Purification of lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts using high-performance liquid chromatography

Glucocerebrosidase from human skin fibroblasts was purified more than 2300-fold to apparent homogeneity with an overall yield of 39% using taurocholate extraction, ammonium sulfate fractionation, and high-performance hydrophobic interaction and gel permeation column chromatography. This relatively high yield is attributed to two modifications from previously published procedures: (i) the elimination of a butanol delipidation step that resulted in substantial loss of enzyme activity; and (ii) the use of 2% (w/v) sodium taurocholate instead of 1-2% sodium cholate that resulted in more than 90% solubilization of total membrane-bound enzyme activity. Confluent monolayers of human cultured skin fibroblasts (approximately 3.6 x 10(8) cells) were harvested from 10 roller bottles. Glucocerebrosidase in the cell pellet was solubilized with 2% (w/v) sodium taurocholate, fractionated in 14% ammonium sulfate, and applied to a high-performance hydrophobic interaction phenyl-5PW column. After an ammonium sulfate descending linear gradient step, glucocerebrosidase was eluted from the column at 4% cholate concentration using a 0-5% linear cholate gradient. There was a 36-fold purification and 80% recovery. In the subsequent step, concentrated glucocerebrosidase fractions from the phenyl column were injected into two Bio-Sil TSK-250 gel permeation columns joined in series. Glucocerebrosidase peak activity was eluted at 263 ml corresponding to Mr 76,000. There was an 18-fold purification and 78% recovery. The enzyme preparation was then recycled through the phenyl-5PW column in order to remove a remaining contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of cytochrome b-245 from human neutrophils.

The low potential cytochrome b (b-245) of the microbicidal oxidase of phagocytic cells has been purified from neutrophils from patients with chronic myeloid leukaemia. Cells were homogenized in the presence of proteinase inhibitors and centrifuged to remove the cytoplasm. The pellets containing membranes, granules and other organelles (15 mg/ml) were then washed with buffered sodium cholate (5 mg/ml). Residual pellets were subsequently solubilized with the non-ionic detergent Triton N 101 (10 mg/ml) which extracted about 60% of the cytochrome b. About 10% of the cytochrome b was of mitochondrial origin which was removed on a column of n-amino-octyl-Sepharose that did not adsorb cytochrome b-245. Cytochrome b-245 was chromatographed on a column of heparin-agarose and eluted with NaCl to give a peak specific content of 11-16 nmol of cytochrome b-245/mg of protein, representing a 140-200-fold purification with a recovery of 15%. This technique results in the purification of approx. 100-150 nmol of highly purified cytochrome b-245 from (3-5) X 10(11) cells within 4 days. The most purified material gave a broad band with an apparent Mr of between 68 000 and 78 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, but gel filtration indicated an aggregated form of the protein in Triton N101 . Purified protein (14 nmol of haem/mg of protein) did not contain FAD or FMN and had no NADPH-dependent O2--generating activity.     

Purification and the effect of peptide N-glycosidase F on lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts.

Glucocerebrosidase was purified from human cultured dermal fibroblasts more than 2200-fold to apparent homogeneity using high performance Alkyl-Superose HR 5/5 hydrophobic interaction and Bio-Sil TSK-250 gel permeation column chromatography. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis and protein staining of the catalytically active and concentrated enzyme fractions from the gel permeation columns revealed the presence of one band of Mr 64,000. The glucocerebrosidase preparation purified to homogeneity was digested with peptide N-glycosidase F that cleaves N-linked oligosaccharide structures from glycoproteins. The molecular weight of glucocerebrosidase after digestion with peptide N-glycosidase F was reduced to Mr 57,000, suggesting that the mature enzyme is a glycoprotein and that N-linked oligosaccharide constitutes a minimum of about 10% of the total molecular weight of the polypeptide. These findings are compatible with the hypothesis that glucocerebrosidase was initially synthesized as a precursor polypeptide which was subsequently glycosylated to become the mature enzyme.     

Purification of isopenicillin N synthetase from Streptomyces clavuligerus

Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 X 10(-3) units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.     

Purification of xanthine dehydrogenase from rat liver: a rapid procedure with high enzyme yields

Xanthine dehydrogenase (EC 1.2.1.37) was purified approximately 1000-fold from liver homogenates of adult male Sprague-Dawley rats. Enzyme recovery was good (greater than 20% of the starting activity was obtained), and the homogeneously pure enzyme had a molecular mass of approximately 300,000 Da. The purified protein exhibited a specific activity of 2470 units/mg protein and spectral properties identical to those of the best preparations of this enzyme reported by other investigators. Routine preparations of this enzyme also possess higher dehydrogenase:oxidase ratios (typically between 5 and 6) than do other xanthine dehydrogenase preparations so far reported in the literature. Maximum dehydrogenase:oxidase ratios, greater than 10, could be obtained from this procedure if only peak dehydrogenase fractions from the chromatography columns were saved. The present small-scale purification method, which can be completed in 48-60 h, utilizes ammonium sulfate fractionation, Sephadex G-200 column chromatography, Blue Dextran-Sepharose column chromatography, and preparative gel electrophoresis.     

Reconstitution and purification of the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain

The sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain has been highly purified. Synaptic plasma membranes from rat brain were extracted with cholate in the presence of 10% ammonium sulfate. The soluble extract was incorporated into liposomes consisting of asolectin and crude brain lipids. Brain lipids markedly enhanced the transport activity. The resulting proteoliposomes catalyzed sodium- and chloride-coupled gamma-aminobutyric acid transport which, in the presence of internal potassium, was greatly (up to 20-fold) stimulated by valinomycin. Using this transport of the reconstituted system as an assay, the transporter was purified by the following steps. The cholate extract was fractionated by ammonium sulfate. The activity was not precipitated by 50% but could be precipitated by 70% ammonium sulfate. The cholate and ammonium sulfate were removed on a Sephadex G-50 column. Subsequently, the transporter was partially purified on DEAE-cellulose in a mixture of Triton X-100 and octyl glucoside. The active fractions were chromatographed on a hydroxylapatite column in the presence of Triton X-100. Although the increase in specific activity was only up to 100-fold, this was due to partial inactivation. The actual purification was at least 1000-fold. The purified transporter exhibited the same features of the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity, and a similar affinity. The sodium dodecyl sulfate gel pattern indicated that a major protein ran as a 24-kDa band. This band may represent the gamma-aminobutyric acid transporter.     

Purification of the muscarinic acetylcholine receptor from porcine brain

The muscarinic acetylcholine receptor of porcine cerebrum has been purified to apparent homogeneity by affinity chromatography, with conjugated 3-(2'-aminobenzhydryloxy)tropane (ABT) as described previously (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). In a single step purification using 900 ml of digitonin/cholate-solubilized preparations and 300 ml of the ABT-agarose gel, we obtained, in a yield of 10-15%, more than 250 pmol of muscarinic receptors which bind [3H]N-methylscopolamine with a specific activity of 1,000-5,000 pmol/mg of protein (1,000-5,000-fold purification). The muscarinic receptors eluted from the ABT-agarose gel with 0.1 mM atropine were adsorbed to hydroxylapatite and then recovered as a concentrated solution. Muscarinic receptors were further purified by rechromatography with the same gel or by gel permeation high pressure liquid chromatography. The amino acid composition of the purified receptor was determined, and the specific activity of the purified preparation was estimated to be 13,100 pmol/mg of protein on the basis of amino acid composition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptors with or without radioiodination revealed a single, major band with an apparent Mr of 70,000 either by silver staining or radioautogram. The major band corresponded to the band which specifically bound [3H]propylbenzylcholine mustard (irreversible muscarinic ligand). The purified receptor showed essentially the same specificity for muscarinic ligands as unpurified receptors.     

Purification of human gamma-interferon to essential homogeneity and its biochemical characterization

A multistep procedure has been developed which enables human gamma-interferon (HuIFN-gamma) to be purified to essential homogeneity. The procedure takes advantage of a modification of a previously described sequential chromatographic technique [Braude, I.A. (1983) Prep. Biochem. 13, 177-190] and the high isoelectric point of HuIFN-gamma (pH 9.5-9.8). The steps include Controlled Pore Glass adsorption chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, cation-exchange chromatography, and gel filtration chromatography. The purified HuIFN-gamma had a specific activity of 5.9 X 10(7) units/mg. This represents a purification of more than 70 000-fold and a 33% recovery. In addition, one gel filtration fraction had a specific activity of 2.5 X 10(8) units/mg. This represents a purification of greater than 300 000-fold and a recovery of greater than 17%. This fraction, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was shown to be composed of one major 26-kilodalton (kDa) species and four minor species of 74, 67, 56, and 22 kDa. Analysis of this material with anti-HuIFN-gamma monoclonal antibody immunoabsorbent columns indicates that both the 26- and the 22-kDa species are HuIFN-gamma. Thus, the final product is essentially homogeneous (90-92% HuIFN-gamma), and the specific activity of pure HuIFN-gamma is approximately (2.7-2.8) X 10(8) units/mg of protein. Finally, the 26- and 22-kDa moieties are shown to be similar, if not identical, proteins as judged by amino acid and sequence analyses.     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.     

A simple and efficient method for the purification of human gamma interferon

A preparative, sequential chromatographic procedure has been developed for the purification of human gamma interferon (HuIFN-gamma). The four steps in the procedure are Controlled Pore Glass-adsorption chromatography, Concanavalin-A affinity chromatography, Heparin-Sepharose affinity chromatography and gel-filtration. By virtue of the development of a coordinated effluent-affluent buffer scheme, eluants can also serve as loading buffers for the succeeding column. Consequently, crude HuIFN-gamma preparations can be purified rapidly (approximately one week), easily, and is amenable to a semi-automated process. The procedure has also been shown to be efficient. Here, as an example, it is reported that an overall purification of greater than 75,000-fold can be achieved, yielding a specific activity of 5.2 X 10(7) units/mg, and a recovery of 95.5%. In addition, the peak fraction, representing 37.8% of the applied activity, had a specific activity of 1.0 X 10(8) units/mg protein and represents a purification of more than 145,000-fold. An SDS-PAGE analysis of one such fraction indicated that approximately 40% of the final material was HuIFN-gamma.     

Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-monooxygenase

7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase was purified from liver microsomes of phenobarbital-treated rabbits. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography on cholate-Sepharose 4B column, hydroxylapatite column chromatography, chromatography on DEAE-Sepharose CL-6B column, and a second hydroxylapatite column chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 9.0 nmol of cytochrome P-450/mg of protein, which corresponded to 5.3-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed as enzyme activity per mg of enzyme protein was increased 315-fold from microsomes. The molecular weight of the enzyme was estimated to be 56,000 from calibrated polyacrylamide gel electrophoresis. The enzyme-pH curve gave a peak at pH 7.0. The Michaelis constant for 7 alpha-hydroxy-4-cholesten-3-one was 27 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 418 nm. 7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase activity was reconstituted from the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 25-hydroxylase activity and that of 26- or 27-hydroxylase but revealed some activity for benzphetamine N-demethylation. The enzyme activity was not inhibited by metapyrone, aminoglutethimide, and KCN, but was seriously inhibited by nonionic detergents such as Emulgen 913. The enzyme was labile under low buffer concentrations but was stabilized at least for 4 weeks under higher buffer concentration such as 300 mM phosphate buffer.     

Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells

A glycoprotein that stimulates the proliferation of multiple hemopoietic stem and progenitor cell types was purified to apparent homogeneity. The factor, termed P cell-stimulating factor (PSF), was assayed by its ability to support the growth of murine factor-dependent hemopoietic cell lines operationally termed persisting cells (P cells). PSF was purified 50,000-fold from serum-free medium conditioned by the myelomonocytic cell line WEHI-3B by sequential ammonium sulfate precipitation, phenyl boronate chromatography, gel filtration on Sephadex G-100, neuraminidase treatment, Mono Q anion exchange chromatography, reverse phase high performance liquid chromatography on a C18 silica column, and two steps of high performance gel permeation chromatography on a TSK 3000 SW column operated under first neutral and then acidic solvent conditions. Although purified PSF could not be detected on sodium dodecyl sulfate-polyacrylamide gels stained with silver, following electrophoresis of purified PSF labeled with iodine-125, autoradiography showed only a single broad band of Mr = 30,000. This labeled band corresponded to the profile of PSF activity eluted from polyacrylamide gel slices. After reduction, labeled PSF had a slightly higher Mr of 32,000, although reduction resulted in loss of 98% of PSF activity, thus suggesting that the integrity of internal disulfide bond(s) was required for activity. When purified PSF was chromatographed on a TSK 3000 SW column under denaturing conditions in 0.1% sodium dodecyl sulfate, the single peak of absorbance at 280 nm coincided with a sharp peak of biological activity. The following unique NH2-terminal amino acid sequence of the purified PSF was obtained: NH2ALA -SER-Ile-Ser-X-X-Asp-Thr-His-Arg-Leu-Thr-Arg-. The concentration of PSF required for half-maximal stimulation of P cell growth was estimated as 1.3 X 10(-13) M or 4 pg/ml. The availability of purified PSF will allow rigorous examination of the hypothesis that a single molecule acts on multiple hemopoietic cell lineages.     

Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase

Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30min to purify BChE from 1ml plasma. The method uses a microfuge spin column to build a 0.2ml procainamide affinity column. The eluted BChE contains 3-4mug of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.     

CDP-diglyceride:inositol transferase from rat liver. Purification and properties.

CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.     

Purification and some properties of an extracellular ribonuclease with antiviral activity against tobacco mosaic virus from <Emphasis Type="Italic">Bacillus cereus</Emphasis>

A new ribonuclease (RNase) with tobacco mosaic virus inhibition was isolated and purified from Bacillus cereus ZH14 through ammonium sulfate precipitation, ultrafiltration, ion-exchange chromatography of DEAE-Sephadex A-50 column, and gel chromatography of Sephacryl S-200HR column. The enzyme was purified approximately 134-fold with a recovery of 9.2%. The RNase had an MW of 75.6 kDa in SDS-PAGE, which differed from RNases reported previously. The inhibitory activity of the RNase in the purification process against tobacco mosaic virus was tested, and the percentage inhibition of the purified RNase (48 U/ml) reached 90%. The protein could tolerate 90°C and pH 4.0.     

Purification and characterization of bovine brain glucocerebrosidase

Glucocerebrosidase was isolated from bovine brain by cholate extraction, ammonium sulfate fractionation, acid precipitation at pH 5.35, and hydrophobic chromatography. The purification is about 2400-fold with a specific activity of about 286,000 nmole/hr/mg protein. Molecular weight as determined by chromatography on Bio-Gel P-200 was 138,000. On SDS-polyacrylamide gel electrophoresis the enzyme protein resolved into two bands with apparent molecular weights of 63,000 and 56,000. These bands are cross-reactive to monospecific polyclonal antibody to homogeneous human placental glucocerebrosidase. The enzyme was found to be a complex glycoprotein based on its lectin binding specificity. Brain enzyme was found to be similar to placental glucocerebrosidase in its pH optima, heat stability at 52 degrees C, and substrate affinity. Enzyme kinetics were measured in the presence of conduritol-beta-epoxide, an irreversible inhibitor, and gluconolactone, a competitive inhibitor.     

Purification of glucoamylase by acarbose (BAY g-5421) affinity chromatography

Aspergillus niger and Rhizopus sp. glucoamylases were purified on an affinity chromatography column from commercially available, impure enzyme preparations. Up to 2 mg of glucoamylase protein was bound without leakage to a 1-ml affinity gel column (0.7 X 2.5 cm) possessing a covalently linked acarbose ligand (1 mg acarbose/g wet gel), and the bound enzyme was specifically released by irrigation of the column with a solution of maltose. A complete cycle of purification was accomplished in about 8 h. Glucoamylases were recovered, in more than 80% yield, free of alpha-amylase activity and possessing specific activities comparable to those of preparations obtained by time-consuming, multistep procedures involving several ion-exchange and hydrophobic column fractionations. Thus, acarbose affinity chromatography provides a general method for the rapid and efficient purification of the glucoamylases, and seems to be ideally suited for scale-up for the commercial purification of these enzymes.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

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.