Mutational Modulation of Substrate Bond-Type Specificity and Thermostability of Glucoamylase from Aspergillus awamori by Replacement with Short Homologue Active Site Sequences and Thiol/Disulfi
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- 作者：Henri-Pierre Fierobe ; Bjarne B. Stoffer ; Torben P. Frandsen ; and Birte Svensson
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：July 2, 1996
- 年：1996
- 卷：35
- 期：26
- 页码：8696 - 8704
- 全文大小：419K
- 年卷期：v.35,no.26(July 2, 1996)
- ISSN：1520-4995
文摘
Rational protein engineering based on three-dimensional structure,sequence alignment, andprevious mutational analysis served to increase thermostability andmodulate bond-type specificity inglucoamylase from Aspergillus awamori. The single freecysteine, Cys320, became disulfide bonded inthe Ala246
Cys mutant, thus enhancing T50 by 4
C to 73 C. Compared to wild-type, Ala246Cyswas roughly twice as active at 66 C, but half as active at 45 C.The alternative, elimination of the thiolgroup in Cys320Ala, barely improved thermostability or alteredactivity. Secondly, to acquireexceptionally high specificity toward 
pha.gif" BORDER=0>-1,6 glucosidic linkages,characteristic of Hormoconis resinaeglucoamylase, two short sequential mutants,Val181Thr/Asn182Tyr/Gly183Ala (L3 glucoamylase)and Pro307Ala/Thr310Val/Tyr312Met/Asn313Gly (L5glucoamylase), were made. Thesehomologue mutants are located in the (pha.gif" BORDER=0>/pha.gif" BORDER=0>)6-fold of thecatalytic domain in segments that connect pha.gif" BORDER=0>-helices5 and 6 and pha.gif" BORDER=0>-helices 9 and 10, respectively. The kinetics ofmalto- and isomaltooligosaccharides hydrolysisclearly demonstrated that combination of the mutations in L3L5compensated adverse effects of the singlereplacements in L3 or L5 glucoamylases to yield wild-type or higheractivity. On pha.gif" BORDER=0>-1,4-linked substrates,typically Km increased 2-fold for L3, andkcat decreased up to 15-fold for L5glucoamylase. In contrast,on pha.gif" BORDER=0>-1,6-linked substrates L3 showed both a 2-fold increase inKm and a 3-fold decrease inkcat, while L5GA caused a similar kcat reduction, but up to9-fold increase in Km. L3L5 glucoamylasehad remarkablylow Km for isomaltotriose throughisomaltoheptaose and elevated kcat onisomaltose, resulting in anapproximately 2-fold improved catalytic efficiency(kcat/Km). Rationalloop replacement thus provedpowerful in achieving variants with enhanced properties of a highlyevolved enzyme.
Cys mutant, thus enhancing T50 by 4C to 73 C. Compared to wild-type, Ala246Cyswas roughly twice as active at 66 C, but half as active at 45 C.The alternative, elimination of the thiolgroup in Cys320Ala, barely improved thermostability or alteredactivity. Secondly, to acquireexceptionally high specificity toward pha.gif" BORDER=0>-1,6 glucosidic linkages,characteristic of Hormoconis resinaeglucoamylase, two short sequential mutants,Val181Thr/Asn182Tyr/Gly183Ala (L3 glucoamylase)and Pro307Ala/Thr310Val/Tyr312Met/Asn313Gly (L5glucoamylase), were made. Thesehomologue mutants are located in the (pha.gif" BORDER=0>/pha.gif" BORDER=0>)6-fold of thecatalytic domain in segments that connect pha.gif" BORDER=0>-helices5 and 6 and pha.gif" BORDER=0>-helices 9 and 10, respectively. The kinetics ofmalto- and isomaltooligosaccharides hydrolysisclearly demonstrated that combination of the mutations in L3L5compensated adverse effects of the singlereplacements in L3 or L5 glucoamylases to yield wild-type or higheractivity. On pha.gif" BORDER=0>-1,4-linked substrates,typically Km increased 2-fold for L3, andkcat decreased up to 15-fold for L5glucoamylase. In contrast,on pha.gif" BORDER=0>-1,6-linked substrates L3 showed both a 2-fold increase inKm and a 3-fold decrease inkcat, while L5GA caused a similar kcat reduction, but up to9-fold increase in Km. L3L5 glucoamylasehad remarkablylow Km for isomaltotriose throughisomaltoheptaose and elevated kcat onisomaltose, resulting in anapproximately 2-fold improved catalytic efficiency(kcat/Km). Rationalloop replacement thus provedpowerful in achieving variants with enhanced properties of a highlyevolved enzyme.