Studies on alpha-amylase from a thermophilic bacterium. II. Thermal stability of the thermophilic alpha-amylase.

The effect of pH, mental ions, and denaturing reagents on the thermal stability of thermophilic alpha-amylase [EC 3.2.1.1] were examined. The enzyme was most stable at around pH 9.2, which is coincident with the isoelectric point of the enzyme. The stability of the enzyme was increased by the addition of calcium, strontium, and sodium ions. The addition of calcium ions markedly stabilized the enzyme. The protective effects of calcium and sodium ions were additive. At room temperature, no detectable destruction of the helical structure of the enzyme was observed after incubation for 1 hr in the presence of 1% sodium dodecylsulfate, 8 M urea or 6 M guanidine-HC1. The addition of 8 M urea or 6 M guanidine-HC1 lowered the thermal denaturation temperature of the enzyme. The enzyme contained one atom of tightly bound intrinsic calcium per molecule which could not be removed by electrodialysis unless the enzyme was denatured. The rate constants of inactivation and denaturation reactions in the absence and presence of calcium ions were measured and thermodynamic parameters were determined. The presence of calcium ions caused a remarkable decrease in the activation entropy.     

A chaperonin from a thermophilic bacterium, Thermus thermophilus, that controls refoldings of several thermophilic enzymes.

A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus. It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate. ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. The T. thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria. At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner. No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution. An excess amount of the chaperonin is inhibitory for refolding. At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented. At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding.     

M(III)-facilitated recovery and concentration of enzymes from mesophilic and thermophilic organisms

Six enzymes isolated from organisms of widely differing thermal growth optima were flocculated from solution at constant pH by addition of Fe(III) solution. In all cases the enzyme concentration was 1 g.l-1 or less. Flocculation profiles were generated for each enzyme over a range of Fe(III) levels. The concentrated enzymes were recovered from the Fe(III)/protein complex by solubilisation with citrate and dithionite followed by precipitation with ammonium sulphate. In all cases approximately 70-80% enzyme recovery was achieved. Enzyme thermal stability did not appear to be important and protein concentration had no effect on the efficiency of enzyme recovery over the range of 0.01-1 g.l-1. Approximately 30 mmol Fe(III)/l of enzyme solution facilitated optimal enzyme recovery for all solutions studied. For protein concentrations up to 1 g.l-1 a 100-fold enzyme concentration factor can be expected.     

Xylan-hydrolysing enzymes from thermophilic and mesophilic fungi

Screening of 40 mesophilic and 13 thermophilic fungi indicated that enzyme activities capable of degrading oat spelt xylan extensively were produced by only a few of the mesophilic species investigated. The relatively low degree of hydrolysis effected by the enzymes from thermophilic organisms could be explained, in part, by their lack of -xylosidase. Several strains of Aspergillus awamori and Aspergillus phoenicis were notable in producing high xylanase and -xylosidase and low protease activities. Of the fungl tested, 13 produced activities capable of removing O-acetyl, arabinosyl, 4-O-methylglucuronyl, feruloyl and coumaroyl substituents from the backbone of xylan polysaccharides as well as endo-1,4--d-xylanase and -1,4-xylosidase. When the growth medium contained oat spelt xylan as carbon source, higher levels of xylanase, -xylosidase and acetyl xylan esterase were found than in cultures containing meadow fescue grass but the latter were richer in ferulic acid and coumaric acid esterases and 4-O-methylglucuronidase. No single organism or carbon source used was capabie of producing high levels of all the debranching enzymes as well as high levels of enzymes capable of cleaving the glycosidic linkages of the xylan backbone. The best ballnce of enzymes was obtained in cultures of A. awamori IMI 142717 and NRRL 2276 and A. phoenicis IMI 214827. Either of these would be suitable for strain improvement studies.The authors are with The Rowett Research Institute. Bucksburn, Aberdeen, AB2 9SB, UK.T.M. Wood is the corresponding author.     

Starch-hydrolyzing enzymes from thermophilic archaea and bacteria

Extremophlic microorganisms have developed a variety of molecular strategies in order to survive in harsh conditions. For the utilization of natural polymeric substrates such as starch, a number of extremophiles, belonging to different taxonomic groups, produce amylolytic enzymes. This class of enzyme is important not only for the study of biocatalysis and protein stability at extreme conditions but also for the many biotechnological opportunities they offer. In this review, we report on the different molecular properties of thermostable archaeal and bacterial enzymes including alpha-amylase, alpha-glucosidase, glucoamylase, pullulanase, and cyclodextrin glycosyltransferase. Comparison of the primary sequence of the pyrococcal pullulanase with other members of the glucosyl hydrolase family revealed that significant differences are responsible for the mode of action of these enzymes.     

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.     

Heat-stable enzymes from extremely thermophilic and hyperthermophilic microorganisms

Only in the last decade have microorganisms been discovered which grow near or above 100°C. The enzymes that are formed by these extremely thermophilic (growth temperature 65 to 85°C) and hyperthermophilic (growth temperature 85 to 110°C) microorganisms are of great interest. This review covers the extracellular and intracellular enzymes of these exotic microorganisms that have recently been described. Polymer-hydrolysing enzymes, such as amylolytic, cellulolytic, hemicellulolytic and proteolytic enzymes, will be discussed. In addition, the properties of the intracellular enzymes involved in carbohydrate and amino-acid metabolism and DNA-binding and chaperones and chaperone-like proteins from hyperthermophiles are described. Due to the unusual properties of these heat-stable enzymes, they are expected to fill the gap between biological and chemical processes.The authors are with the Technical University Hamburg-Harburg, Institute of Biotechnology, Department of Technical Microbiology, Denickestrasse 15, D-21071 Hamburg, Germany     

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.     

Very stable enzymes from extremely thermophilic archaebacteria and eubacteria

Summary Thirty-six thermophilic archaebacteria and nine extremely thermophilic eubacteria have been screened on solid media for extracellular amylase, protease, hemicellulase (xylanase), cellulase, pectinase and lipase activities. Extracellular enzymes were detected in 14 archaebacteria belonging to three different orders. Twelve of these were able to degrade starch and casein and the two Thermofilum strains were able to degrade starch, xylan and carboxymethylcellulose. Three of the eubacteria could degrade only starch. The other six (including four Thermotoga strains) all had activity against starch, xylan and carboxymethylcellulose, and one also had activity against casein. Some of the amylolytic archaebacteria released -glucosidase, -glucosidase, amylase and transglucosylase activities into liquid media containing starch or maltose. Thermotoga strain FjSS3B.1 released amylase, xylanase, cellulase and -glucosidase activities into the medium when grown in the presence of substrates. When the partially purified enzymes from Thermotoga and some of the archaebacteria were compared with known thermostable enzymes the majority were found to be the most thermostable of their type. The -glucosidase, xylanase and cellulase from Thermotoga and two -glucosidases, a -glucosidase, an amylase and a pullulanase from archaebacteria all have half-lives of at least 15 min at 105°C.     

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 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.     

Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts

Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case individually.     

Structure and stability of thermophilic enzymes. Studies on thermolysin

The molecular mechanisms responsible for the unusual stability of enzymes isolated from thermophilic microorganisms are much more complex and subtle than was originally thought. In particular, a general mechanism cannot be proposed, since individual enzymes can be stabilized by specific molecular interactions and forces. The results of studies on thermophilic enzymes obtained in recent years in our laboratory will be summarized, with particular emphasis being placed on those obtained with thermolysin, a stable metalloendopeptidase isolated from Bacillus thermoproteolyticus. Fragmentation of thermolysin by limited proteolysis by added protease (subtilisin) or autolysis mediated by heat or the ion-chelating agent EDTA leads to quite selective peptide bond fissions, allowing isolation of 'nicked' thermolysin species. Correlation of the sites of proteolytic cleavage with the known three-dimensional structure of thermolysin allowed us to infer some of the key characteristics of the structure, folding, dynamics and stability of the thermolysin molecule. The potential utility of these and other studies on thermophilic enzymes in devising strategies for enhancing the stability of mesophilic enzymes using genetic engineering techniques is discussed.