title: [Introduction of additional thiol groups into glucoamylase in Aspergillus awamori and their effect on the thermal stability and catalytic activity of the enzyme].
abstract: Five mutant forms of glucoamylase (GA) from the filamentous fungus Aspergillus awamori with artificial disulfide bonds (4D-G137A\A14C, 6D-A14C\Y419C\G137A, 10D-V13C\G396C, 11D-V13C\G396C\A14C\Y419C\G137A, and 20D-G137A\A246C\A14C) were constructed using computer simulation and experimentally tested for thermostability. The introduction of two additional disulfide bonds between its first and thirteenth alpha-helices and that of the loop located close to a catalytic residue--E400--made it possible to assess the effects of disulfide bridges on protein thermostability. The mutant proteins with combined amino acid substitutions G137A\A14C, V13C\G396C\A14C\Y419C\G137A, and G137A\A246C\A14C showed higher thermal stability as compared to the wild-type protein. At the same time, new disulfide bridges in the mutant A14C\Y419C\G137A and V13C\G396C proteins led to the destabilization of their structure and the loss of thermal stability.
authors.forename: M A
authors.initials: MA
authors.lastname: Surzhik
authors.forename: A E
authors.initials: AE
authors.lastname: Shmidt
authors.forename: E A
authors.initials: EA
authors.lastname: Glazunov
authors.forename: D L
authors.initials: DL
authors.lastname: Firsov
authors.forename: M G
authors.initials: MG
authors.lastname: Petukhov
mesh_terms: D001230:Aspergillus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D003198:Computer Simulation; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D004220:Disulfides; D005656:Fungal Proteins; D011994:Recombinant Proteins; D005087:Glucan 1,4-alpha-Glucosidase