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Lactic acid bacteria (LAB) are known to produce large amounts of α-glucan exopolysaccharides. Family GH70 glucansucrase (GS) enzymes catalyze the synthesis of these α-glucans from sucrose. The elucidation of the crystal structures of representative GS enzymes has advanced our understanding of their reaction mechanism, especially structural features determining their linkage specificity. In addition, with the increase of genome sequencing, more and more GS enzymes are identified and characterized. Together, such knowledge may promote the synthesis of α-glucans with desired structures and properties from sucrose. In the meantime, two new GH70 subfamilies (GTFB- and GTFC-like) have been identified as 4,6-α-glucanotransferases (4,6-α-GTs) that represent novel evolutionary intermediates between the family GH13 and “classical GH70 enzymes”. These enzymes are not active on sucrose; instead, they use (α1 → 4) glucans (i.e. malto-oligosaccharides and starch) as substrates to synthesize novel α-glucans by introducing linear chains of (α1 → 6) linkages. All these GH70 enzymes are very interesting biocatalysts and hold strong potential for applications in the food, medicine and cosmetic industries. In this review, we summarize the microbiological distribution and the structure–function relationships of family GH70 enzymes, introduce the two newly identified GH70 subfamilies, and discuss evolutionary relationships between family GH70 and GH13 enzymes. Electronic supplementary material The online version of this article (doi:10.1007/s00018-016-2245-7) contains supplementary material, which is available to authorized users.

4-alpha-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of alpha-glucans by cleaving and reforming alpha-1,4 glycosidic bonds in alpha-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57) studied in the current work catalyzes both disproportionation and cyclization reactions. Amylose was converted into cyclic amylose (with a minimum size of 17 glucose monomers) as well as to a spectrum of maltodextrins, but in contrast to glycoside hydrolase family 13 cyclodextrin glucanotransferases (CGTases), no production of cyclodextrins (C6-C8) was observed. GTase57 also effectively produced alkyl-glycosides with long alpha-glucan chains from dodecyl-beta-D-maltoside and starch, demonstrating the potential of the enzyme to produce novel variants of surfactants. Importantly, the GTase57 has excellent thermostability with a maximal activity at 95 A degrees C and an activity half-life of 150 min at 90 A degrees C which is highly advantageous in this manufacturing process suggesting that enzymes from this relatively uncharacterized family, GH57, can be powerful biocatalysts for the production of large head group glucosides from soluble starch.

The common saprophyte Aspergillus niger may experience carbon starvation in nature as well as during industrial fermentations. Starvation survival strategies, such as conidiation or the formation of exploratory hyphae, require energy and building blocks, which may be supplied by autolysis. Glycoside hydrolases are key effectors of autolytic degradation of fungal cell walls, but knowledge on their identity and functionality is still limited. We recently identified agnB and cfcA as two genes encoding carbohydrate-active enzymes that had notably increased transcription during carbon starvation in A. niger. Here, we report the biochemical and functional characterization of these enzymes. AgnB is an α-1,3-glucanase that releases glucose from α-1,3-glucan substrates with a minimum degree of polymerization of 4. CfcA is a chitinase that releases dimers from the nonreducing end of chitin. These enzymes thus attack polymers that are found in the fungal cell wall and may have a role in autolytic fungal cell wall degradation in A. niger. Indeed, cell wall degradation during carbon starvation was reduced in the double deletion mutant ΔcfcA ΔagnB compared to the wild-type strain. Furthermore, the cell walls of the carbon-starved mycelium of the mutant contained a higher fraction of chitin or chitosan. The function of at least one of these enzymes, CfcA, therefore appears to be in the recycling of cell wall carbohydrates under carbon limiting conditions. CfcA thus may be a candidate effector for on demand cell lysis, which could be employed in industrial processes for recovery of intracellular products.

The glucansucrase GTFA of Lactobacillus reuteri 121 produces an α-glucan (reuteran) with a large amount of alternating (α1 → 4) and (α1 → 6) linkages. The mechanism of alternating linkage formation by this reuteransucrase has remained unclear. GTFO of the probiotic bacterium Lactobacillus reuteri ATCC 55730 shows a high sequence similarity (80%) with GTFA of L. reuteri 121; it also synthesizes an α-glucan with (α1 → 4) and (α1 → 6) linkages, but with a clearly different ratio compared to GTFA. In the present study, we show that residues in loop977 (970DGKGYKGA977) and helix α4 (1083VSLKGA1088) are main determinants for the linkage specificity difference between GTFO and GTFA, and hence are important for the synthesis of alternating (α1 → 4) and (α1 → 6) linkages in GTFA. More remote acceptor substrate binding sites (i.e.+3) are also involved in the determination of alternating linkage synthesis, as shown by structural analysis of the oligosaccharides produced using panose and maltotriose as acceptor substrate. Our data show that the amino acid residues at acceptor substrate binding sites (+1, +2, +3…) together form a distinct physicochemical micro-environment that determines the alternating (α1 → 4) and (α1 → 6) linkages synthesis in GTFA.

Cyclodextrin glycosyltransferase (CGTase) is an enzyme of the α-amylase family, which uses a double displacement mechanism to process α-linked glucose polymers. We have determined two X-ray structures of CGTase complexes, one with an intact substrate at 2.1 A resolution, and the other with a covalently bound reaction intermediate at 1.8 A resolution. These structures give evidence for substrate distortion and the covalent character of the intermediate and for the first time show, in atomic detail, how catalysis in the α-amylase family proceeds by the concerted action of all active site residues.

A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes. The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly measured using liquid cultures grown in microtiter plates without the need of separation or purification steps.

Various factors controlling dihydroxyacetone (DHA) and glycerol production from methanol by resting cell suspensions of a mutant of Hansenula polymorpha, blocked in DHA kinase and glycerol kinase, were investigated. The presence of methanol (250 mM) and an additional substrate (0.5%, w/v) to replenish the xylulose-5-phosphate required for the assimilation reaction (DHA synthase) was essential for significant triose production by this double mutant. A number of sugars were tested as additional substrates and C5 sugars gave the highest triose accumulation (ca. 20 mM after 45 h). Glucose was the poorest additional substrate and triose production only started after its exhaustion, which occurred in the first few hours. Other sugars were metabolized at a much lower rate and accumulation of trioses began right at the start of the experiments and gradually increased with time. The production rate of total trioses increased, and the relative amount of glycerol diminished with higher oxygen supply rates. The data suggest that conversion of DHA into glycerol, catalysed by reduced nicotine adenine dinucleotide (NADH)-dependent DHA reductase, is partly regulated via intracellular NADH levels. Further support for this hypothesis was obtained in experiments with antimycin A, an inhibitor of the electron transport chain. Addition of higher amounts of methanol and xylose, either by increasing the initial concentrations or by repeated addition of these substrates, resuited in considerably enhanced productivity and a switch towards glycerol formation. After reaching a level of approximately 25 mM the DHA concentration remained constant while the glycerol level gradually increased with time. After an incubation period of 350 h, a total of 3.9 M methanol and 0.62 M xylose had been converted, which resulted in accumulation of 0.76 M trioses, mostly glycerol.

This editorial introduces the special issue of Antonie van Leeuwenhoek, which brings together a selection of invited papers that reflect the breadth and depth of contributions made in the 14th ISBA (International Symposia on the Biology of Actinomycetes) programme held from August 26th to 30th 2007 in Newcastle upon Tyne, UK.

In the yeast Hansenula polymorpha an oxygen-requiring enzyme, alcohol oxidase, catalyzes the conversion of methanol into formaldehyde. After growth on methanol cells of the organism were harvested and entrapped in barium-alginate gels. The diffusion of oxygen towards these cells is seriously hindered by the polymer network. This caused significant changes in the kinetics of methanol oxidation by the immobilized cells as compared to that observed with cells free in suspension. The apparent K S(O2) of the immobilized cells was dependent upon the density of the cells in the alginate beads and the bead radius. Using a well-known theoretical model, originally developed to describe the kinetics of oxygen diffusion in mold pellets, the diffusion coefficient for oxygen in barium-alginate gels was estimated. This coefficient was only 25% of that in water. The model also allowed an adequate simulation of the observed kinetics of methanol oxidation by the immobilized cells.

Diauxic growth was observed in batch cultures of Pseudomonas oxalaticus when cells were pregrown on acetate and then transferred to mixtures of acetate and oxalate. In the first phase of growth only acetate was utilized. After the exhaustion of acetate from the medium enzymes involved in the metabolism of oxalate were synthesized during a lag phase of 2 h, followed by a second growth phase on oxalate. When the organism was pregrown on oxalate, oxalate utilization from the mixture with acetate completely ceased after a few hours during which acetate became the preferred substrate. Similar observations were made with formate/oxalate mixtures in which formate was the preferred substrate. Until formate was exhausted, it completely suppressed oxalate metabolism, again resulting in diauxic growth. However, when the organism was pregrown on oxalate and then transferred to mixtures of oxalate and formate, both substrates were utilized simultaneously although the initial rate of oxalate utilization from the mixture was strongly reduced as compared to growth on oxalate alone. Since both preferred substrates cross the cytoplasmic membrane by diffusion, whereas oxalate is accumulated by an inducible, active transport system, the effect of acetate and formate on oxalate transport was studied at different external pH values. At pH 5.5 both substrates completely inhibited oxalate transport. However, at pH 7.5, the pH at which the diauxic growth experiments were performed, formate and acetate did not affect oxalate transport. Growth patterns and enzymes profiles suggest that, at higher pH values, formate and acetate possibly affect oxalate utilization via an effect on the internal pool of oxalyl-CoA, the first product of oxalate metabolism.

Gluconobacter spp. possess the enzymic potential for two pathways of direct glucose oxidation. It has been proposed that the major part of glucose is oxidized to gluconate via NADP-dependent glucose dehydrogenase and that reoxidation of NADPH under these conditions proceeds via recycling of gluconate through ketogluconates. This hypothesis was tested in experiments in which Gluconobacter oxydans ATCC 621-H was grown in glucose-yeast extract medium containing [14C]2-ketogluconate. As expected, glucose was almost quantitatively oxidized to gluconate, without further accumulation of 2- and 5-ketogluconate. Interestingly, the total amount of neither [14C]2-ketogluconate nor [14C]gluconate did change significantly during this oxidation phase, indicating that recycling of gluconate through ketogluconates did not occur. An analysis of enzyme activities in cell-free extracts of glucose-grown cells of G. oxydans ATCC 621-H showed that the membrane-bound glucose dehydrogenase was far more active than the NADP-linked glucose dehydrogenase. The activity of the latter enzyme constituted only 10-15% of that of quinoprotein glucose dehydrogenase and was far too low to match the in vivo rates of gluconate production in batch cultures of G. oxydans. It is concluded that under these conditions glucose is mainly oxidized to gluconate via the membrane-bound glucose dehydrogenase. Implications of these results for the regulation of ketogluconate formation are discussed.

Metabolic control associated with diauxic growth of Pseudomonas oxalaticus in batch cultures on mixtures of formate and oxalate was investigated by measuring intracellular enzyme and coenzyme concentrations and QO2 values during transition experiments from oxalate to formate and vice versa. In transition from oxalate to formate oxalyl-CoA reductase concentration declined after the exhaustion of oxalate and ribulose-1,5-diphosphate carboxylase and 14CO2 fixation appeared upon addition of formate. In the reciprocal transition, ribulose-1,5-diphosphate carboxylase and 14CO2 fixation rate declined sharply after formate exhaustion, and oxalyl-CoA reductase appeared only after addition of oxalate. The intracellular NAD and NADP concentrations measured in the same experiments are reported. At substrate exhaustion the proportion of NAD in the reduced form fell from 15-20 % to 2 %. On addition of formate to an oxalate-starved culture there was an immediate increase in the proportion of NADH to 50 %; such an increase was not observed in the reverse experiment.

A study of enzyme profiles in Hansenula polymorpha grown on various carbon substrates revealed that the synthesis of the methanol dissimilatory and assimilatory enzymes is regulated in the same way, namely by catabolite repression and induction by methanol. Mutants of H. polymorpha blocked in dihydroxyacetone (DHA) synthase (strain 70 M) or DHA kinase (strain 17 B) were unable to grow on methanol which confirmed the important role attributed to these enzymes in the biosynthetic xylulose monophosphate (XuMP) cycle. Both mutant strains were still able to metabolize methanol. In the DHA kinase-negative strain 17 B this resulted in accumulation of DHA. Although DHA kinase is thought to be involved in DHA and glycerol metabolism in methylotrophic yeasts, strain 17 B was still able to grow on glycerol at a rate similar to that of the wild type. DHA on the other hand only supported slow growth of this mutant when relatively high concentrations of this compound were provided in the medium. This slow but definite growth of strain 17 B on DHA was not based on the reversible DHA synthase reaction but on conversion of DHA into glycerol, a reaction catalyzed by DHA reductase. The subsequent metabolism of glycerol in strain 17 B and in wild type H. polymorpha, however, remains to be elucidated.

In Nocardia sp. 239 d-phenylalanine is converted into l-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent l-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on l- or d-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and l-phenylalanine. Highest rates of l-phenylalanine dehydrogenase production were observed with increasing ratios of l-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from l- and d-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42 000 and consisted of a single subunit; it showed activity with l-phenylalanine, phenylpyruvate, 4-hydroxyphenyl-pyruvate, indole-3-pyruvate and α-ketoisocaproate, but not with imidazolepyruvate, d-phenylalanine and other l-amino acids tested. Maximum activities with phenylpyruvate (310 μmol min-1 mg-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.

Membrane vesicles isolated from oxalate-grown cells of Pseudomonas oxalaticus accumulated oxalate by an inducible transport system in unmodified form against a concentration gradient. This accumulation was dependent on the presence of a suitable electron donor system such as ascorbate-phenazine-methosulphate. In the presence of this energy source, steady state levels of accumulation of oxalate were 10--20-fold higher than in its absence. The oxalate transport system involved showed a high affinity for oxalate (Km = 11 micron) and was highly specific. Oxalate transport was not affected by the presence of other dicarboxylic acids, such as malate, succinate and fumarate and only partly inhibited by acetate. The energy requirement for oxalate transport is discussed and it is concluded that this requirement is most likely equivalent to 1 mole of ATP per mole of oxalate.