Purification and characterization of an l-amino acid oxidase from Pseudomonas sp. AIU 813

An l-amino acid oxidase was found from a newly isolated strain, Pseudomonas sp. AIU 813. This enzyme was remarkably induced by incubation with l-lysine as a nitrogen source, and efficiently purified using an affinity chromatography with l-lysine as ligand. The enzyme oxidized l-lysine, l-ornithine and l-arginine, but not other l-amino acids and d-amino acids. The oxidase activity for l-lysine was detected in a wide pH range, and its optimal was pH 7.0. In contrast, the oxidase activity for l-ornithine and l-arginine was not shown in acidic region from pH 6.5, and optimal pH for both substrates was 9.0. The enzyme was a flavoprotein and composed of two identical subunits with molecular mass of 54.5?kDa. The N-terminal amino acid sequence was similar to that of putative flavin-containing amine oxidase and putative tryptophan 2-monooxygenase, but not to that of l-amino acid oxidases.     

Cloning and sequencing of an exoglucanase gene from Streptomyces sp. M 23, and its expression in Streptomyces lividans TK-24

A gene encoding exoglucanase (CBHII) of Streptomyces sp. M 23 was cloned and sequenced. The cbhII gene consisted of 1359 bp capable of encoding a polypeptide of 453 amino acids with a calculated molecular mass of 45,175 Da. The deduced amino acid sequence showed homology with those of cellulases belonging to family 6 of the glycosyl hydrolases. The cbhII gene was subcloned into the plasmid pSEV1 and expressed in Streptomyces lividans TK-24. The transformed cells were able to secrete the enzyme efficiently in an active form. The CBHII expressed by S. lividans TK-24 was purified to homogeneity by SDS-polyacrylamide gel and characterized. The recombinant CBHII was stable up to 50 degrees C and more than 30% of the original activity remained after heating at 100 degrees C for 10 min. The amino-terminal amino acid sequence of the recombinant CBHII was identified as GPAAPTARVD. These results agreed well with the properties of the authentic CBHII.     

Cloning and sequencing of a gene encoding of aldehyde oxidase in Pseudomonas sp. AIU 362

We have cloned a gene encoding an aldehyde oxidase (ALOD) oxidized glyoxal but not glyoxylic acid from Pseudomonas sp. AIU 362. The ALOD gene contained an open reading frame consisting of 888 nucleotides corresponding to 295 amino acid residues. The deduced amino acid sequence exhibited a high similarity to those of 3-hydroxyisobutyrate dehydrogenases (3-HIBDHs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant ALOD expressed in E.?coli BL21 was 20,000-fold higher than that of ALOD in Pseudomonas sp. AIU 362. The recombinant ALOD exhibited ALOD activity and 3-HIBDH activity. The 3-HIBDH from Pseudomonas putida KT2440 also exhibited ALOD activity. Thus, the ALOD from Pseudomonas sp. AIU 362 and 3-HIBDH from P.?putida KT2440 were classified into the same enzyme group.     

Cloning, sequencing and expression of an alpha-L-arabinofuranosidase from Aspergillus sojae

The arabinofuranosidase gene was cloned from the cDNA of Aspergillus sojae. It was found to contain an open reading frame composed of 984 base pairs (bp) and to encode 328 amino acid residues (aa). The cDNA sequence suggested that the mature enzyme is preceded by a 26-aa signal sequence and the molecular mass was predicted to be 32,749 Da. The A. sojae arabinofuranosidase consists of a single catalytic domain; it does not have a specific substrate-binding domain such as the xylan-binding domain reported in an arabinofuranosidase from Streptomyces lividans (Vincent, P. et al.: Biochem. J., 322, 845-852, 1997). The deduced amino acid sequence of the catalytic domain of the mature enzyme exhibits extensive identity with the catalytic domains of Streptomyces coelicolor (74%), Aspergillus niger (75%), S. lividans (74%), and Aspergillus tubingensis (75%), which are enzymes that belong to family 62 of the glycosyl hydrolases. The cloned AFdase gene was expressed in Escherichia coli BL21 (DE3) pLysS as a cellulose-binding domain tag fusion protein. The specific activity of the purified recombinant enzyme was 18.6 units/mg protein, which is one-fourth that of the enzyme purified from a solid-state culture of A. sojae.     

Cloning,sequencing and expression analysis of a gene encoding alcohol oxidase in Paenibacillus sp. AIU 311

We have cloned a gene encoding an alcohol oxidase (AOD) specific to aldehyde alcohols from Paenibacillus sp. AIU 311. The AOD gene contains an open reading frame consisting of 618 nucleotides corresponding to 205 amino acid residues. The deduced amino acid sequence exhibits a high similarity to that of manganese superoxide dismutases (SODs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant AOD expressed in E. coli BL21 is 26,000-fold higher than that of AOD in Paenibacillus sp. AIU 311. The recombinant AOD also exhibits aldehyde alcohol oxidase activity and SOD activity. The recombinant cells described in this study have utility for the production of glyoxal from glycolaldehyde.     

L-谷氨酸氧化酶的分离纯化及酶学性质的研究

采用硫酸铵分级沉淀、HPLC脱盐、离子交换、Superdex G-200凝胶层析等步骤从华美链霉菌(Streptomyces sp.)发酵液中分离纯化L-谷氨酸氧化酶(GLOD),经过SDS-PAGE电泳检测,样品达到电泳级纯,分子量为140ku。对纯化的GLOD研究发现,该酶的最适反应温度为50℃,最适pH值为7.0,温度稳定范围为0℃～50℃,pH稳定范围在6.0～9.0,GLOD催化L-谷氨酸的米氏常数Km为2.1×10-4mol/L,Na+、K+、Mg2+对酶活几乎没有影响,而Hg2+、Cu2+、Ag+均不同程度的抑制酶的活性。     

Cloning and expression of the N-acetylmuramidase gene from Streptomyces rutgersensis H-46

The N-acetylmuramidase SR1 gene from Streptomyces rutgersensis H-46 was cloned in Escherichia coli JM109 and expressed in E. coli BL21(DE3)pLysS. An open reading frame included the leader peptide region encoding a polypeptide of 65 amino acid residues and the mature SR1 enzyme region encoding a polypeptide of 209 amino acid residues. The overall G + C content of the mature enzyme gene was 67.6%, with 98.1% of G or C in the third position of the codons. The calculated molecular weight of the mature enzyme was 23,057 Da. The amino acid sequence of the mature enzyme showed a significant level of identity with bacteriolytic enzymes from Streptomyces globisporus (50.9% identity), Chalaropsis species (40.2% identity) and Saccharopolyspora erythraea (31.0% identity). The mature enzyme gene cloned into plasmid pET26b carrying a signal peptide, peIB, was expressed in E. coli BL21(DE3)pLysS. The signal peptide region was cleaved during the production of the enzyme. Specific activity of the enzyme purified from the transformant was almost identical to that of the native enzyme. Furthermore, the SR1 enzyme gene cloned with the leader peptide gene into plasmid pET28a was also expressed in E. coli. In this case, a proform-like protein was partially processed; 35 amino acid residues were cleaved but 30 amino acid residues remained. This proform like protein has approximately one-nineteenth the activity of the native enzyme. These results indicated that the native SR1 enzyme was produced in the following manner in the cells of S. rutgersensis H-46. The SR1 enzyme gene was translated to a pre-proform protein followed by the deletion of a signal peptide. Finally, the proform-like protein was processed by deletion of the remaining leader peptide.     

Cloning and sequencing of the deacetylase gene from Vibrio alginolyticus H-8

A gene encoding deacetylase DA1 that is specific for N, N'-diacetylchitobiose was cloned using the shot-gun method with pUC118 and sequenced. The open reading frame encoded a protein of 427 amino acids including the signal peptide. The molecular mass of the mature enzyme estimated from the amino acid sequence data was 44.7 kDa, which is approximately similar to that, estimated by SDS-PAGE (48.0 kDa), of the purified enzyme reported previously. The N-terminal amino acid sequence deduced from the cloned deacetylase gene showed partial sequence homology with the Nod B protein from Rhizobium sp. (37% identity) and chitin deacetylase from Mucor rouxii (28%). It contained a domain, which showed homology with a chitin-binding domain of chitinase A from Bacillus circulans (39%).     

Cloning and expression of an ether-bond-cleaving enzyme involved in the metabolism of polyethylene glycol

A gene encoding an ether-bond-cleaving enzyme, diglycolic acid dehydrogenase (DGADH) from polyethylene glycol-utilizing Pseudonocardia sp. strain K1, was cloned and expressed in Escherichia coli. The deduced amino acid sequence had a high homology with superoxide dismutases (SODs) from various bacteria. The recombinant protein showed the same activities as those of DGADH from Pseudonocardia sp. strain K1, namely, SOD activity and ether-bond-cleaving activity.     

Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae

Isoamyl alcohol oxidase (IAAOD) is a novel enzyme that catalyzes the formation of isovaleraldehyde, which is the main component of mureka that gives sake an off-flavor (Yamashita et al. Biosci. Biotechnol. Biochem., 63, 1216-1222, 1999). We cloned the genomic DNA sequence encoding IAAOD from a koji mold, Aspergillus oryzae, using a PCR-amplified DNA fragment corresponding to the partial amino acid sequences of the purified protein as a probe. The cloned gene comprises 1903 bp of an open reading frame with three putative introns and encodes 567 amino acids with a presumed signal peptide consisting of 24 amino acids at the N-terminus. Moreover, nine potential N-glycosylation sites were present. Homology search on amino acid sequence showed that IAAOD has a region significantly similar to those conserved in FAD-dependent oxidoreductases. Southern hybridization analysis revealed that the cloned gene exists as a single copy in the A. oryzae RIB 40 chromosome. The cloned gene was overexpressed under the control of the amyB promoter in A. oryzae. The isovaleraldehyde-producing activity in the culture supernatant of one transformant was over 800 times as high as that of transformant with the control vector. This result demonstrates that the cloned gene encodes IAAOD. We named this novel alcohol oxidase gene "mreA".     

Purification, bacteriolytic activity, and specificity of beta-lytic protease from Lysobacter sp. IB-9374

Lysobacter sp. IB-9374, which was isolated from soil as a high lysyl endopeptidase-producing strain (Chohnanet al., FEMS Microbiol. Lett., 213, 13-20, 2002), was found to produce a beta-lytic protease capable of lysing gram-positive bacteria such as Staphylococcus aureus, Microccocuseus, and Bacillus subtilis. The Lysobacter strain secreted the beta-lytic protease into the culture medium at a 2.4-fold higher level than Achromobacter lyticus. The enzyme was highly purified through a series of six steps with a high yield. The enzyme was strongly inhibited by tetraethylene-pentamine and 1,10-phenanthroline. The purified enzyme lysed more efficiently almost all the gram-positive bacteria tested than lysozyme, lysostaphin, and mutanolysin. The enzyme was very similar to Achromobacter beta-lytic protease containing one zinc atom in terms of amino acid composition and N-terminal sequence. The nucleotide sequence revealed that the mature enzyme was composed of 179 amino acid residues with additional 198 amino acids at the amino-terminal end of the enzyme. The deduced amino acid sequence of the mature enzyme coincided with that of the Achromobacter enzyme, although the prepro-region showed a 41% sequence identity with the counterpart. These results indicate that Lysobacter sp. is a useful strain for an efficient large-scale preparation of beta-lytic protease capable of lysing bacteria.     

链霉菌MY0504纤溶酶YG4基因的克隆及生物信息学分析

本研究获得了海洋链霉菌MY0504纤溶酶YG4基因,并对其进行生物信息学分析。利用依据同源序列设计的兼并引物,对提取的链霉菌MY0504的基因组DNA进行PCR扩增,将得到的DNA片段连接到pMD18-T载体后转化感受态Trans10细胞,然后对阳性克隆进行测序,并对序列进行生物信息学分析。最终克隆了1083bp的海洋链霉菌MY0504纤溶酶YG4基因。将获得的YG4基因输入Gen Bank网站,进行检索比对,结果显示与丝氨酸蛋白酶基因碱基序列同源性100%。对16s rDNA序列分析结果表明,该菌株与达格斯地链霉菌、氢化链霉菌、嫩白黄链霉菌、浅紫链霉菌的亲缘关系较近;通过对YG4基因编码蛋白的一级结构、二级结构、亚细胞定位和三级结构的预测和分析,结果表明:该基因编码360个氨基酸,其编码产物为稳定的亲水蛋白;二级结构以α螺旋和β折叠为主,无信号肽和跨膜结构域,有40个磷酸化位点;高级结构以无规则卷曲为主。本研究结果为研究海洋链霉菌MY0504纤溶酶YG4基因的表达机制及目的蛋白表达水平的提高提供了重要信息。     

Physiological role of the D-amino acid oxidase gene, DAO1, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii

A methylotrophic yeast, Candida boidinii, exhibits D-amino acid oxidase activity (DAO, EC 1.4.3.3) during its growth on D-alanine as a sole carbon or a nitrogen source. The structural gene (DAO1), encoding DAO, was cloned from a genomic library of C. boidinii. The 1035-bp gene encoded 345 amino acids and the predicted amino acid sequence showed significant similarity to those of DAOs from other organisms. The DAO1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The DAO1-deleted strain did not grow on D-alanine as a carbon source but did grow on D-alanine as a sole nitrogen source (with glucose as the carbon source). These results suggested that, while DAO is critically involved in growth on D-alanine as a carbon source, there should be another enzyme system which metabolizes D-alanine as a nitrogen source in C. boidinii. We also showed that the three C-terminal amino acid sequence of DAO, -AKL was necessary and sufficient for the import of DAO into peroxisomes.     

Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1

A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequence was determined. The cloned fragment contained a 1353 bp open reading frame. The initiation codon was estimated to be an unusual codon, GTG. The gene encoded a signal peptide (40 amino acid residues) for secretion. The molecular mass of the native enzyme was calculated as 43,400 Da from the sequencing data. The deduced amino acid sequence of the enzyme had 74.0 % homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacter sp. H65-7. It also had 45.1% homology with that of inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. The enzyme produced in the culture supernatant of an Escherichia coli clone was purified to the electrophoretically homogeneous stage. The N-terminal amino acid sequence of the cloned enzyme secreted in the broth was the same as that of the native enzyme from A. globiformis C11-1. Therefore, on this enzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.     

Functional analysis of genes encoding putative oxidoreductases in Aspergillus oryzae, which are similar to fungal fructosyl-amino acid oxidase

We found 11 genes (FAO1-11) encoding putative oxidoreductases in the Aspergillus oryzae genome, which are similar to fungal fructosyl-amino acid oxidases. The cDNAs corresponding to the genes were cloned and expressed in Escherichia coli. rFao2 had fructosyl-amino acid oxidase activity, whereas rFao1 did not show any enzyme activity, even though the deduced amino acid sequence of Fao1 is identical to that of one of the fructosyl-amino acid oxidase isozymes from Aspergillus oryzae. rFao7 and rFao8 showed oxidase activity toward sarcosine, L-pipecolate, and L-proline. rFao10 was active toward only sarcosine, of the substrates tested. The functions of the other proteins were also predicted from a phylogenetic analysis.     

Purification and characterization of a dehydrogenase catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde to N alpha-benzyloxycarbonyl-L-aminoadipic acid from rhodococcus sp. AIU Z-35-1

The enzyme catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde (N alpha-Z-L-AASA) to N alpha-benzyloxycarbonyl-L-aminoadipic acid (N alpha-Z-L-AAA) in Rhodococcus sp. AIU Z-35-1 was identified, and its characteristics were revealed. This reaction was catalyzed by a dehydrogenase with a molecular mass of 59 kDa. The dehydrogenase exhibited enzyme activity on not only N alpha-Z-L-AASA but also N alpha-Z-D-AASA and short chain aliphatic aldehydes, but not on aromatic aldehydes and alcohols. The apparent Km values for N alpha-Z-L-AASA, N alpha-Z-D-AASA and NAD+ were estimated to be 3.8 mM, 14.1 mM and 0.16 mM, respectively. The NH2 terminal amino acid sequence of this enzyme exhibited a similarity to those of a piperidein-6-carboxylate dehydrogenase from Streptomyces clavuligerus and a putative dehydrogenase from Rhodococcus sp. RHA 1, but not to those of other microbial aldehyde dehydrogenases.     

Molecular cloning and nucleotide sequencing of organophosphorus insecticide hydrolase gene from Arthrobacter sp. strain B-5

The organophosphorus insecticide hydrolase (OPH) gene of Arthrobacter sp. strain B-5, isolated from turf green soil was cloned into Escherichia coli JM109. Three clones, termed EpB511, EpB521 and EpB531, exhibiting OPH activity were obtained. However, these three clones showed lower OP-degrading ability than strain B-5. A 7.7-kb inserted fragment of the plasmid pB521 harbored by EpB521 was subcloned, resulting in construction of a plasmid, pB526, carrying the 2.6-kb inserted fragment with OP-degrading ability. In this sequence, an open reading frame (ORF) that encodes a 43,607 Da polypeptide composed of 415 amino acids was identified. The N-terminal amino acid sequence deduced from the nucleotide sequence was identical to that of purified OPHs. The deduced amino acid sequence was compared with the sequences in the data bank and a 58.1% amino acid identity was found with the aryldialkylphosphatase from Nocardia sp. strain B-1, an enzyme that possesses catalytic functions similar to OPH.     

Cloning, nucleotide sequence and expression of the beta-N-acetylglucosaminidase gene from Aeromonas sp. no. 10S-24

The beta-GlcNAcase gene of Aeromonas sp. no. 10S-24 was cloned and sequenced. The gene consisted of 2505 bp encoding 835 amino acid residues. The amino acid sequence of the gene shares sequence homology with beta-GlcNAcases of Vibrio vulnificus (37.4%), Serratia marcescens (36.4%), V. harveyi (36.4%), V. parahemolyticus (33.5%), Alteromonas sp. strain O-7 (27.0%), and Candida albicans (28.6%). The beta-GlcNAcase gene of Aeromonas sp. no. 10S-24 was about 630-fold overexpressed in Escherichia coli compared to that in Aeromonas sp. no. 10S-24. The cloned beta-GlcNAcase had almost the same enzymatic properties as beta-GlcNAcase from no. 10S-24. The carbohydrate chain of beta-GlcNAcase from Aeromonas sp. no. 10S-24 was found to contain mannose, galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine and several other unknown saccharides.     

A novel flavin adenine dinucleotide (FAD) containing d-lactate dehydrogenase from the thermoacidophilic crenarchaeota Sulfolobus tokodaii strain 7: purification, characterization and expression in Escherichia coli

Dye-linked D-lactate dehydrogenase activity was found in the crude extract of a continental thermoacidophilic crenarchaeota, Sulfolobus tokodaii strain 7, and was purified 375-fold through four sequential chromatography steps. With a molecular mass of about 93 kDa, this enzyme was a homodimer comprised of identical subunits with molecular masses of about 48 kDa. The enzyme retained its full activity after incubation at 80 degrees C for 10 min and after incubation at pHs ranging from 6.5 to 10.0 for 30 min at 50 degrees C. The preferred substrate for this enzyme was D-lactate, with 2,6-dichloroindophenol serving as the electron acceptor. Using high-performance liquid chromatography (HPLC), the enzyme's prosthetic group was determined to be flavin adenine dinucleotide (FAD). Its N-terminal amino acid sequence was MLEGIEYSQGEEREDFVGFKIKPKI. Using that sequence and previously reported genome information, the gene encoding the enzyme (ST0649) was identified. It was subsequently cloned and expressed in Escherichia coli and found to encode a polypeptide of 440 amino acids with a calculated molecular weight of 49,715. The amino acid sequence of this dye-linked D-lactate dehydrogenase showed higher homology (39% identity) with that of a glycolate oxidase subunit homologue from Archaeoglobus fulgidus, but less similarity (32% identity) to D-lactate dehydrogenase from A. fulgidus. Taken together, our findings indicate that the dye-linked D-lactate dehydrogenase from S. tokodaii is a novel type of FAD containing D-lactate dehydrogenase.     

Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae

Isoamyl alcohol oxidase (IAAOD) is a novel enzyme that catalyzes the formation of isovaleraldehyde, which is the main component of mureka that gives sake an off-flavor (Yamashita et al. Biosci. Biotechnol. Biochem., 63, 1216–1222, 1999). We cloned the genomic DNA sequence encoding IAAOD from a koji mold, Aspergillus oryzae, using a PCR-amplified DNA fragment corresponding to the partial amino acid sequences of the purified protein as a probe. The cloned gene comprises 1903 bp of an open reading frame with three putative introns and encodes 567 amino acids with a presumed signal peptide consisting of 24 amino acids at the N-terminus. Moreover, nine potential N-glycosylation sites were present. Homology search on amino acid sequence showed that IAAOD has a region significantly similar to those conserved in FAD-dependent oxidoreductases. Southern hybridization analysis revealed that the cloned gene exists as a single copy in the A. oryzae RIB 40 chromosome. The cloned gene was overexpressed under the control of the amyB promoter in A. oryzae. The isovaleraldehyde-producing activity in the culture supernatant of one transformant was over 800 times as high as that of transformant with the control vector. This result demonstrates that the cloned gene encodes IAAOD. We named this novel alcohol oxidase gene “mreA”.