Analysis of the gene for β-fructosidase (invertase, inulinase) of the hyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed in Escherichia coli

This is the first report describing the gene structure and the enzymatic properties of a β-fructosidase of a hyperthermophilic organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other β-fructosidases. On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the β-anomeric configuration of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis of sucrose and inulin with k _cat/K _m values (at 75 °C, pH 5.5) of about 4.1 × 10⁴M⁻¹s⁻¹ and 3.1 × 10⁴M⁻¹s⁻¹ respectively. BfrA had an optimum temperature of 90–95 °C (10-min assay) and was extremely insensitive to thermo-inactivation. During 5 h at temperatures up to 80 °C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the most thermostable β-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-β-d-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability recommend it for use in biotechnology. Received: 28 August 1997 / Received revision: 19 January 1998 / Accepted: 24 January 1998     

The β-fructofuranosidase activities of a strain of Clostridium acetobutylicum grown on inulin

Summary The -fructofuranosidase activities of a strain of Clostridium acetobutylicum, selected for its capacity to grow on inulinic substrates, were investigated. When grown on inulin, this strain produced extracellular and intracellular -fructofuranosidases, both of which hydrolysed inulin (inulinase activity) and sucrose (invertase activity). Inulinase activity was higher than invertase activity in the extracellular preparation, the opposite being observed for the cellular preparation. The effects of pH and temperature, substrate specificity and the kinetic constants for inulin and sucrose were studied on both preparations, as well as induction by inulin and repression by glucose and fructose of inulinase and invertase activities. The overall results were consistent with the existence of a least one inulinase, (EC 3.2.1.7), mainly but not entirely released in the extracellular medium, and an invertase (3.2.1.26) localized within the cell.Time course hydrolysis experiments of dalhia inulin and Jerusalem artichoke inulofructans by extracellular inulinase showed that this preparation had a remarkably high specificity for hydrolysis of long chain inulofructans.     

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source. This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30. Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity. The optimal hydrolysis temperature for sucrose, raffinose and inulin was 55°C and the optimal pH for sucrose was 4.75. The apparent K _m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively. Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides. The results obtained suggest the hypothesis that enzyme production was constitutive. Journal of Industrial Microbiology & Biotechnology (2000) 25, 63–69. Received 17 November 1999/ Accepted in revised form 30 May 2000     

General properties of extracellular bacterial inulinase

The extracellular inulinase system of a strain of Arthrobacter sp. consists of a β -fructofuranosidase active on inulin raffinose and sucrose with a relative rate inulin/sucrose (I/S) of 0.2.
Crude enzyme preparations were obtained by fractionation of the liquid culture at stationary phase of growth with ammonium sulphate. Purification was carried out by DEAE cellulose chromatography and ultrogel ACA 34. Only one protein band was observed by electrophoresis. The enzyme was stable at high temperatures and was active at neutral or slightly alkali pH. Fructose is liberated as the sole reaction product of inulin hydrolysis, suggesting that the enzyme was an exoinulinase. The Michaelis constant (calculated at 40°C and pH 6) was 0.25 × 10^-2 mol/l for the inulin and 0.12 × 10^-2 mol/l for sucrose.
The enzyme was suitable for fructose production from root extracts of plants rich in polyfructosans or sucrose.     

Characterization and properties of an inulinase from a thermophilic bacteria

The inulinase of the thermophilic bacterial strain LCB41 (Bacillus sp.) was produced in fermentor using a mineral medium containing inulin as carbon source. The enzyme content was as high as the known inulinase producers and most of the activity was found in the culture medium. The enzyme was stable at high temperature and active at neutral and slightly basic pH. Fructose is liberated as the sole reaction product of inulin hydrolysis, classifying the enzyme as an exoinulinase. Inulin and sucrose were both hydrolyzed at appreciable rates with an (I/S) ratio of 0·40 and (V_m/K_m)₁/(V_m/K_m)_S = 9·9. The enzyme was less inhibited than yeast invertase or Kluyveromyces fragilis inulinase at high sucrose concentrations. The inulinase of strain LCB41 is a good candidate for industrial hydrolysis of inulin or sucrose.     

A study of inulinase activity in theClostridium acetobutylicum strain ABKn8

Summary Several strains ofClostridium acetobutylicum, isolated from sugar beet pulps or Jerusalem artichokes, are able to utilize inulin, a -polyfructosane polymer of fructose with glucose as the terminal residue. Inulin-degrading activity, which was detected in cultures of one such strain, ABKn8, grown in Basol-medium containing inulin, reached a maximum at the end of exponential phase. Most of the enzyme activity was detected in the supernatant. It was stably maintained in 0.1 M acetate buffer pH 5.0, and was optimal at pH 4.6. The enzyme, inulinase was induced by inulin, but not by xylose, fructose or sucrose and was repressed by glucose. Inulinase was active against inulin, sucrose and raffinose, but not melezitose. It had a higher affinity for inulin (K _m : 1.2×10^-2 mM) than all the other known inulinases.     

The cell wall-associated inulinase of Kluyveromyces fragilis

The yeast Kluyveromyces fragilis (ATCC 12424) was grown on a 2% inulin-1% yeast extract medium for 36 h and subsequently fixed with 0.5% glutaraldehyde. The glutaraldehyde treatment did not affect the -fructofuranosidase (inulinase, EC 3.2.1.7) activity of the cells but it did make the cells resistant to chemical and physical treatments that normally release -fructofuranosidase from untreated cells. The enzyme in the treated cells exhibited K_m values for sucrose and raffinose identical to those obtained for the free enzyme. The cell wall of the treated cells exhibited the same diffusion properties for sucrose, raffinose, and inulin as those observed for untreated cells. The -fructofuranosidase was not bound covalently to the cell by the glutaraldehyde treatment. The results support the permeability barrier model for the enzyme retention in the yeast cell wall.     

Purification and properties of an extracellular inulinase-like β-fructosidase from Bacillus stearothermophilus

A novel extracellular β-fructosidase produced by Bacillus stearothermophilus has been identified and purified. The purified enzyme, obtained by using successive QEAE Sepharose fast flow and Sephacryl S300 HR columns, has a 600 kDa relative molecular weight (M_r) and is composed of 60 kDa subunits indicating a multimeric structure. The pH and temperature for optimal activity are 6.5 and 65°C respectively, the enzyme being thermostable at this temperature. The apparent K_m values for sucrose and inulin are 3.56 mmol l^-1 and 1 mmol l^-1 respectively, the total invertase/total inulinase ratio being 4.     

Production and properties of a bacterial thermostable exo-inulinase

Inulinase and Invertase Activities, Thermophilic Bacilli, Enzyme Thermostability Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp. 11 strain was studied by batch cultivation in a fermentor. The achieved inulinase and invertase activities after a short growth time (4.25 h) were similar or higher compared to those reported for other mesophilic aerobic or anaerobic thermophilic bacterial producers and yeasts. The investigated enzyme belonged to the exo-type inulinases and splitted-off inulin, sucrose and raffinose. It could be used at temperatures above 65 degrees C and pH range 5.5-7.5. The obtained crude enzyme preparation possessed high thermostability. The residual inulinase and invertase activities were 92-98% after pretreatment at 65 degrees C for 60 min in the presence of substrate inulin.     

Inulinase activity ofDebaromyces cantarellii

Debaromyces cantarellii Capriotti contains an inulinase activity which is inducible by growth on inulin but not on other β-fructosides. The induction is inhibited by glucose and fructose. The system is situated in the cell wall and can be best extracted with a 20 mm phosphate buffer at pH 8.5. The inulinase activity shows pH optima at 4 and 6, suggesting the presence of two enzymes, the latter being more tightly bound to the cell wall. Both enzymes degrade inulin from the nonreducin end. The cells also contain a constitutive β-fructofuranosidase with a specificity partly overlapping with that of the inulinase(s).     

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.     

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.     

Extracellular invertase of Rhizobium japonicum and its role in free sugar metabolism in the developing root nodules of Sesbania grandiflora

Extracellular invertase of Rhizobium japonicum and its role in free sugar metabolism in the developing root nodules of Sesbania grandiflora L. was studied. The enzyme hydrolysed sucrose extracellularly, and its release was substrate inducible. 0.1 Mβ-mercaptoethanol released the cell-bound form of this enzyme. The production of invertase was low when glucose, galactose, mannose, fructose and raffinose were used as carbon sources in the growth medium. In the developing nodules sucrose was the major sugar. The content of fructose was low in comparison with that of glucose – suggesting that in the nodules, fructose is converted to glucose prior to its entry into the bacterial cell. The content of glucose synchronised with the pattern of change in the activity of invertase in the nodules.     

Carbohydrates,invertase activity,growth and dimorphism inSporisorium reilianum

Sporisorium reilianum, the fungus that causes sorghum head smut, was grown with sucrose, lactose, trehalose or raffinose in liquid suspension or on a solid medium. Liquid culture media were analyzed for hydrolysis products of these carbohydrates to determine extracellular enzyme activity of the fungus. Increased amounts of glucose and fructose in the culture medium ofS. reilianum grown with sucrose or raffinose indicated that invertase (-fructofuranosidase, 3.2.1.26) activity was present. No evidence of extracellular galactosidase or trehalase activity was found. Enhanced sporidial colony formation on carbohydrates that can be hydrolyzed to hexoses, and specific forms of mycelial growth on lactose, trehalose or on a carbohydrate-deficient medium might suggest that mycelial growth is a way of foraging for food sources. However, the rapid and profuse mycelial growth on the host cell wall glycoprotein appears to be in response to abundant food supply (probably of a different type). Therefore availability of different kinds of carbon sources in the environment of the growing fungus might determine dimorphism and associated pathogenesis byS. reilianum.Technical Article No: 30699 from the Texas Agricultural Experiment Station.     

Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of high-fructose syrup

An extracellular exoinulinase (2,1-beta-D fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable (100%) for 3 h at the optimum temperature of 50 degrees C. Mn2+ and Ca2+ produced a 2.4-fold and 1.2-fold enhancement in enzyme activity, whereas Hg2+ and Ag2+ completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6 mg/ml and 41.3 mg/ml, respectively.     

Changes in Sugar Content and Activities of Sucrose Metabolizing Enzymes in Roots and Nodules of Lentil

Activities of acid and alkaline invertases and sucrose synthase were determined in roots and nodules of lentil at various stages of development. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodule cytosol, but there was only a small amount of acid invertase present. Activity of sucrose metabolizing enzymes in roots was significantly less than that observed in the nodules. Amongst sugars, sucrose was found to be the main component in the host cytosol. Lentil neutral invertase (LNI) was partially purified from nodules at 50 days after sowing (DAS). Two forms of invertase were identified, i.e., a major form of 71 kDa which was taken for enzyme characterization and a minor form of 270 kDa which was not used for further studies. The purified enzyme exhibited typical hyperbolic saturation kinetics for sucrose hydrolysis. It had a Km of 11.0 to 14.0 mM for sucrose depending upon the temperature, a pH optimum of 6.8 and an optimum temperature of 40 °C. Compared with raffinose and stachyose, sucrose was better substrate for LNI. The enzyme showed no significant hydrolysis of maltose and p-nitrophenyl--D-glucopyranoside, showing its true -fructosidase nature. LNI is completely inhibited by HgCl₂, MnCl₂ and iodoacetamide but not by CaCl₂, MgCl₂ or BaCl₂.     

Localization of inulinase and invertase in Kluyveromyces species

In vivo hydrolysis of inulin and sucrose was examined in selected yeasts of the genus Kluyveromyces. Cells, grown in sucrose-limited chemostat cultures, were subjected to treatments for the removal of inulinase, the enzyme responsible for the hydrolysis of both inulin and sucrose. The effects of these treatments were studied by measurement of inulin-dependent and sucrose-dependent oxygen consumption by cell suspensions. In Kluyveromyces marxianus var. marxianus, inulinase was partially secreted into the culture fluid. Removal of culture fluid inulinase by washing had no effect on sucrose-dependent oxygen consumption by this yeast. However, this treatment drastically reduced inulin-dependent oxygen consumption. Treatment of washed cells with sulfhydryls removed part of the cell wall-retained inulinase and reduced inulin-dependent oxygen consumption by another 80%. Sucrose-dependent oxygen consumption was less affected, decreasing by 40%. Cell suspensions of K. marxianus var. drosophilarum, K. marxianus var. vanudenii, and Saccharomyces kluyveri rapidly utilized sucrose but not inulin. This is in accordance with the classification of these yeasts as inulin negative. Supernatants of cultures grown at pH 5.5 did not catalyze the hydrolysis of inulin and sucrose. This suggested that these yeasts contained a strictly cell-bound invertase, an enzyme not capable of inulin hydrolysis. However, upon washing, cells became able to utilize inulin. The inulin-dependent oxygen consumption further increased after treatment of the cells with sulfhydryls. These treatments did not affect the sucrose-dependent oxygen consumption of the cells. Apparently, these treatments removed a permeability barrier for inulin that does not exist for sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359)

This is the first report describing the purification and enzymatic properties of a native invertase (β-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85°C at pH 6.0. At temperatures of 80–85°C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K _m and k _cat /K _m values (at 85°C and pH 6.0) of about 14 mM and 5.2 × 10⁸ M⁻¹ s⁻¹, respectively. Dedicated to the memory of Prof. R. A. Nicolaus, founder of the Institute (1968).     

Fructanolytic and saccharolytic enzymes of the rumen bacterium <Emphasis Type="Italic">Pseudobutyrivibrio ruminis</Emphasis> strain 3 — <Emphasis Type="Italic">preliminary study</Emphasis>

P. ruminis strain 3 was isolated from the ovine rumen and identified on the basis of comparison of its 16S rRNA gene with GenBank. The bacterium was able to grow on Timothy grass fructan, inulin, sucrose, fructose and glucose as a sole carbon source, reaching absorbance of population in a range of 0.4–1.2. During 1 d the bacteria exhausted 92–97 % of initial dose of saccharides except for inulin (its utilization did not exceed 33 %). The bacterial cell extract catalyzed the degradation of Timothy grass fructan, inulin and sucrose in relation to carbon source present in growth medium. Molecular filtration on Sephadex G-150, polyacrylamide gel electrophoresis combined with zymography technique and TLC was used to identify enzymes responsible for the digestion of sucrose and both polymers of fructose. Two specific endolevanases (EC 3.2.1.65), nonspecific β-fructofuranosidase (EC 3.2.1.80 and/or EC 3.2.1.26) and sucrose phosphorylase (EC 2.4.1.7) were detected in cell-free extract from bacteria grown on Timothy grass fructan.     

Expression of exo‐inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta‐gami B (DE3) and its characterization

Inulin is a linear carbohydrate polymer of fructose subunits (2‐60) with terminal glucose units, produced as carbon storage in selected plants. It cannot directly be taken up by most microorganisms due to its large size, unless prior hydrolysis through inulinase enzymes occurs. The hydrolyzed inulin can be taken up by microbes and/or recovered and used industrially for the production of high fructose syrup, inulo‐oligosaccharides, biofuel, and nutraceuticals. Cell‐free enzymatic hydrolysis would be desirable for industrial applications, hence the recombinant expression, purification and characterization of an Aspergillus niger derived exo‐inulinase was investigated in this study. The eukaroyototic exo‐inulinase of Aspergillus niger 12 has been expressed, for the first time, in an E. coli strain [Rosetta‐gami B (DE3)]. The molecular weight of recombinant exo‐inulinase was estimated to be ～81 kDa. The values of K_m and V_max of the recombinant exo‐inulinase toward inulin were 5.3 ± 1.1 mM and 402.1 ± 53.1 µmol min^?1 mg^?1 protein, respectively. Towards sucrose the corresponding values were 12.20 ± 1.6 mM and 902.8 ± 40.2 µmol min^?1 mg^?1 protein towards sucrose. The S/I ratio was 2.24 ± 0.7, which is in the range of native inulinase. The optimum temperature and pH of the recombinant exo‐inulinase towards inulin was 55°C and 5.0, while they were 50°C and 5.5 towards sucrose. The recombinant exo‐inulinase activity towards inulin was enhanced by Cu²⁺ and reduced by Fe²⁺, while its activity towards sucrose was enhanced by Co²⁺ and reduced by Zn²⁺. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:629–637, 2016