Purification and Some Properties of β-Fructofuranosidase from Bifidobacterium adolescentis G1

A unique β-fructofuranosidase was purified from the extract of Bifidobacterium adolescentis G1 by anion-exchange, hydrophobic, and gel filtration chromatographies, and preparative electrophoresis. The molecular mass was 74kDa by SDS–PAGE, and the isoelectric point was pH 4.5. The enzyme was a monomeric protein. The pH optimum was at 6.1. The enzyme was stable at pH from 6.5 to 10.0, and up to 45°C. The neutral sugar content was 1.2%. The enzyme hydrolyzed 1-kestose faster than sucrose or inulin. The hydrolytic activity was strongly inhibited by Cu²⁺, Ag⁺, Hg⁺, and ρ-chloromercuribenzoic acid. The K_m (mM) and k₀ (s^?1) were: 1-kestose, 1.1 and 231; sucrose, 11 and 59.0; inulin, 8.0 and 149, respectively. From the kinetic results, β-fructofuranosidase from B. adolescentis G1 was concluded to have a high affinity for 1-kestose, thus differing from invertases and exo-inulinases in substrate specificity.     

Optimization of extracellular xylanase production by Dictyoglomus sp. B1 in continuous culture

The thermophilic, xylanolytic, anaerobic organism, Dictyoglomus sp. B1, was cultivated in batch and continuous cultures in media containing insoluble beech-wood xylan. The extracellular xylanase activity levels obtained for the two cultivation methods were compared. Experiments were performed separately to determine the optimum substrate concentration, dilution rate, pH and temperature for xylanase production. Maximum xylanase activity was found at a substrate concentration of 1.5 g xylan/l, a dilution rate of 0.112 h^–1, pH 8.0 and at 7°C. Different combinations of these optimum values were used in a 2³ factorial experiment to investigate whether an increase in the xylanase production/activity could be achieved. A maximum xylanase activity of 2312 U/l was found when fermentors were operated at 73°C with a substrate concentration of 1.5 g xylan/l, pH 8.0, and a dilution rate of 0.112 h^–1. Thus, the optimum xylanase activity in the factorial experiment was obtained when the conditions that gave the maximum xylanase activities in the individual experiments were combined. Optimum xylanase activity obtained in the 2³ factorial experiment was 6.2 times higher than the activity found in the initial batch culture (373 U/l) and 3.0 times higher than the activity of a batch culture (783 U/l) grown at the same optimum conditions as the factorial experiment. The higher specific xylanase activity (217 U/mg protein) found in the 2³ factorial experiment was 4.1 times higher than the specific activity in the initial batch culture (53 U/mg protein).     

Purification and characterization of an exo-polygalacturonase from Pycnoporus sanguineus

The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42 kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50–60 °C, with some enzyme activity retained up to 80 °C. Its activation energy was 5.352 cal mol⁻¹. PGase I showed a higher affinity towards PGA than citric pectin (Km = 0.55 ± 0.02 and 0.72 ± 0.02 mg ml⁻¹, respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.     

Studies on the growth of a thermotolerant yeast strain,Kluyveromyces marxianus IMB3, on sucrose containing media

A thermotolerant alcohol-producing yeast strain, Kluyveromyces marxianus IMB3 was shown to grow on sucrose (10% [w/v]) containing media at 45 °C. Under such conditions the organism reached stationary phase within 20 hours and yielded ethanol concentrations in the region of 33g/L. During growth on sucrose containing media the organism was found to produce a cell- associated activity capable of hydrolysing sucrose. This activity was shown to have a Km of 5.0mM when sucrose was used as the substrate. In addition the enzyme was shown to have a pH optimum of 5.0 and a temperature optimum of 50–55 °C and under those conditions the enzyme was shown to be relatively thermostable.     

Structural analysis and optimised production of fructo- oligosaccharides by levansucrase from Acetobacter diazotrophicus SRT4

Major fructo-oligosaccharides (FOS) produced by levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 were characterised as 1-kestose and nystose by acid hydrolysis and 13C-NMR spectroscopy. The highest yields of 1-kestose (481 mM; 241 g/l) and nystose (81 mM; 54 g/l) were achieved at initial sucrose concentration of 1754 mM (600 g/l), pH 5.5 and 40°C. The synthesized FOS reached 50% (w/w) of total sugars in the reaction mixture, with a conversion efficiency over 70% (w/w) based on the amount of sucrose converted to 1-kestose.     

Continuous production of neo-fructooligosaccharides by immobilization of whole cells of <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

To produce neo-fructooligosaccharides (neo-FOSs) in a 500ml continuous packed-bed reactor using whole cell immobilization of Penicillium citrinum KCCM 11663, the optimum reaction conditions were 50 °C, pH 6 with 600 g sucrose l^-1 being fed as substrate at 1.3 ml min^-1 . Under these conditions, the maximum neo-FOSs production was 49 g l^-1. In a packed-bed reactor, continuous production of neo-FOSs was possible for 50 d indicating a potential for industrial production. Revisions requested 6 September 2004/14 October 2004; Revisions received 7 October 2004/29 November 2004     

Kinetic studies of acetate fermentation by Methanosarcina sp. MSTA-1

Summary The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp. MSTA-1. The optimum temperature for growth ranged around 55° C, and optimum pH was 6.5–7.5, giving a minimum generation time of 12.6–13.9 h (µ_max = 0.050–0.055 h^–1) and a maximum value of the specific acetate consumption rate (q _infs ^supps ) of 14–20 mmol/g cells per hour. Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature. Methanosarcina sp. MSTA-1 showed a low affinity for acetate substrate. Growth at 55° C and at constant pH 7 resulted in a K _m value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively. Offprint requests to: R. Moletta     

Purification and properties ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524

Summary Purification and properties of two -fructofuranosidases, which produce 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, fromAureobasidium sp. ATCC 20524 are reported. The enzymes were purified to homogeneity by fractionations involving ethanol, calcium acetate and ammonium sulfate and DEAE-Cellulofine and Sephadex G-200 chromatography. Molecular weights of the enzymes were estimated to be about 318000 (P-1) and 346000 (P-2) daltons by gel filtration. The enzymes were glycoproteins that contained about 30% (w/v) (P-1) and 53% (w/v) (P-2) carbohydrate. The optimum pH for the enzymatic reactions were 4.5–5.5 (P-1) and 4.5–6 (P-2). The enzymes were stable over a wide pH range (4–9). The optimum reaction temperatures for both enzymes were 50–55°C and they retained more than 94% (P-1) and 98% (P-2) activities at 50°C after 15 min. TheK _m values for sucrose were 0.47 M (P-1) and 0.65 M (P-2). The enzymes were inhibited by mercury, copper and lead ions as well asp-chloromercuribenzoate.     

Xylanase production by fungal strains on spent sulphite liquor

Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and d-xylose as carbon substrates. An SSLc-based medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml⁻¹, respectively; these values were higher than those obtained on xylan or d-xylose with the same fungal strains. The highest xylanase activity of 322 U ml⁻¹ was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5–10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20–30% without sacrificing brightness.     

Production of high-content fructo-oligosaccharides by an enzymatic system from Penicillium rugulosum

Summary The production of high-content fructo-oligosaccharides from sucrose by a crude FTF from a new strain of Penicillium isolated in our Laboratory was investigated. The optimum conditions for the production of the enzyme and for the enzymic reaction have been determined. It has been demonstrated that the crude enzyme acts as a mixed enzyme system of fructosyl transferase (FTF; Class 2 of Enzyme Nomenclature) and glycosidases (Class 3 of Enyme Nomenclature). Under optimum conditions: pH 5.5, temperature 55°C, sucrose concentration 750 g/l, enzyme concentration 5 FTF units/g sucrose, conversion yield up to 80% were obtained and high concentration of nystose (412 g/l) and fructofuranosyl-nystose (176 g/l) were accumulated.     

Factors Affecting Yield and Safety of Protein Production from Cassava by Cephalosporium eichhorniae

The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45° to 47°C, maximum protein yield at 45°C, and no growth at 25°C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45°C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10⁶ to 10⁸ per animal) injected by various routes into normal and γ-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.     

Production of 1-kestose by Scopulariopsis brevicaulis

A 1-kestose-producing fungus, strain N-01, was isolated and identified as Scopulariopsis brevicaulis. The optimal conditions for 1-kestose production by strain N-01 were studied and the following conditions were established: the strain was cultivated in a 500-ml conical flask containing 25 ml of medium composed (per liter) of 150 g sucrose, 15 g yeast extract, 0.6 g urea, 1 g K₂HPO₄, and 0.3 g MgSO₄·7H₂O (pH 7.0) at 30°C for 72 h. When the strain was cultivated in a 2.5-l jar fermentor, 95 g of 1-kestose was produced with a theoretical yield of 85%. From this culture broth 1-kestose was crystallized and then recrystallized, giving purities of 98.0 and 99.8% with yields of 71.0 and 78.0%, respectively. In particular, the first crystals in the recrystallization gave over 99.9% purity. By using these crystals, the general properties of 1-kestose were determined; most of these properties coincided with the data in the literature, though the melting point range was narrower. Finally a scheme for the large-scale purification of 1-kestose is proposed.     

Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

Summary A β-galactosidase from Thermotoga maritima produced galacto-oligosaccharides (GOS) from lactose by transgalactosylation when expressed in Escherichia coli. The enzyme activity for GOS production was maximal at pH 6.0 and 90 °C. In thermal stability experiments, the enzyme followed first-order kinetics of pH and thermal inactivation, and half-lives at pH 5.0, pH 8.0, 80 °C, and 95 °C were 27 h, 82 h, 41 h, and 14 min, respectively, suggesting that the enzyme was stable below 80 °C and in the pH range of 5.0–8.0. Mn²⁺ was the most effective divalent cation for GOS production. Cu²⁺ and EDTA inhibited more than 84% of enzyme activity. GOS production increased with increasing lactose concentrations and peaked at 500 g lactose/l. Among tested enzyme concentrations, the highest production of GOS was obtained at 1.5 units enzyme/ml. Under the optimal conditions of pH 6.0, 80 °C, 500 g lactose/l, and 1.5 units enzyme/ml, GOS production was 91 g/l for 300 min, with a GOS productivity of 18.2 g/l · h and a conversion yield of GOS to lactose of 18%.     

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hveragerdi and Fluir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO₂, H₂, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring     

The isolation and characterisation of a salicylate-hydroxylase-producing strain of Pseudomonas putida

Summary A salicylate-hydroxylase-producing strain of Pseudomonas putida with an unusual capability to grow at toxic levels of salicylate up to 10 g l^–1 has been isolated. It grew well under continuous culture conditions, with optimum growth at pH 6.5 and a temperature of 25° C. The use of an ammonium salt as a nitrogen source, instead of nitrate, resulted in a 30–40% increase in its biomass yield coefficient. Optimum growth under continuous culture conditions was achieved using 4 g l^–1 salicylate at 25° C, pH 6.5 and 0.2 h^–1 dilution rate. High salicylate hydroxylase enzyme activity [236 units (U) l^–1] and productivity (424.8 U h^–1) were obtained at a dilution rate of 0.45 h^–1 using a mineral medium containing 4 g l^–1 of salicylate. Operating under continuous culture conditions with oxygen limitation and a slight accumulation of residual salicylate (0.2 g l^–1) resulted in a decrease in culture performance and enzyme productivity. Correspondence to: R. Marchant     

Purification and characterisation of 1F-fructosyltransferase from the roots of asparagus (asparagus officinalis L.)

A fructosyltransferase that transfers a terminal d-fructosyl group from a (2→1)-β-linked fructosaccharide to HO-1 of another d-fructosyl group has been purified from an extract of asparagus roots by successive chromatography with DEAE-cellulose, octyl-Sepharose, Sephadex G-200, and raffinose-coupled Sepharose 6B. The disc-electrophoretically homogeneous enzyme was free from β-d-fructofuranosidase, sucrose:sucrose 1-fructosyltransferase, and 6^G-frutosyltransferase activity, and catalysed the d-fructosyl transfer from 1-kestose more rapidly to saccharides of the neokestose series [1^F(1-β-d-fructofuranosyl)_m-6^G(1-β-d-fructofuranosyl)_nsucrose] than to those of the 1-kestose series [1^F(1-β-d-fructofuranosyl)_nsucrose]. The enzyme was tentatively termed 1^F-fructosyltransferase. The general properties of the enzyme were as follows: mol. wt., ~64,000; optimum pH, ~5.0; stable at pH 5.0–5.5 at 45° for 20 min; stable at 30–45° for 10 min; inhibited by Hg²⁺, p-chloromercuribenzoate, and Ag⁺.     

Mycelia-associated β-galactosidase activity in microbial pellets of Aspergillus and Penicillium strains

Pellet formation and production of mycelia-associated -galactosidase were investigated in 15 Aspergillus and Penicillium strains. Mycelia-associated enzyme activity was measured in sonicated homogenates. The properties of the mycelia-associated -galactosidase of A. phoenicis QM 329 was investigated. The pH optimum of the mycelia-associated enzyme was 4.0. The optimum temperature under assay conditions was 70°C and the optimum temperature for repeated lactose hydrolysis was 60°C. Repeated batch hydrolysis of lactose was made with pellets from five Aspergillus strains. A. phoenicis QM 329 showed the least enzyme leakage from the pellets during hydrolysis. From repeated lactose hydrolysis experiments it was estimated that 50% of the mycelia-associated -galactosidase activity remained after 1300 h. Correspondence to: F. Tjerneld     

Lactosucrose production by various microorganisms harboring levansucrase activity

Production of the artificial sweetener, lactosucrose, by various microorganisms containing levansucrase activity was investigated. Of the tested bacteria, Bacillus subtilis was the most effective producer using lactose as an acceptor and sucrose as a fructosyl donor. Lactosucrose production by this strain was optimal at pH 6.0 and 55 °C whereupon 181 g lactosucrose l^–1 was produced from 225 g lactose l^–1 and 225 g sucrose l^–1 in 10 h.     

Optimal conditions for levan formation by an overexpressed recombinant levansucrase

Summary A genetically modified levansucrase, which contained His-affinity tag in its C-terminal, was constructed by PCR reaction using two synthetic primers. This modified protein was produced up to 30 % in total cell protein of E. coli, and was purified by a one-step affinity chromatography. The optimum pH for levan production was pH 5 and the optimum temperature was 0 °C. The higher velocity of levan formation within shorter enzyme reaction times was achieved by increasing the levels of enzyme concentration. The optimal sucrose concentration for levan production was around 20 %. Under these conditions, more than 50 g levan/l was produced.