عنوان

Computational studies of glucoamylase selectivity

Number

TLpq304307032

.Language of Text, Soundtrack etc

انگلیسی

Title Proper

Computational studies of glucoamylase selectivity

General Material Designation

[Thesis]

First Statement of Responsibility

P. M. Coutinho

Subsequent Statement of Responsibility

P. J. Reilly

Name of Publisher, Distributor, etc.

Iowa State University

Date of Publication, Distribution, etc.

1996

Specific Material Designation and Extent of Item

233

Dissertation or thesis details and type of degree

Ph.D.

Body granting the degree

Iowa State University

Text preceding or following the note

1996

Text of Note

Glucoamylase (GA) is an industrial enzyme involved in the production of glucose and fructose syrups from starch. Exhibiting a large spectrum of selectivities, GAs are able to cleave glucose from the nonreducing ends of usd\alphausd-(1,4) glycosidic bonds of maltooligosaccharide chains and the usd\alphausd-(1,6) bonds initiating their branches. The high temperatures and glucose concentrations occurring in saccharification lead to the formation of condensation products, reducing the overall glucose yield. Modified versions of Aspergillus GA that better satisfy industrial requirements are desirable. To achieve this, a structure-based multisequence alignment of the primary sequences of the catalytic domains, linkers, and starch-binding domains of GAs from filamentous fungal, yeast, eubacterial, and archaeal origin has been made to correlate structure to GA stability and selectivity. Evolutionary interpretation of the alignment has elucidated the improvements undergone naturally by GA to improve its catalytic properties. To understand the molecular interactions between GA and its substrates, a protocol was formulated to combine the conformational characterization of substrates using MM3(92) and the Monte Carlo-based docking software AutoDock that allows the interaction of flexible ligands with proteins. This method was applied to study GA active-site interactions with (1) different inhibitors and monosaccharide substrates, yielding very good correspondence with results obtained by X-ray crystallography and so verifying the validity of the approach; (2) several isomaltose analogues, giving a structural basis for some unexplained kinetic properties; and (3) ten glucopyranosyl-based disaccharides, identifying the different requirements that substrates need to satisfy for GA hydrolysis to occur, and revealing that flexibility of the second subsite explains GA properties and preferences. The combined method constitutes a successful approach to study protein-carbohydrate interactions. Finally, an extensive table of GA kinetic and inhibition properties of different natural and genetically modified GAs has been compiled. An assessment of A. niger GA properties reveals that at higher temperatures larger amounts of condensation products are expected.

Applied sciences

Biochemistry

Chemical engineering

condensation products

glucopyranosyl

Pure sciences

P. J. Reilly

P. M. Coutinho

Electronic name

p

[Thesis]

276903

a

Y