Purification and characterization of the catabolic α-acetolactate synthase from Leuconostoc mesenteroides subsp. cremoris

The -acetolactate synthase from Leuconostoc mesenteroides subsp. cremoris was purified to homogeneity in SDS-PAGE. The enzyme is a trimer of 3×55,000 Da. It was unstable but could be preserved by addition of pyruvate and thiamine pyrophosphate in the buffer. The enzyme exhibits Michaelis-Menten kinetics, and K _m for pyruvate is 10 mM. Three intermediates in glucose metabolism (ATP, 3-phosphoglycerate, and phosphoenolpyruvate) exhibit a noncompetitive inhibition towards the enzyme. This enzyme does not require any divalent metal ion for activity. The -acetolactate synthase from Leuconostoc mesenteroides subsp. cremoris is not inhibited by the branched-chain amino acids (valine, leucine, and isoleucine), is FAD independent, and displays an optimal activity at pH 5.3. Therefore, it can be concluded that the purified enzyme belongs to the catabolic -acetolactate synthases, involved in the 2,3-butanediol pathway but not in branchedchain amino acids biosynthesis.     

Quantification of extracellular α-acetolactate oxidative decarboxylation in diacetyl production by an α-acetolactate overproducing strain of Lactococcus lactis sp. lactis bv. diacetylactis

The quantification of -acetolactate (AAL) extracellular oxidative decarboxylation during an AAL overproducing strain culture shows that this reaction is at the origin of about 90% of the diacetyl production and that only a small proportion of extracellular AAL is readily transformed to diacetyl. These results, compared with previous ones obtained with a non AAL accumulating strain, allow research options to be put forward for the improvement of microbiological diacetyl production.     

Characterization of 2,3-butanediol-forming and valine-sensitive α-acetolactate synthase of Enterobacter cloacae

-Acetolactate synthase (-ALS) of Enterobacter cloacae ATCC 27613 was purified to homogeneity by ammonium sulphate precipitation, Sephadex G-200 gel filtration and hydroxyapatite affinity chromatography. The molecular weight of the enzyme was found to be 60 kDa by SDS–polyacrylamide gel electrophoresis and 200 kDa by gel filtration through Sephadex G-200, showing that the enzyme is a homotrimer. The K _m and V _max of the enzyme were 20 mM and 200 mol min^–1 mg (protein)^–1 respectively. The enzyme was optimally active at pH 6.0–8.0, 37 °C and showed concentration-dependent sensitivity to cofactors viz. FAD, NADP and NADPH and branched chain amino acids: leucine, isoleucine and valine. Substances like sodium formate, sodium acetate and sodium propionate, sugars and the selected intermediates of glycolytic pathway inhibited the enzyme. Glycerol, BSA and pyruvate-TPP stabilized the -ALS. The enzyme showed the properties of both a catabolic as well as an anabolic -ALS.     

Kinetic study of the chemical reactivity of α-acetolactate as a function of pH in water, and in fresh and fermented culture media used for Lactococcus lactis spp. lactis bv. diacetylactis cultivation

Summary Spontaneous oxidative decarboxylation of -acetolactic acid (ALA) to diacetyl has been assessed under anaerobiosis as a function of pH in water, and in fresh and filtered Lactococcus lactis spp. lactis bv. diacetylactis SD 933 fermented culture media. Whatever the reaction medium, ALA was shown to be potentially reactive, depending on the pH of medium. Diacetyl production mechanism by this strain is discussed on the basis of these kinetic data.     

Nucleoids and coated vesicles of “Epulopiscium” spp.

We describe here aspects of the anatomy of two “Epulopiscium” morphotypes, unusually large bacteria that are not yet cultured and that reproduce by the internal generation of two or more vegetative daughter cells. Two morphotypes, A and B, which are enteric symbionts of several species of herbivorous surgeonfish (Acanthuridae), were collected around the Great Barrier Reef of Australia, preserved there, and later stained for light microscopy. Some samples were examined by electron microscopy. In both morphotypes, countless discrete nucleoplasms or nucleoids were found to occupy a single shallow layer just beneath the surface all around these organisms. At each end of the morphotype B cells, a membrane-bound compartment containing dense cords of chromatin was observed. When these were found at each end of growing daughter cells, no polar compartments were then found in their mother organism. Electron micrographs of sections of morphotype A symbionts show that their outermost region is composed of tightly packed coated vesicles, each surrounded by a thin, dense, spacious capsule. Near the surface of type A organisms the remains of broken vesicles, broken capsules, and a finely fibrous matrix fuse to form a fabric that serves as the cell wall. Morphotype B organisms, however, were observed to have a distinct, morphologically continuous outer wall. Received: 3 December 1997 / Accepted: 11 June 1998     

Occurrence of α-acetolactate decarboxylases among lactic acid bacteria and their utilization for maturation of beer

Summary Acetolactate decarboxylase activity has been detected among three genera, nine species and 263 strains of lactic acid bacteria tested in the course of a screening for acetolactate decarboxylases amenable for use in brewing as maturation aid. Streptococcus diacetylactis strain FD-64-D was found to generate a decarboxylase exhibiting a satisfactory activity and an excellent stability at the pH prevailing in beer and wort. This decarboxylase could not be solubilized but enzymatically active, freeze-dried cells were effective for satisfactory flavour maturation of beer although difficulties were encountered during attempts to remove the applied cell material by filtration of the beer. Lactobacillus casei DSM 2547 was likewise found to produce a decarboxylase exhibiting a satisfactory activity and stability at the low pH of beer and which, in addition, was readily solubilized. A method has been developed for pilot scale production of preparations of this decarboxylase suitable for use in brewing.Abbreviations DSM Deutsche Sammlung von Microorganismen - EDTA Ethylene diaminetetra-acetic acid     

Molecular cloning of the gene encoding α-acetolactate decarboxylase from Enterobacter aerogenes

Enterobacter aerogenes genomic library has been constructed using cosmid pJB8 in Escherichia coli. The gene encoding α-acetolactate decarboxylase (ALDC) has been isolated from this library by direct measurement of enzyme activity. The expression of the ALDC gene in E. coli appears to originate from the own promoter. Subsequent subcloning revealed that the ALDC gene locates within 1.7 kb BamHI-PstI fragment.     

Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate

With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.     

Production and localisation of carboxymethylcellulase,xylanase and β-glucosidase fromCellulomonas andMicrococcus spp.

Summary Cellulomonas and Micrococcus spp. grew well at 30°C, pH 7.0, and produced carboxymethylcellulase (CMCase) and xylanase enzymes. Only one species of Micrococcus was able to produce an appreciable amount of -glucosidase. This is the first report where Micrococcus sp., isolated from termite gut, was able to produce all three enzymes (i.e. CMCase, xylanase and -glucosidase) required for degradation of cellulosic and hemicellulosic substrates. Offprint requests to: A. Varma     

Variation analysis of pathogenic Vibrio spp. and Pseudomonas spp. in Changhai mollusc farming waters using real-time PCR assay during 2011–2014

Bacterial disease has caused high mortality of breeding molluscs from 2009 to 2011 in the Changhai area (Dalian, China). Vibrio spp. and Pseudomonas spp. have been detected as major pathogenic agents for aquatic animals in this area. In the present study, four virulence genes including vsm, toxR, aprX and carA were targeted to develop a real-time PCR assay for the quantitative detection of Vibrio splendidus, V. parahaemolyticus, Pseudomonas fluorescens and P. putida, respectively. The sensitivity and specificity of the assays were verified by experimental samples, and the variation tendencies of pathogenic V. splendidus, V. parahaemolyticus, P. fluorescens and P. putida strains from June to September were also detected in mollusc farming waters during 2011–2014. The concentration of V. splendidus increased from June to July, reduced in August, and then increased again in September. The highest count of V. parahaemolyticus appeared in July, and then dramatically decreased from August to September. Conversely, the counts of P. fluorescens and P. putida remained at lower levels from June to August, and then dramatically peaked in September. All four pathogenic bacteria displayed similar fluctuation tendencies of count variation in each year, and their concentrations were found to have a correlation with the average annual temperature. The variation tendency of pathogenic bacteria with temperature suggested that temperature was one of the most important factors to regulate the bacterial growth in a farming area, which could further provide information for the early warning of disease outbreak by using a convenient real-time PCR assay.     

Production of β-glycosidases (linamarase and amygdalase) and pectolytic enzymes by Penicillium spp.

Fifty-eight strains, representing 31 species of Penicillium, were screened for extracellular -glycosidase (amygdalase/linamarase) and pectolytic (polygalacturonase, pectin lyase) enzymes. One strain each of P. turbatum, P. piceum and P. paxilli showed very high -glycosidase activity and slightly lower activities were found in P. crustosum, P. expansum, P. oxalicum and P. aurantiogriseum. Generally, maximum -glycosidase activity showed reached during the stationary phase of growth. The seven species with highest -glycosidase activity showed different patterns of pectolytic activities, indicating that different species or combinations of species could be selected for different potential applications.L. Brimer is with the Department of Pharmacology and Pathobiology, Royal Veterinary & Agricultural University, 13 Bulowsvej, DK 1870 Frederiksberg C, Denmark; A.R. Cicalini and F. Federici are with the Dipartimento Agrobiologia e Agrochimica, University of Tuscia, Via S.C. de Lellis, I-01100 Viterbo, Italy. M. Petruccioli is with the Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, University of Basilicata, Via N. Sauro, 85, I-85100 Potenza, Italy.     

Fusarium spp. and Fusarium mycotoxins in maize: a problem for Flanders?

Fusarium species cause not only root, stem and ear rot with severe reductions in crop yield, they produce also toxic secondary metabolites (mycotoxins) such as deoxynivalenol (DON) and zearalenone (ZEA). During several growing seasons the presence of Fusarium spp was followed up. DON and ZEA were determined and related to infection levels. The distribution of DON and ZEA in the different plant parts was studied as well as the influence of the ensiling process on the mycotoxin content. More or less important varietal differences in susceptibility for Fusarium spp. could be detected. DON and ZEA were clearly present in most of the analysed samples. No clear relationship could be detected between visual disease symptoms and mycotoxin content. The accumulation of DON and ZEA was different for the analysed aerial plant parts. The ensiling process gave no reduction of the mycotoxin content.     

α-Pyrone and seiricuprolide derivatives from the mangrove-derived fungi Pestalotiopsis spp. PSU-MA92 and PSU-MA119

Investigation of the mangrove-derived fungi Pestalotiopsis spp. PSU-MA92 and PSU-MA119 resulted in the isolation of three new α-pyrones, pestalotiopyrones A–C (1–3), and two new seiricuprolides, pestalotioprolides A (4) and B (5), together with two known compounds. Their structures were identified by analysis of spectroscopic data. Compound 5 was isolated as its diacetate derivative (6). The antibacterial and antifungal activities of 2 were evaluated.     

Optical Resolution and Biological Activities of α-Ionylideneacetic Acid.

Nuclease activity associated with cells and protoplasts was analyzed by agarose gel electrophoresis. Datura innoxia protoplasts were found to possess a high exonuclease activity. On the other hand, Datura innoxia cells had an endonuclease activity, but no apparent exonuclease. The exonucleases from the protoplasts were active at pH 5 and 6, but not at pH 9. Endonuclease activity from the cells was also inhibited at pH 9. Cultured cells of Daucus carota, Glycine max, Pisum sativum and Vicia hajastana had endonuclease activity, but did not exhibit exonuclease activity. Nicotiana suaveolens cells had both types of nuclease activity. On the other hand, cells from cereals such as Triticum monococcum, Oryza sativa, and Zea mays had active exonuclease activity.     

Differences between α- and β-Chain Mutants of Human Haemoglobin and between α- and β-Thalassaemia. Possible Duplication of the α-Chain Gene

Human adult haemoglobin consists of two unlike pairs of polypeptide chains, and can be described as α₂β₂. Amino-acid substitutions in either of the two types of chain result in α- and β-chain variants. In thalassaemia, which causes a lowered production of haemoglobin, the α or the β chain can be affected, the result being α- or β-thalassaemia. There is a quantitative difference in the proportion of α- and β-chain variants to normal haemoglobin in the respective heterozygotes, and there is also a difference in the pattern of inheritance of α- and β-thalassaemia: these could possibly be explained by assuming that man has one gene for the β- and two for the α-chain.     

α-Acetolactate overexpression from glucose in the diacetyl producer Lactococcus lactis ssp. lactis bv. diacetylactis B2103, a natural mutant lacking α-acetolactate decarboxylase

We investigated the effects of the oxygen supply rate on the activity of pyruvate metabolic pathways and their end products, the lactatedehydrogenase (LDH), pyruvateformiatelyase (PFL), pyruvatedehydrogenase (PDH) and acetolactatesynthase (ALS) pathways, in the Lactococcus lactis ssp. lactis bv. diacetylactis strain B2103/74. We found that this culture, apart from inactivated α-acetyldecarboxylase, also possesses a unique natural capacity to overexpress α-acetolactate (AL) up to 25–28 mM. Our search for similar properties among the diacetilicus bv. strains showed that this ability is quite rare. We identified a single additional strain, 7590 from the National Russian Collection of Industrial Microorganisms (NRCIM-7590), which displayed a similar capacity. However, unlike B2103/74, NRCIM-7590 has an active α-acetolactate decarboxylase and therefore can only produce acetoin. AL overexpression took place under conditions of intense aeration (K _L a ≥ 90–120 h^?1), and the composition of the medium played a decisive role in AL productivity. We found that AL overproduction is determined by a diversion of a portion of pyruvate flow from the LDH to the PDH and ALS pathways. We further found that all additional pyruvate, supplied from LDH, is utilized exclusively by the ALS pathway because of the restricted capacity of the PDH pathway. This shift in pyruvate metabolism in the B2103/74 strain, from LDH to PDH and ALS pathways, is associated with the initiation of an oxidation reaction that reduces oxygen to H₂O and sequesters NADH from the LDH pathway in the process. A specific manifestation of this reaction in B2103/74 and NRCIM-7590 cultures, which results in a profound shift of the pyruvate metabolism towards the production of α-acetolactate, is due to the function of a potent oxidative system that shifts 75–80% of NADH flow from LDH to the oxidative pathway, resulting in the regeneration of NAD⁺. The nature of this oxidative system is not known. Based on our studies, we propose that the structure of the newly discovered oxidative system is similar to a simple transmembrane electron transport chain.     

New polyhydroxytriterpenoid derivatives from fruits of Terminalia chebula Retz. and their α-glucosidase and α-amylase inhibitory activity

Three new polyhydroxytriterpenoid derivatives, 23-O-neochebuloylarjungenin 28-O-β-d-glycopyranosyl ester (1), 23-O-4′-epi-neochebuloylarjungenin (2), and 23-O-galloylpinfaenoic acid 28-O-β-d-glucopyranosyl ester (17) were isolated from the fruits of Terminalia chebula Retz. along with fourteen known ones. Their structures were elucidated by 1D and 2D NMR spectroscopic data and acid hydrolysis. After evaluating for Baker’s yeast α-glucosidase, rat intestinal α-glucosidase, and porcine pancreatic α-amylase inhibitory activities of all the isolated compounds, 23-O-galloylarjunolic acid (11, IC₅₀ 21.7 μM) and 23-O-galloylarjunolic acid 28-O-β-d-glucopyranosyl ester (12, IC₅₀ 64.2 μM) showed potent inhibitory activities against Baker’s yeast α-glucosidase compared to the positive control, acarbose (IC₅₀ 174.0 μM). However, all the tested compounds except for the positive control, acarbose, had no or only weak inhibitory activity against rat intestinal α-glucosidase and porcine pancreatic α-amylase.