Toyopearl HW-55S Research Articles

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.).

BackgroundWe have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.ResultsA cDNA, named aleh1, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The aleh1 encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (pI) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of aleh1 was produced in Pichia pastoris, and was purified by ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic chromatography with Toyopearl HW55S and gel filtration chromatography with Toyopearl HW55S. Purified recombinant protein showed hydrolyzing activity against β-2, 1 type fructans such as 1-kestose, nystose, fructosylnystose and inulin. On the other hand, sucrose, neokestose, 6-kestose and high DP levan were poor substrates.The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that aleh1 encoded 1-FEH.

Purification, Cloning and Functional Characterization of Fructan: Fructan 1-Fructosyltransferase from Edible Burdock (Arctium lappa L.)

A fructan:fructan 1-fructosyltransferase (1-FFT) was purified from edible burdock for the first time, and a cDNA encoding 1-FFT was isolated from the plant. Two 1-FFTs named 1-FFTa and 1-FFTb were purified from extract of edible burdock by ammonium sulfate precipitation, followed by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-55S and Sephadex G-100 columns. Inulin-type fructan such as nystose and fructooligosaccharides with higher DP were produced from 1-kestose by purified 1-FFTs. The general properties of both purified enzymes were very similar to each other. The purified 1-FFTa and 1-FFTb showed a single band by native PAGE and two bands, relative molecular masses of about 46,000 and 17,500 for 1-FFTa and 46,000 and 17,000 for 1-FFTb, by SDS-PAGE, respectively. The N-terminal sequences of the 46,000 peptides of both enzymes were the same, and those of the 17,500 and the 17,000 peptides were also identical. Based on the sequences, the 1-FFT cDNA named alft1 was cloned. The alft1 encoded a polypeptide of 617 amino acids. The relative molecular mass and pI of the mature protein region of deduced polypeptide were calculated to be 60,213 and 4.89, respectively. The characteristics of recombinant protein produced by Pichia pastoris closely resembled those of the native 1-FFTs purified from edible burdock. In this study, we demonstrated that alft1 encoding edible burdock 1-FFT was involved in the elongation of fructosyl chains of fructooligosaccharides in edible burdock.

A Synthetic Peptidoglycan Fragment as a Competitive Inhibitor of the Melanization Cascade

Melanin synthesis is essential for defense and development but must be tightly controlled because systemic hyperactivation of the prophenoloxidase and excessive melanin synthesis are deleterious to the hosts. The melanization cascade of the arthropods can be activated by bacterial lysine-peptidoglycan (PGN), diaminopimelic acid (DAP)-PGN, or fungal beta-1,3-glucan. The molecular mechanism of how DAP- or Lys-PGN induces melanin synthesis and which molecules are involved in distinguishing these PGNs are not known. The identification of PGN derivatives that can work as inhibitors of the melanization cascade and the characterization of PGN recognition molecules will provide important information to clarify how the melanization is regulated and controlled. Here, we report that a novel synthetic Lys-PGN fragment ((GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala)2, T-4P2) functions as a competitive inhibitor of the natural PGN-induced melanization reaction. By using a T-4P2-coupled column, we purified the Tenebrio molitor PGN recognition protein (Tm-PGRP) without causing activation of the prophenoloxidase. The purified Tm-PGRP recognized both Lys- and DAP-PGN. In vitro reconstitution experiments showed that Tm-PGRP functions as a common recognition molecule of Lys- and DAP-PGN-dependent melanization cascades.

Purification and Characterization of a Beta-Glucosidase from Citrus sinensis var. Valencia Fruit Tissue

A preliminary survey demonstrated activity for alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-arabinosidase, beta-D-glucosidase, beta-D-xylosidase, and beta-D-galactosidase in orange fruit flavedo and albedo tissue. alpha-L-Rhamnosidase was not detected. Subsequently, a beta-glucosidase was purified from mature fruit rag tissue (composed of intersegmental septa, squeezed juice sacs, and fruit core tissue) of Citrus sinensis var. Valencia. The beta-glucosidase exhibited low levels of activity against p-nitrophenyl-beta-D-fucopyranoside (13.5%) and p-nitrophenyl-alpha-D-glucopyranoside (7.0%), compared to its activity against p-nitrophenyl-beta-D-glucopyranoside (pNPG, 100%). The enzyme was purified by a combination of ion exchange (anion and cation) and gel filtration (Superdex and Toyopearl HW-55S) chromatography. It has an apparent molecular mass of 64 kDa by denaturing electrophoresis or 55 kDa by gel filtration chromatography (BioGel P-100). Hydrolysis of pNPG demonstrated a pH optimum between 4.5 and 5.5. At pH 5.0 the temperature optimum was 40 degrees C. At pH 5.0 and 40 degrees C the K(m) for pNPG was 0.1146 mM and it had a V(max) of 5.2792 nkatal x mg(-1) protein (katal = 0.06 International Units = the amount of enzyme that produces, under standard conditions, one micromol of product per min). Of the substrates tested, the enzyme was most active against the disaccharide cellobiose (1-->4), but was not active against p-nitrophenyl-beta-D-cellobioside. High levels of activity also were observed with the disaccharides laminaribiose (1-->3), gentiobiose (1-->6), and sophorose (1-->2). Activity greater than that observed with pNPG was obtained with the flavonoids hesperetin-7-glucoside and prunin (naringenin-7-glucoside), salicin, mandelonitrile-beta-D-glucoside (a cyanogenic substrate), and sinigrin (a glucosinolate). The enzyme was not active against amygdalin, coniferin, or limonin glucoside.

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel.

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Enzyme responsible for egg envelope (chorion) hardening in fish: purification and partial characterization of two transglutaminases associated with their substrate, unfertilized egg chorion, of the rainbow trout, Oncorhynchus mykiss.

From our previous studies, we have suggested that "egg envelope (chorion) hardening-enzyme" (first proposed by Zotin [J. Embriol. Exp. Morph. 6, 546-568 (1958)]) in the rainbow trout, Oncorhynchus mykiss, is transglutaminase (TGase), and that it coexists with its substrate, the unfertilized egg chorion, and forms epsilon-(gamma-glutamyl)lysine cross-links between the chorion proteins. In the present study, we extracted the TGase activity from the isolated chorions by homogenization with isotonic saline (143 mM NaCl-10 mM Tris.HCl, pH 7.2) and fractionated the extract by Toyopearl HW55S gel filtration with the isotonic saline containing 5 mM CaCl2 and 5 mM 2-mercaptoethanol (2-ME). One peak of TGase activity (P2) was obtained. When the eluates were dialyzed against 5 mM CaCl2/5 mM 2-ME/10 mM Tris.HCl (pH 7.2), another peak of the activity (P1) appeared. P1 TGase activity, which becomes apparent in a medium of low ionic strength, is involved in acceleration of the chorion hardening after egg activation in fresh water, so-called water activation. We purified the two TGases, P1 and P2, by SP-Sepharose, Q-Sepharose, and TSK-gel column chromatography. The molecular mass of the native form of P1 TGase was estimated as 103 kDa by Toyopearl HW55S gel filtration and as 100 kDa by the TSK-gel filtration. SDS-PAGE analysis showed that it consisted of heterogeneous 86- and 76-kDa proteins. However, these proteins closely resembled each other in amino acid composition, which was characterized by high content of Thr, Gly, and Pro residues as compared with P2 TGase. In contrast, the P2 TGase was isolated as a homogeneous 76-kDa protein and characterized by high content of Glx (Glu/Gln) and His residues. Neither of the chorion TGases of rainbow trout, P1 and P2, was similar to the liver-type TGase of red sea bream or the tissue-type TGase of chum salmon in amino acid composition. Examination of susceptibility to various inhibitors, reactivation by CaCl2, pH dependency, and activity of the polymerization of chorion proteins suggested that the P1 and P2 TGases were essentially similar to each other in enzymatic properties.

Studies on evodiasaccharide-B purified from Evodiae fructus: Part I. Its properties and effects on cerebral blood flow

A polysaccharide, evodiasaccharide-B (ES-B), was purified by precipitation with 80 % ethanol, centrifugal ultrafiltration, dialysis, gel-filtration on Toyopearl HW-55S, anionexchange chromatography and again gel-filtration on Toyopearl HW-55S from the H 2 O-soluble fraction of the fruits of Evodia rutaecarpa (Juss.) Benth. (Rutaceae). Homogeneity of the ES-B was determined by gel-filtration and electrophoresis. ES-B was composed of nearly equal amount of uronic acids and neutral sugars. Galacturonic acid is the main uronic acid and galactose, rhamnose, mannose and arabinose are present in the neutral sugars. ES-B (0.5 mg/kg i.v.) increased the cerebral blood flow (CBF) of anesthetized cats for 20 min. It had however, negligible effects on other cardiorespiratory functions. These results suggest that ES-B is an active substance in the hot-water extract from the fruits of E. rutaecarpa which selectively increases the CBF.

Purification and Characterization of a New Anti-Nucleating Protein Isolated from <I>Acinetobacter calcoaceticus</I> KINI-1

Strain KINI-1 capable of producing an anti-nucleating agent was isolated from a camphor leaf. Strain KINI-1 was identified as Acinetobacter calcoaceticus from its characteristics and taxonomics; the optimum temperature and pH for the production of the anti-nucleating agent were 30°C and 7.0, respectively. As the activity of this agent disappeared with the application of protease treatment, this agent, which was an anti-nucleating protein (ANP) was partially purified from the culture to an electrophoretically main band state by ultrafiltration, acetone precipitate, QA-52 column chromatography and Toyopearl HW 55S and Superose 12 gel filtration chromatography. The molecular weight of the ANP estimated by sodium dodecylsulfatepolyacrylamide gel electrophoresis was 55 kDa. The ANP had various substrate specificities for ice nuclei from various ice-nucleating bacteria and AgI. It was demonstrated that the antinucleating activity of ANP increased in proportion to the protein concentration and the antinucleating activity of ANP against ice nucleus from the cells of Erwinia uredovora KUIN-3 was 2.2°C at a concentration of 12.5μg/ml. Furthermore, we found that this ANP was bound to the surface of the ice nuclei, thereby causing anti-nucleating activity. The surface hydrophobicities of the ice nuclei were increased after binding with ANP, whereas the surface charge was changed from 6.6 to 6.1. Also, we confirmed that this action by ANP could give rise to a supercooling point at -5.0°C in the cell suspension of Erwinia uredovora KUIN-3.

Purification and characterization of thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius ATCC 33909 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatographies on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, Toyopearl HW-55S and Ultrogel AcA44. The enzyme had a molecular mass of 59,000 by SDS-polyacrylamide gel electrophoresis and a pI of 6.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The enzyme showed the highest activity from pH 5.5 to 6.0 and at 75 degrees C, and was stable from pH 5.5 to 9.5 and up to 85 degrees C. The activity was inhibited by Hg2+, Cu2+, Fe2+, Pb2+, and Zn2+. The Km values of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose were 16.7 mM, 2.7 mM, 3.7 mM, and 4.9 mM, respectively.

Purification and characterization of the 3-ketosteroid-delta 1-dehydrogenase of Arthrobacter simplex produced in Streptomyces lividans.

The 3-ketosteroid-delta 1-dehydrogenase (KS1DH) gene of Arthrobacter simplex IFO12069 cloned in Streptomyces lividans was overexpressed, resulting in production of the enzyme both extracellularly and intracellularly. The enzyme was purified by ammonium sulfate fractionation and chromatographies using DEAE-Toyopearl, Butyl-Toyopearl and Toyopearl HW55S from the supernatant of culture broth and cell-free extracts of S. lividans, and both preparations showed the same characteristics. The N-terminal amino acid sequence of both KS1DHs was M-D-W-A-E-E-Y-D, which coincided with the amino acid sequence deduced from the nucleotide sequence. Thus, the extracellular enzyme may derived from leakage of S. lividans cells during cultivation rather than secretion by processing of the signal sequence. The molecular weight of the enzyme was about 55,000, identical with the size deduced from the nucleotide sequence (M(r) 54,329). The optimum conditions for its activity were pH 10.0 and 40 degrees C. The enzyme catalyzed the conversion of several 3-keto-steroids, but those containing 11 alpha- or 11 beta-hydroxyl group were converted at low rates. The amino acid sequence of KS1DH from A. simplex is similar to those of KS1DH of Pseudomonas testosteroni and fumarate reductase from Shewanella putrefaciens.

Purification and characterization of a novel enzyme, maltooligosyl trehalose trehalohydrolase, from Arthrobacter sp. Q36.

A novel enzyme, maltooligosyl trehalose trehalohydrolase from Arthrobacter sp. Q36 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatography on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, and Toyopearl HW-55S. The enzyme specifically catalyzed the hydrolysis of the alpha-1,4-glucosidic linkage that bound the maltooligosyl and trehalose moieties of maltooligosyl trehalose. The Km of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose was 5.5 mM, 4.6 mM, 7.0 mM, and 4.2 mM, respectively. The enzyme had a molecular mass of 62,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was threonine. The enzyme showed the highest activity at pH 6.5 and 45 degrees C, and was stable from pH 5.0 to 10.0 and up to 45 degrees C. The activity was inhibited by Hg2+, Cu2+, Fe2+, and Zn2+.

Purification and Characterization of a Xylanase from Robillarda sp. Y-20 Grown on Xylan.

An endo-1, 4-β-xylanase (1, 4-β-Xylanohydrolase, EC 3.2.1.8) has been easily purified from culture filtrate of Robillarda sp. Y-20 by combination of specific affinity chromatography on Bio-Gel P-6DG and gel filtration chromatography on Toyopearl HW-55S. The yield of the purified enzyme was about 40% of that in the culture filtrate. The molecular weight of the xylanase determined by SDS-PAGE was 23400Da. The pI value was 9.5. The optimum pH and temperature were pH 4.5-5.5 and 55°C, respectively. The HPLC analysis showed that the main hydrolysate of xylooligosaccharide was xylobiose, and the enzyme had the transxylosidase activity. Km and Vmax values for xylotriose, xylotetraose, xylopentaose and xylohexose were obtained and from these data the enzyme was shown to have 5 subsites. It was found that the enzyme showed a specific affinity toward P-6DG column in the presence of xylooligosaccharides.

Purification and Properties of Cyclodextrin Glucanotransferase fromBrevibacteriumsp. No. 9605

Cyclodextrin glucanotransferase (EC 2.4.1.19) from Brevibacterium sp. No. 9605 was purified to homogeneity by chromatography on butyl-Toyopearl 650M, γ-cyclodextrin-Sepharose 4B, and Toyopearl HW-55S. The molecular weight of the purified enzyme was estimated to be 75,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the purified enzyme was 2.8. The optimum pH and temperature were pH 10 and 45°C, respectively. The enzyme was stable at the range of pH 6–8 and at temperatures 50°C or less in the presence of CaCl2. The enzyme produced mainly γ-cyclodextrin from starch in the initial stage of reaction, but later, the proportion of β-cyclodextrin was increased.

Purification of a Latent Form of Polyphenoloxidase from La France Pear Fruit and Its Pressure-activation

A latent form, mostly soluble, of polyphenoloxidase of La France pear fruit (Pyrus communis) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography with DEAE-Sephadex A-25 and then DEAE-Toyopearl 650M, followed by gel permeation chromatography with Toyopearl HW-55s. The addition of 10% glycerol to the eluting buffer was needed for purification. The purified latent enzyme seemed to be a monomeric protein; the molecular weight was estimated to be 70,000 by gel permeation chromatography and 65,000 by SDS–polyacrylamide gel electrophoresis. The enzyme was activated by pressurization at 400 MPa or higher or by treatment with SDS. The highest activity was obtained by pressurization at 600 MPa and 20°C for 6 h.

Comparison of the multiple molecular forms of bovine adrenocortical P450 scc with those of corpus luteum P450 scc

1. 1. Bovine adrenocortical P450 scc was resolved into several fractions by chromatography on AH-Sepharose 4B followed by gel filtration on Toyopearl HW55S. All fractions contained P450 scc of the same molecular size and the P450 scc could be resolved into 3–4 major and more than 10 minor isoelectric point forms by isoelectric focusing on polyacrylamide gel in the presence of Emulgen 913. 2. 2. Both the AH-Sepharose chromatography profile and the isoelectric focusing pattern of the adrenocortical P450 scc were more complex than those of the corpus luteum P450 scc. The corpus luteum P450 scc was practically devoid of the neutral to acidic isoelectric point forms. 3. 3. Three to four P450 scc subfractions with different isoelectric focusing pattern were obtained from a purified preparation of adrenocortical P450 scc by ion-exchange chromatography on DEAE-Toyopearl 650S or DEAE-Sephadex A25. These P450 scc subfractions showed essentially the same spectral properties, catalytic activity, molecular weight and N-terminal amino acid sequence. 4. 4. The most acidic (the latest eluting) subfraction was composed mostly of the neutral to acidic isoelectric point forms. The sedimentation characteristics of this subfraction was also studied. 5. 5. The structural basis of the multiple molecular forms was discussed.

Preparation and some properties of a novel maltotetraose-forming enzyme of Pseudomonas saccharophila.

It was found that Pseudomonas saccharophila produced a maltotetraose-forming enzyme . The enzyme was purified by ammonium sulfate fractionation and column chromatography on DEAE-Toyopearl 650M and Toyopearl HW-55s. The activity recovery was 19% at the final step of the purification . The purified enzyme was homogeneous electrophoretically and its molecular weight was 62, 000. The optimum pH and temperature were 6.7 and 55°C, respectively. The enzyme was stable up to 40°C in the pH range of 5.5 to 10.5 for 1 hr, and thermostable in the presence of 2 mM CaCl2, up to 45°C . The isoelectric point of the enzyme was 4.7. The enzyme activity was inhibited by metalions such as Ag+, Hg2+, Co2+, Cu2+, Fe3+, Al3+, Zn2+, and enhanced by Sr2+. The enzyme specifically produced maltotetraose from starch, did not act on glucose, maltose, maltotriose, and maltotetraose. The enzyme action proceeded from non-reducing ends of the substrates and seemed to be difficult skipping over the branches of amylopectin.

Immunolabeling of vitellogenin in the oocyte of viviparous fish, Poecilia reticulata

The guppy is viviparous and fertilization is internal. The spermatozoa are introduced by gonopodium of the male and the fertilization may occur upon the completion of maturation. The maturation of the primary oocyte probably starts immediately after puturition. The present study is to observe the changes of the follicular layer and egg cortex in the oocyte of the guppy and also to observe the accumulation of vitellogenin in the oocyte by use of immunolabeling method.Highly purified vitellogenin from the fish was used as immunogen to obtain rabbit anti-vitel 1ogenin antibody. Immunoglobulin (IgG) fraction was prepared by precipitation of the antiserum with 50% ammonium sulfate followed by gel filtration through Toyopearl HW55S column chromatography.The oocyte of the guppy, Poecilia reticulata were obtained by dissecting the female. Pre-embedding technigue was applied to immunolabeling of vitellogenin. The oocytes fixed for five hours in chille d 1% g l utaraldehyde and 2 % paraformaldehyde solution diluted with 0.1 M phosphate buffer (pH: 7.0)

Studies on enzymes produced by Bacillus. Part III. Purification and some properties of a protopectin-solubilizing enzyme that has potent activity on sugar beet protopectin.

We found a novel protopectin-solubilizing enzyme that had potent activity on the protopectin in sugar beet pulp, in the culture filtrate of Bacillus subtilis IFO 3134. The enzyme was purified by treatment with EDTA and chromatography on CM-Cellulofine CH, Butyl-Toyopearl 650, and Toyopearl HW-55S, and was isolated as a homogeneous protein. The enzyme had an apparent molecular weight of 30,000 in SDS-polyacrylamide gel electrophoresis, and 27,000 in permeation chromatography on Toyopearl HW-55S, with its isoelectric point around pH 9.0. The enzyme was stable from pH 5.0 to 9.0 and up to 60°C. The optimum pH for enzyme action was 6.0 at 37°C and pH 8.0 at 60°C. The enzyme catalyzed the release of pectin from protopectin of various citrus fruit peels as well as sugar beet pulp, but much less from that of lemon peel, without catalyzing the degradation of polygalacturonic acid. The enzyme catalyzed the degradation of arabinan in sugar beet pulp, and it seemed to release pectin by splitting the arabinan connect...

Purification and properties of a β-galactosidase with high galactosyl transfer activity from Cryptococcus laurentii OKN-4

β-Galactosidase of Cryptococcus laurentii OKN-4 was solubilized from a cell wall preparation by Zymolyase-20T, and purified by column chromatographies on DEAE-Sephadex A-50, TSK gel Toyo Pearl HW-55S and TSK gel DEAE-5PW. The purified β-galactosidase was homogenous on polyacrylamide disc gel electrophoresis, and the molecular weight was estimated to be about 200,000 by gel filtration on Toyo Pearl HW-55S and about 100,000 by SDS-PAGE. The enzyme showed the optimum pH at 4.3, and was stable at pH's between 2.8 and 9.3. The optimum temperature of the enzyme was 60°C, and it was stable at temperatures below 57.5°C for 10 min incubation. The K m values of the enzyme were 18.2 and 11.4 mM, and those of V max 76.9 and 5.3 μmol/min/mg protein for O- nitrophenyl-β- d - galactoside and lactose, respectively. The enzyme was strongly inhibited by Hg 2+, Ag +, 2-mercaptoethanol, glucose, maltose and maltotriose. It produced 4'GL ( O-β- d- galactopyranosyl-(1→4)-O-β- d- galactopyranosyl-(1→4)- d- glucopyranose) in a yield of 28.2% from 2.5% lactose solution by galactosyl transfer reaction. The enzyme also produced other galactooligosaccharides, including di-, tri-, and tetrasaccharides. The galactosyl transfer reaction was also observed in a 1% lactose solution. The enzyme had several acceptors and gave transfer products from lactose, lactitol, xylose, arabinose, ribose, glucose and galactose.

Preparation and some properties of a novel maltopentaose-forming enzyme of a Pseudomonas species.

A novel enzyme, of a Pseudomonas species, was purified by ammonium sulfate fractionation and column chromatographies on DEAE-Toyopearl 650M and Toyopearl HW-55s.The activity recovery was 87% at the ammonium sulfate fractionation step and 44% at the final step of the purification, respectively. The purified enzyme was homogeneous electrophoretically and its molecular weight was 72,000. The optimum pH and temperature were 6.5 and 55°C, respectively. The enzyme was stable up to 45°C in the pH range of 6.5 to 9.0 for 1 hr, and thermostable in the presence of 2 mm CaCl2 up to 50°C. The isoelectric point of the enzyme was 6.5. The enzyme activity was inhibited by metal ions such as Zn2+, Cu2+, Ag+ and Hg2+, and enhanced by Rb+, Mg2+, Co2+ and Ca2+.The enzyme specifically produced maltopentaose from starch in the initial stage of the reaction, finally producing maltose and maltotriose. The enzyme did not act on glucose, maltose or maltotriose, and maltohexaose was considerably resistant to the enzyme’s action. I...