Purification and Properties of NADP-Dependent Maltose Dehydrogenase Produced by Alkalophilic Corynebacterium sp. No. 93–1

NADP-dependent maltose dehydrogenase (NADP-MalDH) was completely purified from the cell free extract of alkalophilic Corynebacterium sp. No. 93–1. The molecular weight of the enzyme was estimated as 45,000~48,000. The enzyme did not have a subunit structure. The isoelectric point of the enzyme was estimated as pH 4.48. The pH optimum of the enzyme activity was pH 10.2, and it was stable at pH 6 to 8. The temperature optimum was 40°C, and the enzyme was slightly protected from heat inactivation by 1 mm NADP. The enzyme oxidized d-xylose, maltose and maltotriose, and the Km values for these substrates were 150mm, 250 mm and 270 mm, respectively. Maltotetraose and maltopentaose were suitable substrates. The Km value for NADP was 1.5 mm with 100mm maltose as substrate. The primary product of this reaction from maltose was estimated as maltono-δ-lactone, and it was hydrolyzed non-enzymatically to maltobionic acid. The enzyme was inhibited completely by PCMB, Ag⁺ and Hg²⁺.     

Corynebacterium antarcticum sp. nov., Corynebacterium marambiense sp. nov., Corynebacterium meridianum sp. nov., and Corynebacterium pygosceleis sp. nov., isolated from Adélie penguins (Pygoscelis adeliae)

A taxonomic study was conducted on 16 bacterial strains isolated from wild Adélie penguins (Pygoscelis adeliae) from Seymour (Marambio) Island and James Ross Island. An initial screening by repetitive sequence-based PCR fingerprinting divided the strains studied into four coherent groups. Phylogenetic analysis based on 16S rRNA gene sequences assigned all groups to the genus Corynebacterium and showed that Corynebacterium glyciniphilum and Corynebacterium terpenotabidum were the closest species with 16S rRNA gene sequence similarities between 95.4 % and 96.5 %. Further examination of the strains studied with ribotyping, MALDI-TOF mass spectrometry, comprehensive biotyping and calculation of average nucleotide identity and digital DNA–DNA hybridisation values confirmed the separation of the four groups from each other and from the other Corynebacterium species. Chemotaxonomically, the four strains P5828^T, P5850^T, P6136^T, P7210^T representing the studied groups were characterised by C_16:0 and C_18:1 ω9c as the major fatty acids, by the presence of meso-diaminopimelic acid in the peptidoglycan, the presence of corynemycolic acids and a quinone system with the predominant menaquinone MK-9(H₂). The results of this study show that the strains studied represent four new species of the genus Corynebacterium, for which the names Corynebacterium antarcticum sp. nov. (type strain P5850^T = CCM 8835^T = LMG 30620^T), Corynebacterium marambiense sp. nov. (type strain P5828^T = CCM 8864^T = LMG 31626^T), Corynebacterium meridianum sp. nov. (type strain P6136^T = CCM 8863^T = LMG 31628^T) and Corynebacterium pygosceleis sp. nov. (type strain P7210^T = CCM 8836^T = LMG 30621^T) are proposed.     

Purification and Characterization of α-Hydroxyglutarate Dehydrogenase from Alcaligenes sp.

NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD⁺ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation.     

Purification,Crystallization and Some Enzymatic Properties of Acid Protease of Cladosporium sp. No. 45–2

An acid protease of Cladosporium sp. No. 45–2 was purified and crystallized by precipitation with ammonium sulfate, fractional precipitation with acetone, and pH adjustment. About 600 mg of third crystallized preparation was obtained from one liter of culture broth. The purified enzyme was chromatographically homogeneous and confirmed to be monodispersive by physicochemical criteria such as uhracentrifugal and electrophoretical analysis. The enzyme was most active at pH values between 2.5 and 2.7 toward both casein and hemoglobin and was stable at pH values from 2.5 to 7.0 on twenty hour incubation at 30°C.

Millimolar concentration of sodium lauryl sulfate markedly inhibited the enzyme, wheares diisopropyl phosphorofluoridate, sulfhydryl reagents, ethylenediaminetetra acetic acid, and divalent metal ion relatively little affected the activity. The enzyme was most resistant toward S-PI among the acid proteases tested.     

Purification and Characterization of a Liquefying α-Amylase from Alkalophilic Thermophilic Bacillus sp. AAH-31

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS–PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4–10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.     

Some Physicochemical Properties and Substrate Specificity of Acid Protease Isolated from Cladosporium sp. No. 45–2

Some physicochemical properties and substrate specificity of crystalline acid protease obtained from Cladosporium sp. No. 45–2 were investigated.

The molecular weight determined by the sedimentation equilibrium method and Sephadex G–75 gel filtration was 32,000 and 30,000, respectively. The isoelectric point was determined as 4.6 by using the Tiselius electrophoresis apparatus and the amino terminal amino acid was found to be alanine. The enzyme did not contain histidine and was composed of 294 amino acid residues. Substrate specificity against synthetic peptides was similar to that of pepsin. The enzyme was remarkably inactivated by a synthetic pepsin inhibitor such as α-Diazo-p-bromo acetophenone, Diazo acetyl glycine ethylester and N-Diazo acetyl norleucine methylester.     

The Isolation,Purification, and Some Properties of NAD-Dependent Isocitrate Dehydrogenase from the Organic Acid–Producing Yeast Yarrowia lipolytica

The NAD⁺-dependent isocitrate dehydrogenase of the organic acid–producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The K _m for NAD⁺ is 136 M, and that for isocitrate is 581 M. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD⁺) in the overproduction of citric and keto acids is discussed.     

Utilization of Alcohols by Rhodopseudomonas sp. No. 7 Isolated from n-Propanol–Enrichment Cultures

A purple non-sulfur bacterium, Rhodopseudomonas sp. No. 7, was isolated from n-propanol–enrichment cultures under anaerobic-light conditions. Strain No. 7 can produce hydrogen from alcohols. The rate of hydrogen production from n-propanol was 34 μl/hr/mg dry cells. Strain No. 7 showed multiplication by budding and the best growth on n-propanol among other organic compounds tested. But its growth on n-propanol was poor under aerobic-dark conditions. NAD-linked alcohol dehydrogenase, NAD-linked aldehyde dehydrogenase, acyl-CoA synthetase and malate synthetase were found in strain No. 7. These enzymes were constitutive. On the other hand, isocitrate lyase was induced in cells grown on ethanol but not on n-propanol. No activity of phenazine methosulfate-linked alcohol dehydrogenase was detected in strain No. 7.     

Malate Dehydrogenase, Alcohol Dehydrogenase, and 6-Phosphogluconate Dehydrogenase Isozymes of Zea mays L. × Tripsacum dactyloides L. Hybrids and Parents

Electrophoretic patterns of malate dehydrogenase (Mdh), alcohol dehydrogenase (Adh), and 6-phosphogluconate dehydrogenase (Pgd) of Zea mays L. × Tripsacum dactyloides L. hybrids and their parents were compared. The components of enzymes specific to T. dactyloides may be used as markers to identify the following T. dactyloides chromosomes in the hybrids: Tr 16 (Mdh 2 and Pdg 1), Tr 7, and/or Tr 13 (Adh 2). The isozymes of Mdh 2 are supposed as a possible biochemical marker to evaluate the introgression of genes, determining an apomictic mode of reproduction from T. dactyloides (localized on Tripsacum 16 chromosome) into Z. mays. The isozymes may be used as markers for the identification of maize chromosomes 1 and 6 in the hybrids as well. Chromosome count taken on the examined hybrids showed the addition of 9 to 13 chromosomes of T. dactyloides to maize chromosome complement.     

Purification and Properties of an Exocellular γ-Glutamyl Arylamidase from Bacillus sp. Strain No. 12

An exocellular γ-glutamyl arylamide-hydrolyzing enzyme was produced by a Bacillus sp. in L-glutamate-containing medium. This enzyme was a tetrameric simple protein composed of two heavy subunits (Mr 56,000) and two light subunits (Mr 46,000). It hydrolyzed γ-amido, acyl and aryl bonds in L- and D-glutamyl compounds, and the activity on L-glutamic acid γ-p-nitroanilide was inhibited by the addition of glutamate and γ-glutamyl compounds but not by α-glutamyl compounds. The activity was stimulated by various dipeptides but not by free amino acids, L-Alanine, glycine, L-serine and L-cysteine inhibited the enzyme competitively. Addition of hy-droxylamine had no effect on the activity.     

Chemical Selection of Mutants That Affect Alcohol Dehydrogenase in Drosophila II. Use of 1–PENTYNE–3–Ol

We describe a procedure for the selection of alcohol dehydrogenase negative mutants in Drosophila. The method consists of exposing eggs and larvae to low concentrations of 1-pentyne-3-ol dissolved in the culture medium. Only those flies with greatly reduced levels of alcohol dehydrogenase activity survive. In addition, genotypically negative flies die if their mothers are alcohol dehydrogenase positive. Using this procedure and formaldehyde to generate mutants, we were able to detect seven alcohol dehydrogenase negative mutants out of 350,000 individuals subjected to selection. At least five of the mutants contain small deletions that include the alcohol dehydrogenase locus.     

Purification and characterization of a cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3

Purification of a cis-epoxysuccinic acid hydrolase was achieved by ammonium sulfate precipitation, ionic exchange chromatography, hydrophobic interaction chromatography followed by size-exclusion chromatography. The enzyme was purified 177-fold with a yield of 14.4%. The apparent molecular mass of the enzyme was determined to be 33 kDa under denaturing conditions. The optimum pH for enzyme activity was 7.0, and the enzyme exhibited maximum activity at about 45 °C in 50 mM sodium phosphate buffer (pH 7.5). EDTA and o-phenanthrolin inhibited the enzyme activity remarkably, suggesting that the enzyme needs some metal cation to maintain its activity. Results of inductively coupled plasma mass spectrometry analysis indicated that the cis-epoxysuccinic acid hydrolase needs Zn²⁺ as a cofactor. Eight amino acids sequenced from the N-terminal region of the cis-epoxysuccinic acid hydrolase showed the same sequence as the N-terminal region of the beta subunit of the cis-epoxysuccinic acid hydrolase obtained from Alcaligenes sp.     

β-1, 3-Glucanase Produced by Alkalophilic Bacteria Bacillus No. K-12-5

Bacillus No. K–12–5 isolated from soil produced a β-1,3-glucanase in alkaline media. The characteristic point of this bacteria was especially good growth in alkaline media, and no growth was observed in neutral media such as nutrient broth. The β-1,3-glucanase of Bacillus No. K–12–5 was purified by DEAE-cellulose, Sephadex G–100 and hydroxyl apatite columns. The enzyme was most active at pH 5.5 ~ 8.0 which was much broader and higher than those of Bacillus criculans enzyme. The sedimentation constant was about 3.6 and molecular weight was about 40,000. The isoelectric point was about pH 3.5 and the enzyme was most stable at pH 7. Calcium ion was not effective to stabilize the enzyme. The enzyme did not hydrolyse laminaritriose. Laminaritetraose was hydrolysed, and glucose and laminaritriose were detected in the hydrolysate. The enzyme split laminaran at random and yielded glucose, laminaribiose, laminaritriose and higher oligosaccharides. If the enzyme is a single entity, it is a type of endo-β-1,3-glucanase. However, activity of hydrolysis of fungal cell walls was lower than that of B. circulans enzyme.     

Colonization and Biodegradation of Photo-Oxidized Low-Density Polyethylene (LDPE) by New Strains of Aspergillus sp. and Lysinibacillus sp.

The primary objective of this study was the isolation of low-density polyethylene (LDPE)-degrading microorganisms. Soil samples were obtained from an aged municipal landfill in Tehran, Iran, and enrichment culture procedures were performed using LDPE films and powder. Screening steps were conducted using linear paraffin, liquid ethylene oligomer, and LDPE powder as the sole source of carbon. Two landfill-source isolates, identified as Lysinibacillus xylanilyticus XDB9 (T) strain S7-10F and Aspergillus niger strain F1-16S, were selected as super strains. Photo-oxidation (25 days under ultraviolet [UV] irradiation) was used as a pretreatment of the LDPE samples without pro-oxidant additives. The PE biodegradation process was performed for 56 days in a liquid mineral medium using UV-irradiated pure LDPE films without pro-oxidant additives in the presence of the bacterial isolate, the fungal isolate, and the mixture of the two isolates. The process was monitored by measuring the fungal biomass, the bacterial growth, and the pH of the medium. During the process, the fungal biomass and the bacterial growth increased, and the pH of the medium decreased, which suggests the utilization of the preoxidized PE by the selected isolates as the sole source of carbon. Carbonyl and double bond indices exhibited the highest amount of decrement and increment, respectively, in the presence of the fungal isolate, and the lowest indices were obtained from the treatment of a mixture of both fungal and bacterial isolates. Fourier transform infrared (FT-IR), x-ray diffraction (XRD), and scanning electron microscopy (SEM) analyses showed that the selected isolates modified and colonized preoxidized pure LDPE films without pro-oxidant additives.     

LC–MS-based chemotaxonomic classification of wild-type Lespedeza sp. and its correlation with genotype

In this study, 39 specimens belonging to Lespedeza species (Lespedeza cyrtobotrya, L. bicolor, L. maximowiczii, and Lespedeza cuneata) (Leguminosae) were classified phenotypically and genotypically. We constructed a phylogenetic tree based on the combined nrDNA (internal transcribed spacer; ITS) and cpDNA (trnL-trnF) sequences with the aim of classifying the genotypes. Samples were mainly divided into three genotypes. Samples of L. cyrtobotrya and L. bicolor were mixed in a single branch, whereas samples of L. maximowiczii and L. cuneata were clustered within species, respectively. We performed a liquid chromatography–electrospray ionization–mass spectrometry-based metabolite profiling analysis to classify the phenotypes. Multivariate statistical analyses such as principal component analysis (PCA) and hierarchical clustering analysis (HCA) were used for the clustering pattern analysis and distance analysis between species, respectively. According to the PCA and HCA results, leaves were classified into four phenotypes according to species. In both the genetic and chemotaxonomic classification methods, the distance between L. cyrtobotrya and L. bicolor was the closest between species, and L. cuneata was the farthest away from the other three species. Additionally, orthogonal partial least squares-discriminant analysis was employed to identify significantly different phytochemicals between species. We classified L. cyrtobotrya and L. bicolor by identifying significantly different phytochemicals. Interestingly, leaves and stems showed different phenotypic classifications based on the chemotaxonomic classification. Stem samples of the other three species were mixed regardless of species, whereas L. cyrtobotrya stem samples were clustered within species. The phenotypic classification of leaves coincided more with the genotypic classification than that of stems. Key message We classified four wild-type Lespedeza sp. by analyzing the combined nrDNA (ITS) and cpDNA (trnL-trnF) sequences. We also classified leaves and stems of Lespedeza sp. by applying liquid chromatography–mass spectroscopy-based metabolite profiling.