Isolation and Characterization of Bacterial Strains with Inulinase Activity

Thirty-two bacterial strains growing on inulin as the sole carbon and energy source were isolated from soil samples by enrichment culture on a mineral medium. Twenty of the strains were identified as Flavobacterium multivorum. All the bacteria contained a β-fructosidase that was active on both inulin and sucrose. The enzyme activity was cell bound and was produced at the end of the growth phase. These enzymes have potential uses in the preparation of fructose syrups from inulin and invert sugar from sucrose.     

Fermentation of Inulin by Clostridium thermosuccinogenes sp. nov., a Thermophilic Anaerobic Bacterium Isolated from Various Habitats

Four closely related strains of thermophilic bacteria were isolated via enrichment in batch and continuous culture with inulin as the sole source of carbon and energy by using inoculations from various sources. These new strains were isolated from beet pulp from a sugar refinery, soil around a Jerusalem artichoke, fresh cow manure, and mud from a tropical pond in a botanical garden. The cells of this novel species of strictly anaerobic, gram-positive bacteria were rod shaped and nonmotile. Growth on inulin was possible between 40 and 65°C, with optimum growth at 58°C. All strains were capable of fermenting a large number of sugars. Formate, acetate, ethanol, lactate, H₂, and succinate were the main organic fermentation products after growth on fructose, glucose, or inulin. Synthesis of inulinase in batch culture closely paralleled growth, and the enzyme was almost completely cell bound. Strain IC is described as the type strain of a new species, Clostridium thermosuccinogenes sp. nov., with a G+C content of 35.9 mol%.     

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556

From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45°C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight⁻¹ at D = 0.1 h⁻¹ to 2 U mg of cell dry weight⁻¹ at D = 0.8 h⁻¹. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.     

Activity of fructanase in batch cultures of oral streptococci

Several strains of oral streptococci produced fructanase when grown in the absence of d-fructan in a complex medium supplemented with d-glucose. The major part of the activity was extracellular, and only 1–5% was associated with the cells. Release of fructanase began early in the exponential phase and the enzyme was stable in the stationary phase for several h if the pH did not fall below 6. Among the strains of Streptococcus mutans, serotypes a, d, and g released the highest amount of fructanase, and the low level of enzyme produced by strains of serotype c was increased when d-fructose replaced d-glucose as carbon source for growth. Fructanase of S. mutans readily hydrolysed (2 → 6)-β-d-fructans, but (2 → 1)-β-d-fructans and inulin were more resistant. Adsorption of fructanase to (2 → 6)-β-d-fructan, or inhibition with Tris buffer, provided effective means of eliminating fructanase activity from culture filtrates. This procedure should permit a more accurate determination of fructosyltransferase activity of S. mutans strains.     

Bioconversion of inulin to 2,3-butanediol by a newly isolated Klebsiella pneumoniae producing inulinase

Inulin could be converted to bio-based chemicals by an inulinase producer without external inulinase, and the production of 2,3-butanediol was less than 50 g/L. In this work, a novel inulinase producer of Klebsiella pneumoniae H3 was isolated, and inulinase catalytic properties as well as 2,3-butanediol fermentation were investigated. The enzyme was an intracellular inulinase with an optimal pH of 6 ∼ 7 and a temperature of 30 °C. The use of inulin by H3 was dependent on the degree of polymerization (DP), and the average DP of inulin in fermentation broth increased from 2.82 to 8.08 in 24-h culture of batch fermentation. Acidic pretreatment was developed to increase inulin utilization by adjusting medium pH to 3.0 prior to sterilization. In batch fermentation with optimized medium and fermentation conditions, the concentration of target product (2,3-butanediol and acetoin) was 80.4 g/L with a productivity of 2.23 g/(L⋅h), and a yield of 0.426 g/g inulin.     

Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10⁷) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.     

Invertase SUC2 Is the Key Hydrolase for Inulin Degradation in Saccharomyces cerevisiae

Specific Saccharomyces cerevisiae strains were recently found to be capable of efficiently utilizing inulin, but genetic mechanisms of inulin hydrolysis in yeast remain unknown. Here we report functional characteristics of invertase SUC2 from strain JZ1C and demonstrate that SUC2 is the key enzyme responsible for inulin metabolism in S. cerevisiae.     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Growth characteristics of selected thermophilic strains of cyanobacteria using crossed gradients of temperature and light

Growth requirements of 10 possibly thermophilic strains of cyanobacteria were compared under a wide spectrum of light and temperature conditions (7–80Wm^?2, 12–40°C). The strains were isolated from different localities: six of thermal springs in Slovakia (4 from Pie??any, 2 from Sklené Teplice), one from thermal waters in Rupite, Bulgaria, 2 strains from a hypersaline lake Chott-el-Djerid, Tunisia, and one strain from the tropical island of Cebu, Philippines. Although the crossed gradient unit allowed only sub-optimal temperature range with respect to thermophile definition, i.e. optimum temperature above 45°C, there were difference among the strains. The most thermophilic and high-light tolerant strain was Synechococcus bigranulatus strain Lukavsky 2005/66, with a peak above 45°C; the second was Chroococcidiopsis thermalis strain Hindák 2008/9, and Isocystis sp. strain Hindák 2006/1. The temperature requirements of other strains were similar; the least thermophilic were both Slovakian strains of the genus Hapalosiphon. Growth was not limited below 80 W m^?2, except for H. fontinalis strain Hindák 2008/3, which prefers lower irradiance. Hapalosiphon delicatulus strain Hindák 2007/20, isolated from a bark tree on the tropical island of Cebu was rather mesophilic than thermophilic and shade-preferring. The CCA revealed that the ecologically similar strains originated from the same or nearby localities. There were no significant correlations between temperature optima in culture and in nature. Bulgarian and Tunisian strains preferred higher irradiances.     

Impairment of inulin assimilation and β-fructosidase activity due to a petite mutation in Kluyveromyces marxianus

A respiratory deficient mutant of Kluyveromyces fragilis was isolated using ethidium bromide mutagenesis. It was characterized by a loss of cytochromes a+a3 and deficiency in cytochrome b. This petite mutant has brought about modifications in the excretion pattern of -fructosidase active on saccharose and inulin. The mutant practically no longer excretes the enzyme, and is incapable of growth and fermentation in the presence of inulin. The study of the activities of different enzyme extracts (culture medium, whole and disrupted cells) on inulin and saccharose suggests the existence of an unique enzyme system capable of taking several form, and also shows the influence of the growth substrate on the I/S activity ratio.     

Growth of and trehalase activity in the thermophilic fungusThermomyces lanuginosus

The thermophilic fungus,Thermomyces lanuginosus, was grown in a glucose-asparagine liquid medium. Optimal mycelial growth occurred at 50°C. The conditions for sporulation were different from those required for vegetative growth. the former being favoured by lower nitrogen level and temperature. Trehalase (α, α-glu coside-l-glucohydrolase, EC 3.2.1.28) was one of the most active glycosidases at 50°C. Non-sporulating mycelium had higher levels of this enzyme than the sporulating mycelium. Trehalase was synthesized constitutively and its activity appears to be controlled by catabolite repression.     

Studies on a beta-fructosidase (inulinase) produced bySaccharomyces fragilis

Summary A study was made of a β-fructosidase, which is produced extracellularly and intracellularly bySaccharomyces fragilis. The enzyme catalyzes the hydrolysis of inulin, bacterial levans, sucrose, and the fructose portion of raffinose, by splitting off terminal fructosyl units. It attacks β-2,1 as well as β-2,6 linkages. The enzyme content of inulin-grown cells is sufficient to allow fermentation of inulin at the same rate as glucose. The ratio of hydrolysis rates with sucrose and inulin was about 25 for the β-fructosidase ofS. fragilis and about 14,000 for invertase.S. fragilis does not contain significant amounts of invertase and it ferments inulin, sucrose and raffinose with the aid of a related, but different enzyme, inulinase. Conditions of growth were established which favor inulinase synthesis. Highest yields were obtained with inulin as the carbon source, and somewhat lower yields with raffinose. Glucose, fructose and sucrose were poor inducers of inulinase. The pH of the medium during growth on inulin had to be in the range where inulinase could act, otherwise growth was tardy and poor. In an inulin containing medium aeration favored enzyme production as a result of stimulation of growth. The inulinase content of the cells in a unit volume was generally greater than that in the culture medium. The intracellular inulinase could be solubilized quantitatively by autolysis. The intra-and extracellular inulinases were concentrated and purified to the same extent. Comparison of the two preparations with respect to substrate specificity, rate of inactivation by heat, pH optima with sucrose (4.2) and with inulin (5.0), and elution patterns from a column of diethylaminoethyl cellulose, indicated that the intra-and extracellular enzymes were identical.     

Fermentation of Fructooligosaccharides and Inulin by Bifidobacteria: a Comparative Study of Pure and Fecal Cultures

The utilization of fructooligosaccharides (FOS) and inulin by 55 Bifidobacterium strains was investigated. Whereas FOS were fermented by most strains, only eight grew when inulin was used as the carbon source. Residual carbohydrates were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection after batch fermentation. A strain-dependent capability to degrade fructans of different lengths was observed. During batch fermentation on inulin, the short fructans disappeared first, and then the longer ones were gradually consumed. However, growth occurred through a single uninterrupted exponential phase without exhibiting polyauxic behavior in relation to the chain length. Cellular β-fructofuranosidases were found in all of the 21 Bifidobacterium strains tested. Four strains were tested for extracellular hydrolytic activity against fructans, and only the two strains which ferment inulin showed this activity. Batch cultures inoculated with human fecal slurries confirmed the bifidogenic effect of both FOS and inulin and indicated that other intestinal microbial groups also grow on these carbon sources. We observed that bifidobacteria grew by cross-feeding on mono- and oligosaccharides produced by primary inulin intestinal degraders, as evidenced by the high hydrolytic activity of fecal supernatants. FOS and inulin greatly affected the production of short-chain fatty acids in fecal cultures; butyrate was the major fermentation product on inulin, whereas mostly acetate and lactate were produced on FOS.     

Pectinase production by a Brazilian thermophilic fungus <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> N31 in solid-state and submerged fermentation

Thermophilic organisms produce thermostable enzymes, which have a number of applications, justifying the interest in the isolation of new thermophilic strains and study of their enzymes. Thirty-four thermophilic and thermotolerant fungal strains were isolated from soil, organic compost, and an industrial waste pile based on their ability to grow at 45°C and in a liquid medium containing pectin as the only carbon source. Among these fungi, 50% were identified at the genus level as Thermomyces, Aspergillus, Monascus, Chaetomium, Neosartoria, Scopulariopsis, and Thermomucor. All isolated strains produced pectinase during solid-state fermentation (SSF). The highest polygalacturonase (PG) activity was obtained in the culture medium of thermophilic strain N31 identified as Thermomucor indicae-seudaticae. Under SSF conditions on media containing a mixture of wheat bran and orange bagasse (1 : 1) at 70% of initial moisture, this fungus produced the maximum of 120 U/ml of exo-PG, while in submerged fermentation (SmF) it produced 13.6 U/ml. The crude PG from SmF was more thermostable than that from SSF and exhibited higher stability in acidic pH.     

The thermophilic bacteria hydrolyzing agar: Characterization of thermostable agarase

Three strains of anaerobic thermophilic bacteria capable of growing on agarose as a source of energy and carbon were isolated from hot springs near Lake Baikal (Barguzin National Park) and the caldera Uzon (Kamchatka). Cells of all the three strains were spore bacilli with peritrichous flagellation. These isolates grew at a temperature of 55–60°C and pH 6.5–7.0 and fermented a wide range of organic substrates. Analysis of the 16S rRNA sequences allowed us to ascribe the strains B5 and K14 to the genus Thermoanaerobacter and the strain K67 to the genus Caldoanaerobacter. According to the results of DNA-DNA hybridization, B5 was determined as belonging to the species Thermoanaerobacter wiegelii. Agarase was isolated by preparative PAGE and subsequent gel chromatography from the culture liquid of strain B5 grown on the medium containing 0.5% agarose and 0.3% galactose. The molecular weight of this enzyme amounted to 67 kDa and pI, to 4.2. The T. wiegelii B5 agarase was active in the pH range of 3.5 to 7.0 (optimum, 5.2) and temperature range of 50 to 80°C (optimum, 70°C). The preincubation of this enzyme at 90° C for 60 min did not reduce the agarase activity. This activity increased in the presence of metal ions; the maximal effect was observed in the presence of 5 mM Mg²⁺ and 25 mM Co²⁺.     

Xylanolytic enzyme activities produced by mesophilic and thermophilic actinomycetes grown on graminaceous xylan and lignocellulose

Mesophilic and thermophilic strains of actinomycetes were grown on media containing graminaceous xylan or lignocellulose. Aliquots of the culture fluids were sampled and assayed for enzyme activities involved in the degradation of hemicellulose. Xylanase, acetyl esterase and α-arabinofuranosidase activities could be detected after different times of incubation; their production was also dependent on the growth medium. The highest levels of xylanase activity were found in cultures of strains of Streptomyces, Actinomadura sp. and Saccharomonospora viridis. Streptomyces cyaneus produced the highest amount of arabinofuranosidase whereas acetyl esterase activities were highest in S. cyaneus, S. viridis and Pseudonocardia thermophila .     

Enzymology of the thermophilic ascomycetous fungus Thermoascus aurantiacus

Different strains of the thermophilic ascomycetous fungus Thermoascus aurantiacus have been reported in the literature to produce high levels of a variety of industrial interest enzymes (i.e. amylases, cellulases, pectinases and xylanases), which have been shown to be remarkably stable over a wide range of temperatures and appear to have tremendous commercial potential. Most studies on enzyme production by T. aurantiacus are carried out in chemically defined liquid medium, under conditions suitable for induction of a particular enzyme. A few studies have investigated the production of some enzymes by T. aurantiacus by solid-state fermentation, using lignocellulosic materials. The present review focuses on the enzymes produced by T. aurantiacus, their main kinetic parameters, and the effect of different culture conditions on production and enzyme activity. It also provides a view of the possible applications of T. aurantiacus enzymes, considering that this thermophilic fungus could comprise a potential source of thermostable enzymes.     

Strain variability of <Emphasis Type="Italic">Irpex Lacteus</Emphasis> basidiomycetes in the synthesis of specific milk-clotting proteinases

The ability to synthesize milk-clotting (rennet) proteinases was studied in eight strains of Irpex Lacteus basidiomycetes. It was found that the studied I. lacteus strains are characterized by different enzyme activities in their culture liquid. For I. lacteus strains 2425, 2426, and 2427, the maximum milk-clotting activity was detected during the exponential growth phase on the 15th day of cultivation on glucose–peptone medium. These I. lacteus strains are the most prospective milk-clotting enzyme producers for further research and practical application. I. lacteus strains 2421, 2422, 2423, 2424, and 2428 showed lower values of the enzyme activity and require additional research to determine the optimal culture conditions.     

Thermoalkalophilic lipase-producing Bacillus selected by continuous cultivation

Abstract Several lipase-producing thermophilic bacteria were isolated by continuous culture from samples collected near Bulgarian hot springs. Most of them had lipase activity of about 0.5 U ml⁻¹ when activated in shaken flasks. Three strains, Gram-positive sporeforming rods, possess higher enzyme activity in a Tween-80 containing medium. The highest lipase activity (2.0–3.0 U ml⁻¹) was observed in the newly-isolated thermoalkalophilic Bacillus sp. strain MC7.     

Calculation of the molecular masses of two newly synthesized thermostable enzymes isolated from thermophilic microorganisms

Two thermostable enzymes synthesized by thermophilic microorganisms were isolated and purified. A thermostable ß-galactosidase was produced in a continuous fermentation process by Bacillus stearothermophilus TP 32 as an intracellular enzyme. After applying different concentration procedures the raw extract enzyme was prepurified on a Sephadex G-200 size exclusion column. The isolated ß-galactosidase fraction was then separated with HPLC on a TSK G 3000 SW size exclusion column to determine the molecular mass based on calibration curves of standard proteins. The other enzyme, a thermostable protease, was synthesized by Bacillus stearothermophilus TP 26 as an extracellular enzyme. After its concentration, the enzyme was purified on a classical size exclusion column (Sephacryl S-200) and on a HPLC size exclusion column (BIO-SIL TSK-250). The micropreparatively isolated fraction was separated again on this HPLC column to determine its molecular mass. The optimum temperature of both enzymes was approximately 75°C.