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     

Purification and characterization of extracellular inulinase from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the purified inulinase

The extracellular inulinase of the marine yeast Pichia guilliermondii strain 1 was purified to homogeneity resulting in a 7.2-fold increase in specific inulinase activity. The molecular mass of the purified enzyme was estimated to be 50.0 kDa. The optimal pH and temperature for the purified enzyme were 6.0 and 60°C, respectively. The enzyme was activated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Fe³⁺, Fe²⁺, Cu²⁺, and Co²⁺, but Mg²⁺, Hg²⁺, and Ag⁺ inhibited activity. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1, 10-phenanthroline. The K _m and V _max values of the purified inulinase for inulin were 21.1 mg/mL and 0.08 mg/min, respectively. A large number of monosaccharides were detected after the hydrolysis of inulin. The deduced protein sequence from the cloned P. guilliermondii strain 1 inulinase gene contained the consensus motifs R-D-P-K-V-F-W-H and W-M-N-D-P-N-G, which are conserved among the inulinases from other microorganisms.     

Effect of inulin on yeast inulinase production in sauerkraut brine

Kluyveromyces marxianus NRRL Y-1196 produced the highest inulinase activity (38 U/mg protein) of six yeasts examined after 24 h growth in sauerkraut brine in shaking flasks at 30°C with 0.3% inulin as an enzyme inducer. The enzyme was recovered by acetone fractionation, with a yield of 81%. It had maximum activity at pH 4.4 and 55°C with K _m values for inulin and sucrose of 3.92 mm and 11.9 mm, respectively. The yeast raised the pH from 3.4 to above 7.0, using all the lactic acid in the brine. Growth of K. marxianus in sauerkraut brine with a small amount of inulin may usefully decrease the BOD and concomitantly produce inulinase.The authors are with the Department of Food Science and Technology, Cornell University, Geneva, New York 14456, USA     

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     

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.     

Inulinase production by the marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the crude inulinase

The marine yeast strain G7a isolated from sediment of China South Sea was found to secrete a large amount of inulinase into the medium. This marine yeast strain was identified to be a strain of Cryptococcus aureus according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast showed the highest activity at pH 5.0 and 50 °C. The optimal medium for inulinase production was artificial seawater containing inulin 4.0% (w/v), K₂HPO₄ 0.3% (w/v), yeast extract 0.5% (w/v), KCl 0.5% (w/v), CaCl₂ 0.12% (w/v), NaCl 4.0% (w/v) and MgCl₂·6H₂O 0.6% (w/v), while the optimal cultivation conditions for inulinase production were pH 5.0, a temperature of 28 °C and a shaking speed of 170 rpm. Under the optimal conditions, over 85.0 U/ml of inulinase activity was produced within 42 h of fermentation at shake flask level. This is very high level of inulinase activity produced by yeasts. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.     

Inulinase ofDebaryomyces cantarellii

Debaryomyces cantarellii excretes into a buffered medium an inulinase of β-fructofuranosidase type, its synthesis being induced by inulin. The enzyme has a pH optimum at 4 and its optimum temperature is 50°C. ItsK _m for inulin is 15mm.     

Immobilization of thermostable exo-inulinase from mutant thermophilic Aspergillus tamarii-U4 using kaolin clay and its application in inulin hydrolysis

In this study, attempts were made to immobilize purified exo-inulinase from mutant thermophic Aspergillus tamarii-U4 onto Kaolinite clay by covalent bonding cross-linked with glutaraldehyde with an immobilization yield of 66% achieved. The free and immobilized inulinases were then characterized and characterization of the enzymes revealed that temperature and pH optima for the activity of the free and immobilized enzymes were both 65?°C and pH 4.5 respectively. The free inulinase completely lost its activity after incubation at 65?°C for 6 h while the immobilized inulinase retained 16.4% of its activity under the same condition of temperature and incubation time. The estimated kinetic parameters K_m and V_max for the free inulinase as estimated from Lineweaver-Burk plots were 0.39?mM and 4.21?µmol/min for the free inulinase and 0.37?mM and 4.01?µmol/min for the immobilized inulinase respectively. Inulin at 2.5% (w/v) and a flow rate of 0.1?mL was completely hydrolysed for 10?days at 60?°C in a continuous packed bed column and the operational stability of the system revealed that the half-life of the immobilized inulinase was 51?days. These properties make the immobilized exo-inulinase from Aspergillus tamarii-U4 a potential candidate for the production of fructose from inulin hydrolysis.     

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.     

β-Fructofuranosidase and sucrose phosphorylase of rumen bacterium <Emphasis Type="Italic">Pseudobutyrivibrio ruminis</Emphasis> strain 3

The subject of this study was the fructan and sucrose degrading enzymes of bacterium Pseudobutyrivibrio ruminis strain 3. It was stated that cell extract from bacteria growing on inulin contained β-fructofuranosidase (EC 3.2.1.80 and/or EC 3.2.1.26) and sucrose phosphorylase (EC 2.4.1.7), while the bacteria maintained on sucrose showed only phosphorylase. Partially purified β-fructofuranosidase digested inulooligosaccharides and sucrose to fructose or fructose and glucose, respectively, but was unable to degrade the long chain polymers of commercial inulin and Timothy grass fructan. Digestion rate of inulooligosaccharides fit Michaelis–Menten kinetics with V_max 5.64 μM/mg/min and K_m 1.274%, respectively, while that of sucrose was linear. Partially purified sucrose phosphorylase digested only sucrose. The digestion products were fructose, glucose-1P and free glucose. The reaction was in agreement with Michaelis–Menten kinetics. The V_max were 0.599 and 0.584 μM/mg/min, while K_m were 0.190 and 0.202% for fructose release and glucose-1P formation, respectively, when bacteria grew on inulin. The V_max were, however, 1.37 and 1.023 μM/mg/min, while K_m were 0.264 and 0.156%, if bacteria were grown on sucrose. The free glucose was hardly detectable for the enzyme originated from inulin grown bacteria, but glucose levels ranged from 0.05 to 0.25 μM/mg/min, when cell extract from bacteria grown on sucrose was used. Release of free glucose was observed when no inorganic phosphate was present in reaction mixture.     

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.     

Synthesis of wall glucan from sucrose by enzyme preparations from Pisum sativum

Radioactive sucrose, supplied through the cut base to Pisum sativum epicotyls, was transported to the growing apex (plumule and hook) and used there for the synthesis mainly of uridine diphosphoglucose (UDP- glucose), fructose and cell wall glucan. Enzyme extracts of the apical tissue contained sucrose synthetase activity which was freely reversible, i.e. formed UDP-glucose and fructose from sucrose (pH optimum = 6·6 for the cleavage reaction, K_m for sucrose = 63 mM). Particulate fractions of the same tissue contained a β-glucan synthetase which utilized UDP-glucose for formation of alkali-soluble and -insoluble products (pH optimum = 8·4, K_m for UDP-glucose = 1·9 mM). Values for V_max and yields of these two synthetase activities were sufficient to account for observed rates of cellulose deposition during epicotyl growth (15–25 μg/hr/epicotyl). When soluble pea enzyme was supplied with sucrose and UDP at pH 6·6 and then the preparation was supplemented with particles bearing β-glucan synthetase at pH 8·4, the glucose moiety of sucrose was converted to glucan in vitro. The results indicate that it is feasible for these synthetases to co-operate in vivo to generate β-glucan for expanding cell walls.     

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.     

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 1. Preparation and enzymatic properties

Kluyveromyces marxianus cells with inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) activity have been immobilized in open pore gelatin pellets with retention of > 90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6.0. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mol^?1, respectively. K_m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.     

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.     

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)     

Characterization of the spinach leaf phosphorylases

The chloroplastic and the cytoplasmic phosphorylases were purified and their kinetic properties characterized. The cytoplasmic enzyme was purified to homogeneity via affinity chromatography on a glycogen-Sepharose column. Subunit molecular weight studies indicated a value of 92,000, whereas a native molecular weight value of 194,000 was obtained by sucrose density gradient centrifugation. The chloroplast enzyme's native molecular weight was determined to be 203,800. The cytoplasmic enzyme shows the same V_max for maltopentaose, glycogen, amylopectin, amylose, and debranched amylopectin but is only slightly active toward maltotetraose. The K_m for phosphate at pH 7.0 is 0.9 millimolar and for glucose-1-phosphate, 0.64 millimolar. The K_m values for phosphorolysis of amylopectin, amylose, glycogen, and debranched amylopectin are 26, 165, 64, and 98 micrograms per milliliter, respectively. In contrast, the relative V_max values for the chloroplast enzyme at pH 7.0 are debranched amylopectin, 100, amylopectin, 63.7, amylose, 53, glycogen, 42, and maltopentaose, 41. K_m values for the above high molecular weight polymers are, respectively, 82, 168, 122 micrograms per milliliter, and 1.2 milligrams per milliliter. The K_m value for inorganic phosphate is 1.2 millimolar. The chloroplastic phosphorylase appears to have a lower apparent affinity for glycogen than the cytoplasmic enzyme. The results are discussed with respect to previous findings of multiple phosphorylase forms found in plant tissues and to possible regulatory mechanisms for controlling phosphorylase activity.     

Partial purification and characterization of exoinulinase produced from Bacillus sp.

Inulinase are industrial food enzymes which have gained much attention in recent scenario. In this study, Inulinase producing eight bacterial colonies were isolated and screened from three different plant root tubers soil sample. Among 8 inulinase producing colonies, the higher yielding colony was selected with 25.10?U/mL for further studies. The best inulinase producing colony was identified by partial 16S rRNA gene sequence as Bacillus sp. The crude inulinase was purified by using ammonium sulphate precipitation, dialysis and ion exchange chromatography on DEAE – sephacel and obtained 1.9 purification fold with total activity 293 U. The purified enzyme was subjected to characterization studies and it was found to be stable at 30–60?°C and optimum temperature was at 55?°C. The enzyme was stable at pH 3.0–7.0 and optimum pH was at 6.5. The K_m and V_max value for inulinase was found to be 0.117?mg/mL and 4.45?μmol?min?mg^?1 respectively, demonstrate its greater affinity. Hence, this enzyme can be widely used for the production of fructose, and fructooligosaccharides, which are important ingredients in food and pharmaceutical industry.     

Optimization of simultaneously enzymatic fructo- and inulo-oligosaccharide production using co-substrates of sucrose and inulin from Jerusalem artichoke

Prebiotic substances are extracted from various plant materials or enzymatic hydrolysis of different substrates. The production of fructo-oligosaccharide (FOS) and inulo-oligosaccharide (IOS) was performed by applying two substrates, sucrose and inulin; oligosaccharide yields were maximized using central composite design to evaluate the parameters influencing oligosaccharide production. Inulin from Jerusalem artichoke (5–15% w/v), sucrose (50–70% w/v), and inulinase from Aspergillus niger (2–7 U/g) were used as variable parameters for optimization. Based on our results, the application of sucrose and inulin as co-substrates for oligosaccharide production through inulinase hydrolysis and synthesis is viable in comparative to a method using a single substrate. Maximum yields (674.82?mg/g substrate) were obtained with 5.95% of inulin, 59.87% of sucrose, and 5.68 U/g of inulinase, with an incubation period of 9?hr. The use of sucrose and inulin as co-substrates in the reaction simultaneously produced FOS and IOS from sucrose and inulin. Total conversion yield was approximately 67%. Our results support the high value-added production of oligosaccharides using Jerusalem artichoke, which is generally used as a substrate in prebiotics and/or bioethanol production.