Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C. It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.     

运用定点突变提高重组木聚糖酶在毕赤氏酵母中的表达

运用PCR介导的定点突变对米曲霉(Aspergillus　oryzae)来源的木聚糖酶在毕赤酵母中的重组表达进行了研究,获得一表达量远远高于亲本的突变株I156A,对其进行了提纯并研究其酶学特性,除热稳定性外其余与亲本基本一致。突变株I156A所产木聚糖酶XynFl的分子量为35kD,在pH 4～9范围内稳定,最适pH为70,最适温度为45℃,在50℃以下稳定性略高于亲本。     

米曲霉耐热木聚糖酶纯化及酶学特性研究

对米曲霉原始发酵液中耐热木聚糖酶进行纯化和酶学特性研究,利用甘蔗渣为碳源培养米曲霉,通过超滤和阴离子交换柱两步纯化得到木聚糖酶XynH1,分子量35．402kDa,利用飞行时间质谱和SDS—PAGE分析,推断XynH1为XylanaseXynF1,分子量为35．402kDa。XynH1属于糖苷水解酶家族10,酶活为442．2IU／nag,最适pH和温度分别为pH6．0和65℃,80℃以下及pH4．0～10．5范围内较稳定。     

Molecular cloning, overexpression, and purification of a major xylanase from Aspergillus oryzae

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58 degrees C. It had a Km of 5.1 mg/ml and a Vmax of 123 micromol/min/mg when birch wood xylan was used as a substrate.     

Molecular cloning and overexpression of fleA gene encoding a fucose-specific lectin of Aspergillus oryzae

A protein from the cell lysate of Aspergillus oryzae was purified by column chromatography immobilized with a ferrichrysin (Fcy), which is one of the siderophores of A. oryzae. It is produced only in an iron-deficient culture and its molecular weight is estimated as 35,000 by SDS-PAGE. Two internal amino acid sequences of the protein obtained by lysylendopeptidase digestion were analyzed. Molecular cloning shows that it encodes 310 putative amino acid residues separated by 4 introns and is designated as fleA. It shows approximately 26% similarity with the gene encoding a fucose-specific lectin of Aleuria aurantia (AAL). The gene was overexpressed under control of the melO promoter in a submerged culture of A. oryzae. The fleA gene product showed hemagglutination activity against rabbit erythrocytes. A hemagglutination inhibition assay of monosaccharides showed that this lectin specifically binds to L-fucose and weakly reacts with mannose and N-acetyl-neuraminic acid.     

Sequence analysis and overexpression of a pectin lyase gene (pel1) from Aspergillus oryzae KBN616

A gene (pel1) encoding pectin lyase (Pel1) was isolated from a shoyu koji mold, Aspergillus oryzae KBN616, and characterized. The structural gene comprised 1,196 bp with a single intron. The ORF encoded 381 amino acids with a signal peptide of 20 amino acids. The deduced amino acid sequence showed high similarity to those of Aspergillus niger pectin lyases and Glomerella cingulata PnlA. The pel1 gene was successfully overexpressed under the promoter of the A. oryzae TEF1 gene. The molecular mass of the recombinant pectin lyase substantially coincided with that calculated based on nucleotide sequence.     

Characterization of a neutral ceramidase orthologue from Aspergillus oryzae

Ceramide is an important molecule not only structurally but also regulationally as a modulator of various cellular events. Ceramidase (CDase) are classified into three different types (acid, alkaline, and neutral CDases). Neutral CDase could play an important role in the regulation of ceramide levels in the extracellular space. In this study, we describe the characterization of a neutral CDase orthologue from the filamentous fungus Aspergillus oryzae . The gene encoding the neutral CDase orthologue was cloned and overexpressed in A . oryzae . The purified recombinant enzyme was optimally active at pH 4.0–4.5 and 40 °C. The apparent K _m and V _max values of the enzyme for C12-NBD-ceramide were 3.32 μM and 0.085 μmol min⁻¹ mg⁻¹, respectively.     

Molecular cloning of a gene encoding endo-beta-D-1,4-glucanase PCE1 from Phycomyces nitens

We previously cloned three endoglucanase genes, rce1, rce2, and rce3, from Rhizopus oryzae as the first cellulase genes from the subdivision Zygomycota. In this study, an endoglucanase gene, designated a pce1 gene, was cloned by plaque hybridization with the codon usage-optimized rce1 gene as a probe from Phycomyces nitens, a member of the subdivision Zygomycota. The pec1 gene had an open reading frame of 1,038 nucleotides encoding an endoglucanase (PCE1) of 346 amino acid residues. The amino acid sequence deduced from the pce1 gene consisted of a cellulose-binding domain (CBD) at the N terminus and of a catalytic domain belonging to family 45 glycoside hydrolase at the C terminus. PCE1 was purified to apparent homogeneity from the culture supernatant of P. nitens and the molecular mass was found to be 45 kDa. The optimum pH for the CMCase activity of PCE1 was 6.0, and the optimum temperature was 50 degrees C, the lowest among the family 45 endoglucanases.     

Pectin methylesterase gene (pmeA) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene product

A gene (pmeA) encoding pectin methylesterase was isolated from a shoyu koji mold, Aspergillus oryzae KBN616, and characterized. The structural gene comprised 1,370 bp with six introns. The PMEA protein consisted of 331 amino acids with a putative signal peptide of 17 amino acids. The deduced amino acid sequence was very similar to those of Aspergillus niger PMEA and Aspergillus aculeatus PME1. The pmeA gene was efficiently expressed under control of the A. oryzae TEF1 gene promoter for purification and characterization of the ezymatic properties. PMEA had a molecular mass of 38.5 kDa, a pH optimum of 5.0, and a temperature optimum of 55 degrees C.     

Sequence Analysis, Overexpression, and Antisense Inhibition of a β-Xylosidase Gene, xylA, from Aspergillus oryzae KBN616

β-Xylosidase secreted by the shoyu koji mold, Aspergillus oryzae, is the key enzyme responsible for browning of soy sauce. To investigate the role of β-xylosidase in the brown color formation, a major β-xylosidase, XylA, and its encoding gene were characterized. β-Xylosidase XylA was purified to homogeneity from culture filtrates of A. oryzae KBN616. The optimum pH and temperature of the enzyme were found to be 4.0 and 60°C, respectively, and the molecular mass was estimated to be 110 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The xylA gene comprises 2,397 bp with no introns and encodes a protein consisting of 798 amino acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced amino acid sequence shows high similarity to Aspergillus nidulans XlnD (70%), Aspergillus niger XlnD (64%), and Trichoderma reesei BxII (63%). The xylA gene was overexpressed under control of the strong and constitutive A. oryzae TEF1 promoter. One of the A. oryzae transformants produced approximately 13 times more of the enzyme than did the host strain. The partial-length antisense xylA gene expressed under control of the A. oryzae TEF1 promoter decreased the β-xylosidase level in A. oryzae to about 20% of that of the host strain.     

嗜热木糖异构酶在大肠杆菌中的表达、纯化及性质研究

为获得具有高热稳定性的木糖异构酶,运用基因工程技术,从嗜热栖热菌Thermus thermophilus HB8中克隆到嗜热木糖异构酶基因xylA。测序结果表明,该基因与GenBank数据库中相比271位的碱基A突变为G,导致氨基酸序列中N91D突变。将该基因克隆到载体pET22b(+),并在E. coli BL21(DE3)中进行高效表达。通过热变性和强阴离子交换两步对该酶进行纯化,并对酶学性质进行了研究。结果表明,该酶最适温度为80 °C,最适pH为8.0,80 °C下半衰期为225 min。在60 °C,pH 7.5该酶的Km为15.20 mmol·L-1,Vmax为69.54 μmol·min-1,kcat为50.62 s-1,kcat/Km为3.33 L·s-1·mmol -1。研究结果为嗜热木糖异构酶的进一步工业应用奠定了基础。     

Cloning and enhanced expression of the cytochrome P450nor gene (nicA; CYP55A5) encoding nitric oxide reductase from Aspergillus oryzae

We cloned and characterized the gene and cDNA of Aspergillus oryzae cytochrome P450nor (Anor). The Anor gene (nicA; CYP55A5) has a different gene structure from other P450nor genes in that it has an extra intron. There were not only two kinds of mRNA but also two sets of TATA-box and CCAAT-box, and it appears that this gene has two expression patterns, like CYP55A1 of Fusarium oxysporum. A reporter analysis using the uidA gene indicated that gene expression of CYP55A5 was induced under anaerobic conditions, like CYP55A1. When the CYP55A5 gene was overexpressed in A. oryzae, a large amount of active Anor were accumulated as intracellular protein. Anor employed both NADH and NADPH as electron donors for reducing nitric oxide to nitrous oxide. Anor measured the amount of NO generated from 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOC5) with a spectrophotometer. The sensitivity was 10 nmol/ml.     

Characterization of methylglyoxal synthase from Clostridium acetobutylicum ATCC 824 and its use in the formation of 1, 2-propanediol.

A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The Km and Vmax values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min(-1) microgram(-1), respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.     

淀粉结合域与α-淀粉酶的融合表达及其酶学性质

利用RT-PCR从Rhizopus oryzaeGX-08总RNA中克隆到糖化酶的淀粉结合域(SBD)基因(sbd),将该基因片段插入α-淀粉酶(CN7A)基因cn7a的5′端构建融合表达质粒pSE-sbdcn7a。嵌合酶SBD-CN7A在Escherichia coliJM109表达,并经Ni-NTA、Sephacryl S300纯化。酶学性质研究表明:嵌合酶在最适作用条件方面与原始酶并无明显差别;在以生玉米粉为底物时,其比酶活提高了8.7倍,而以可溶性淀粉为底物时其比酶活是原始酶的1.8倍,Km也从3.784 g/L降低为2.234 g/L;嵌合酶在65℃下的半衰期从10 min缩短为4 min。结果表明,淀粉结合域SBD的融合赋予了α-淀粉酶CN7A水解生淀粉的能力。     

Biological activities of purified harpin(Xoo) and harpin(Xoo) detection in transgenic plants using its polyclonal antibody

Many harpins have been found in plant pathogen bacteria that can elicit disease and insect resistance in plants, and promote plant growth. In this work, we overexpressed and purified Xanthomonas oryzae pv. oryzae harpin, harpinxoo, in Escherichia coli BL21/pGEX-hpa1. Harpinxoo was fused to the Cterminus of glutathione S-transferase （GST） and purified using the Bulk GST purification module and thrombin cleavage capture kit. Purified harpinxoo protein was sensitive to protease K and stable to heat treatment, and could not induce a hypersensitive response after treatment with various plant metabolic inhibitors; these characteristics were similar to harpinEa of Erwinia amylovora. The purified harpinxoo showed a similar ability to induce tobacco mosaic virus resistance in tobacco as harpinEa. Its antibody worked well in detecting the purified harpinxoo, harpinxoo in the total protein of E. coli BL21/pGEX-hpa1 and an hpal transgenic rice.     

Cloning, expression, and crystallization of a hyperthermophilic protein that is homologous to the eukaryotic translation initiation factor, eIF5A.

A gene coding for a protein homologous to a translation initiation factor of eukaryotes, eIF5A, was cloned from Methanococcus jannaschii, a hyperthermophile with an optimum growth temperature of 85 degrees C. The protein was overexpressed, purified and crystallized. The crystals were obtained by vapor diffusion method with 8% PEG 4000 as precipitant and belong to space group P4(1)22 with unit cell dimensions a = b = 45.52 A and c = 155.59 A. These crystals diffract to at least 2.2 A resolution.     

Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli

Plant chitinases are pathogenesis-related proteins, which are believed to be involved in plant defense responses to pathogen infection. In this study, chitinase gene from barley was cloned and overexpressed in Escherichia coli. Chitinase (35 kDa) was isolated and purified. Since the protein was produced as insoluble inclusion bodies, the protein was solubilized and refolded. Purified chitinase exerted broad-spectrum antifungal activity against Botrytis cinerea (blight of tobacco), Pestalotia theae (leaf spot of tea), Bipolaris oryzae (brown spot of rice), Alternaria sp. (grain discoloration of rice), Curvularia lunata (leaf spot of clover) and Rhizoctonia solani (sheath blight of rice). Due to the potential of broad-spectrum antifungal activity barley chitinase gene can be used to enhance fungal-resistance in crop plants such as rice, tobacco, tea and clover.     

Molecular cloning,purification and characterization of two endo-1,4-β-glucanases from Aspergillus oryzae KBN616

Two endo-1,4-β-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and temperature optimum of 45 °C. Received: 3 July 1996 / Accepted: 15 July 1996     

Development of an efficient production method for beta-mannosidase by the creation of an overexpression system in Aspergillus aculeatus

AIM: To develop an overexpression system in Aspergillus aculeatus in order to establish an efficient overproduction method of beta-mannosidase (MANB). METHODS AND RESULTS: An overexpression plasmid for the manB gene, encoding A. aculeatus MANB, was constructed and introduced into A. aculeatus cells. The gene was overexpressed under an improved promoter containing 12 copies of Region III cis-elements of Aspergillus oryzae in the transformant, and it secreted 2.56 mg MANB ml(-1) in liquid culture, which obtained a 9.4-fold higher productivity than that achieved in an overexpression system in A. oryzae. Most of the secreted protein in the cultured medium of the transformed A. aculeatus was the overproduced enzyme. CONCLUSIONS: Aspergillus aculeatus with the introduced overexpression plasmid produced 2.56 mg MANB ml(-1) in cultured medium. The improved promoter with A. oryzae Region III functioned in A. aculeatus; thus the strain is an expectant host for recombinant protein productions. SIGNIFICANCE AND IMPACT OF THE STUDY: The overexpression system with the improved promoter in A. aculeatus brought the highest productivity of MANB reported to date. The expression system would be a strong bioindustrial tool for protein production.