Purification, characterization and gene cloning of thermostable O-acetyl-L-homoserine sulfhydrylase forming gamma-cyano-alpha-aminobutyric acid

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable gamma-cyano-alpha-aminobutyric acid synthase. The enzyme was purified and characterized. The purified enzyme has a molecular mass of approximately 180 kDa and consists of four identical subunits. It was stable in the pH range of 6.0 to 10.5 and up to 60 degrees C. The enzyme catalyzed the gamma-replacement reaction of O-acetyl-L-homo-serine with cyanide ions. The gene encoding the gamma-cyano-alpha-aminobutyric acid synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the gamma-cyano-alpha-aminobutyric acid synthase from the bacterium is O-acetyl-L-homoserine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, was introduced into Escherichia coli JM109. The transformed E. coli cells overexpressed gamma-cyano-alpha-aminobutyric acid synthase. The heat stable gamma-cyano-alpha-aminobutyric acid synthase can be applied to the synthesis of [5-11C]L-glutamic acid used as a tracer for positron emission tomography.     

Purification, characterization, and gene cloning of sphingomyelinase C from Streptomyces griseocarneus NBRC13471

Sphingomyelinase C (SMC) was purified to homogeneity from the culture supernatant of Streptomyces griseocarneus NBRC13471. The purified enzyme appeared as a single band of 38 kDa by using an electropherogram trace. The molecular mass of the enzyme as determined by MALDI-TOF MS was 32,102 Da, indicating that SMC is monomeric in nature. Under experimental conditions, the highest enzyme activity was found at pH 9.0 and 50–55 °C, and the enzyme was stable from pH 5 to 10 and up to 37 °C. The SMC activity requires Mg²⁺ or Mn²⁺ and the order of potency to enhance the activity was Zn²⁺ ≥ Mn²⁺ > Cu²⁺ ≥ Fe²⁺. Phenylmethylsulfonyl fluoride and EDTA inhibited the enzyme activity, showing that SMC belongs to a group of metalloenzymes and a class of serine hydrolases. The enzyme activity was inhibited by DTT, but not by mercaptoethanol and iodoacetamide. SDS inhibited the enzyme activity; by contrast, Triton X-100 stimulated the activity. The N-terminal and internal amino-acid sequences were determined as H₂N-APAAATPSLK, AREIAAAGFFQGND, and NTVVQETSAP. The gene encoding SMC consisted of 1020 bp encoding a signal peptide of 42 amino acids and a mature protein of 297 amino acids with a calculated molecular mass of 32,125 Da. The conserved region of DNase I-like family enzymes and the amino acid residues that are highly conserved in the active center of other bacterial SMCs were also found in the deduced amino acid sequence of S. griseocarneus SMC.     

Purification, characterization and functional cloning of inositol oxygenase from Cryptococcus

The enzyme inositol oxygenase (myo-inositol : oxygen oxidoreductase; E.C. 1.13.99.1) is a monooxygenase that converts inositol into glucuronic acid in the presence of molecular oxygen. This enzyme is integrated into a pathway leading to either degradation and energy production or the biosynthesis of precursors for polysaccharides. The enzyme was purified from the yeast Cryptococcus lactativorus by a five-step chromatography procedure. The purified enzyme shows a molecular mass of 37 kDa on SDS-PAGE, similar to the estimation of the size of the native enzyme determined by size exclusion chromatography. Peptides of the inositol oxygenase protein derived from a tryptic digest were sequenced de novo by nanoelectrospray tandem mass spectrometry. Using degenerate oligonucleotides, the corresponding gene was cloned from first strand cDNA. The open reading frame encodes a 315 amino acid polypeptide with a predicted molecular mass of 36.9 kDa. Inositol oxygenase is a single copy gene in C. lactativorus. It has close homologues in other fungi such as Cryptococcus neoformans and Neurospora crassa. Biochemical characterization of the enzyme showed a pH optimum of 6-6.5 and a temperature optimum of 30 degrees C. Myo-inositol is the only accepted substrate with a Km of ca. 5 mM. The enzyme contains a Fe-centre but the enzyme activity is resistant to KCN.     

Purification, characterization, and gene cloning of thermophilic cytochrome cd1 nitrite reductase from Hydrogenobacter thermophilus TK-6

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6 can grow autotrophically under anaerobic conditions by denitrification. One of the denitrification enzymes, cytochrome cd(1) nitrite reductase, was isolated and its gene was cloned from strain TK-6. The subunit molecular mass of the purified enzyme was 61.5 kDa and the isoelectric point was determined to be 9.3. The optimum temperature and pH for the enzymatic reaction were 70-75 degrees C and 6.5-7.0, respectively. The structural gene for the enzyme, nirS, is probably transcribed as a hexacistronic operon with the following genes encoding a putative diheme cytochrome c and the proteins required for biosynthesis of heme d(1). The NirS sequence was phylogenetically distinct from those of proteobacteria. The consensus -35 and -10 sequences were found in the putative nirS promoter region, but the consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide sensing regulators were not found in this region.     

Isolation, characterization, and molecular cloning of a thermostable xylitol oxidase from Streptomyces sp. IKD472

A thermophilic bacterium, Streptomyces sp. IKD472, that can oxidize xylitol was isolated from a hot spring and was found to produce xylitol oxidase. The purified enzyme was a monomeric protein with an apparent molecular weight of 43 k as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. This novel enzyme is capable of catalyzing the oxidation of one mole of xylitol to form one mole each of xylose and hydrogen peroxide. Since the V(max)K(m) value for xylitol was two and four times higher than those for galactitol and n-sorbitol, respectively, the enzyme was designated as xylitol oxidase. The enzyme was stable in the pH range from 5.5 to 10.5 and at temperatures up to 65 degrees C. The optimal temperature and pH were 55 degrees C and pH 7.5, respectively. Xylitol oxidase bound one mole of FAD as a coenzyme per mole of protein. The amino acid sequence of the NH2 terminus and the fragments obtained by lysylendpeptidase digestion of xylitol oxidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 2.8-kb chromosomal fragment hybridizing to the probes was cloned into pUC18 in Escherichia coli. The gene consists of an open reading frame of 1245 by that encodes a protein containing 415 amino acids with a molecular weight of 44,730 but without the conserved nucleotide-binding sequence, Gly-X-Gly-X-X-Gly. The amino acid sequence has 70% identity to putative oxidoreductase from Streptomyces coelicolar, 51% to sorbitol oxidase from Streptomyces sp., and 26% to L-gulonolactone oxidase from rat in terms of the overall amino acid sequence. DNA manipulation of the cloned gene in E. coli, by alteration of a strong promoter and a synthesized ribosome-binding sequence at an appropriate position, resulted in overproduction of xylitol oxidase 100 times more than that produced in the original Streptomyces sp. IKD472. The enzyme properties of recombinant xylitol oxidase were the same as those of the authentic enzyme. Stable xylitol oxidases, which allow easier quantitative analysis of xylitol, are useful for clinical applications.     

Purification, characterization, and gene cloning of glycerol dehydrogenase from Hansenula ofunaensis, and its expression for production of optically active diol

Optically active alcohol is an important building block as a versatile chiral synthon for the asymmetric synthesis of pharmaceuticals and agrochemicals. We purified and characterized glycerol dehydrogenase from Hansenula ofunaensis and prepared optically active 1,2-octanediol using a recombinant Escherichia coli harboring the glycerol dehydrogenase gene. The deduced amino acid sequence was investigated for identities with those of other alcohol dehydrogenases in the NCBI databank. The identification of the unknown product of a resting-cell reaction was performed by GC-MS. In the deduced amino acid sequence composed of 376 residues, the NAD(H) binding pattern and cysteine residues that correspond to the cysteine ligands at the zinc atom were conserved as they are in alcohol dehydrogenases from other origins. Glycerol dehydrogenase from Hansenula polymorpha DL-1 (Pichia angusta, DDBJ/EMBL/GenBank accession no. BAD32688) had the highest identity to our enzyme, showing 73% identity. Our glycerol dehydrogenase catalyzed the NAD(+)-dependent oxidation of long-chain secondary alcohols such as 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol. Activities toward 2,4-pentanediol and 2,5-hexanediol were hardly detected. From these results, it was confirmed that our enzyme requires two hydroxyl groups on adjacent carbon atoms for oxidation. 2,3-Pentanedione, 2,3-hexanedione, and 3,4-hexanedione were significantly reduced. The transformants oxidized only (R)-1,2-octanediol in 50 mM racemate (R:S=52:48), and produced (S)-1,2-octanediol (24 mM, <99.9% e.e.) after 24 h of incubation. The reaction product was suggested to be 1-hydroxy-2-octanone by GC-MS, which showed secondary hydroxyl groups oxidized. Glycerol dehydrogenase from H. ofunaensis could be useful for the production of long-chain optically active secondary alcohols.     

Molecular cloning and characterization of thermostable beta-lactam acylase with broad substrate specificity from Bacillus badius

The gene (pac) encoding beta-lactam acylase from Bacillus badius was cloned and expressed in Escherichia coli. The pac gene was identified by polymerase chain reaction (PCR) using degenerated primers, on the basis of conserved amino acid residues. By using single specific primer PCR (SSP-PCR) and direct genome sequencing, a complete pac gene with its promoter region was obtained. The ORF consisted of 2415 bp and the deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a signal sequence, an alpha-subunit, a spacer peptide and a beta-subunit. The pac gene was expressed with its own promoter in different E. coli host strains and a maximum recombinant PAC (1820 U l(-1)) was obtained in E. coli DH5alpha. The recombinant PAC was purified by Ni-NTA chromatography and the purified PAC had two subunits with apparent molecular masses of 25 and 62 kDa. This enzyme exhibited a high thermostability with a maximum activity at 50 degrees C. This enzyme showed stability over a wide pH range (pH 6.0-8.5) with a maximum activity at pH 7.0 and activity on a wide beta-lactam substrate range. The K(m) values obtained for the hydrolysis of penicillin G and a chromogenic substrate, 6-nitro-3-phenylacetylamidobenzoic acid, from B. badius PAC were 39 and 41 microM, respectively. The PAC activity was competitively inhibited by PAA (K(i), 108 microM) and noncompetitively by 6-APA (K(i), 17 mM). The constitutive production of B. badius PAC in E. coli and its easier purification together with the advantageous properties, such as thermostability, pH stability and broad substrate specificity, make this as a novel enzyme suitable for beta-lactam industry.     

Purification and characterization of a novel thermostable phytase from the thermophilic Geobacillus sp. TF16

A novel phytase from thermophilic Geobacillus sp. TF16 was puri?ed approximately 5-fold using ammonium sulfate precipitation and ion exchange chromatography, and determined as a single band 106.04 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimum temperature and optimum pH were found to be 85°C and 4.0, respectively. The enzyme is highly thermostable and V_max and K_m values were calculated as 526.28 U/mg and 1.31 mM, respectively. It was also found that the enzyme exhibited a broad substrate selectivity and resistance toward proteases and effectively hydrolyzed soymilk phytate. These results suggest that this study provides an alternative phytase enzyme with enhanced properties.     

Purification and characterization of intracellular and extracellular, thermostable and alkali-tolerant alcohol oxidases produced by a thermophilic fungus, Thermoascus aurantiacus NBRC 31693

Intracellular and extracellular alcohol oxidases (AO int and AO ext) were purified from the liquid and solid cultures of a thermophilic fungus, Thermoascus aurantiacus NBRC 31693, as electrophoretically and isoelectrophoretically homogeneous proteins, respectively. Both enzymes contained a flavin adenine dinucleotide (FAD) cofactor and were stained with Schiff's reagent. The molecular weight of AO int was estimated to be about 320 kDa and its subunit was 75 kDa. The molecular weight of AO ext was about 560 kDa, and it was composed of two types of subunits (75 kDa and 59 kDa). The pIs of AO int and AO ext were 5.88 and 6.08, respectively. AO int and AO ext were stable up to 60 degrees C and 55 degrees C, respectively. The enzymes were stable over a wide range of pH from 6 to 11. AO int oxidized short straight-chain alcohols (K(m) for methanol, 13.5 mM and K(m) for ethanol, 15.8 mM). On the other hand, AO ext could oxidize secondary alcohols and aromatic alcohols (veratryl alcohol and benzyl alcohol) in addition to straight-chain alcohols (K(m) for methanol, 0.5 mM and K(m) for ethanol, 10.2 mM).     

Purification and gene cloning of a chitosanase from Bacillus ehimensis EAG1

Bacillus ehimensis EAG1 (IFO15659) produced and secreted chitosanase in the presence of exogenous chitosan. The chitosanase was purified from the culture filtrate of the bacterium to apparent homogeneity in SDS-polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 31,000. A 1.9-kbp DNA fragment containing the chitosanase gene was cloned and the complete nucleotide sequence was determined. The sequence was found to contain a single open reading frame encoding a protein of 302 amino acids. The deduced amino acid sequence showed significant homology with the chitosanase from Bacillus circulans MH-K1.     

热稳定金刚石聚晶的制备及表征

在高温高压条件下（5.6GPa,1200℃～1480℃）,以含硼金刚石微粉为原料,镍基合金为烧结助剂,采用熔渗法成功制备了热稳定金刚石聚晶（Thermally stable PCD）。通过X射线衍射（XRD）和扫描电镜（SEM）,研究了烧结温度对热稳定金刚石聚晶的物相成分、微观组织形貌的影响;并与普通金刚石聚晶进行了差热（DTA）、热重（TG）的对比分析测试,结合X射线光电子能谱（XPS）测试结果给出了相应解释。实验结果表明：在压强为5.6GPa条件下,温度在1300℃～1450℃区间内,才能实现热稳定PCD的烧结;此时形成的热稳定PCD的耐热性和抗氧化性相比普通PCD均有较大幅度提高。     

甘蓝型油菜BnPexl6p基因cDNA的克隆与表达

利用模式植物拟南芥AtPexl6p／SSEl基因序列与油菜数据库BBSRCBrassicaDB比对,得到甘蓝型油菜（Brassica napus）同源EST序列,拼接出油菜Pexl6p基因全长eDNA电子克隆,然后设计全长引物,以甘蓝型油菜种子eDNA为模板,克隆Rexl6p基因,得到全长为1101bp的eDNA,编码366个氨基酸的蛋白,命名为BnPexl6p。Bn-Pexl6p基因序列与拟南芥AtPexl6p／SSE1同源,其蛋白与拟南芥同源蛋白具有完全相同的vrs2型过氧化物酶体定位肽,进化关系相近。半定量PCR（RT—PCR）发现BnPexl印基因在油菜幼嫩的根、茎、叶和种子中均有高丰度表达,在种子发育过程的中期和后期表达水平增加,暗示该基因在油脂的积累中有重要作用。     

褐色喜热裂孢菌Thermobifida fusca产麦芽糖α-淀粉酶基因的克隆表达及酶学性质研究

以T.fusca基因组DNA为模板,PCR分别扩增不包括和包括其基因前段信号肽的产麦芽糖淀粉酶的基因片段tfa和sptfa,并克隆至表达载体pSE380上,获得重组质粒pSE380-tfa和pSE380-sptfa,以大肠杆菌JM109为宿主细胞大量表达融合蛋白,利用金属镍亲和层析对菌株JM109/pSE380-tfa表达的α-淀粉酶进行纯化,SDS-PAGE显示纯化蛋白的分子量约为64ku,K m值为1.305mg/mL,最适反应温度为60℃,最适pH为7.0;检测到菌株JM109/pSE380-sptfa的培养基上清有相当一部分酶活。本研究麦芽糖α-淀粉酶与可溶性淀粉反应,经HPLC检测产物均为麦芽三糖、麦芽糖和葡萄糖的混合物。因此麦芽糖α-淀粉酶在生产高麦芽糖浆上起到了一定的作用。      

Molecular cloning and characterization of a thermostable carboxylesterase from an archaeon, Sulfolobus shibatae DSM5389: non-linear kinetic behavior of a hormone-sensitive lipase family enzyme

A gene coding for an esterase (SshEstI, 915 bp in length) of the thermoacidophilic archaeon Sulfolobus shibatae DSM5389 was cloned, sequenced, and overexpressed in Escherichia coli JM109 cells as a soluble, catalytically active protein. The deduced amino acid sequence of SshEstI was consistent with a protein containing 305 amino acid residues with a molecular mass of 33 kDa. Sequence comparison studies indicated that SshEstI could be a member of the hormone-sensitive lipase family, in that it had the highest sequence similarity to esterases from Sulfolobus solfataricus (90% identity) and Archaeoglobus fulgidus (42%) and a lipase from Pseudomonas sp. B11-1 (38%). The recombinant enzyme was highly thermostable and retained more than 70% of its initial activity after incubation at 90 degrees C and pH 7.0 for 30 min. The recombinant enzyme catalyzed the hydrolysis of p-nitrophenyl (p-NP) esters with C2-C16 acyl chains but not the hydrolysis of triacylglycerides such as tributyrin and triolein. The enzymatic hydrolysis of p-NP acetate proceeded in a linear manner with time, whereas that of p-NP esters with acyl chains of C5 or longer showed a biphasic profile, where a rapid release of p-nitrophenol ( approximately 3 min) was followed by a slow, sustained release. These non-linear kinetics may be explained in terms of a very slow, presteady-state burst phenomenon of p-nitrophenol release or a hysteretic behavior of SshEstI with these substrates.