Bacillus stearothermophilus enzymes

Enzymes. Termamyl, AMG, and Dextrozyme were obtained from Novo Nordisk Bioindustrials, Inc., Danbury, CT. Bacillus stearothermophilus alpha-amylase was supplied by Enzyme Bio-Systems Ltd., Englewood Cliffs, NJ. 

Thermostability of Thermoanaerohacter sp. CGTase. The addition of 40ppm Ca+ + to the CGTase preparation during incubation at high temperatures in the absence or presence of starch substrate provided no enhancement of the thermostability of the enzyme. A comparison of the thermostable CGTase was made to other thermostable enzymes used in starch liquefaction including Termamyl Bacillus licheniformis) and Bacillus stearothermophilus alpha-amylase. 

The standard CGTase employed in the cyclodextrin industry is produced by Bacillus mascerans. This enzyme is reported to be stable only at temperatures around 50 C and loses activity rapidly above 50 C (19), A more thermostable CGTase compared to the B, mascerans CGTase is produced by Bacillus stearothermophilus with a temperature optimum of 1(PC (20). However, the Thermoanaerobaaer CGTase is far more thermostable with its optimum of 950C, and is to our knowledge, the most thermostable CGTase. 

In the case of enzymes, the rate of the catalysed reaction increases regularly with increasing temperature. However, the probability of the unfolding of the threedimensional conformation of the protein molecule also increases, as there is more energy available to split the non-covalent interactions between side chains. In some cases it has been demonstrated that such noncovalent interactions play a dominant role in the stability of the native conformation. For example, Brosnan, Kelly and Fogarty (1992) demonstrated that the irreversible thermoinactivation of -amylase of Bacillus stearothermophilus at 90°C is related to changes of the hydrophobic interactions in the molecule. 

Fig. 2.6. Regulation of Phosphofructokinase from Bacillus stearothermophilus. The tetrameric phosphofructokinase is aUostericaUy regulated by ADP, Frc-6-phosphate, and phosphoenolpyruvate (PEP). The binding of ADP and Frc-6-phosphate converts the enzyme into the active R state. PEP binds to the T state and inhibits phosphofructokinase. The circles represent the R state, and the squares represent the T state of the enzyme.

FIGURE 25-8 Large (Klenow) fragment of DNA polymerase I. This polymerase is widely distributed in bacteria. The Klenow fragment, produced by proteolytic treatment of the polymerase, retains the polymerization and proofreading activities of the enzyme. The Klenow fragment shown here is from the thermophilic bacterium Bacillus stearothermophilus (PDB ID 3BDP). The active site for addition of nucleotides is deep in the crevice at the far end of the bound DNA. The dark blue strand is the template. 

Figure 2-13 (A) Stereoscopic view of the nucleotide binding domain of glyceraldehyde phosphate dehydrogenase. The enzyme is from Bacillus stearothermophilus but is homologous to the enzyme from animal sources. Residues are numbered 0-148. In this wire model all of the main chain C, O, and N atoms are shown but side chains have been omitted. The large central twisted P sheet, with strands roughly perpendicular to the page, is seen clearly hydrogen bonds are indicated by dashed lines. Helices are visible on both sides of the sheet. The coenzyme NAD+ is bound at the end of the P sheet toward the viewer. Note that the two phosphate groups in the center of the NAD+ are H-bonded to the N terminus of the helix beginning with RIO. From Skarzynski et al.llla (B) Structural formula for NAD+.

The tyrosyl-tRNA synthetase from Bacillus stearothermophilus crystallizes as a symmetrical dimer of Mr2 X 47 316. It catalyzes the aminoacylation of tRNA1 in a two-step reaction. Tyrosine is first activated (equation 15.1) to form a very stable enzyme-bound tyrosyl adenylate complex. Tyrosine is then transferred to tRNA (equation 15.2).6... 

This behavior could easily be mistaken for half-of-the-sites reactivity, but all four sites appear to be independent.55,57 Coenzymes also bind independently to each site. In contrast, the enzyme from Bacillus stearothermophilus is allosteri-cally regulated.44,45 A tetrameric form is stabilized by the binding of two effector molecules of fructose 1,6-bisphosphate and binds pyruvate 50 times more tightly than the dimeric form does. The turnover numbers of tetramer and dimer are the same, and so they form a V-system (Chapter 8). The build up of the glycolytic intermediate fructose 1,6-bisphosphate under anaerobic conditions thus stimulates the regeneration of NAD+. 

Philip Evans and his coworkers have determined the crystal structures of phosphofructokinase from two species of bacteria, E. coli and Bacillus stearothermophilus. By crystallizing the enzyme in the presence and absence of the substrate and several allosteric effectors, they obtained detailed views of both the T and R conformations. This work has led to an explanation of why phosphofructokinase appears to be constrained largely to all-or-nothing transitions between these two states, rather than adopting a series of intermediate conformations. 

Computer-generated structure of two of the four subunits of phosphofructokinase from Bacillus stearothermophilus. The enzyme, shown as yellow and light blue tubes, was crystallized in the R conformation in the presence of the substrate fructose-6-phosphate (dark blue) and the allosteric activator ADP (pink). The magnesium ions (white/silver spheres), Mg2+, bound to the ADP molecules are also shown. (Copyright 1994 by the Scripps Research Institute/Molecular Graphics Images by Michael Pique using software by Yng Chen, Michael Connolly, Michael Carson, Alex Shah, and AVS, Inc. Visualization advice by Holly Miller, Wake Forest University Medical Center.)... 

Enzyme (Ref.) (Organism) Sorbitol dehydrogenase [83] (Rhodobacter sphaeroides) Benzyl-ADH [84] (Acinetobacter calcoacticus) Glycerol dehydrogenase [85] (Bacillus stearothermophilus)... 

I n this way we have shown that phosphoryl transfer catalysed by Bacillus stearothermophilus and rabbit skeletal muscle phosphofructokinase (6), and rabbit skeletal muscle pyruvate kinase occurs with inversion of configuration at phosphorus (7). The simplest interpretation of these stereochemical results is that phosphoryl transfer occurs by an in-line mechanism in the enzyme substrate ternary complexes. Stereochemical analysis is thus proving to be of considerable importance for delineating the mechanism adopted by phosphokinases. ... 

CF Hawkins, A Borges, RN Perham. Cloning and sequence analysis of the genes encoding the a and (3 subunits of the El component of the pyruvate dehydrogenase multi enzyme complex of Bacillus stearothermophilus. Eur JBiochem 191 337-346, 1990. 

Catalase I of Bacillus stearothermophilus shows 95 % specificity to the catalase reaction and 5 % specificity to peroxidase activity [177]. In early examples of evolution of a heme enzyme, Trakulnaleamsai et al. [178] and later Matsuura et al. [179] generated catalase libraries by random mutagenesis using sodium nitrite [180] and isolated variants with improved peroxidase activity. The second round of mutagenesis generated a triple mutant with 58 % specificity to peroxidase activity, but decreased thermostability. The authors further evolved their catalase mutant to improve thermostability back to that of wild-type by adding random peptide tails to the C-terminus [181]. 

The bacterial formamidopyrimidine-DNA glycosylase (alias Fpg or MutM) is a bifunctional base-repair enzyme (DNA glycosylase/AP lyase) that removes a wide range of oxidized purines from oxidatively damaged DNA. The crystal structures of the Fpg (MutM) enzymes from Thermus thermophilus, Lactococcus lactis Escherichia coli Bacillus stearothermophilus and the sequence similar endonuclease VIII (Nei) from E. all share the same... 

Elymodavine 0-a-glucoside Enzyme a-glucosidase from Bacillus stearothermophilus or that from rice... 

A spectacular example of stability enhancement through immobilization has been reported for the enzyme catechol-2,3-dioxygenase.27 This enzyme, isolated from the thermophilic bacterium Bacillus stearothermophilus, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde (which can be monitored by absorbance at 375 nm). The soluble enzyme exhibits maximal activity at 50 °C, but following immobilization on glyoxyl agarose beads with a borohydride reduction step, the optimum reaction temperature shifted to 70 °C. At a total protein concentration of 0.010 mg/mL and a temperature of 55 °C, the half-life of the soluble enzyme was 0.08 h, while the enzyme-modified beads had a half-life of 68 h. This represents a 750-fold enhancement of stability that has been attributed to the prevention of subunit dissociation upon immobilization. 

Manganese compounds of biologic importance are examined by pulse radiolysis e.g., the rate of dismutation of radiation-generated Of is catalyzed by Escherichia coli, Mn-containing superoxide dismutase involving electron transfer in which enzymes with Mn(IV), Mn(IIl), Mn(II) and Mn(I) oxidation states are involved. A kinetic model for the reaction mechanism of an Mn dismutase from Bacillus stearothermophilus accounts for the variation of the rate of decay on the concentrations of Oj, enzyme, HjOj, NaNj, KCN and H+. 

---