5344

A 1319 bp long cDNA encoding for a polygalacturonase (EC 3.2.1.15) from Aspergillus niger RH5344 comprises a single open reading frame of 1089 bp which includes the mature protein of 362 amino acids and an NH2-terminal signal peptide of 27 amino acids. The directly determined peptides of the mature polygalacturonase confirmed the sequence information deduced from the cDNA.     

Cloning and sequence analysis of an intron-containing domain from a peptide synthetase-encoding gene of the entomopathogenic fungus Metarhizium anisopliae

A gene encoding a putative peptide synthetase has been cloned and partially sequenced from the filamentous fungus, Metarhizium anisopliae. The deduced amino acid sequence of one entire domain and the following spacer is typical of fungal peptide synthetases, showing good conservation of the six expected core sequences. There are two introns within this region, the first interrupting core 5 (RLDLTDIE) of the domain and the second in a conserved area of the spacer region.     

Cloning of Clostridium thermocellum endoglucanase genes in Escherichia coli

Six independent and distinct cel genes coding endoglucanases have been selected from C. thermocellum pUC19-based gene bank in E. coli TG1. E. coli-derived Cel-proteins possessing Mr from 39,000 to 61,000 are able to cleave lichenan, as well as xylan and carboxymethyl cellulose. Cel 7- and Cel 8-endoglucanases are characterized by cellobiohydrolase type substrate specificity, being able to cleave model fluorogenic aryldisaccharide substrate MU-G2. The clone pCU110 (cel 7) produces about 10-fold more endoglucanase activity than other clones.     

Molecular cloning and sequence analysis of an inulinase gene from an Aspergillus sp

Selected endophytic fungi have been report to be inulin degraders to produce fructose or other oligosaccharides. In this study, the Aspergillus sp. producing inulinase were isolated from selected plant species at Serdang area in Malaysia. Fungal isolates were screened solely based on inulin degrading enzymes production and two isolates named Asf1 and Onf1 were selected as the best inulinase enzyme producers. Genomic DNA of these two isolates were extracted and amplified by polymerase chain reaction (PCR). A 1,341 bp DNA fragment containing inulinase gene was successfully amplified from Asf1 fungal isolate and was named as inu2 gene in this study. Based on the morphological characteristics, rDNA and neighbour-joining phylogenetic analysis, Asf1 fungal isolate could display closely-related to the genus of Aspergillus. The complete sequence designated Asf1 Inu2 gene was successfully obtained via rapid-amplification of cDNA ends-polymerase chain reaction (RACE-PCR). A 2.3 kb DNA fragment encoding endoinulinase, inu2, from Asf1 fungal isolate includes an open reading frame of 1,552 bp with calculated molecular weight of 55,954.1 Da and signal peptide sequence of 23 amino acids. The deduced amino acid sequence of the Asf1 inu2 displayed 97, 96, 69 and 22% identities to that of A. ficuum inu2, A. niger inuB, P. purpurogenum and K. marxianus, respectively. Phylogenetic analysis showed that fungal endo- and exo-inulinases have indepently evolved with the respective hydrolytic activities toward terminal and internal β-(2 → 1)-fructofuranosidic linkages in inulin.     

Cloning and DNA sequence analysis of the glucose oxidase gene from Aspergillus niger NRRL-3

A cDNA library from Aspergillus niger strain NRRL-3 enriched in sequences glucose oxidase was constructed. An 800 bp cDNA clone isolated from this library was used to screen 12,000 recombinant phages from an EMBL3 genomic library. A 15 kbp DNA segment isolated from this library contained the 1815 bp structural gene for glucose oxidase as well as a short 5'- and a longer 3'-noncoding region. The deduced protein sequence was verified by partial peptide sequencing.