Isolation and basic characterization of a beta-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture.

AIMS: To study glycosidase activities of a Lactobacillus brevis strain and to isolate an intracellular beta-glucosidase from this strain. METHODS AND RESULTS: Lactic acid bacteria (LAB) isolated from a commercially available starter culture preparation for malolactic fermentation were tested for beta-glycosidase activities. A strain of Lact. brevis showing high intracellular beta-D-glucosidase, beta-D-xylosidase and alpha-L-arabinosidase activities was selected for purification and characterization of its beta-glucosidase. The pure glucosidase from Lact. brevis has also side activities of xylosidase, arabinosidase and cellobiosidase. It is a homotetramer of 330 kDa and has an isoelectric point at pH 3.5. The K(m) for p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside is 0.22 and 1.14 mmol l(-1), respectively. The beta-glucosidase activity was strongly inhibited by gluconic acid delta-lactone, partially by glucose and gluconate, but not by fructose. Ethanol and methanol were found to increase the activity up to twofold. The free enzyme was stable at pH 7.0 (t(1/2) = 50 day) but not at pH 4.0 (t(1/2) = 4 days). CONCLUSIONS: The beta-glucosidase from Lact. brevis is widely different to that characterized from Lactobacillus casei (Coulon et al. 1998) and Lactobacillus plantarum (Sestelo et al. 2004). The high tolerance to fructose and ethanol, the low inhibitory effect of glucose on the enzyme activity and the good long-term stability could be of great interest for the release of aroma compounds during winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the release of aroma compounds by LAB has been demonstrated by several authors, little information exists on the responsible enzymes. This study contains the first characterization of an intracellular beta-glucosidase isolated from a wine-related strain of Lact. brevis.

Cloning, nucleotide sequence and expression of a mannitol dehydrogenase gene from Pseudomonas fluorescens DSM 50106 in Escherichia coli.

A NAD-dependent mannitol dehydrogenase (MtlD) was purified to homogeneity from P. fluorescens DSM50106 and the N-terminal amino acid sequence was determined. An oligonucleotide deduced from this peptide sequence was used as a probe to isolate the mannitol dehydrogenase gene (mtlD) from a genomic library of P. fluorescens. Nucleotide sequence analysis of a 1.8 kb NruI fragment containing the entire mtlD gene revealed an open reading frame of 1482 bp encoding a protein with a calculated molecular weight of 54.49 kDa. The enzyme shared a high similarity with a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall identity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-1-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activity of the mannitol dehydrogenase synthesized in E. coli was increased from 0.02 U (mg protein)(-1) to 10 U (mg protein)(-1). After fusion of six histidine codons to the 3' end of mtlD gene and expression in E. coli active mannitol dehydrogenase could be purified in a two-step procedure by affinity chromatography using a Ni2+ matrix column. The purified enzyme exhibited a specific activity of 46 U (mg protein)(-1) and was shown to be a polyol dehydrogenase with a broad substrate spectrum oxidizing efficiently mannitol, sorbitol and arabitol.

Control of the association state of tetrameric glucose-fructose oxidoreductase from Zymomonas mobilis as the rationale for stabilization of the enzyme in biochemical reactors.

Tetrameric, NADP-containing glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis catalyzes the oxidation of glucose into glucono-delta-lactone coupled to the reduction of fructose to sorbitol. GFOR is inactivated during substrate turnover in vitro, the long-term stability of the enzyme during conversions in biochemical reactors thereby being drastically reduced. The process of inactivation is triggered by structural transitions that are induced by the lactone product and involves aggregation as the ultimate cause of irreversible inactivation. Guanidinium hydrochloride-induced changes in the conformation of GFOR seem to be similar to those observed in the presence of lactone, and are manifested by incubation time-dependent increases in protein fluorescence and the solvent-exposed hydrophobic surface. The formation of high-order protein associates in solution in the presence of this denaturant proceeds from the native tetramer to a reversibly inactivated octamer and then to a dodecameric form that cannot be reactivated through spontaneous or assisted refolding. Therefore, stabilization of GFOR during turnover requires that the marked tendency of the enzyme to form aggregates is prevented efficiently. This goal has been accomplished in the presence of low urea concentrations (1.0 M), which led to a 10-fold increase in the half-life of GFOR under operational conditions.

Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435.

In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on D-xylose or L-arabinose as well as on the structurally related sugars D-arabinose or D-lyxose. Mixtures of D-xylose with the more rapidly metabolizable sugar D-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of D-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control.

Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite.

Downstream processing by biospecific chromatography of maltodextrin phosphorylase from Escherichia coli, overexpressed in E. coli, was substantially improved by a novel approach using ceramic hydroxyapatite. Wild-type and a less active mutant enzyme were purified from crude bacterial cell extracts in one efficient separation step that yielded phosphorylase in purity > 95% in at least 90% recoveries. At pH 6.9 and 25 degrees C, wild-type and mutant phosphorylases eluted from the hydroxyapatite column at a phosphate concentration of 0.4 M whereas calcium ions failed to displace the enzymes. The dynamic capacity for phosphorylase binding in the presence of bulk proteins was approximately 3 mg enzyme ml-1 matrix. The interaction of E. coli phosphorylase with hydroxyapatite seems to be mediated by surface amino groups, so that the bound enzyme retained almost full catalytic activity. Compared to the soluble enzyme, immobilization onto hydroxyapatite resulted in a more than 30-fold stabilization of wild-type phosphorylase against thermal and proteolytic inactivation and thus could improve the operational stability of phosphorylase during conversion of polysaccharide to glucose 1-phosphate.

A simple assay for measuring cellobiose dehydrogenase activity in the presence of laccase.

The commonly used assay for measuring cellobiose dehydrogenase (CDH) activity, based on the reduction of dichlorophenol-indophenol (DCIP), has been adapted to measure this enzyme activity in the presence of laccase, which is often formed concurrently with CDH by a number of fungi. Laccase interferes with the assay by rapidly reoxidizing the reduced form of DCIP and can mask CDH activity completely. It can be conveniently and completely inhibited by 4 mM fluoride in the assay, while CDH activity is only slightly affected by the addition of this inhibitor. The modified assay enables the detection of low CDH activities even in the presence of very high excesses of laccase. It should be useful for screening culture supernatants of wood-degrading fungi for CDH since the assay is rapid and uses inexpensive and nontoxic reagents. Furthermore, it might be used for the detection of other enzyme activities which are assayed by following the reduction of quinones or analogue compounds such as DCIP.

Aluminum-free oral phosphate binder.

For the purpose of intestinal phosphate binding we have developed aluminum free substances. These substances are natural polymers consisting of heteropolyuronic acid charged with different cations. The in vitro experiments showed an efficacy 2 to 3 times greater than Aludrox. During the clinical application, up to one year, no serious side effects have been detected. Serum phosphate levels could not be lowered in all patients to the desired level of 5 mg% mainly due to problems in patients compliance and to low dosage of the prescribed phosphate binder.

Reaction engineering aspects of alpha-l,4-D-glucan phosphorylase catalysis : comparison of plant and bacterial enzymes for the continuous synthesis of D-glucose-1-phosphate.

Some important process properties of alpha-l,4-D-glucan phosphorylases isolated from the bacterium Corynebacterium callunae and potato tubers (Solatium tuberosum) were compared. Apart from minor differences in their stability and specificity (represented by the maximum degree of maltodextrin conversion) and a 10-fold higher affinity of the plant phosphorylase for maltodextrin (K (M) of 1.3 g/L at 300 mM of orthophosphate), the performances of both enzymes in a continuous ultrafiltration membrane reactor were almost identical. Product synthesis was carried out over a time course of 300-400 h in the presence or absence of auxiliary pullulanase (increasing the accessibility of the glucan substrate for phosphorolytic attack up to 15-20%). The effect of varied dilution rate and reaction temperature on the resulting productivities was quantitated, and a maximum operational temperature of 40 degrees C was identified.

Production of hemicellulose- and cellulose-degrading enzymes by various strains of Sclerotium rolfsii.

A number of wild-type isolates of Sclerotium rolfsii were screened for their capacity to produce lignocellulolytic enzymes when grown on a cellulose-based medium.S. rolfsii proved to be an efficient producer of hemicellulolytic enzymes under the conditions selected for this screening, although there was a great variability in enzyme activities formed by the different isolates. In addition to xylanase and mannanase, which were produced in remarkably high levels, a number of accessory enzymes, which are important for the complete degradation of substituted hemicelluloses and include a-arabinosidase, acetyl esterase, and a-galactosidase, are formed by S. rolfsii. Efficient production of xylanase and mannanase was achieved when cellulose-based media were used for growth. Under these conditions, enhanced levels of endoglucanase were formed as well. Formation of xylanase and mannanase could be more specifically induced when using xylan or mannan as growth substrates, although the enzyme activities thus obtained were significantly lower compared to cultivations on cellulose as main inducing substrate.

Production of a novel pyranose 2-oxidase by basidiomycete Trametes multicolor.

During a screening for the enzyme pyranose 2-oxidase (P2O) which has a great potential as a biocatalyst for carbohydrate transformations, Trametes multicolor was identified as a promising, not-yet-described producer of this particular enzyme activity. Furthermore, it was found in this screening that the enzyme frequently occurs in basidiomycetes. Intracellular P2O was produced in a growth-associated manner by T. multicolor during growth on various substrates, including mono-, oligo-, and polysaccharides. Highest levels of this enzyme activity were formed when lactose or whey were used as substrates. Peptones from casein and other casein hydrolysates were found to be the most favorable nitrogen sources for the formation of P2O. By applying an appropriate feeding strategy for the substrate lactose, which ensured an elevated concentration of the carbon source during the entire cultivation, levels of P2O activity obtained in laboratory fermentations, as well as the productivity of these bioprocess experiments, could be enhanced more than 2.5-fold.

Efficient production of mannan-degrading enzymes by the basidiomycete Sclerotium rolfsii.

Sclerotium rolfsii CBS 191.62 was cultivated on a number of carbon (C) sources, including mono- and disaccharides, as well as on polysaccharides, to study the formation of different mannan-degrading enzyme activities. Highest levels of mannanase activity were obtained when alpha-cellulose-based media were used for growth, but formation of mannanase could not be enhanced by employing galactomannan as the only carbon source. Although both xylanase and cellulase formation was almost completely repressed when S. rolfsii was grown on more readily metabolizable carbohydrates, including glucose or mannose, considerable amounts of mannanase activity were secreted under these growth conditions. Enhanced mannanase production only commenced when glucose was depleted in the medium. The maximal mannanase activity of 240 IU/mL obtained in a laboratory fermentation is remarkable. Mannanase activity formed under these derepressed conditions could be mainly attributed to one major, acidic mannanase isoenzyme with a pI value of 2.75.

Simultaneous enzymatic synthesis of gluconic acid and sorbitol. Continuous process development using glucose-fructose oxidoreductase from Zymomonas mobilis.

The production of sorbitol and gluconic acid by isolated glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis has been studied in a convective, 100-mL loop reactor with tangential ultrafiltration. Using a dilution rate of 0.04/h and 5 kU/L GFOR, substrate conversion (3 M sugar) in a single stage was >85%, and productivities of 126 g sorbitol/(L x d) were obtained. At a constant recycle rate (3/min) and a membrane area of 50 cm2, the dilution rates (and thus productivities) were however limited by a more than 30-fold reduction of the permeate flow in the presence of high sugar and protein concentrations (5 g/L). Protein was added, together with 10 mM dithiothreitol, to improve the stability of GFOR during substrate turnover and crossflow filtration, thus leading to a stable operation of the enzyme reactor for at least 5 d.

Simultaneous enzymatic synthesis of gluconic acid and sorbitol: production, purification, and application of glucose-fructose oxidoreductase and gluconolactonase.

With regard to the enzymatic synthesis of sorbitol and gluconic acid, a screening was carried out to identify promising producers of glucose-fructose oxidoreductase (GFOR) and gluconolactonase (GL). Zymomonas mobilis DSM 473 and Rhodotorula rubra DSM 70403 have been selected for the synthesis of GFOR and GL, respectively. Maximal enzyme production by these organisms has been achieved at D-glucose concentrations of 200 and 150 g/L, respectively. Both GFOR and GL were purified and characterized with respect to some of their catalytic properties. GL showed strict specificity for 1,5-(delta)-lactones and was activated by Mg2+ and Mn2+ ions. The potential use of soluble GFOR is limited by its inactivation during substrate conversion, and the effects of reaction temperature and pH on the "catalytic" stability of GFOR were evaluated. Exogenous addition of auxiliary GL had no effect on oxidoreductase stability and did not improve productivities.

Biotransformation of pineapple juice sugars into dietetic derivatives by using a cell free oxidoreductase from Zymomonas mobilis together with commercial invertase.

An easy procedure for cell free biotransformation of pineapple juice sugars into dietetic derivatives was accomplished using a commercial invertase and an oxidoreductase from Zymomonas mobilis. First, pineapple juice sucrose was quantitatively converted into glucose and fructose by invertase, thus increasing the concentration of each monosaccharide in the original juice to almost twice. In a second step, glucose-fructose oxidoreductase (GFOR) transformed glucose into gluconolactone, and fructose into the low calorie sweetener sorbitol. The advantage of using GFOR is simultaneous reduction of fructose and oxidation of glucose, allowing the continuous regeneration of the essential coenzyme NADP(H), that is tightly bound to the enzyme. The yield of GFOR catalyzed sugar conversion depends on initial pH and control of pH during the reaction. At optimal conditions (pH control at 6.2) a maximum of 80% (w/v) sugar conversion was obtained. Without pH control, GFOR is inactivated rapidly due to gluconic acid formation. Therefore, conversion yields are relatively low at the natural pH of pineapple juice. The application of this process might be more advantageous on juices of other tropical fruits (papaya, jackfruit, mango) due to their naturally given higher pH.