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Forsberg, Zarah, 2009. Hydrolysis and glycosynthase activity studies of laminarinase, Lam16A, wildtype and its catalytic deficient mutants E115G, E115S and E120A from Phanerochaete chrysosporium. SLU, Dept. of Molecular Biosciences, Uppsala. Uppsala: SLU, Dept. of Molecular Biosciences

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Abstract

Laminarinase 16A from Phanerochaete chrysosporium is a 36 kDa enzyme with typical endo-β-1,3(4)-glucanase activity, belonging to family 16 of glycoside hydrolases. The catalytic amino acids in the active site are identified as nucleophile Glu 115 and acid/base Glu 120. Prior to this project, these residues had been mutated to make catalytic deficient mutants E115G, E115S and E120A in order to obtain structures in complex with natural substrates. In the present work, the nucleophile mutant E115G has been expressed and successfully purified and shown to possess glycosynthase activity when using an α-fluoride derivative of laminariheptaose as substrate by the detection of circular laminariheptaose using HPLC. Activity measurements revealed that the E115G mutant had substantial activity in presence of acetate and formiate, and the other nucleophile mutant, E115S, in the presence of formiate, which indicates that those molecules can act as external nucleophiles. Circular β-glucans from two different nitrogen-fixing bacterial species were tested as potential substrates for wildtype Lam16A and the E115G mutant in presence of acetate. HPLC analysis revealed that there was no effect on the preparation with circular β-1,2-glucan, whereas one component of the other β-1,3/1,6-glucan preparation was consumed and new peaks appeared in the chromatogram, although further studies are needed to identify these components. The findings from this project reveals that this enzyme has many interesting capacities that hopefully in the future may be used in the synthesis of interesting oligosaccharides that can be applied in the fields of environmental protection, pre-biotics and medicine.

Main title:Hydrolysis and glycosynthase activity studies of laminarinase, Lam16A, wildtype and its catalytic deficient mutants E115G, E115S and E120A from Phanerochaete chrysosporium
Authors:Forsberg, Zarah
Supervisor:Ståhlberg, Jerry
Examiner:UNSPECIFIED
Series:UNSPECIFIED
Volume/Sequential designation:UNSPECIFIED
Year of Publication:2009
Level and depth descriptor:Other
Student's programme affiliation:BTEMP Biotechnology Programme 240 HEC
Department:(VH) > Dept. of Molecular Biosciences
Keywords:Laminarinase 16A (Lam16A), β-glucans, glycoside hydrolases, catalytic deficient mutants, glycosynthases activity, circular oligosaccharides, HPLC, external nucleophiles
URN:NBN:urn:nbn:se:slu:epsilon-s-7986
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-s-7986
Subjects:SLU > (NL, NJ) > Dept. of Molecular Biology (until 131231)
Physical sciences
Language:English
Deposited On:18 Oct 2017 11:17
Metadata Last Modified:18 Oct 2017 11:17

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