Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis.


Abstract

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific DeltaGmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state. Study holds ProTherm entries: 5645, 5646, 5647, 5648, 5649, 5650, 5651, 5652, 5653, 5654, 5655, 5656, 5657, 5658, 5659, 5660, 5661, 5662, 5663, 5664, 5665, 5666, 5667 Extra Details:

Submission Details

ID: mYBtMV993

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Feller G;d'Amico D;Gerday C,Biochemistry (1999) Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis. PMID:10194383
Additional Information

Number of data points 55
Proteins Pancreatic alpha-amylase ; GLYCOSYLTRANSFERASE ; Alpha-amylase ; Alpha-amylase ; Alpha-amylase 1 ; Alpha-amylase 1 ; Alpha-amylase ; Alpha-amylase ; Alpha-amylase
Unique complexes 6
Assays/Quantities/Protocols Experimental Assay: dHvH pH:7.2, buffers:MOPS: 30 mM ; Experimental Assay: dHcal buffers:MES: 30 mM, pH:6.4 ; Experimental Assay: Tm buffers:MES: 30 mM, pH:6.4 ; Experimental Assay: dHvH buffers:MES: 30 mM, pH:6.4 ; Experimental Assay: dHcal buffers:MES: 30 mM, pH:6.3 ; Experimental Assay: Tm buffers:MES: 30 mM, pH:6.3 ; Experimental Assay: dHvH buffers:MES: 30 mM, pH:6.3 ; Experimental Assay: dHcal pH:6.2, buffers:MES: 30 mM ; Experimental Assay: Tm pH:6.2, buffers:MES: 30 mM ; Experimental Assay: dHvH pH:6.2, buffers:MES: 30 mM ; Experimental Assay: dHcal buffers:MES: 30 mM, pH:6.1 ; Experimental Assay: Tm buffers:MES: 30 mM, pH:6.1 ; Experimental Assay: dHvH buffers:MES: 30 mM, pH:6.1 ; Experimental Assay: dHcal pH:6.0, buffers:MES: 30 mM ; Experimental Assay: Tm pH:6.0, buffers:MES: 30 mM ; Experimental Assay: dHvH pH:6.0, buffers:MES: 30 mM ; Experimental Assay: dHcal buffers:MES: 30 mM, pH:5.9 ; Experimental Assay: Tm buffers:MES: 30 mM, pH:5.9 ; Experimental Assay: dHvH buffers:MES: 30 mM, pH:5.9 ; Experimental Assay: dHcal pH:5.8, buffers:MES: 30 mM ; Experimental Assay: Tm pH:5.8, buffers:MES: 30 mM ; Experimental Assay: dHvH pH:5.8, buffers:MES: 30 mM ; Experimental Assay: dHcal pH:5.7, buffers:MES: 30 mM ; Experimental Assay: Tm pH:5.7, buffers:MES: 30 mM ; Experimental Assay: dHvH pH:5.7, buffers:MES: 30 mM ; Experimental Assay: dHcal pH:7.2, buffers:MOPS: 30 mM ; Experimental Assay: Tm pH:7.2, buffers:MOPS: 30 mM
Libraries Mutations for sequence EKDANFASGRNSIVHLFEWKWNDIADECERFLQPQGFGGVQISPPNEYLVADGRPWWERYQPVSYIINTRSGDESAFTDMTRRCNDAGVRIYVDAVINHMTGMNGVGTSGSSADHDGMNYPAVPYGSGDFHSPCEVNNYQDADNVRNCELVGLRDLNQGSDYVRGVLIDYMNHMIDLGVAGFRVDAAKHMSPGDLSVIFSGLKNLNTDYGFADGARPFIYQEVIDLGGEAISKNEYTGFGCVLEFQFGVSLGNAFQGGNQLKNLANWGPEWGLLEGLDAVVFVDNHDNQRTGGSQILTYKNPKPYKMAIAFMLAHPYGTTRIMSSFDFTDNDQGPPQDGSGNLISPGINDDNTCSNGYVCEHRWRQVYGMVGFRNAVEGTQVENWWSNDDNQIAFSRGSQGFVAFTNGGDLNQNLNTGLPAGTYCDVISGELSGGSCTGKSVTVGDNGSADISLGSAEDDGVLAIHVNAKL ; Mutations for sequence A:ANLNGTLMQYFEWYMPNDGQHWKRLQNDSAYLAEHGITAVWIPPAYKGTSQADVGYGAYDLYDLGEFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKGGADATEDVTAVEVDPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWHWYHFDGTDWDESRKLNRIYKFQGKAWDWEVSNE/B:NGNYDYLMYADIDYDHPDVAAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWQNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNSTVVSKHPLKAVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQR ; Mutations for sequence EYSSNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFNPGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLSGLLDLALGKDYVRSKIAEYMNHLIDIGVAGFRIDASKHMWPGDIKAILDKLHNLNSNWFPEGSKPFIYQEVIDLGGEPIKSSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRYFENGKDVNDWVGPPNDNGVTKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQVAFGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISNSAEDPFIAIHAESKL ; Mutations for sequence MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAVNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKGLSQSDNGYGPYDLYDLGEFQQKGTVRTKYGTKSELQDAIGSLHSRNVQVYGDVVLNHKAGADATEDVTAVEVNPANRNQETSEEYQIKAWTDFRFPGRGNTYSDFKWHWYHFDGADWDESRKISRIFKFRGEGKAWDWEVSSENGNYDYLMYADVDYDHPDVVAETKKWGIWYANELSLDGFRIDAAKHIKFSFLRDWVQAVRQATGKEMFTVAEYWQNNAGKLENYLNKTSFNQSVFDVPLHFNLQAASSQGGGYDMRRLLDGTVVSRHPEKAVTFVENHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGTSPKEIPSLKDNIEPILKARKEYAYGPQHDYIDHPDVIGWTREGDSSAAKSGLAALITDGPGGSKRMYAGLKNAGETWYDITGNRSDTVKIGSDGWGEFHVNDGSVSIYVQK ; Mutations for sequence TPTTFVHLFEWNWQDVAQECEQYLGPKGYAAVQVSPPNEHITGSQWWTRYQPVSYELQSRGGNRAQFIDMVNRCSAAGVDIYVDTLINHMAAGSGTGTAGNSFGNKSFPIYSPQDFHESCTINNSDYGNDRYRVQNCELVGLADLDTASNYVQNTIAAYINDLQAIGVKGFRFDASKHVAASDIQSLMAKVNGSPVVFQEVIDQGGEAVGASEYLSTGLVTEFKYSTELGNTFRNGSLAWLSNFGEGWGFMPSSSAVVFVDNHDNQRGHGGAGNVITFEDGRLYDLANVFMLAYPYGYPKVMSSYDFHGDTDAGGPNVPVHNNGNLECFASNWKCEHRWSYIAGGVDFRNNTADNWAVTNWWDNTNNQISFGRGSSGHMAINKEDSTLTATVQTDMASGQYCNVLKGELSADAKSCSGEVITVNSDGTINLNIGAWDAMAIHKNAKLNTSSAS ; Mutations for sequence QYAPQTQSGRTSIVHLFEWRWVDIALECERYLGPKGFGGVQVSPPNENVVVTNPSRPWWERYQPVSYKLCTRSGNENEFRDMVTRCNNVGVRIYVDAVINHMCGSGAAAGTGTTCGSYCNPGSREFPAVPYSAWDFNDGKCKTASGGIESYNDPYQVRDCQLVGLLDLALEKDYVRSMIADYLNKLIDIGVAGFRIDASKHMWPGDIKAVLDKLHNLNTNWFPAGSRPFIFQEVIDLGGEAIKSSEYFGNGRVTEFKYGAKLGTVVRKWSGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGSSILTFWDARLYKVAVGFMLAHPYGFTRVMSSYRWARNFVNGEDVNDWIGPPNNNGVIKEVTINADTTCGNDWVCEHRWREIRNMVWFRNVVDGEPFANWWDNGSNQVAFGRGNRGFIVFNNDDWQLSSTLQTGLPGGTYCDVISGDKVGNSCTGIKVYVSSDGTAQFSISNSAEDPFIAIHAESKL
Sequence Assay Result Units