Highly active and thermostable amylases and pullulanases from various anaerobic thermophiles

Summary High concentrations of amylases and pullulanases were formed by continuous cultivation of Thermoanaerobacter finnii, Thermobacteroides acetoethylicus, Thermoanaerobacter ethanolicus and Clostridium thermosaccharolyticum in chemostats under starch limitation. 70% to 98% of these enzymes were transported and released into the culture fluid. These extracellular enzymes were extremely thermostable under aerobic conditions and in the absence of substrate and metal ions. The amylases and pullulanases from the first three organisms had an optimal temperature of 90°C. The enzymes from C. thermosaccharolyticum were most active at 75°C. The pH optima of the amylolytic enzymes from the microorganisms investigated ranged between 5 and 6. The addition of calcium ions in vitro significantly enhanced pullulanase activity from T. finnii and C. thermosaccharolyticum. The influence of other metal ions and cyclodextrins on the activities of the amylolytic enzymes is also described.     

Amylases of the Fungus Aspergillus flavipes Associated with Fucus evanescens

A promising producer of extracellular amylases, Aspergillus flavipes, was selected from 245 strains of marine fungi. Depending on the conditions of growth, this strain produced diverse amylolytic complexes. When grown on a medium containing peptone and yeast extract (pH 7.0), A. flavipes synthesized three forms of amylase, differing in pH optimum (5.5, 6.0, and 7.5). A single form of the enzyme was synthesized either in the absence of peptone from the medium or at the initial pH value of the medium, equal to 8.6. The activity of the isolated amylase forms decreased in the presence of proteolytic enzymes. New, highly stable forms of amylase (with pH optima of 5.5 and 7.5 and maximum activity at 60–80°C) were synthesized in the presence of diisopropyl fluorophosphate, an inhibitor of proteases.     

Characterization of amylolytic and pullulytic enzymes from thermophilic archaea and from a newFervidobacterium species

Nine extremely thermophilic archaea and one novel thermophilic bacterium were screened for their ability to produce amylolytic and pullulytic enzymes. Cultivation of these micro-organisms was performed in the absence of elemental sulphur with starch as the major carbon source. Enzymatic activity was mainly detected in two archaea belonging to the order Thermoproteales,Desulfurococcus mucosus andStaphylothermus marinus, in two archaea belonging to the order Thermococcales,Thermococcus celer andT. litoralis and in two novel archaeal strains, TYS and TY previously isolated from the Guaymas Basin in the Gulf of California. Both amylolytic and pullulytic activities were also detected in a newly isolated thermophilic bacterium belonging to the order Thermotogales and previously described asFervidobacterium pennavorans. Best yields for enzyme production were obtained in 1–1 batch cultures with the strains TYS (13 units U/1 of amylase, 6 U/1 of pullulanase),F. pennavorans (2.5 U/l of amylase, 4.5 U/l of pullulanase) andT. litoralis (3.0 U/l of amylase). Enzymes were in general characterized by temperature optima around 90–100°C, pH optima around 5.5–6.5 and a high degree of thermostability. Due to the remarkable properties of these enzymes, they are of interest for biotechnological applications.     

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.     

Molecular cloning and expression of an extracellular α-amylase gene from an Antarctic deep sea psychrotolerant <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain 7193

Psychrotolerant Pseudomonas stutzeri strain 7193 capable of producing an extracellular α-amylase was isolated from deep sea sediments of Prydz Bay, Antarctic. The 59678-Da protein (AmyP) was encoded by 1665-bp gene (amyP). The deduced amino acid sequence was identified with four regions, which are conserved in amylolytic enzymes and form a catalytic domain, and was predicted to be maltotetraose forming extracellular amylase by using the I-TASSER online server. Purification of AmyP amylases from both the recombinant of Escherichia coli Top 10 F′ and strain 7193 was conducted. Biochemical characterization revealed that the optimal amylase activity was observed at pH 9.0 and temperature 40°C. The enzymes were unstable at temperatures above 30°C, and only retain half of their highest activity after incubation at 60°C for 5 min. Thin-layer chromatography analysis of the products of the amylolytic reaction showed the presence of maltotetraose, maltotriose, maltose and glucose in the starch hydrolysate.     

Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25

A bacterium that secretes maltooligosaccharide-forming amylase in a medium containing 12.5% (vol/vol) dimethylsulfoxide (DMSO) was isolated and identified as Brachybacterium sp. strain LB25. The amylase of the strain was purified from the culture supernatant, and its molecular mass was 60 kDa. The enzyme was stable at pH 7.0–8.5 and active at pH 6.0–7.5. The optimum temperature at pH 7.0 was 35°C in the presence of 5 mM CaCl₂. The enzyme hydrolyzed starch to produce maltotriose primarily. The enzyme was active in the presence of various organic solvents. Its yield and product selectivity of maltooligosaccharides in the presence of DMSO or ethanol were compared with those of the industrial maltotriose-forming amylase from Microbacterium imperiale. Both enzymes improved the production selectivity of maltotriose by the addition of DMSO or ethanol. However, the total maltooligosaccharide yield in the presence of the solvents was higher for LB25 amylase than for M. imperiale amylase.     

Screening of yeast strains capable of hyperproducing amylolytic enzymes

Summary Fifteen strains of yeast, which produced an extracellular amylolytic enzymes, were isolated from nature. One of them produced more than 100 times the enzyme activity in comparison with the 14 strains and the extremely hyperproducing strain of yeast was identified asCandida sp. 347. Paper chromatograms of the amylolytic enzyme demonstrated activity of amyloglucosidase. The optimum pH for activity of the enzyme was 5.5–6.0 and optimum temperature was 60°C.     

Properties of an extracellular amylase purified from a Bacillus species

A strain of Bacillus produced an amylase with properties characteristically different from known bacterial amylases. The purified 80 kDa protein of pI 5.1 dextrinized starch, glycogen and pullulan. The temperature and pH optima of the enzyme were 60 °C and 6.6 respectively. In the presence of 0.05 M CaCl₂, the enzyme retained stability for 15 min at 80 °C. Antibodies raised to the amylase protein showed no reaction with -amylases of Bacillus sp. and B. licheniformis. In culture, proteolytic degradation of the enzyme was observed.     

Stabilization of a truncated <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain TS-23 <Emphasis Type="Bold">α</Emphasis>-amylase by replacing histidine-436 with aspartate

Summary Histidine-436 of a truncated Bacillus sp. strain TS-23 α-amylase (His₆-tagged ΔNC) has been known to be responsible for thermostability of the enzyme. To understand further the structural role of this residue, site-directed mutagenesis was conducted to replace His-436 of His₆-tagged ΔNC with aspartate, lysine, tyrosine or threonine. Starch-plate assay showed that all Escherichia coli M15 transformants conferring the mutated amylase genes retained the amylolytic activity. The over-expressed proteins have been purified to near homogeneity by nickel-chelate chromatography and the molecular mass of the purified enzymes was approximately 54 kDa. The specific activity for H436T was decreased by more than 56%, while H436D, H436K, and H436Y showed a higher activity to that of the wild-type enzyme. Although the mutations did not lead to a significant change in the K_m value, more than 66% increase in the value of catalytic efficiency (k_cat/K_m) was observed in H436D, H436K, and H436Y. At 70 °C, H436D exhibited an increased half-life with respect to the wild-type enzyme.     

Amylolytic enzyme production byRhizopus oryzae grown on agricultural commodities

The amylolytic enzyme production byRhizopus oryzae NRRL 395 grown on different agricultural commodities was datermined. The mould produced much higher enzyme activity from barley, corn, bats, and rice than from cassava. The optimal temperature for enzyme production was 30°C. Neutralization with CaCO₃ greatly enhanced the rate of enzyme production. Nitrogen supplementation of cassava resulted in higher enzyme yields.

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Résumé On a déterminé la production d'enzymes amyloytiques parRhizopus oryzae NRRL 395. cultivé sur différents produits agricoies. La moisissure produit une activité enzymatique plus élevée à partir du malt. du maïs, de l'avoine et du riz qu'á partir du manioc. La température optimale pour la production d'enzyme est de 30°C. La neutralisation par al CaCO₂ augmente fortement la vitesse de production de l'enzyme. L'ajout d'azote au manioc résulte en un accroissement du rendement enzymatique.

     

Comparison of amylolytic properties of Lactobacillus amylovorus and of Lactobacillus amylophilus

Partially purified amylases produced by Lactobacillus amylovorus and L. amylophilus were compared and they differed in several properties. The maximum amylase activity of L. amylovorus was higher than that of L. amylophilus. As estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the molecular mass of the enzymes was 140 kDa for L. amylovorus amylase and 100 kDa for L. amylophilus amylase. Maximum enzymatic activities were obtained when the strains were grown in the presence of CaCO₃, on maltose with L. amylovorus and on sucrose with L. amylophilus. Optimal activities were obtained at pH values between 5.0 and 6.0 for both amylases. The L. amylovorus amylase was stable at a higher temperature (50°C) than the L. amylophilus amylase (40°C). Of six substrates examined, greatest activity was obtained by both enzymes on soluble starch. Neither enzymes hydrolysed pullulan or - and \-cyclodextrins. With the exception of Hg²⁺, which partially inhibited both enzymes, various metal ions, such as 1 mm Ca²⁺ and Ba²⁺, stimulated L. amylophilus amylase activity whereas they inhibited L. amylovorus amylase activity. Correspondence to: J. Morlon-Guyot, ORSTOM     

Characterization of a recombinant amylolytic enzyme of hyperthermophilic archaeon Thermofilum pendens with extremely thermostable maltogenic amylase activity

A gene (Tpen_1458) encoding a putative alpha amylase from hyperthermophilic archaeon Thermofilum pendens (TfMA) was cloned and expressed in Escherichia coli. The recombinant amylolytic enzyme was purified by Ni-NTA affinity chromatography and its catalytic properties were examined. Purified TfMA was extremely thermostable with a half-life of 60 min at an optimal temperature of 95°C. TfMA activity increased to 136% in the presence of 5 mM CaCl₂. Maximal activity was measured toward γ-cyclodextrin with a specific activity of 56 U/mg using copper bicinchoninate method. TfMA catalyzed the ring-opening reaction by cleaving one α-1,4-glycosidic linkage of cyclodextrin to produce corresponding single maltooligosaccharide at the initial time. The final products from cyclodextrins, linear maltooligosaccharides, and starch were glucose and maltose, and TfMA could also degrade pullulan and amylase inhibitor acarbose to panose and acarviosine-glucose, respectively. These results revealed that TfMA is a novel maltogenic amylase.     

Medium optimization for glucoamylase production by a yeast,Pichia subpelliculosa ABWF-64, in submerged cultivation

An amylolytic yeast strain Pichia subpelliculosawas shown to produce glucoamylase in submerged cultivation. The yeast strain produced the enzyme optimally at 30 °C and pH 5.6 in shake flasks agitated at 200 rev min^–1 in the optimized glucoamylase production medium containing 1% starch, 0.2% yeast extract, 0.4% K₂HPO₄, 0.035% NaCl and 0.1% MgCl₂. Maximum enzyme production was attained during early growth of 11 h in shake flasks, and 6 h in a laboratory fermenter. By optimizing media components and cultivation parameters, a 15-fold increase in glucoamylase secretion was achieved.     

Extracellular alkaline protease of newly isolatedRhizopus oryzae

Summary A fungal isolate identified asRhizopus oryzae, produces an extracellular alkaline serine protease. Maximum protease formation was after six days in shake flask culture at two different conditions of pH and temperature optimum (pH 5 at 30°C and pH 10 at 37°C). AgNO₃ and Tween 80 increased protease synthesis. The enzyme is stable between pH 3 and pH 11 and has a temperature optimum of 60°C.     

<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.     

Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway

The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.Cell extracts of pyruvate-grown P.furiosus contained the following enzyme activities: phosphoenolpyruvate synthetase (0.025 U/mg, 50 °C), enolase (0.9 U/mg, 80 °C), phosphoglycerate mutase (0.13 U/mg, 55 °C), phosphoglycerate kinase (0.01 U/mg, 50 °C), glyceraldehyde-3-phosphate dehydrogenase reducing either NADP⁺ or NAD⁺ (NADP⁺: 0.019 U/mg, NAD⁺: 0.009 U/mg; 50 °C), triosephosphate isomerase (1.4 U/mg, 50 °C), fructose-1,6-bisphosphate aldolase (0.0045 U/mg, 55 °C), fructose-1,6-bisphosphate phosphatase (0.026 U/mg, 75 °C), and glucose-6-phosphate isomerase (0.22 U/mg, 50 °C). Kinetic properties (V _max values and apparent K _m values) of the enzymes indicate that they operate in the direction of sugar synthesis. The specific enzyme activities of phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADP⁺-reducing) and fructose-1,6-bisphosphate phosphatase in pyruvate-grown P. furiosus were by a factor of 3, 10 and 4, respectively, higher as compared to maltose-grown cells suggesting that these enzymes are induced under conditions of gluconeogenesis. Furthermore, cell extracts contained ferredoxin: NADP⁺ oxidoreductase (0.023 U/mg, 60 °C); phosphoenolpyruvate carboxylase (0.018 U/mg, 50 °C) acts as an anaplerotic enzyme.Thus, in P. furiosus sugar formation from pyruvate involves reactions of the Embden-Meyerhof pathway, whereas sugar degradation to pyruvate proceeds via a modified non-phosphorylated Entner-Doudoroff pathway.     

Purification of lipase from Geotrichum candidum: conditions optimized for enzyme production using Box–Behnken design

The fungus Geotrichum candidum was selected from isolates of oil-mill waste as a potent lipase producer. Factors affecting lipase production by the fungus G. candidum in yeast-extract-peptone medium have been optimized by using a Box–Behnken design with seven variables to identify the significant correlation between effects of these variables in the production of the enzyme lipase. The experimental values were found to be in accordance with the predicted values, the correlation coefficient is 0.9957. It was observed that the variables days (6), pH (7.0), temperature (30 °C), carbon (1.25%), nitrogen (2.0%), Tween (1.0%) and salt concentrations (0.5 mM) were the optimum conditions for maximum lipase production (87.7 LU/ml). The enzyme was purified to homogeneity with an apparent molecular mass of 32 kDa by SDS-PAGE. The optimum pH at 40 °C was 7.0 and the optimum temperature at pH 7.0 was 40 °C. The enzyme was stable within a pH range of 6.5 to 8.5 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, and EDTA. However, the presence of Ca²⁺ and Ba²⁺ ions enhanced the activity of the enzyme.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable a-amylase with temperature optimum at 80 °C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000—20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000—20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of a-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60—75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na₂SO₄.     

Some extracellular enzymes associated with two tomato fruit spoilage molds

The production of amylolytic, cellulolytic and pectinolytic enzymes by Aspergillus flavus and A. fumigatus was investigated. The two fungi were cultured on wheat offal and liquid crystalline carboxymethylcellulose media. A. flavus produced amylases on basal and starch containing media while A. fumigatus could only produce amylases on starch medium. The cellulolytic activities of filtrates from culture or infected fruits showed that A. flavus produced lesser quantities of cellulolytic enzymes than A. fumigatus. At 25 °C and at a pH range of 6–8, A. flavus best produces amylases and cellulases, while A. fumigatus showed highest activities of the two enzymes at 35–40 °C and at pH 7.0. Two pectinolytic enzymes — polymethylgalacturonase and pectinmethyltrans-eliminase — were identified in vivo with the two molds. An endopolygalacturonase in addition to these two pectinolytic enzymes was well associated with A. fumigatus.     

Purification and characterization of a truncatedBacillus subtilis α-amylase produced byEscherichia coli

ABacillus subtilis amylase gene was inserted into a plasmid which transferred toEscherichia coli. During cloning, a 3 region encoding 171 carboxyterminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The transformed cells produced substantial amylolytic activity. The active protein was purified to apparent homogeneity. Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of theB. subtilis complete -amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with aK _m of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50°C, and the purified enzyme was stable at temperatures up to 50°C. While Hg²⁺, Fe³⁺ and Al³⁺ were effective in inhibiting the truncated enzyme Mn²⁺ and Co²⁺ considerably enhanced the activity.