<Emphasis Type="Italic">Rhizopus microsporus</Emphasis> var.<Emphasis Type="Italic"> rhizopodiformis</Emphasis>: a thermotolerant fungus with potential for production of thermostable amylases

The effect of several nutritional and environmental parameters on growth and amylase production from Rhizopus microsporus var. rhizopodiformis was analysed. This fungus was isolated from soil of the Brazilian "cerrado" and produced high levels of amylolytic activity at 45°C in liquid medium supplemented with starch, sugar cane bagasse, oat meal or cassava flour. Glucose in the culture medium drastically repressed the amylolytic activity. The products of hydrolysis were analysed by thin layer chromatography, and glucose was detected as the main component. The amylolytic activity hydrolysed several substrates, such as amylopectin, amylase, glycogen, pullulan, starch, and maltose. Glucose was always the main end product detected by high-pressure liquid chromatography analysis. These results indicated that the amylolytic activity studied is a glucoamylase, but there were also low levels of -amylase. As compared to other fungi, R. microsporus var. rhizopodiformis can be considered an efficient producer of thermostable amylases, using raw residues of low cost as substrates. This information is of technological value, considering the importance of amylases for industrial hydrolysis.     

Production of an amylase-degrading raw starch by Gibberella pulicaris

An endophytic fungus, Gibberella pulicaris, produced an amylase which degraded raw starches from cereals and other crops including raw potato, sago, tapioca, corn, wheat and rice starch. In each case, glucose was the main product. Among the raw starches used, raw potato starch gave the highest enzyme activity (85 units mg^–1 protein) and raw wheat starch the lowest (49 units mg^–1 protein). The highest amylase production (260 units mg^–1 protein) was achieved when the concentration of raw potato starch was increased to 60 g l^–1. Optimum hydrolysis was at 40°C and pH 5.5.     

Purification and properties of the raw starch-degrading α- amylase of Bacillus sp. IMD 434

The raw starch-degrading a-amylase of Bacillus sp. IMD 434 was purified to homogeneity by acetone precipitation, ion- exchange chromatography and hydrophobic interaction chromatography. The enzyme had a relative molecular mass of 69,200, displayed maximum activity at pH 6.0 and 65°C and released large amounts of glucose and maltose on hydrolysis of starch.     

Cloning, sequencing, characterization, and expression of a new α-amylase isozyme gene (amy3) from Pseudomonas sp.

A new gene encoding an -amylase has been cloned, sequenced and expressed in E. coli from an alkaliphilic Pseudomonas sp. KFCC10818. The structural gene is 1356 base pairs long and encodes a protein of 452 amino acids. The recombinant -amylase has been purified and biochemically characterized. Molecular mass of the protein deduced from SDS-PAGE was 50 kDa. The enzyme showed an activity optimum at pH 8 and at 40 °C with complete stability at pH 13 for 3 h. The enzyme released maltose and maltotriose on hydrolysis of soluble starch. Amylose was hydrolysed over 5 times faster than amylopectin by the enzyme while the hydrolysis of cyclodextrin or pullulan was negligible.     

Process optimization for the production of sugar for the bioethanol industry from Tapioca,a non-conventional source of starch

A commercial preparation of -amylase, Biotempase, obtained from Biocon India Pvt. Ltd., and crude glucoamylase produced from Aspergillus sp. NA21 were used to hydrolyse tapioca powder, a non-conventional starchy substrate. Among various concentrations of starch (15–35%, dry weight/volume) tried for maximum liquefaction; slurry made with 25% substrate concentration proved optimal. An economical process of liquefaction was carried out using steam under pressure (0.2–0.3 bar, 104–105 °C) to liquefy a 25% slurry in just 45 min, contrary to a slower process carried out at 95 °C in a water bath. For liquefaction of starch a pH of 5.0 proved to be optimum. The dose of Biotempase as prescribed by the supplier could be reduced by 33% achieving the same degree of liquefaction, by addition of CaCl₂ to the starch slurry at the concentration of 120 mg/l. The conditions for the saccharification of liquefied starch were optimized to be 60 °C and pH 5.0, producing 90% saccharification in 24 h. Supplementation of divalent ions Ca²⁺, Mg²⁺ and Zn²⁺ in the process of saccharification showed no effect. Finally glucose was found to be the main hydrolysis product in the saccharification of tapioca starch.     

Purification and characterization of thermostable β-amylase and pullulanase from high-yielding Clostridium thermosulfurogenes SV2

Thermostable -amylase and pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes strain SV2, were purified by salting out with ammonium sulphate, DEAE-cellulose column chromatography, and gel filtration using Sephadex G-200. Maltose was identified as a major hydrolysis product of starch by -amylase, and maltotriose was identified as a major hydrolysis product of pullulan by pullulanase. The molecular masses of native -amylase and pullulanase were determined to be 180 and 100 kDa by gel filtration, and 210 and 80 kDa by SDS–PAGE, respectively. The temperature optima of purified -amylase and pullulanase were 70 and 75°C, respectively, and both enzymes were completely stable at 70°C for 2h. The presence of starch further increased the stability of both the enzymes to 80°C and both displayed a pH activity optimum of 6.0. The starch hydrolysis products formed by -amylase action had -anomeric form.     

Process optimization for the production of sugar for the bioethanol industry from sorghum,a non-conventional source of starch

A commercial preparation of -amylase, Biotempase, obtained from Biocon India Pvt. Ltd., and crude glucoamylase produced from Aspergillus sp. NA21 were used to hydrolyse sorghum powder, a non-conventional starchy substrate. Among various concentrations of starch (15–35%, dry weight/volume) tried for maximum liquefaction; slurry made with 25% substrate concentration proved optimal. An economical process of liquefaction was carried out using steam under pressure (0.2–0.3 bar, 104–105 °C) to liquefy a 25% slurry in just 45 min, contrary to a slower process carried out at 95 °C in a water bath. For liquefaction of starch a pH of 5.0 proved to be optimum. The dose of Biotempase as prescribed by the supplier could be reduced by 33% achieving the same degree of liquefaction, by addition of CaCl₂ to the starch slurry at the concentration of 200 mg/l. The conditions for the saccharification of liquefied starch were optimized to be 45 °C and pH 5.0, producing 90% saccharification in 24 h. Supplementation of divalent ions Ca²⁺, Mg²⁺ and Zn²⁺ in the process of saccharification showed no effect. Finally glucose was found to be the main hydrolysis product in the saccharification of sorghum starch.     

Symplasmic and apoplasmic radial ion transport in plant roots

Enzymes of starch synthesis and degradation were identified in crude extracts of the unicellular green alga Dunaliella marina (Volvocales). By polyacrylamide gel electrophoresis and specific staining for enzyme activities, 4 multiple forms of starch synthase, 2 amylases, and at least 2 forms of -glucan phosphorylase were visible. Using specific -glucans incorporated into the gel before electrophoresis we have tentatively correlated -amylase and -amylase with both hydrolytic activities. The activities of -glucan phosphorylase and amylase(s) were measured quantitatively in crude extracts, and the concomitant action of -glucan phosphorylase and amylase(s) was found to account for the fastest rate of starch mobilization observed in vivo. Isolated chloroplasts retained both typical plastid marker enzymes and ADPglucose pyrophosphorylase, starch synthase, amylase(s), and -glucan phosphorylase to a similar percentage. Gel electrophoretic analysis followed by staining for enzyme activity of a stromal fraction resulted in a pattern of multiple forms of starch-metabolizing enzymes analogous to that found in a crude extract. We interpret the combined data as indicating the exclusive location in vivo of starch-metabolizing enzymes in chloroplasts of D. marina.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DDT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G1P glucose 1-phosphate - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - P_i inorganic orthophosphate - RuBP carboxylase ribulose-1,5-bisphosphate carboxylase, EC 4.1.1.39     

The high maltose-forming α-amylase ofSaccharomonospora viridis: mechanisms of action

Summary The thermophilic actinomycete,Saccharomonospora viridis produces a thermostable -amylase which forms 63% (w/w) maltose on hydrolysis of starch. Maltotriose and maltotetraose are the only intermediate products observed during this reaction, with maltotriose accumulating to 40% (w/w). Both unimolecular and multimolecular mechanisms (transfers and condensation) have been shown to occur during the concentration-dependent degradation of maltotriose and maltotetraose. Such reactions result in the almost exclusive formation of maltose from maltotriose at high initial concentration. These mechanisms of action result in the production of the high levels of maltose obtained upon hydrolysis of starch and related substrates.     

Rapid and simple purification of a novel extracellular β-amylase from Bacillus sp.

Bacillus sp. KYJ 963, a local isolate, produced an extracellular amylase with M _r=59 kDa. The amylase was easily purified by adsorption on soluble starch. The analyses of TLC and N-terminal amino acid sequence from the purified protein revealed that the enzyme was a novel -amylase which could not hydrolyze maltose or -cyclodextrin and its N-terminal amino acid sequence was A-V-N-G-Q-S-F-N-S-N-Y-K-T-Y-K-.     

Mycelia-associated β-galactosidase activity in microbial pellets of Aspergillus and Penicillium strains

Pellet formation and production of mycelia-associated -galactosidase were investigated in 15 Aspergillus and Penicillium strains. Mycelia-associated enzyme activity was measured in sonicated homogenates. The properties of the mycelia-associated -galactosidase of A. phoenicis QM 329 was investigated. The pH optimum of the mycelia-associated enzyme was 4.0. The optimum temperature under assay conditions was 70°C and the optimum temperature for repeated lactose hydrolysis was 60°C. Repeated batch hydrolysis of lactose was made with pellets from five Aspergillus strains. A. phoenicis QM 329 showed the least enzyme leakage from the pellets during hydrolysis. From repeated lactose hydrolysis experiments it was estimated that 50% of the mycelia-associated -galactosidase activity remained after 1300 h. Correspondence to: F. Tjerneld     

Functional significance of amylase polymorphism in Drosophila melanogaster. III. Ontogeny of amylase and some α-glucosidases

Changes in amylase (E.C. 3.2.1.1), maltase (E.C. 3.2.1.20), sucrase, and PNPGase activities in relation to changes in wet weight and protein content were studied during the development of larvae and adult flies from two strains of Drosophila melanogaster, homozygous for different amylase alleles. All -glucosidase activities increase exponentially during a large part of larval development, parallel to the increase in weight, and drop at the end of the third instar. Amylase activity of the Amy ¹ strain follows the same pattern. In contrast, amylase activity of the Amy ^4,6 strain continues its exponential increase longer. In the third larval instar amylase activity in the Amy ^4,6 strain becomes much higher than in the Amy ¹ strain. During the first hours of adult life amylase activity of the two strains does not differ. Then Amy ^4,6 activity starts to rise and becomes much higher (4–5 times) than Amy ¹ amylase activity, which remains approximately constant. All adult enzyme activities are much higher than in larvae. Comparison of enzyme activity of amylase and -glucosidases in larvae and adults confirms that differences in amylase activities can become important only when starch is a limiting factor in the food.The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Purification and some properties of Α-amylase from an ectomycorrhizal fungus, <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>

-Amylase from a still culture filtrate of Tricholoma matsutake, an ectomycorrhizal fungus, was isolated and characterized. The enzyme was purified to a homogeneous preparation with Toyopearl-DEAE, gel filtration, and Mono Q column chromatography. The -amylase was highly purified (3580 fold) with a recovery of 10.5% and showed a single protein band by SDS-PAGE. The enzyme was most active at pH 5.0–6.0 toward soluble starch and stable within the broad pH range 4.0–10.0. This -amylase was a relatively thermostable enzyme (optimum temperature, 60°C; thermal stability, 50°C). The molecular mass was 34kDa by size-exclusion chromatography and 46kDa by SDS-PAGE. This enzyme was not inhibited by the Hg²⁺ ion. Measurement of viscosity and TLC and HPLC analysis of the hydrolysates obtained from amylose showed that the amylase from T. matsutake is an endo-type (-amylase). Substrate specificity was tested using amylose with different polysaccharides. This -amylase readily hydrolyzed the -1,4 glucoside bond in soluble starch and amylose A (MW, 2900), but did not hydrolyze the -1,6 bond and cyclic polysaccharides such as - and -cyclodextrin.     

Saccharification of indigenous starches by β-Amylase of Bacillus megaterium

Extracellular -amylase from Bacillus megaterium converted indigenous starches from low-grade, raw materials to maltose. The extent of saccharification was higher with gelatinized starches than the raw material. For the gelatinized starches the optima for saccharification were pH 6.9 and 60°C.     

Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis

We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl₂ and MnCl_{_2}.     

Purification and some properties of an α-amylase glucoamylase fusion protein from Saccharomyces cerevisiae

A fusion gene containing the Bacillus subtilis -amylase gene and Aspergillus awamori glucoamylase cDNA was expressed in Saccharomyces cerevisiae. The resulting bifunctional fusion protein having both -amylase and glucoamylase activities secreted into the culture medium was purified to apparent homogeneity by affinity chromatography and gel filtration on Sephadex G-100. The enzyme had an apparent molecular mass of 150 kDa and showed an optimum pH and temperature of 6.0 and 60 °C, respectively. The main hydrolysis products from soluble starch were glucose and maltose.     

Production of amylase by Arthrobacter psychrolactophilus

Arthrobacter psychrolactophilus ATCC 700733 grew with a doubling time of 1.5–2.3 h (22°C) and produced up to 0.2 units/mL (soluble starch assay) of extracellular amylase in tryptic soy broth without dextrose (TSBWD) containing 0.5% or 1.0% (w/v) soluble starch or maltose as the fermentable substrate. Time-course experiments in media containing soluble starch as substrate showed that amylolytic activity appeared in cultures at 24 h (after exponential growth had ceased), reached peak levels in 72–96 h, and declined rapidly after reaching peak levels. Peak levels were highest in TSBWD containing 1.0% soluble starch. Proteolytic activity appeared at about the same time as amylolytic activity and increased during the period of amylase production. Significant amylase production was not observed in cultures in TSBWD with 0.5% glucose or in cultures grown at 28°C, but low levels of amylase were observed in TSBWD cultures grown at 19–23°C which contained no added carbohydrate. A single band of activity was observed after electrophoresis of supernatant fractions in non-denaturing gels, followed by in situ staining for amylolytic activity. The amylase possessed a raw starch-binding domain and bound to uncooked corn, wheat or potato starch granules. It was active in the Phadebas assay for -amylase. Activity was maximum on soluble starch at a temperature between 40°C and 50°C. The amylase after purification by affinity chromatography on raw starch granules exhibited two starch-binding protein bands on SDS gels of 105 kDa and 26 kDa.     

Production of 2-O-α-d-glucopyranosyl l-ascorbic acid using cyclodextrin glucanotransferase from Paenibacillus sp.

Transglycosylation to produce a 2-O--d-glucopyranosyl l-ascorbic acid (AA-2G) was studied using cyclodextrin glucanotransferase (CGTase) from Paenibacillus sp. A series of maltooligosaccharides substituted 2-O-derivatives of l-ascorbic acid (AA) were analyzed by HPLC. The maltooligosaccharides were hydrolyzed by glucoamylase to give AA-2G. CGTase also produced AA-2G using dextrin as a glycosyl donor and AA as an acceptor. CGTase utilized -, -, and -CDs, amylose, soluble starch and corn starch as glycosyl donors but not glucose.     

Syntheses of hydroxybenzyl-α-glucosides by amyloglucosidase-catalyzed transglycosylation

Glycosides were synthesized from soluble starch and hydroxybenzylalcohols (o, m and p) by the transglycosylation activity of amyloglucosidase from Rhizopus sp. in a water system. The synthesized glycosides were identified as hydroxybenzyl--glucosides, of which one molecule of hydroxybenzylalcohol was bound to 1-OH of glucose. The transglycosylation reaction was performed in a reaction system containing 50 mg starch, 50 mg hydroxybenzylalcohol, and 10 units of enzyme in pH 5.0 at 45°C. The glycoside was rapidly hydrolyzed to glucose and hydroxybenzylalcohol by the -glucosidase.     

High-Activity Barley \bold\ralpha-Amylase by Directed Evolution

Barley -amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae. The gene coding for the wild-type enzyme was subjected to directed evolution. Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate. The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity. After three rounds of directed evolution, one mutant (Mu322) showed 1000 times the total activity and 20 times the specific activity of the wild-type enzyme produced by the same yeast expression system. Comparison of the amino acid sequence of this mutant with the wild type revealed five substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B. Two of these mutations, Q44H and R303K, result in amino acids highly conserved in cereal -amylases. R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule.