Agar degradation by microorganisms and agar-degrading enzymes

Agar is a mixture of heterogeneous galactans, mainly composed of 3,6-anhydro-l-galactoses (or l-galactose-6-sulfates) d-galactoses and l-galactoses (routinely in the forms of 3,6-anhydro-l-galactoses or l-galactose-6-sulfates) alternately linked by β-(1,4) and α-(1,3) linkages. It is a major component of the cell walls of red algae and has been used in a variety of laboratory and industrial applications, owing to its jellifying properties. Many microorganisms that can hydrolyze and metabolize agar as a carbon and energy source have been identified in seawater and marine sediments. Agarolytic microorganisms commonly produce agarases, which catalyze the hydrolysis of agar. Numerous agarases have been identified in microorganisms of various genera. They are classified according to their cleavage pattern into three types—α-agarase, β-agarase, and β-porphyranase. Although, in a broad sense, many other agarases are involved in complete hydrolysis of agar, most of those identified are β-agarases. In this article we review agarolytic microorganisms and their agar-hydrolyzing systems, covering β-agarases as well as α-agarases, α-neoagarobiose hydrolases, and β-porphyranases, with emphasis on the recent discoveries. We also present an overview of the biochemical and structural characteristics of the various types of agarases. Further, we summarize and compare the agar-hydrolyzing systems of two specific microorganisms: Gram-negative Saccharophagus degradans 2–40 and Gram-positive Streptomyces coelicolor A3(2). We conclude with a brief discussion of the importance of agarases and their possible future application in producing oligosaccharides with various nutraceutical activities and in sustainably generating stock chemicals for biorefinement and bioenergy.     

Purification and immunochemical characterization of ascitic fluid glycoproteins containing certain tumor-associated and blood group antigen markers

Ascitic fluids from patients with various types of cancer were screened for the CA 19-9 and CA 125 tumor-associated antigenic activities. Two fluids exhibiting the highest activities were tested for their binding to various lectin-Sepharose columns resulting in both being bound best to wheat germ agglutinin (WGA) Sepharose. The WGA column eluate of one fluid was further chromatographed by HPLC and three peaks were obtained with approximate molecular weights of 3.65 MDa, 664 kDa and 330 kDa, of which only the largest fraction contained the CA 19-9 activity. The fluids were also fractionated on a Sephacryl S-400 column with most of the activity being present in or near the void volume.Monoclonal antibodies were used to demonstrate that the purified glycoproteins also contained the blood group A determinant, the four Lewis determinants Le^a, Le^b, Le^x and Le^y, and the sialylated-Le^x determinant, while other antibody analyses failed to detect other blood group and/or carbohydrate sequence determinants. Some of the blood group expressions could be separated from the CA 19-9 and CA 125 active glycoproteins by adsorption with various lectins other than the WGA.Abbreviations used NeuAc N-acetyl-D-neuraminic acid - Gal galactose,D-galactopyranose - Fuc fucose,L-fucopyranose - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine - WGA wheat germ agglutinin - PBS phosphate buffered saline     

Lack of association between the Gc and Lp serum type systems

Two earlier reports (Mohr and Berg, 1963; Berg and Wendt, 1964) have indicated the possibility of an association between the Gc and Lp serum type systems. In the present study the Gc and Lp types have been determined in a material of 796 Norwegian blood donor sera. A X ₂-test failed to verify a statistically significant association between the factors of the two systems.     

Fermentative production of branched chain amino acids: a focus on metabolic engineering

The branched chain amino acids (BCAAs), l-valine, l-leucine, and l-isoleucine, have recently been attracting much attention as their potential to be applied in various fields, including animal feed additive, cosmetics, and pharmaceuticals, increased. Strategies for developing microbial strains efficiently producing BCAAs are now in transition toward systems metabolic engineering from random mutagenesis. The metabolism and regulatory circuits of BCAA biosynthesis need to be thoroughly understood for designing system-wide metabolic engineering strategies. Here we review the current knowledge on BCAAs including their biosynthetic pathways, regulations, and export and transport systems. Recent advances in the development of BCAA production strains are also reviewed with a particular focus on l-valine production strain. At the end, the general strategies for developing BCAA overproducers by systems metabolic engineering are suggested.     

Effect of organic cosolvent on kinetic resolution of tert-leucine by penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) from Kluyvera citrophila immobilized on Amberzyml was used for enantioselective hydrolysis of N-phenylacetylated-dl-tert-leucine (N-Phac-dl-Tle) to produce l-tert-leucine (l-Tle). The effects of various organic cosolvents on hydrolysis of N-Phac-dl-Tle have been investigated in aqueous-cosolvent medium. It was founded that the rate of PGA-catalyzed reaction was significantly affected by the presence of 2% (v/v) organic cosolvent concentration. The initial rate fell with increasing logP of the cosolvent, but for logP values less than −0.24 the rate was faster than in purely aqueous medium. Additionally, the relative rate increases with the increase of dielectric constant (ε) of organic cosolvents. The yields of l-Tle in all aqueous-cosolvent systems were above 95% with the enantiomeric excess (ee) of >99%.     

Microbial l-methioninase: production, molecular characterization, and therapeutic applications

l-Methioninase is ubiquitous in all organisms except in mammals. It mainly catalyzes the, α, γ-elimination of l-methionine to α-ketobutyrate, methanethiol, and ammonia. Unlike normal cells, methionine dependency was reported as a biochemical phenomenon among various types of cancer cells. Thus, l-methioninase is the universal protocol for triggering the majority of tumor cells. This review is an attempt to briefly describe the occurrence of the biochemical and molecular properties of l-methioninase by a comparative manner to the eukaryotic and prokaryotic source for the maximum exploitation in the therapeutic field. The combination of l-methioninase treatment, gene therapy, and chemotherapeutic drugs clearly explores the various therapeutic aspects of this enzyme. Finally, the perspectives for increasing the therapeutic efficacy of this enzyme were described.     

Na+-independent l-alanine uptake by trout cells. Evidence for the existence of at least two functionally different asc systems

The Na⁺-independent uptake of l-alanine has been studied in trout red blood cells, isolated hepatocytes and peripheral blood lymphocytes. The present study shows the existence of two functionally different Na⁺-independent systems for short chain neutral amino acids in these cells. They are designated as asc systems because of their resemblance to systems described in other cell types. Besides their independence of sodium and a rough similarity in substrate preference, the most important property shared by the two carriers is a lack of trans-stimulation, allowing further differentiation from system L. One of them is an unusually stereospecific carrier present in red blood cells, the other is less restrictive and present in hepatocytes and peripheral blood lymphocytes. Extracellular acid pH increases the incorporation to red blood cells, while it slightly depresses the uptake in the other cells. From the data presented, it is not possible, at first, to classify these carriers as asc ₁ or asc ₂ systems. Moreover, the system present in red cells resembles that found in the nonerythroid cells, BSC-1, while there is no clear parallelism between the system found in hepatocytes/lymphocytes and any of those described previously.This work was supported in part by a grant from the DGICYT (PB91-0235) of the Spanish Government and by a grant from the CIRIT (AR91-21) of the Generalitat de Catalunya. M.A.G. is a recipient of a fellowship from the Generalitat de Catalunya. We would like to express our sincere thanks to Mrs. Rosa Marsol and Mr. Antonino Clemente (Medi Natural, Generalitat de Catalunya) for their help and logistical assistance and to Mr. Robin Rycroft for his editorial help. J.L. Albi, P. Canals and M.A. Gallardo contributed equally to this study.     

Serological researches in the south of Moldavia in connection with the problem of the ethnogeny of the Gagauzes, the Moldavians and the Bulgarians

With the aid of data of frequencies of genetically determined blood group systems, the authors have tried to show the basic ethno-genetic directional patterns in Southern Moldavia and in the Dniester-Carpathian-Danubian region in its entirety. Blood Groups A1A27B0, RHESUS, MN, and KELL have been determined in six random samples from four Gagauz villages (n = 330), one Moldavian village (n = 101) and one Bulgarian village (n = 96). The analysis of gene frequencies demonstrates genetic homogeneity of the total Gagauz population. Statistically reliable differentiation is observed only for the RHESUS system. It is possible now to suppose that the haemotological types of modern Gagauz and Bulgarian populations have been developed on the basis of the Balcanic serogenetic types, partially transformed under influence of gene flow from probably Central Asian or other eastern centers. The position of the Moldavians on the serogenetic map of Europe is less certain, due to a considerable ambiguity in the allele frequencies of the various blood group systems. Founder effects may account for these observations. However, the genetic distance and cluster analyses carried out on the frequencies of the surveyed blood group systems have shown the affinity of Moldavians with the Romanians and Eastern European populations.     

A study of the genus leuconostoc

Summary Leuconostocs from herbage and grass silage were compared with strains from culture collections. The organisms were divided into sections, on ability to form detectable amounts of H₂O₂ from glucose and dextran from sucrose, and into groups by various tests of which the most useful were catalase activity, ability to grow at various temperatures, manner of growth in glucose soft agar and action on pentoses. The satisfactory division of all these organisms into species on ability to ferment pentoses and sucrose and to form dextran from sucrose, the basis of Hucker and Pederson's (1931) classification, was not possible. No useful alternative scheme was apparent. The preference of one group of organisms for aerobic conditions coupled with their ability to form catalase when provided with haem compounds, suggests that leuconostocs may have evolved from organisms possessing a respiratory pathway.     

Die Korrelation von Blutgruppenantigenen und-substanzen mit hypertonischer Erkrankung

We have investigated in 383 hypertonics, 151 males and 232 females, the AB0 blood groups, secretion of blood group substances AB0 (H), Rh₀ (D) factor, M and N groups and haptoglobin groups in the serum.Most patients were 40–50 years old and older, suffering from a fixed arterial hypertension with a diastolic blood pressure not lower than 110 mm Hg. All patients with renal, matabolic or nervous disorders were excluded from our series.In comparison with normal values in Czechoslovak population a significantly lower percentage was ascertained of nonsecretors of group substances in both sexes of B group hypertonics. In 72 persons of group B there were 8 nonsecretors i.e. 11.11% as compared with 37.84% nonsecretors in normal population; for 1 degree of freedom P < 0.001, for 4 degrees of freedom P < 0.05.Furthermore, in male hypertonics a preponderance of 0 over A, in female hypertonics that of A over 0 were ascertained. There were 39.07% male hypertonics of blood group 0 in comparison with 33.25% of group 0 in normal population and 28.88% in female hypertonics.In males hypertonics the blood group A appeared in 33.11%, the normal values being 40.79% and the incidence in females hypertonics being 45.26%.These differences achieved statistically significant values only in mutual comparison of the male and female group of hypertonics.In distribution of characters M/N and haptoglobin types no deviations from normal values were observed.

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Technische Mitarbeit: B. Knedlhansová und J. Benetková     

Phage typing ofSalmonella Typhi in the Netherlands

Summary A survey has been given of the results of phage typing of strains ofS. typhi found in Holland. It has been shown, that type A includes a different group of strains in systems drawn up with different Vi phages. An auxiliary system — to be used besides the system ofCraigie andYen — and a few new types, have been described.     

AB0 blood groups and the type of leprosy in an Indian population

In 931 leprosy patients from Bankura, West Bengal, India, the relationship between the AB0 blood group and the immunological type of leprosy (lepromatous vs. nonlepromatous) was examined. Among the 472 lepromatous patients, there was a slight excess of blood group A (and also AB and B) as compared with the nonlepromatous group. This excess was not significant statistically in our material. However, combination of the data with those published by Beiguelman (1964) from Brazil and Yankah (1965) from Ghana renders a significantly higher incidence of group A (and probably B and AB) in lepromatous patients.The examinations were supported by WHO (Grant No. G 3/181/15), by the Deutsche Forschungsgemeinschaft and by the Indian Statistical Institute.     

Entomophages et maladies des insectes

Summary Without discussing the problem of the particular role of Epizootics in natural populations, the author examines the different types of relations existing between entomophagous insects and diseases of their hosts. The consequence for parasites and predators of a possible use of pathogenes as a method of Biological Control of pests is examined with more detail in the case ofP. brassicae L. submitted to sprays with a suspension of spores ofB. thuringiensis Berliner. It is shown that two hymenopterous parasites (Apanteles glomeratus L. Anilastus ebeninus Grav). can survive the death of their host, under certain circumstances. The survival ofA. glomeralus is conditionned by its being in the last larval instar on the day of treatment. The consequence of the disease of the host caterpillar is a shortening of the endoparasitic life of the Braconid. A similar effect can be observed whenP. brassicae caterpillars are treated with a contact insecticide, but in this case,Apanteles larvae are killed by residues on their emergence from the host. There is no direct effect of spore suspensions on the larvae of the parasite. It seems thus possible to adopt bacterial control measures preserving a part of the entomophagous populations.

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I.N.R.A., Laboratoire de lutte biologique de La Minière.     

Fermentation of seaweed sugars by Lactobacillus species and the potential of seaweed as a biomass feedstock

It is known that seaweeds differ greatly from land plants in their sugar composition. The current research on the L-lactic acid fermentation process focuses on land plant sugars as a carbon source, with the potential of seaweed sugars being largely ignored. This study examined the feasibility of seaweed biomass as a possible carbon source for the production of l-lactic acid, by comparing the fermentation of seaweed sugars (d-galactose, d-mannitol, l-rhamnose, d-glucuronic acid, and l-fucose) and land plant sugars (d-glucose, d-xylose, d-mannose, and l-arabinose). The experiments were repeated with 2 sugar acids (d-gluconic acid, d-glucaric acid) in order to investigate the effect of the degree of reduction of carbon source on the fermentation yield. This research also examined the effect of bacterial strain on the characteristics of fermentation reactions, by conducting l-lactic acid fermentation with 7 different Lactobacillus species. Taking into account the sugar composition of seaweed and the levels of lactic acid production from each pure sugar, it was possible to predict the lactic acid production yield of various seaweeds and land plants. From comparative analysis of the predicted lactic acid production yield, it was found that seaweeds are already comparable to lignocellulosics at the current stage of technology. If new technologies for the utilization of non-fermentable seaweed sugars are developed, seaweeds show promise as an even more useful biomass feedstock than lignocellulosics.     

NMDA Receptor Regulation by <Emphasis Type="SmallCaps">D</Emphasis>-serine: New Findings and Perspectives

The N-methyl-d-aspartate (NMDA) receptors play key roles in excitatory neurotransmission and are involved in several important processes, including learning, behavior, and synaptic plasticity. The regulation of NMDA receptor neurotransmission has been extensively studied, but many important questions still remain unsolved. One of the most debated aspects of the NMDA receptor regulation relates to the identity, role, and cellular origin of the NMDA coagonist(s). In addition to glutamate, the NMDA receptor activity was believed to be regulated by the coagonist glycine. More recently, d-serine has also been proposed to play a role as a key coagonist for NMDA receptor activity and neurotoxicity. A surprising unique biosynthetic pathway for d-serine has been demonstrated, indicating the conservation of d-amino acid metabolism in mammals. d-Serine was originally shown to be exclusively made in astrocytes, indicating a possible role as a gliotransmitter. Nevertheless, recent data indicate that d-serine has a neuronal origin as well, which raises several new questions on d-serine disposition. In this review, I discuss recent advances in the field and propose a novel model of d-serine signaling that includes a bidirectional flow of d-serine between astrocytes and neurons. This review is dedicated to the memory of Dr. Marcos Wolosker.     

<Emphasis Type="SmallCaps">l</Emphasis>-Aspartate dehydrogenase: features and applications

l-Amino acid dehydrogenases are a group of enzymes that catalyze the reversible oxidative deamination of l-amino acids to their corresponding 2-oxoacids, using either nicotinamide adenine dinucleotide (NAD⁺) or nicotinamide adenine dinucleotide phosphate (NADP⁺) as cofactors. These enzymes have been studied widely because of their potential applications in the synthesis of amino acids for use in production of pharmaceutical peptides, herbicides and insecticides, in biosensors or diagnostic kits, and development of coenzyme regeneration systems for industrial processes. This article presents a review of the currently available data about the recently discovered amino acid dehydrogenase superfamily member l-aspartate dehydrogenase (l-AspDH), their relevant catalytic properties and speculated physiological roles, and potential for biotechnological applications. The proposed classification of l-AspDH on the basis of bioinformatic information and potential role in vivo into NadB (NAD biosynthesis-related) and non-NadB type is unique. In particular, the mesophilic non-NadB type l-AspDH is a novel group of amino acid dehydrogenases with great promise as potential industrial biocatalysts owing to their relatively high catalytic properties at room temperature. Considering that only a few l-AspDH homologs have been characterized so far, identification and prodigious enzymological research of the new members will be necessary to shed light on the gray areas pertaining to these enzymes.     

Purification and characterization of a lectin,BPL-3, from the marine green alga <Emphasis Type="Italic">Bryopsis plumosa</Emphasis>

A novel lectin was isolated and characterized from Bryopsis plumosa (Hudson) Agardh and named BPL-3. This lectin showed specificity to N-acetyl-d-galactosamine as well as N-acetyl-d-glucosamine and agglutinated human erythrocytes of all blood types, showing slight preference to the type A. SDS-PAGE and MALDI-TOF MS data showed that BPL-3 was a monomeric protein with molecular weight of 11.5 kDa. BPL-3 was a non-glycoprotein with pI value of ∼7.0. It was stable in high temperatures up to 70°C and exhibited optimum activity in pH 5.5–10. The N-terminal and internal amino acid sequences of the lectin were determined by Edman degradation and enzymatic digestion, which showed no sequence homology to any other reported proteins. The full sequence of the cDNA encoding this lectin was obtained from PCR using cDNA library, and the degenerate primers were designed from the N-terminal amino acid sequence. The size of the cDNA was 622 bp containing single ORF encoding the lectin precursor. This lectin showed the same sugar specificity to previously reported lectin, Bryohealin, involved in protoplast regeneration of B. plumosa. However, the amino acid sequences of the two lectins were completely different. The homology analysis of the full cDNA sequence of BPL-3 showed that it might belong to H lectin group, which was originally isolated from Roman snails.     

Some observations on the fine structure of the synaptic area in the ciliary ganglion of the chick

Summary The fine structure of the synaptic area in the ciliary ganglion from 10-days chick embryo to the adult was studied by electron microscopy.The ciliary ganglion cell is unipolar and a considerable surface of which is covered by the calyx terminal. The peripheral part of the calyx divides into several terminal knobs and form a basket terminal.Four types of contact configurations were observed in the calyciform ending: 1) contact area without membrane specialization which occupies the most part of the contact, 2) desmo-some-like structure which is observed in various places of the contact surface, 3) synaptic complex and 4) close apposition of apposing plasma membranes.The presence of the synaptic complex and the close appositon of apposed plasma membranes seems to correspond to the dual natures of the transmission obtained by Martin and Pilar (1963a, b).In addition, some considerations were made on the subsurface cistern and on the possible functional significance of the myelin sheath surrounding the ganglion cell and the calyx.This work was supported in part by Grant NB-03348-03 from the National Institutes of Health, United States Public Health Service.Dr. Takahashi wishes to express his sincere thanks to Professor S. Watanabe, Department of Anatomy, Sapporo Medical College, who offered an opportunity for doing work at the Department of Anatomy, Hiroshima University.     

Fine structure of the Paraventricular organ of Xenopus laevis tadpoles

Summary The ultrastructure of the Paraventricular organ in the hypothalamus of Xenopus laevis tadpoles is described. It appeares that the Paraventricular organ of this anuran species is homologous with the Organon vasculosum hypothalami or the Paraventricular organ of other vertebrates.The Paraventricular organ of Xenopus laevis is characterized by an ependymal lining with only few cilia and by two types of nerve cells. Both types of nerve cells have ventricular processes, protruding into the lumen of the third ventricle and forming a network. The protrusions bear cilia of the 8+1 pattern. It has been possible to distinguish both types of nerve cells on account of their dense-core vesicles. A secretory function of both cell types is suggested.In a region close to the Paraventricular organ, another granulated type of nerve cell has been observed. A relationship between these cells and the preoptic nucleus is discussed.The author thanks Prof. Dr. P. G. W. J. van Oordt for his helpful comments and criticism, Mr. H. van Kooten for photographic assistance and Mr. F. Dijk for technical assistance.     

Microencapsulated bacterial cells can be used to produce the enzyme feruloyl esterase: preparation and in-vitro analysis

Biotechnological production of ferulic acid, a precursor of vanillin, is an attractive alternative for various industries due to the high price and demand for natural ferulic acid. Feruloyl esterase has been identified as a key enzyme involved in microbial transformations of ferulic acid to vanillin. Several fungal feruloyl esterases have been purified and characterized for their use in the production of ferulic acid. This paper, for the first time, discusses the use of lactic acid bacteria for the production of ferulic acid. Specifically, we have used Lactobacillus cells and microencapsulation so that ferulic acid can be produced continuously using various types of fermentation systems. Bacteria were encapsulated in alginate-poly-l-lysine-alginate (APA) microcapsules, and the production of ferulic acid by lactobacilli was detected using a real-time high-performance liquid chromatography (HPLC)-based assay. Results show that ferulic acid can be produced using microencapsulated Lactobacillus fermentum (ATCC 11976) with significant levels of biological feruloyl esterase activity.