Stereocaulon ramulosum (SW.) Raüsch, a lichen that grows in Southern Brazil, was treated with hot aqueous alkali, the extract fractionated with Fehling solution, and the resulting insoluble copper complex regenerated. The polysaccharide was fractionated stepwise in the presence of Cetavlon, and at pH 8.5, in the presence of borate, the predominant component was precipitated. From this, galactomannan B was obtained and shown to contain a main chain of (1→6)-linked α-D-mannopyranosyl units, which were unsubstituted (1), monosubstituted with single-unit side-chains of β-D-Galp-(1→4)-(2), and disubstituted with β-D-Galp-(1→4)- and α-D-Manp-(1→2)- (3). These principal components were arranged sequentially, with the structure 3→2 making a large contribution.

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Bacillus circulans WL-12, a yeast and fungal cell wall lytic bacterium, secretes a variety of polysaccharide degrading enzymes into the culture medium. When β-1, 3-glucanase was induced with pachyman, a β-1, 3-glucose polymer obtained from the tree fungus Poria cocus Wolf, six distinct active molecules of the enzyme with different molecular weights were detected in the culture supernatant of this bacterium. Molecular cloning of one of the β-1, 3-glucanase genes into E. coli was achieved by transforming E. coli HB101 cells with recombinant plasmids composed of chromosomal DNA fragments prepared from B. circulans WL-12 and the plasmid vector pUC 19. A recombinant plasmid containing 4.4kb of inserted DNA in the PstI site of pUC 19, designated as pNT003, conferred the ability to degrade pachyman on E. coli cells. The presence of pNT003 was harmful for E. coli cells and caused cell lysis, especially at higher temperatures of cultivation. β-1, 3-Glucanase activity detected in E. coli was mainly recovered in the periplasmic fraction when cell lysis did not occur. SDS-PAGE analysis revealed that the periplasmic fraction contained four active molecules of β-1, 3-glucanase which corresponded to four of the six active molecules produced by B. circulans WL-12.

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Neurospora sitophila produced extracellular and cell wall-associated lectins. The addition of L-sorbose to a culture resulted in a decrease in the production of the former lectin and complete abolition of the latter. The lectin in the culture filtrate was purified by bovine submaxillary mucin-conjugated Sepharose chromatography. The molecular weight of the lectin was calculated to be approx. 40, 000 by Sephacryl S-200 gel filtration, and that of the subunit to be approx. 22, 000 by SDS/polyacrylamidegel electrophoresis. The lectin was not inhibited by simple sugars or their homopolymers. It was inhibited strongly by glycoproteins from human erythrocyte membrane and bovine submaxillary mucin, and moderately by α1-acid glycoprotein from human plasma, human IgA and IgM, and fetal calf fetuin. The lectin agglutinated human type A, B and O erythrocytes to the same degree. Erythrocytes from chick, horse, rabbit and sheep were more efficiently agglutinated.

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Hormone-like substances corresponding to β-myrcene from pine trees were found from Botrytis cinerea, on which the pine wood nematode multiplied very rapidly. The hormone-like substances of B. cinerea were identified as 3-octanol and 1-octen-3-ol. The racemates and optically active compounds of these alcohols exhibited attracting, molting and multiplication activities similar to β-myrcene. These compounds may play an important role in the growth of the pine wood nematode.

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The general hypothesis of enhanced membrane permeability as a cause of increased production of primary and secondary metabolites (the "leak model") was disproved for the nucleotide (NAD) fermentation with Brevibacterium ammoniagenes ATCC 6872, as manganese deficient producer cells displayed a high membrane potential, like non-producing cells supplied with 10μM Mn²⁺.
However, the protein composition of the membrane was affected by a manganese deficiency. A manganese deficiency-like protein pattern of the membrane could be induced, in the presence of 10μM Mn²⁺, by either inhibition of DNA precursor biosynthesis (ribonucleotide reduction) with 20HIM hydroxyurea (HU) or inhibition of DNA replication with 30μM mi torn vein C.

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An activator stimulating the enzymatic hydrolysis of phospholipids was purified to a homogeneous state from autolyzed Torulaspora delbrueckii cell washings. Autolyzed cell washings were extracted with chloroform and ethanol, and the activator was purified about 130-fold by sequential column chromatographies on DEAE-Sephacel, Sephacryl S-300, and TSK gel G 3000 SW (high performance liquid chromatography, HPLC). The molecular weight of the activator was about 175, 000 as estimated by gel filtration on HPLC. However, the purified activator gave two protein bands corresponding to molecular weights from 102, 000 to 129, 000 and from 71, 000 to 88, 000, respectively, on SDS-polyacrylamide gel electrophoresis, when stained with silver stain reagent and periodic acid-Sniff (PAS) reagent. The activator was sensitive to heat treatment at 70 °C for 10min. The purified activator had no enzymatic activity, but stimulated the hydrolysis of phospholipids by water-soluble and membrane-bound phospholipases B if the substrates were pre-incubated with the activator. No stimulation of hydrolysis by the enzyme was observed when the activator was pre-incubated with the enzyme. The hydrolytic rate of phosphatidylcholine by the enzyme at acidic pH (pH 2.6) depended upon the amount of activator added. On the other hand, the hydrolytic rate at alkaline pH (pH 7.6) was stimulated greatly by more than 0.04 nmol of the activator.

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To characterize the novel macromolecular antitumor antibiotic SN-07 and the role of DNA in SN-07, we reconstructed SN-07 in vitro from SN-07 chromophore and calf thymus DNA. Reconstructed SN-07 was identical with the native SN-07 in the ultraviolet absorption spectrum, nuclease-resistance characteristics, and biological activities (antibacterial activity and cytotoxicity). The DNA moiety promoted the cytotoxic activity of SN-07 chromophore against KB and HeLa cells, but suppressed the activity against some bacteria and L1210 cells. DNA containing guanine suppressed the activity more strongly than DNA not containing guanine against some bacteria and L1210 cells. The antibacterial activity of SN-07 chromophore was affected by dosage only when DNA was supplied, but not RNA or BSA. Thus the DNA composition of the SN-07 chromophore-DNA complex affected the activity of SN-07 chromophore and guanine had an important role of the binding of SN-07 chromophore to DNA.

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SN-07 chromophore is an anthracycline antibiotic, but is atypical in the group because it contains an eight-membered ring with the carbinolamine structure. We constructed the SN-07 chromophore-DNA complex and investigated the binding mode of SN-07 chromophore to DNA. SN07 chromophore was released from the SN-07 chromophore-poly(dl-dC)•poly(dl-dC) complex, but not from the SN-07 chromophore-poly(dG-dC)•poly(dG-dC) complex, and carminomycin III was released from both DNA complexes by the HPLC analysis. It was suggested that the carbinolamine structure of SN-07 chromophore had a specific selectivity to DNA binding and the 2-amino group of guanine had an important role, so SN-07 chromophore was very different from the related anthracycline antibiotics. SN-07 chromophore-λDNA complex was resistant to digestion by an excess of a number of restriction enzymes, but the carminomycin III-λDNA complex was sensitive. We showed that the restriction enzyme recognized GC, the HhaI reaction was strongly inhibited by SN-07 chromophore in the complex, but the restriction enzyme recognized AT, and the DraI reaction was not. Inhibition intensity was due to the recognition sequence and its neighboring sequence. It was evidenced that SN-07 chromophore had the sequence specificity of the binding to DNA and its preferred sequence was 5'-GC-3'.

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Liposidomycin C (C₄₂H₆₇N₅O₂₁S, M.W. 1009) is a novel nucleoside antibiotic containing uracil, a sulfated aminosugar, and a fatty acid. It is a specific inhibitor of peptidoglycan synthesis of bacteria, inhibiting the formation of the lipid intermediates from uridine 5'-diphospho-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelyI-D-[¹⁴C]alanyl-D-[¹⁴C] alanine and uridine 5'-diphospho-N-acetylglucosamine with a particulate enzyme from Escherichia coli Y-10. It also inhibited the formation of MurNAc(-pentapeptide)-P-P-lipid in the absence of UDP-GlcNAc. On the other hand, it inhibited the activity of N-acetylglucosamine transglycosylase and peptidoglycan transglycosylase only weakly using the same system from E. coli. Thus, it is concluded that the site of action of liposidomycin C is phospho-N-acetylmuramyl-pentapeptide transferase in peptidoglycan synthesis.

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In relation to 5'-mononucleotide production through RNA degradation with a crude enzyme preparation, containing 5'-nucleotidase as well as nuclease H, from a moderate halophile, Micrococcm varians subsp. halophilus, the selective inhibitory effects on S'-nucleotidase of various metal ions, especially Zn²⁺, and that of EDTA were examined. The results indicate that the yield of 5'-mononucleotides on RNA degradation in a column bioreactor containing flocculated cells with the crude enzymes adsorbed on them was greatly improved in the presence of ZnSO₄.

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The resolution of a membrane-bound quinoprotein alcohol dehydrogenase (ADH), a key enzyme in vinegar making, was examined in Gluconobacter suboxydans. ADH in membranes of the organism was found to be converted to the apo-form, which is free from pyrroloquinoline quinone (PQQ), during cultivation or incubation at acid pH. The apoenzyme appeared at the stationary phase when cells were grown in medium exhibiting an acid pH at the end of the cultivation. The incubation of resting cells containing the holoenzyme in acid buffer also caused apoenzyme formation. On the other hand, the enzyme in cells grown or incubated at neutral pH was almost all of the holo-form. The apoenzyme was reactivated by exogenous PQQ and calcium ions. Of the tested metal ions, only the calcium ion was effective. The recovery of ADH activity was attained on incubation of the apoenzyme with PQQ and calcium ions at pH 6.0 and 25°C for 10 min.

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Strains producing higher levels of cellulolytic enzymes were selected from among 520 strains of plant pathogenic fungi, Fusarium species, and F. oxysporum strain SUF850 was found to be the best producer. When strain SUF850 was cultured using one of three polysaccharides, Avicel, carboxymethyl cellulose (CMC) or xylan, as a carbon source, the culture filtrate contained degrading activities toward all three substrates, i.e., irrespective of the carbon source used. From the culture filtrate of Avicel-grown cells, four distinct enzymes were purified to homogeneity, as judged on SDS-PAGE. They were designated as CMCase I, CMCase II, p-nitrophenyl-β-D-cellobiosidase and xylanase, and the characteristics of the individual enzymes were examined and compared.

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To prepare a chemically modified urokinase that does not dissociate into two peptide fragments upon reduction of its disulfide bridge, we cross-linked the enzyme intramolecular!) with various bifunctional imidoesters. The enzyme underwent the intramolecular cross-linking most moderately by the reaction at 4 °C for 5 hr with 3 mW dimethyl suberimidate in 0.1 M potassium phosphate buffer (pH 9.0). The cross-linked urokinase isolated by gel filtration with a yield of 25 % showed a specific activity of 76, 000 International Units/mg protein, which corresponds to 53 % of that of the native enzyme. Although the modified enzyme was similar to the native urokinase in some properties such as the autocatalytic self-digestion and the low affinity to fibrin, it showed higher in vivo and in vitro stabilities than the native one.

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A dual enzyme system of exo-maltotetraohydrolase [EC 3.2.1.60] and pullulanase [EC 3.2.1.41] was studied for the continuous production of maltotetraose. Porous chitosan beads were selected from among many carriers as the best carrier to immobilize both enzymes.
The properties of the immobilized enzymes were examined and compared with those of the native enzymes. For exo-maltotetraohydrolase, the optimum pH of the immobilized enzyme shifted slightly to the acidic side and the pH stability was improved on the alkaline side. The optimum temperature of the immobilized enzyme increased by about 15°C and thermostability was improved by about 10°C. As for pullulanase, very little difference in thermostability was observed.
The effects of operating conditions on the continuous production of maltotetraose using exo-maltotetraohydrolase immobilized on the porous chitosan beads were examined. Porous chitosan beads were recognized to be superior to Diaion HP-50.
The continuous production of maltotetraose was accomplished using the dual immobilized enzyme system. The dual enzyme system proved to be effective to increase the maltotetraose content in the product. A stable operation was successfully continued for more than 60 days.

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Partially purified antibody specific to the antitumor, O-6 branched β-(1→ 3)-D-glucan (WG), isolated from the cold alkali-extract of the fruiting body of Volvariella volvacea (Fukurotake) was obtained by immunization of rabbits with the conjugate of WG with bovine serum albumin (BSA). Hapten inhibition studies of the precipitation reaction of the antibody and the β-D-glucan with various (1→6)-linked and branched (1→3)-linked β-D-gluco-oligosaccharides showed that the antibody recognizes the sequence involving the non-reducing terminal glucosyl groups and possibly the branch points. The WG antibody also interacted with other branched β-(1→ 3)- D-glucans, but the reactivity differed depending on the degree of branching. In connection with the specificity of the antibody, the antibody to glucan polv alcohol (WG polyol), raised by immunization with WG polyol-BSA, recognized mainly the polyol groups in the side chains and a part of (1→3)-linked glucose residues in the main chain. In relation to the antitumor action of WG on mouse-implanted Sarcoma 180, the serum of the mouse, after 12-23hr intraperitoneal administration of WG, had potent antitumor activity in another group of tumor-bearing mice. When this serum was put onto the antibody-conjugated immunpadsorbent column, the tumor-inhibiting factor was mostly retained on the column, suggesting that the factor is closely related to the glucan or glucan conjugate. Thus, the antibody-conjugated affinity column was shown to be useful in studies of the mechanism of antitumor action.

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Rats were fed for one day on a diet containing nondialyzable ¹⁵N-melanoidin prepared in a model system of glucose and ¹⁵N-glycine, and the excreta were collected over the following 7-day period. The total amounts of ¹⁵N excreted in the feces and in the urine were 80.9 % and 1.95%, respectively, of ¹⁵N in the melanoidin fed to the rats, indicating that melanoidin was difficult to excrete. The urea-¹⁵N content in the ¹⁵N of the urine was found to vary from 2.4 to 14.0 %, and the ratio of ¹⁵N in the urea to that in the urine was found to be large on the early and late days after the administration of ¹⁵N-melanoidin. Compared to the nondialyzable melanoidin fed to the rats, the melanoidin excreted in the feces was characterized by elevated percentages of melanoidin components with pi from 2.73 to 2.90 and of those retained at the charged area, and was difficult to be electroforesed by electrofocusirig.

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An aminopeptidase. Ey, is a newly discovered enzyme of hen's egg yolk. About 95 % of the total activity was found in the yolk plasma fraction, with most of the remainder in the yolk granule fraction. The highly purified fraction from plasma showed about 13, 000-fold higher specific activities than that of yolk plasma. Polyacrylamide gel electrophoresis revealed that the enzyme was homogeneous. The bound enzyme in the granule was extracted with 3 % NaCl and was partially purified. The soluble and bound forms of aminopeptidase differ in solubility, molecular weight, heat stability, optimal pH, and Km.

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A model system was employed to investigate the role of glutaminase in soy sauce fermentation. In this system, a soybean protein was used as the starting material and a water extract of wheat bran koji was used as the enzyme preparation containing proteinases, peptidases, glutaminases and so on. A soybean protein was digested with the enzyme preparation. γ-Glutamylserine, γ-glutamylglutamic acid and γ-glutamylalanine were isolated from an acidic fraction of the soybean protein digest.
The addition of the purified glutaminase from Aspergillus oryzae led to more production of glutamic acid and γ-glutamyl peptide and less production of glutamine and pyroglutamic acid, γ-Glultamyltranspeptidase activity and γ-glutamyl peptide(s) were also detected in a practical soy sauce mash.

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A kinetic resolution of [1, 1-binaphthyl]-2, 2'-diol (binaphthol) and its esters was first accomplished by lipase-catalyzed transesterification in an organic solvent. Acylation of binaphthol with enol esters in diisopropyl ether-acetone (9:1, v/v) gave solely (R)-2-acyloxy-2'-hydroxy-1, 1'-binaphthyl (binaphthyl monoesters) having 90 - 95 % optical purities. The unreacted binaphthol, which was also recovered in high chemical yields, was the S enantiomer with 69-89% e.e. On the other hand, the lipase-catalyzed deacylation or alcoholysis of racemic binaphthyl monoesters gave (S)-monoesters and (R)-binaphthol in high chemical and optical yields (>90% e.e.). In deacylation, the reaction period was much shortened by introducing the more electronegative chlorine atom into the acetyl group of the substrate.

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The effects of fat hydroperoxides on the reaction rate and enzyme stability in glycerolysis of safflower oil catalyzed by Pseudomonas fluorescens lipase were investigated. In the batch glycerolysis, the initial reaction rate, r_i, decreased linearly with the increase in the safflower oil's peroxide value (POV).
In the continuous glycerolysis using a microporous hydrophobic membrane bioreactor, the outlet conversions decreased monomolecularly with the elapsed time due to inactivation. The first-order decay constants, K_a, increased with the increase in the safflower oil's POV.
The courses of sodium dodecyl suit ate-pol yacrl lamide gel electrophoresis (PAGE) of the enzyme in the glycerol phase in the continuous glycerolysis showed that the high hydroperoxide content caused polymerization of the lipase. From the activity staining of the bands of the native proteins developed in the PAGE, the highly polymerized lipase was found to have no lipolytic activity.

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The volatile extract obtained by steam distillation of the oil from roasted sesame seeds was separated into its neutral, weakly acidic, acidic and basic fractions. The neutral fraction was further separated by preparative TLC. All fractions were analyzed by means of a gas chromatograph (GC) equipped with FID and FPD, and by gas chromatography/mass spectrometry (GC/MS) and/or gas chromatography/Fourier transform-infrared (GC/FT-IR) spectrometry. Two hundred and twenty one constituents were identified. One hundred and forty six of these compounds are being reported for the first time in the aroma of roasted sesame seeds, 7 of which were newly identified as naturally occurring flavor components. Two dithioketones, 1-methyldithio-2-propanone and 1-methyldithio-2-butanone, are new compounds.

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A conformational analysis of cycloartenol, 24-methylenecycloartanol and their derivatives was carried out in the solution and the solid state by an NMR study and X-ray crystallographic analysis, respectively. Complete assignments of the ¹H NMR spectra of these compounds were made in order to elucidate the conformation involving the ring system and side chain. Rings A to C had a chairhalfchair-boat conformation, and the side chain had a zig-zag conformation.

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Microorganisms capable of producing high amounts of α-acetolactate decarboxylase (ALDC; EC 4.1.1.5) were screened for with stock type cultures. Brevibacterium acetylicum had the most potent enzyme activity among the strains tested. The productivity of ALDC by B. acetylicum was elevated by adding Zn²⁺ to the medium. ALDC was purified from the cell-free extract of B. acetylicum by a procedure involving ammonium sulfate fractionation, Sephadex G-100 gel filtration, and DEAE-cellulose and FPLC-MonoQ^TM column chromatographies. The purified enzyme was homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was 62, 000 by TSK-gel filtration and the subunit molecular weight was 31, 000 by SDS polyacrylamide gel electrophoresis. The enzyme activity was inhibited by metal chelators such as diethyldithiocarbamate, 8-oxyquinoline, and o-phenanthroline. Analysis by atomic absorption spectrophotometry showed that zinc atoms were involved in the purified enzyme preparation.

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A new enzyme, N-acetyl-D-hexosamine dehydrogenase (N-acetyl-α-D-hexosamine:NAD⁺ 1-oxidoreductase), was purified to homogeneity on polyacrylamide gel electrophoresis from a strain of Pseudomonas sp. about 900-fold with a yield of 12%. The molecular weight of the enzyme was about 124, 000 on gel filtration and 30, 000 on SDS-polyacrylamide gel electrophoresis, respectively. Its isoelectric point was 4.7. The optimum pH was about 10.0. The enzyme was most stable between pH 8.0 and pH 10.5. The highest enzyme activity was observed with N-acetyl-D-glucosamine (Km=5.3mM) and N-acetyl-D-galactosamine (Km=0.8mM) as the sugar substrate. But it was not so active on N-acetyl-D-mannosamine. NAD⁺ was used specifically as the hydrogen acceptor. The anomeric requirement of the enzyme for N-acetyl-D-glucosamine was the α-pyranose form, and the reaction product was N-acetyl-D-glucosaminic acid. The enzyme activity was inhibited by Hg²⁺ and SDS, but many divalent cations, metal-chela ting reagents, and sulfhydryl reagents had no effect.

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To find the primary sequence of Irpex lacteus aspartic proteinase (ILAP), a cDNA library of I. lacteus mRNA in pBR322 was constructed. A clone, which had an insert size of about 1.2 kilobase pairs, was found to contain the coding region of the mature enzyme. The deduced amino acid sequence showed that the enzyme consisted of 340 amino acid residues with a molecular weight of 35, 000. Cysteine and methionine were not found in the enzyme and two putative N-glycosylation sites were indicated. The lack of S-S bridges in the molecule is a striking feature of the enzyme. The alignment of the sequence of the enzyme against other aspartic proteinases revealed homology around the active site aspartic acid residues.

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The production of deoxyribonucleoside triphosphates (dNTPs) from deoxyribonucleoside monophosphates (dNMPs) was investigated using dried baker's yeast in the presence of a high concentration of inorganic phosphate. The amount of dATP formed by 150mg/ml of yeast cells was 127 mM, with a 84% yield of the substrate, dAMP, after 18 hr incubation at 28°C with 150 mM dAMP, 800mM glucose, 800 mM potassium phosphate buffer (pH 7.0) and 10mM MgSO₄. The amount of dGTP or dCTP formed was in the range of 26-34mM, from 100 mM of either dGMP or dCMP, respectively, after 8 hr incubation, but dTTP was not formed during the incubation period.
In a reaction mixture containing a dNMP-mixture, which was prepared through the hydrolysis of salmon testes DNA or herring sperm DNA (100mg/ml) with Nuclease P₁, 32-49 HIM dNTP was obtained.

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The non-Newtonian behavior and dynamic viscoelasticity of deacylated xanthan solution were measured with a rheogoniometer. The flow curves for the deacylated xanthan at concentrations below 0.5% approximated to shear-thinning behavior, and to plastic behavior above 0.8%. The apparent viscosity of a 0.5% solution of deacylated xanthan increased with increasing temperature up to 25°C, which was estimated to be a transition temperature, then it decreased rapidly. The dynamic modulus of deacylated xanthan increased a little with increase of temperature up to 25 and 30°C in 0.8 and 1.0% solutions, then it decreased rapidly with further increases of temperature. The specific rotation of deacylated xanthan stayed constant up to 25°C, then it decreased rapidly with further increases in temperature. Possible mode of intramolecular associations between an alternate hydroxyl group at C-3 and the adjacent hemiacetal oxygen atom of the D-glucosyl residues, and between the methyl group of the acetyl residue and the adjacent hemiacetal oxygen atom of the D-glucosyl residue were proposed.

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Scedosporium sp. A-4 used gaseous hydrocarbons as a sole source of carbon and energy, and oxidation of the gaseous hydrocarbons was studied with propane-grown resting mycelia of this organism. The gaseous n-alkanes (C₂ to C₄) were oxidized to their corresponding primary alcohols and secondary alcohols by the resting mycelia. Methane was not oxidized. Propylene was oxidized but not used for growth. In the primary oxidative reaction of gaseous n-alkanes, an incorporation experiment of ¹⁸O₂ gas demonstrated that one atom of molecular oxygen is incorporated into them. The oxidation of the gaseous hydrocarbons was inhibited by carbon monoxide, potassium cyanide, sodium azide, and Hg₂₊.

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A series of 3-acyl-2, 4, 6-trihydroxybenzamides was synthesized, and the compounds' PET inhibitory activities were examined in isolated chloroplasts. In general, the PET inhibitory activity was found to depend on the overall lipophilicity of the molecule. Low activities of the mono and dihydroxy derivatives indicated that the three hydroxyl groups on the nucleus were essential for high activity. The PET inhibition study, on chloroplasts isolated from an atrazine resistant biotype of Brassica napus and using thermoluminescence analysis, suggested that the trihydroxybenzamide derivatives would be classified as a urea type rather than a phenol type of PET inhibitor. However the trihydroxybenzamide derivatives, like the phenol type of PET inhibitor, showed a lag time before inhibition started, which was followed by constant activity. These results indicate that the binding domain for the trihydroxybenzamide derivatives overlapped with those of both the urea type and phenol type of PET inhibitors.

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A raw-starch-digesting amylase, Dabiase K-27, was immobilized covalently on an enteric coating polymer (hydroxypropyl methylcellulose acetate succinate: AS) as a carrier which is autoprecipitating in an insoluble state below pH 4 as well as reversibly soluble-insoluble depending on pH. Dabiase immobilized on AS (D-AS) showed a sharp response of solubility to slight changes of pH without decrease in enzymatic activity. Moreover, D-AS in an insoluble state had good properties of sedimentation and a large portion of D-AS spontaneously precipitated after 10 min at pH 4.
D-AS was used successively for repeated ethanol production from raw starch, in which D-AS and flocculating yeast cells were separated simultaneously from a product solution by sedimentation in a reactor with a conical bottom. In the five batches of 10% raw starch, the total amount of ethanol produced from 150 g of raw starch was 61 g, a value of which corresponds to the average ethanol productivity of 0.85 g/l/hr. The repeated ethanol production by a combination of D-AS and flocculating yeast cells is a promising procedure for effectively using the enzyme and recovering the product solution economically in a heterogeneous culture system containing a solid substrate.

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