Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum

Glucoamylase produced byScytalidium thermophilum was purified 80-fold by DEAE-cellulose, ultrafiltration and CM-cellulose chromatography. The enzyme is a glycoprotein containing 9.8% saccharide, pI of 8.3 and molar mass of 75 kDa (SDS-PAGE) or 60 kDa (Sepharose 6B). Optima of pH and temperature with starch or maltose as substrates were 5.5/70 °C and 5.5/65 °C, respectively. The enzyme was stable for 1 h at 55 °C and for about 8 d at 4 °C, either at pH 7.0 or pH 5.5. Starch, amylopectin, glycogen, amylose and maltose were the substrates preferentially hydrolyzed. The activity was activated by 1 mmol/L Mg²⁺ (27%), Zn²⁺ (21%), Ba²⁺ (8%) and Mn²⁺ (5%).K _m and {ie11-1} values for starch and maltose were 0.21 g/L, 62 U/mg protein and 3.9 g/L, 9.0 U/mg protein, respectively. Glucoamylase activity was only slightly inhibited by glucose up to a 1 mol/L concentration.     

Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme. Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities at alkaline pH, of 7-9 and 8-10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 50-75 degrees C, with optimal activity at 70 degrees C and were stable at 70 degrees C for at least 120 min. Both PLP and Est1 exhibited high V(max) toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C(4) and C(5)) and long chain (C(14) and C(16)) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which usually show no activity for substrates longer than C(10). We suggest that PLP and EstA enzymes are novel and have a; b potential use in industrial applications.     

Thermostable conidial and mycelial alkaline phosphatases from the thermophilic fungus Scytalidium thermophilum

An extracellular (conidial) and an intracellular (mycelial) alkaline phosphatase from the thermophilic fungus Scytalidium thermophilum were purified by DEAE-cellulose and Concanavalin A-Sepharose chromatography. These enzymes showed allosteric behavior either in the presence or absence of MgCl₂, BaCl₂, CuCl₂, and ZnCl₂. All of these ions increased the maximal velocity of both enzymes. The molecular masses of the conidial and mycelial enzymes, estimated by gel filtration, were 162 and 132 kDa, respectively. Both proteins migrated on SDS-PAGE as a single polypeptide of 63 and 58.5 kDa, respectively, suggesting that these enzymes were dimers of identical subunits. The best substrate for the conidial and mycelial phosphatases was p-nitrophenylphosphate, but β-glycerophosphate and other phosphorylated compounds also served as substrates. The optimum pH for the conidial and mycelial alkaline phosphatases was 10.0 and 9.5 in the presence of AMPOL buffer, and their carbohydrate contents were about 54% and 63%, respectively. The optimum temperature was 70–75°C for both activities. The enzymes were fully stable up to 1 h at 60°C. These and other properties suggested that the alkaline phosphatases of S. thermophilum might be suitable for biotechnological applications. Journal of Industrial Microbiology & Biotechnology (2001) 27, 265–270. Received 10 January 2001/ Accepted in revised form 10 July 2001     

Thermostable amylolytic enzymes of thermophilic microorganisms from deep-sea hydrothermal vents

Microorganisms-grauling above 60 °C isolated from deep-sea hydrothermal vents were screened for amylolytic activity. Of the 269 strains tested, 70 were found to be positive. Nine archaea (including Thermococcus hydrothermalis AL662 and Thermococcus fumicolans ST557) and one thermophilic bacterium were selected for the determination of thermostability, and the temperature and pH optima of their amylolytic enzymes. Pullulanase, α-glucosidase and α-amylase activities were detected in four archaeal strains (including AL662 and ST557) related to the genus Thermococcus. The anaerobic hyperthermophilic archaeon, Thermococcus hydrothermalis was chosen for the further study of the α-glucosidase activity, and a preliminary characterization of this enzyme was carried out. The small number of highly thermostable α-glucosidases that has been described to date, combined with the very interesting properties of this enzyme, suggest a use for this enzyme in biotechnological processes.     

Thermostable lipolytic enzymes production in batch and continuous cultures of Thermus thermophilus HB27

Several studies in laboratory-scale bioreactors are undertaken in order to verify the beneficial effect of thermal spring water in the culture medium of Thermus thermophilus HB27. Two bioreactor configurations, stirred tank and airlift, are investigated to determine the most suitable one to develop a continuous process. Water mineral composition affects the lipolytic enzyme secretion and growth of T. thermophilus HB27 in both bioreactor configurations. Furthermore, the lipolytic activity is strongly enhanced when stirred tank bioreactor is used. Moreover, operation in a stirred tank at an agitation rate of 650 rpm leads to the highest total lipolytic activity (intra- and extracellular enzyme) around 280 U/L after 32 h. Continuous cultures operating in the optimised conditions determined in batch cultures are carried out. It is noticeable that the stirred tank bioreactor was able to operate in a continuous flow mode without operational problems. In addition, the lipolytic activity obtained is about 2-fold higher than that attained in batch cultures.     

Properties of proteolytic enzymes isolated from a thermophilic strain of Micromonospora vulgaris 42.

Two proteolytic enzymes, protease A and protease B, were isolated in homogeneous state from the cultural broth of the thermophilic actinomycete Micromonospora vulgaris 42. Their physicochemical properties were studied, i.e., molecular weight (50 000 for protease A and 30 000 for protease B), amino acid composition, N-terminal amino acids (phenylalanine for protease A and alanine for protease B). The specificity of the action of these enzymes was assayed by splitting the B chain of oxidized insulin. Both enzymes are neutral proteases of the thermolysine type.     

Characterization of an extracellular thermophilic alkaline esterase produced by Bacillus subtilis DR8806

This work is a report of the characterization of an alkaline lipolytic enzyme isolated from Bacillus subtilis DR8806. The extracellular extract was concentrated using ammonium sulfate, and ultrafiltration. The active enzyme was purified by Q-sepharose ion exchange chromatography. The molecular mass of the enzyme was estimated to be 60.25 kDa based on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The optimum pH and temperature of this enzyme were observed to be 8.0 and 50 °C, respectively. The enzyme exhibited a half-life of 72 min at its optimum temperature. It was stable in the presence of metal ions (10 mM) such as Ca²⁺, K⁺ and Na⁺, whereas Cu²⁺, Fe²⁺, Zn²⁺, Mn²⁺, Co²⁺, Mg²⁺ and Hg²⁺ were found to have inhibitory effects. However, the enzyme activity was not affected significantly by 1% Triton X-100. The study of substrate specificity showed that the purified enzyme has a preferential specificity for small ester of p-nitrophenyl acetate (C₂), and it was the most efficiently hydrolyzed substrate as compared to the other esters. The kinetic parameters showed that the enzyme has K_m of 4.2 mM and V_max of 151 μmol min⁻¹ mg⁻¹ for p-nitrophenyl acetate. The hydrolysis rates of the fluorescence substrates were increased in the presence of the purified enzyme. Regarding the features of the enzyme, it may be utilized as a novel candidate for industrial applications.     

Thermostable amylase production by immobilized thermophilic Bacillus sp.

Agar, agarose and alginate immobilized cells of thermophilic Bacillus sp. WN11 produced 7.0, 6.2, and 10.5 U/ml of thermostable amylase in shake flasks, respectively. Alginate entrapped cells released high level of amylase (10.5 U/ml) than freely suspended cells (8.0 U/ml). Amylase production was stable in five successive batches with productivity of 9-12 U/ml for alginate, 5.5-8.8 U/ml for agar, and 4.8-8.0 for agarose immobilized cells.     

Heat-stable extracellular proteolytic enzyme produced by Thermus caldophilus strain GK24, an extremely thermophilic bacterium

Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T. caldophilus sp. n. strain GK24. The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth. Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about 11S. The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2). The enzyme was stable in the pH range of 5 to 11 for 1 day at 4 degrees C or for 1 h at 70 degrees C. The enzyme sample showed a maximal activity at 90 degrees C and had an extreme stability toward treatment by heat and denaturing reagents. The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin. The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE).     

Formation of lipolytic enzymes by Brevibacterium linens

When Brevibacterium linens ATCC 9172 was grown in shake flasks, it produced a cell-associated lipase with a specific activity of 152 to 188 U g^–1 cells depending on the composition of the growth medium. There was no growth in media containing tributyrine as the sole carbon source. The cell-associated lipase had maximum activity at pH 8.0 and 37 °C and was strongly inhibited by 3,4-dichloroisocoumarin, an inhibitor specific for serine esterases. Cell-associated activity was released from the cells by treatment with lysozyme. The kinetics of lipase formation was closely related to the amount of biomass formed during growth.     

Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT-1

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH2-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin I was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80 degrees C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine.HCl and 1% SDS) at 23 degrees C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.     

Thermostable alkaline lipase from a newly isolated thermophilic Bacillus,strain A30-1 (ATCC 53841)

A thermophilic bacterium was isolated from a hot spring area of Yellowstone National Park. The organism grew optimally at 60–65°C and in the pH range of 6–9. It was characterized as Bacillus sp. In the presence of corn or olive oil (1.0%) as the growth substrate, this Bacillus produced an extracellular lipolytic activity (EC 3.1.1.3). The enzyme activity could be efficiently recovered by ultrafiltration of cell-free culture supernatant. The partially purified lipase preparation had an optimum temperature of 60°C, at an optimum pH of 9.5. It retained 100% of the original activity after being heated at 75°C for half an hour. The half life of the enzyme was 8 h at 75°C. The enzyme retained at least 90% of the original activity after it was incubated at 60°C for 15 h at pH's in the range of 5 to 10.5. The enzyme was active on triglycerides containing fatty acids having a carbon chain length of C16 : 0 to C22 : 0 as well as on natural fats and oils. The enzyme activity was stable to both hydrogen peroxide and alkaline protease which are detergent ingredients. The purified enzyme had an isoelectric point of 5.15 and an approximate molecular weight of 65,000.     

嗜热解烃菌DM-2产生的生物乳化剂

【目的】生物乳化剂是一类由微生物代谢产生的大分子生物表面活性物质,从胜利油田中1区N3块地层环境中筛选到一株能产生一种生物乳化剂的嗜热解烃菌DM-2,经鉴定为嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus),研究其产生的生物乳化剂的化学组成和理化性质。【方法】采用化学显色、红外光谱、高效液相色谱和氨基酸自动分析等方法确定乳化剂的化学组成;根据乳化剂在不同条件下的乳化指数(EI-24)确定其理化性质。【结果】菌株DM-2产生的乳化剂主要由多糖(71.4%,质量比)和蛋白(27.75%,质量比)组成,对柴油、苯、二甲苯和煤油等石油烃均有很好的乳化效果。理化性质分析显示它是一种耐高温、耐盐、耐酸碱的高效乳化剂。【结论】菌株DM-2产生的乳化剂是一种新型的生物乳化剂,在石油开采、原油集输、油罐清洗和石油污染治理等领域具有潜在的应用价值。     

Gas phase transesterification reactions catalyzed by lipolytic enzymes

Porcine pancreatic lipase and Fusarium solani cutinase were used to catalyze transesterification reactions between methyl propionate, ethyl propionate, and a series of primary alcohols at high temperatures in a continuous packed-bed gas-solid reactor, in which the solid phase is composed of the enzyme and the substrates and products are in a gaseous form. In this type of system, enzyme activity was found to depend essentially on the water activity (A(w)) of the enzyme preparation.     

Effect of proteolytic and lipolytic enzymes on the adenylate cyclase activity from brain membranes of Ceratitis capitata

1. The effect of various proteolytic enzymes was assayed on the adenylate cyclase activity in purified brain membrane preparations from the insect Ceratitis capitata. Trypsin, chymotrypsin, papain, thermolysin, elastase, subtilisin and prot. XIV were examined. 2. Trypsin treatment, at 37 degrees C, decreased the adenylate cyclase activity even in the presence of GppNHp that protects the activity from the thermal inactivation. 3. Residual basal, GppNHp- and F(-)-stimulated activities were similar when membrane preparations were preincubated either in the presence or in the absence of GppNHp and F-. 4. All proteolytic activities assayed on the brain membrane preparations, excepting papain, exerted an inhibition of adenylate cyclase in basal conditions. 5. The inhibition was stronger in the presence of F- than in the presence of other regulators. 6. Papain showed also a notable inhibition of adenylate cyclase in the presence of F-. 7. Phospholipase A2 treatment decreased both basal and stimulated activity; however, F(-)-sensitive activity was less affected than basal and GppNHp-sensitive activity. F(-)-stimulated activity was less affected by phospholipase A2 than either basal or GppNHp-stimulated activities. 8. Phospholipids are, then, essential for the highest basal activity, although the relationship between catalytic and nucleotide-regulatory components was unaffected by this treatment.     

Inactivation of proteolytic enzymes by cestodes

Inhibitor activity of cestodes from intestines of different hosts (sea birds, salt-water fish, and freshwater fish) was investigated. Alcataenia larina, Arctotaenia tetrabothrioides, Tetrabothrius erostris, T. minor, Wardium cirrosa, Bothriocephalus scorpii, Eubothrium rugosum, and Triaenophorus nodulosus were able to inhibit the activity of the commercial trypsin and activity of proteinase homogenates of the intestinal mucosa of the hosts. It was suggested that the inhibitor produced by the cestodes is specific for trypsin and protects them from the digestive enzymes of the host.     

Purification and some properties of two extracellular proteolytic enzymes produced byVibrio SA1

The purification and characterisation of an extracellular endo and aminopeptidase of the marineVibrio SA1 is described. The endopeptidase was purified by ammonium sulphate precipitation, gel filtration and affinity chromatography. It had a molecular weight of approximately 31 000, a pH optimum at 7.8 and a temperature optimum at 50 C. The enzyme was rapidly inactivated at 65 C.The aminopeptidase was purified by ammonium sulphate precipitation, gel filtration and preparative polyacrylamide gel electrophoresis. This enzyme had a molecular weight of approximately 21 000, a pH optimum at 8.6 and a temperature optimum at 60 C. Both proteases were inactivated by EDTA while reactivation occurred by Ca²⁺, Zn²⁺ and Mg²⁺ ions.The endopeptidase hydrolysed several peptide bonds in the oxidized B-chain of insulin, particularly those involving amino groups of hydrophobic amino acid residues with bulky side chains. It was unable to hydrolyse synthetic dipeptides, but a number of tripeptides were hydrolysed at a low rate. The aminopeptidase hydrolysed leucinamide and di- and tripeptides containing hydrophobic bulky amino acids as the N-terminal residue. It was concluded that the endopeptidase and the aminopeptidase ofVibrio SA1 possess complementary specificities.Abbreviations T= Triethylenetetramine - S= Succinic acid - PIPES= Piperazine-N,Nbis(2-ethane sulfonic acid) - HEPES= N-2-hydroxy-ethylpiperazine-N-2-ethane sulfonic acid Part of this study was supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

A β-lactamase produced by a thermophilic Bacillus

Abstract A β-lactamase was purified from a thermophilic Bacillus strain, that had been isolated from a traditional hot bath in the Meknes area (Morocco). The properties of the enzyme were very similar to those of the β-lactamase produced by Bacillus licheniformis 749C but it exhibited a somewhat increased thermostability and a higher activation energy with cefazolin as substrate. These properties were expected for an enzyme produced by a thermophilic strain.