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An alkaline active maltooligosaccharide forming alpha-amylase from Bacillus halodurans

Hashim, Suhaila LU (2004)
Abstract
The East African Rift Valley contains a number of soda lakes, which represent some of the most stable, naturally occurring alkaline environments on earth. Alkaliphilic microorganisms found in these habitats have attracted a great interest due to their ability to produce extracellular enzymes that are active and stable at high pH values for industrial applications. Starch hydrolysing alkaliphilic bacteria were isolated from samples collected from hot springs around Lake Bogoria, Kenya and identified as <i>Bacillus halodurans</i>. These isolates were found to produce extracellular amylolytic enzymes active at pH 10.0 and 55 ºC. The gene encoding Amy 34, a maltohexaose forming á-amylase from <i>Bacillus halodurans</i>... (More)
The East African Rift Valley contains a number of soda lakes, which represent some of the most stable, naturally occurring alkaline environments on earth. Alkaliphilic microorganisms found in these habitats have attracted a great interest due to their ability to produce extracellular enzymes that are active and stable at high pH values for industrial applications. Starch hydrolysing alkaliphilic bacteria were isolated from samples collected from hot springs around Lake Bogoria, Kenya and identified as <i>Bacillus halodurans</i>. These isolates were found to produce extracellular amylolytic enzymes active at pH 10.0 and 55 ºC. The gene encoding Amy 34, a maltohexaose forming á-amylase from <i>Bacillus halodurans</i> isolate LBK 34, was sequenced, cloned and expressed in <i>E. coli</i>. From sequence analysis, the four conserved regions within the á-amylase family of enzymes and a carbohydrate binding module (CBM family 25) at the C- terminal were identified, while a structural similarity search revealed that the enzyme is related to cyclodextrin glycosyltransferases (CGTases). Recombinant Amy 34 is a monomer of 119 kDa, exhibiting optimum activity at pH 10.5 - 11.5 and 60 °C. The enzyme hydrolyses starch to form a mixture of maltooligosaccharides, the main product being maltohexaose. The enzyme could also hydrolyse ã-cyclodextrin but not á- and â-cyclodextrins. Thermal unfolding of Amy 34 is irreversible with four transitions, as determined by differential scanning calorimetry. A transition (denaturation) temperature, Tm of 70.8 °C is obtained at pH 9.0, which increases by 5 °C and decreases by 10.4 °C when Amy 34 is heated in presence of 100-fold molar excess of CaCl<sub>2</sub> and metal chelator, EDTA, respectively.These observations, together with the inhibitory effect of calcium ions observed at high concentrations on enzyme activity suggests that calcium plays a role in stabilisation rather than having a direct role in the catalytic activity of Amy 34.Amy 34 amylase was also used to generate starch hydrolysate for subsequent use as a carbon source for <i>Halomonas boliviensis</i> LCI, a moderate halophile, to produce a bioplastic, poly(â-hydroxybutyrate). Finally, maltohexaose yield from soluble starch using Amy 34 was improved by <i>in situ</i> product recovery as compared to the process run under conventional batch conditions. (Less)
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author
opponent
  • Dr Feller, Georges, Laboratory of Biochemistry, Institute of Chemistry B6, University of Liège, Liège, Belgium.
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Biokemisk teknik, Biochemical technology, differential scanning calorimetry, soda lakes, Bacillus halodurans, alkaliphile, á-amylase
pages
128 pages
publisher
Suhaila Hashim, Department of Biotechnology, Box 124, SE- 221 00, Lund, Sweden,
defense location
Lecture Hall B, Center for Chemistry and Chemical Engineering, Lund Institute of Technology.
defense date
2004-12-14 10:30
ISBN
91-89627-25-3
language
English
LU publication?
yes
id
43a59d58-e334-454a-b0f8-a15c57e61d4a (old id 467633)
date added to LUP
2007-10-13 13:41:04
date last changed
2016-09-19 08:45:06
@misc{43a59d58-e334-454a-b0f8-a15c57e61d4a,
  abstract     = {The East African Rift Valley contains a number of soda lakes, which represent some of the most stable, naturally occurring alkaline environments on earth. Alkaliphilic microorganisms found in these habitats have attracted a great interest due to their ability to produce extracellular enzymes that are active and stable at high pH values for industrial applications. Starch hydrolysing alkaliphilic bacteria were isolated from samples collected from hot springs around Lake Bogoria, Kenya and identified as &lt;i&gt;Bacillus halodurans&lt;/i&gt;. These isolates were found to produce extracellular amylolytic enzymes active at pH 10.0 and 55 ºC. The gene encoding Amy 34, a maltohexaose forming á-amylase from &lt;i&gt;Bacillus halodurans&lt;/i&gt; isolate LBK 34, was sequenced, cloned and expressed in &lt;i&gt;E. coli&lt;/i&gt;. From sequence analysis, the four conserved regions within the á-amylase family of enzymes and a carbohydrate binding module (CBM family 25) at the C- terminal were identified, while a structural similarity search revealed that the enzyme is related to cyclodextrin glycosyltransferases (CGTases). Recombinant Amy 34 is a monomer of 119 kDa, exhibiting optimum activity at pH 10.5 - 11.5 and 60 °C. The enzyme hydrolyses starch to form a mixture of maltooligosaccharides, the main product being maltohexaose. The enzyme could also hydrolyse ã-cyclodextrin but not á- and â-cyclodextrins. Thermal unfolding of Amy 34 is irreversible with four transitions, as determined by differential scanning calorimetry. A transition (denaturation) temperature, Tm of 70.8 °C is obtained at pH 9.0, which increases by 5 °C and decreases by 10.4 °C when Amy 34 is heated in presence of 100-fold molar excess of CaCl&lt;sub&gt;2&lt;/sub&gt; and metal chelator, EDTA, respectively.These observations, together with the inhibitory effect of calcium ions observed at high concentrations on enzyme activity suggests that calcium plays a role in stabilisation rather than having a direct role in the catalytic activity of Amy 34.Amy 34 amylase was also used to generate starch hydrolysate for subsequent use as a carbon source for &lt;i&gt;Halomonas boliviensis&lt;/i&gt; LCI, a moderate halophile, to produce a bioplastic, poly(â-hydroxybutyrate). Finally, maltohexaose yield from soluble starch using Amy 34 was improved by &lt;i&gt;in situ&lt;/i&gt; product recovery as compared to the process run under conventional batch conditions.},
  author       = {Hashim, Suhaila},
  isbn         = {91-89627-25-3},
  keyword      = {Biokemisk teknik,Biochemical technology,differential scanning calorimetry,soda lakes,Bacillus halodurans,alkaliphile,á-amylase},
  language     = {eng},
  pages        = {128},
  publisher    = {ARRAY(0xabe67c0)},
  title        = {An alkaline active maltooligosaccharide forming alpha-amylase from <i>Bacillus halodurans</i>},
  year         = {2004},
}