Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.     

Characterization of a novel hydroxynitrile lyase from Nandina domestica Thunb

The leaves of Nandina domestica Thunb. exhibited high hydroxynitrile lyase (HNL) activity in (R)-mandelonitrile synthesis. The specific activity of young leaves was significantly higher than that of mature leaves. We isolated two HNLs with molecular mass of 24.9 kDa (NdHNL-S) and 28.0 kDa (NdHNL-L) from the young leaves. Both NdHNLs were composed of two identical subunits, without FAD and carbohydrates. We purified NdHNL-L and revealed its enzymatic properties. The whole deduced amino acid sequence of NdHNL-L was not homologous to any other HNLs, and the specific activity for mandelonitrile synthesis by NdHNL-L was higher than that by other plant HNLs. The enzyme catalyzed enantioselective synthesis of (R)-cyanohydrins, exhibited high activity at pH 4.0, and high stability in the pH range of 3.5–8.0 and below 55°C. Thus, NdHNL-L is a novel HNL with novel amino acid sequence and has a potential for the efficient production of (R)-cyanohydrins.     

Cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Acidovorax sp. strain SA1 and purification of the enzyme

The gene of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase (i3HBOH) was cloned and sequenced from a poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Acidovorax sp. strain SA1. The i3HBOH gene has 876 nucleotides corresponding to the deduced sequence of 292 amino acids. In this amino acid sequence, the general lipase box sequence (G-X₁-S-X₂-G) was found, whose serine residue was determined to the active sites serine by site-directed mutagenesis. An i3HBOH was purified to electrophoretical homogeneity from SA1. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE. The N-terminal amino acid sequence of the purified enzyme corresponded to the deduced N-terminal amino acid sequence in the cloned i3HBOH gene. This is the first cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase gene to date. Received: 19 October 2001 / Accepted: 7 December 2001     

Purification and biochemical characterization of a fibrinolytic enzyme from Bacillus subtilis BK-17

A fibrinolytic enzyme from Bacillus subtilis BK-17 has been purified to homogeneity by gel-filtration and ion-exchange chromatography. Compared to the crude enzyme extract, the specific activity of the enzyme increased 929-fold with a recovery of 29%. The subunit molecular mass of the purified enzyme was estimated to be 31 kDa by SDS–PAGE. The N-terminal amino acid sequence of the purified fibrinolytic enzyme was: A-Q-S-V-P-Y-G-V-S-Q-I-K-A-P-A-A-H-N. The sequence was highly homologous to the fibrinolytic enzymes nattokinase, subtilisin J and subtilisin E from Bacillus spp. However, there was a substitution of three amino acid residues in the N-terminal sequence. The amidolytic activity of the purified enzyme for several substrates was assessed. In comparison with nattokinase and CK (fibrinolytic enzyme from a Bacillus spp.), which showed strong fibrinolytic activity, the amidolytic activity of the enzyme for the synthetic substrate, kallikrein (H-D-Val-Leu-Arg-pNA, S-2266) increased 2.4- and 11.8-fold, respectively.     

The hydroxynitrile lyase from almond: a lyase that looks like an oxidoreductase

BACKGROUND: Cyanogenesis is a defense process of several thousand plant species. Hydroxynitrile lyase, a key enzyme of this process, cleaves a cyanohydrin into hydrocyanic acid and the corresponding aldehyde or ketone. The reverse reaction constitutes an important tool in biocatalysis. Different classes of hydroxynitrile lyases have convergently evolved from FAD-dependent oxidoreductases, alpha/beta hydrolases, and alcohol dehydrogenases. The FAD-dependent hydroxynitrile lyases (FAD-HNLs) carry a flavin cofactor whose redox properties appear to be unimportant for catalysis. RESULTS: We have determined the crystal structure of a 61 kDa hydroxynitrile lyase isoenzyme from Prunus amygdalus (PaHNL1) to 1.5 A resolution. Clear electron density originating from four glycosylation sites could be observed. As concerns the overall protein fold including the FAD cofactor, PaHNL1 belongs to the family of GMC oxidoreductases. The active site for the HNL reaction is probably at a very similar position as the active sites in homologous oxidases. CONCLUSIONS: There is strong evidence from the structure and the reaction product that FAD-dependent hydroxynitrile lyases have evolved from an aryl alcohol oxidizing precursor. Since key residues implicated in oxidoreductase activity are also present in PaHNL1, it is not obvious why this enzyme shows no oxidase activity. Similarly, features proposed to be relevant for hydroxy-nitrile lyase activity in other hydroxynitrile lyases, i.e., a general base and a positive charge to stabilize the cyanide, are not obviously present in the putative active site of PaHNL1. Therefore, the reason for its HNL activity is far from being well understood at this point.     

Screening hydroxynitrile lyases for (R)-pantolactone synthesis

The application of (R)-hydroxynitrile lyases ((R)-HNLs) enables a simple chemo-enzymatic approach towards (R)-pantolactone synthesis. For the first time, several new recombinant almond (R)-HNL isoenzymes were compared with native HNLs from different Prunus species with respect to cyanohydrin formation from hydroxypivalaldehyde providing the chiral key precursor in HNL based (R)-pantolactone synthesis. Recombinant PaHNL5 (R-selective hydroxynitrile lyase, isoenzyme 5, from Prunus amygdalus) surpasses all other tested natural and recombinant HNL variants. At low pH even very low amounts of crude enzyme catalysed stereoselective hydroxypivalaldehyde cyanohydrin formation in water based reaction systems.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Cloning and characterization of EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes

By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated. High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv. syringae. The sequenced ORF encodes for a protein of 298 amino acids. The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities. The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed. Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized. Received: 8 January 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Hydroxynitrile lyases from prunus seeds in the preparation of cyanohydrins

The hydroxynitrile lyase (HNL) activity of nine defatted Prunus seeds was compared for catalyzing the addition of HCN to aromatic, heteroaromatic and α,β-unsaturated aldehydes. Although the conversion and enantiomeric excess (ee) of the corresponding cyanohydrins were both influenced by the HNL source and the chemical structure of the aldehyde, Prunus HNLs were all suitable for the enantioselective preparation of cyanohydrins.     

NADP+-dependent isocitrate dehydrogenase from a psychrophilic bacterium,Psychromonas marina

The gene encoding NADP⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) of a psychrophilic bacterium, Psychromonas marina, was cloned and sequenced. The open reading frame of the gene encoding IDH of P. marina (PmIDH) was 2229 bp in length and corresponded to a polypeptide composed of 742 amino acids. The molecular mass of IDH was calculated as 80,426 Da. The deduced amino acid sequence of PmIDH exhibited high degrees of homology with the monomeric IDH from other bacteria such as Colwellia maris (62% identity) and Azotobacter vinelandii (AvIDH) (64%). His-tagged PmIDH overexpressed in Escherichia coli cells was purified and characterized. The optimum temperature of PmIDH activity was about 35 °C; however, the enzyme lost 74% of the activity after incubation for 10 min at 30 °C, indicating that this enzyme is thermolabile. Chimeric enzymes produced through domain swapping between PmIDH and mesophilic AvIDH were constructed and their optimum temperatures and thermostability were determined. The results suggest that regions 2 and 3, especially region 3, of the two IDHs are involved in their catalytic activities and optimum temperature and thermostability for activity.

     

Purification of Cytochrome P-450«/A from n-Alkane-grown Cells of Candida maltosa,and Cloning and Nucleotide Sequencing of the Encoding Gene

When the cells of an n-alkane-assimilating yeast, Candida maltosa I AM12247, were transferred from a glucose medium to an n-alkane medium, various enzymes are induced in the endoplasmic reticulum and peroxisome. Cytochrome P-450alk, one of these enzymes in the endoplasmic reticulum, was purified after mild solubilization of the membrane, followed by a few steps of chromatography. The enzyme was characterized spectrophotometrically and its N-terminal amino acid sequence (12 residues) was determined.

Using oligonucleotide probes prepared to match parts of the N-terminal amino acid sequence and of the partial cDNA sequence of cytochrome P-450alk of C. maltosa EH 15, we isolated from a gene library of C. maltosa I AM 12247 a clone which had a gene encoding cytochrome P-450a/Ar. By nucleotide sequencing of this gene, the amino acid sequence of this enzyme was deduced. It consisted of 523 amino acids (59,838 daltons), with a non-cleavable signal sequence in the N-terminal region. The structure of this enzyme was compared with some other members of the cytochrome P-450 superfamily.     

Purification of a novel extracellular laccase from solid-state culture of the edible mushroom <Emphasis Type="Italic">Lentinula edodes</Emphasis>

The laccases (EC 1.10.3.2) secreted into solid-state culture by Lentinula edodes were analyzed. The fungus secreted at least two laccases in the solid-state culture. One laccase was purified to a homogeneous preparation using anion-exchange, hydrophobic, and size-exclusion chromatography. SDS-PAGE analysis showed that the purified laccase, Lcc6, was a monomeric protein of 58.5 kDa. The optimum pH for enzyme activity was about 3.5, and the laccase was most active at 40°C. The N-terminal amino acid sequence of Lcc6 did not correspond to the sequence of Lcc1, which was previously purified from L. edodes. Lcc6 had decolorization activity to some chemical dyes.     

Molecular cloning and characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101

A chitosanase was purified from the culture fluid of the chitino- and chitosanolytic bacterium Burkholderia gladioli strain CHB101. The purified enzyme (chitosanase A) had a molecular mass of 28 kDa, and catalyzed the endo-type cleavage of chitosans having a low degree of acetylation (0–30%). The enzyme hydrolyzed glucosamine oligomers larger than a pentamer, but did not exhibit any activity toward N-acetyl-glucosamine oligomers and colloidal chitin. The gene coding for chitosanase A (csnA) was isolated and its nucleotide sequence determined. B. gladioli csnA has an ORF encoding a polypeptide of 355 amino acid residues. Analysis of the N-terminal amino acid sequence of the purified chitosanase A and comparison with that deduced from the csnA ORF suggests post-translational processing of a putative signal peptide and a possible substrate-binding domain. The deduced amino acid sequence corresponding to the mature protein showed 80% similarity to the sequences reported from Bacillus circulans strain MH-K1 and Bacillus ehimensis strain EAG1, which belong to family 46 glycosyl hydrolases. Received: 30 July 1999 / Revised revision: 17 February 2000 / Accepted: 25 February 2000     

Purification,Characterization, and Molecular Cloning of Tyrosinase from Pholiota nameko

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.     

Sequence analysis of the gene encoding a novel l-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases

The gene (ddc) encoding a novel enzyme, l-2,4-diaminobutyrate decarboxylase (DABA-DC; EC 4.1.1.-) in Acinetobacter baumannii was sequenced, and an open reading frame of 1,530 nucleotides was detected. The sequence of 20 N-terminal amino acids of purified DABA-DC and of its proteolytic peptide fragments coincided with those deduced from the nucleotide sequence determined. Comparison of the predicted amino acid sequence of the A. baumannii enzyme with those of other pyridoxal 5′-phosphate-dependent decarboxylases revealed significant similarity to the group II amino acid decarboxylases and conservation of the putative pyridoxal 5′-phosphate-binding domain. Received:20 February 1996 / Accepted 15 April 1996     

Molecular cloning and characterization of the gene encoding a fibrinolytic enzyme from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Strain A1

A fibrinolytic metalloprotease gene from Bacillus subtilis has been cloned in Escheridria coliXL1-Blue and the bacterial expressed enzyme was purified. The nucleotide sequence of the cloned fibrinolytic enzyme gene revealed a single open reading frame of 1023 bp coding for 341 amino acids (M _r 37708.21 Da). N-terminal amino acid sequencing of the fibrinolytic enzyme excreted from E. coli host cells revealed that the mature fibrinolytic enzyme consists of 288 amino acids (M _r 31391.1 Da). The deduced amino acid sequence showed significant homology with Erwina carotovora neutral metalloprotease and Serratia marcescens minor metalloprotease by 65 and 58% amino acid sequence identity, respectively. The protein showed significant alignments with the conserved domain of catalytic activity and the -helix domain in Bacillus anthracisthermolysis metalloprotease. The biochemical properties of the purified enzyme suggested that the enzyme is a fibrinolytic metalloprotease, which has optimal activity at pH 7.0 and 50 °C.     

N-terminal amino acid sequence of Brevibacterium sp. R312 wide-spectrum amidase

Summary A wide-spectrum amidase from Brevibacterium sp. R312 was partially purified. The enzyme subunit was purified by reversed phase HPLC and the N-terminal amino acid sequence was found to be identical to that of Pseudomonas aeruginosa aliphatic amidase. Offprint requests to: A. Arnaud     

Potent Fibrinolytic Enzyme from the Lysate of Katsuwonus pelamis Digestive Tract (Shiokara): Purification and Characterization

Strong fibrinolytic enzyme was purified from the lysate of Katsuwonus pelamis digestive tract (Japanese traditional fermented food, “shiokara”). The enzyme was an alkaline trypsin-like serine protease, and a pH- and salt-resistant protein. The N-terminal amino acid sequence of the enzyme showed similarity with those of trypsin from other organisms. The molecular weight and isoelectric point of the enzyme were estimated to be 38,000 and 4.65, respectively. The enzyme is probably useful as a thrombolytic agent.     

Purification,N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.