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Maltogenic amylase of Geobacillus thermoleovorans (Gt-MamyIII), which has the highest thermostability among bacterial maltogenic amylases, has been used as a model enzyme to understand the role of networked salt bridges in thermoadaptation. The role of intra-chain cross-domain salt bridge networks in the thermostabilization of maltogenic amylase of G. thermoleovorans was confirmed by site-directed mutagenesis and CD analysis. The amino acid pairs in seven salt bridges have been mutated singly and pair-wise, and their free energy of thermal inactivation has been calculated. Among seven, single and double mutations in the amino acids corresponding to four salt bridges (lys306.glu336, arg403.asp65, arg497.glu523 and lys524.glu523) decrease T 1/2 and T m of Gt-MamyIII significantly. Moreover, glu523 forms networked salt bridges with arg497 and lys524. OE1 of glu523 forms electrostatic interactions with NH1 of arg497, NH2 of arg497 and NZ of lys524 at a distance of 2.33, 2.02 and 0.33 Å, respectively. The mutations in three buried amino acids led to a decline in T 1/2 and T m . The buried as well as networked cross-domain salt bridges thus appear to play a significant role in the thermostabilization of Gt-MamyIII. The salt bridges lys306.glu336 and arg403.asp65, which are isolated and partially accessible, play a minor role in its thermostabilization. [ABSTRACT FROM AUTHOR]

Abstract: A gene encoding acidic, thermostable and raw starch hydrolysing α-amylase was cloned from an extreme thermophile Geobacillus thermoleovorans and expressed. The ORF of 1650bp encodes a 515 amino acid protein (Gt-amy) with a signal peptide of 34 amino acids at the N-terminus. Seven conserved sequences of GH-13 family have been found in its sequence. The specific enzyme activity of recombinant Gt-amy is 1723Umg−1 protein with a molecular mass of 59kDa. It is optimally active at pH 5.0 and 80°C with t 1/2 values of 283, 184 and 56min at 70, 80 and 90°C, respectively. The activation energy required for its temperature deactivation is 84.96kJmol−1. Ca2+ strongly inhibits Gt-amy at 10mM concentration, and inhibition kinetics with Ca2+ reveals that inhibition occurs as a result of binding to a lower affinity secondary Ca2+ binding site in the active centre in a mixed-type inhibition manner. The K m and k cat of the Gt-amy are 0.315mgmL−1 and 2.62×103 s−1, respectively. Gt-amy is Ca2+-independent at the concentration used in industrial starch saccharification, and hydrolyses raw corn and wheat starches efficiently, and thus, is applicable in starch saccharification at the industrial sub-gelatinization temperatures. [Copyright &y& Elsevier]

Abstract: A xylanase gene (xyl-gt) of 1.224kbp was cloned from the extremely thermophilic bacterium Geobacillus thermoleovorans that encodes a protein containing 408 amino acid residues. Eight conserved regions (signature sequences) of GH family 10 xylanases have been found in the xylanase. When the xylanase gene was cloned and expressed in Escherichia coli BL21 (DE3), the recombinant strain produced xylanase titer of 270Umg−1 which is 27-fold higher than the wild strain. It is optimally active at 80°C and pH 8.5 with a high thermostability over broad range of pH (6–12) and temperature (40–100°C). The end products of the hydrolysis of birch wood xylan and agro-residues included xylobiose, xylotriose, xylotetraose and xylopentaose. The xylanase of G. thermoleovorans is one of the rare xylanases that exhibits thermo-alkali-stability, and thus, it is a suitable candidate for pre-bleaching of paper pulps and generating xylooligosaccharides from agro-residues for use as prebiotics. [Copyright &y& Elsevier]

j.l. uma maheswar rao and t. satyanarayana. 2004.Effect of polyamines and their biosynthesis inhibitors on the production of hyperthermostable and Ca2+-independentα-amylase byGeobacillus thermoleovoransMTCC 4220Theα-amylase was produced in starch-yeast extract-tryptone (SYT) broth with different polyamines (PA) and polyamine biosynthesis inhibitors, methylglyoxal-bis-guanylhydrazone (MGBG) and cyclohexylammonium sulphate (CHA) at 70°C. The bacterial pellets were obtained after growingG. thermoleovoransat different temperatures, and used in determining total PA. The cell-free culture filtrates were used inα-amylase assays.During growth, total polyamines in biomass increased till 2 h, and thereafter, decreased gradually. The total polyamine content was very high in the biomass cultivated at 55°C when compared with that of higher temperatures. Enzyme titre enhanced up to 70°C, and thereafter declined. Extracellular enzyme and protein levels declined in the presence of exogenously added PA. The intracellular enzyme titres, however, were higher in putrescine (put) and spermidine (spd) than in spermine (spm). Polyamine biosynthesis inhibitor, MGBG enhanced secretion ofα-amylase in a laboratory fermentor as well as shake flasks, although CHA did not affect it.The intracellular accumulation ofputin the presence of MGBG appeared to enhance synthesis and secretion ofα-amylase. Extracellular enzyme and protein levels were low in the presence of exogenously added PA, but their intracellular levels, however, were higher inputandspdthan inspm.A substantial increase in the synthesis and secretion ofα-amylase was attained inG. thermoleovoransin the presence of polyamine biosynthesis inhibitor MGBG. [ABSTRACT FROM AUTHOR]

Abstract Aim: Statistical optimization for maximum production of a hyperthermostable, Ca[sup 2+] -independent and high maltose-forming α -amylase by Geobacillus thermoleovorans . Methods and Results: G. thermoleovorans was cultivated in 250 ml flasks containing 50 ml of chemically defined glucose–arginine medium (g l[sup -1]: glucose 20; arginine 1·2; riboflavin 150 μ g ml[sup -1] ; MgSO[sub 4] . 7H[sub 2] O 0·2; NaCl 1·0; pH 7·0). The medium was inoculated with 5 h-old bacterial inoculum (1·8 × 10[sup 8] CFU ml[sup -1] ), and incubated in an incubator shaker at 70°C for 12 h at 200 rev min[sup -1] . The fermentation variables optimized by ‘one variable at a time’ approach were further optimized by response surface methodology (RSM). The statistical model was obtained using central composite design (CCD) with three variables: glucose, riboflavin and inoculum density. An over all 24 and 70% increase in enzyme production was attained in shake flasks and fermenter because of optimization by RSM, respectively. A good coverage of interactions could also be explained by RSM. The end products of the action of α -amylase on starch were maltose (62%), maltotriose (31%) and malto-oligosaccharides (7%). Conclusions: RSM allowed optimization of medium components and cultural parameters for attaining high yields of α -amylase, and further, a good coverage of interactions could be explained. The yield of maltose was higher than maltotriose and malto-oligosaccharides in the starch hydrolysate. Significance and Impact of the Study: By applying RSM, critical fermentation variables were optimized rapidly. The starch hydrolysate contained a high proportion of maltose, and therefore, the enzyme can find application in starch saccharification process for the manufacture of high maltose syrups. The use of this enzyme in starch saccharification eliminates the addition of Ca[sup 2+] . [ABSTRACT FROM AUTHOR]

Abstract Aims: Selection of suitable surfactants for enhancing and stabilizing α -amylase of Geobacillus thermoleovorans . Methods and Results: Geobacillus thermoleovorans was cultivated in shake flasks containing 50 ml of starch-yeast extract-tryptone (SYT) medium with/without surfactants. Titres of the enzyme in media were monitored. The enzyme was also preserved at 4°C with/without surfactants and enzyme activities were determined. Among polyethylene glycol (PEGs) of different molecular weights, PEG 8000 (0·5%, w/v) caused a slight increase in the enzyme titre, while Tween-20, Tween-40 and Tween-60 (0·03%, w/v) exerted a significant stimulatory effect on enzyme secretion. In the presence of SDS, Tween-80 and cholic acid (0·03%, w/v), the enzyme production was nearly twofold higher than that in the control. The anionic (SDS, cholic acid) and non-ionic (Tweens) detergents increased the cell membrane permeability, and thus, enhanced α -amylase secretion. Furthermore, anionic surfactants exhibited stabilizing effect on the enzyme during preservation at 4°C. Conclusions: PEG 8000 and the ionic detergents (SDS, cholic acid and Tween-80) were more effective in the solubilization of cell membrane components, and enhancing enzyme yields than the cationic detergents such as CTAB (N,Cetyl-N,N,N-trimethyl ammonium bromide). Further, these surfactants were found to stabilize the enzyme at 4°C. Significance and Impact of the Study: The secretion of Ca2+ -independent hyperthermostable α -amylase was enhanced in the presence of certain anionic and non-ionic detergents in the medium. Furthermore, the surfactants stabilized the enzyme during preservation at 4°C. The use of this enzyme in starch hydrolysis eliminates the addition of Ca2+ in starch liquefaction and its subsequent removal by ion exchange from sugar syrups. [ABSTRACT FROM AUTHOR]