LANNY HARTANTI, 090970206 (2013) PERAN PENTING RESIDU ASP121 PADA AKTIVITAS KATALITIK b-D-XILOSIDASE DARI Geobacillus thermoleovorans IT-08 (The Crucial Role of Asp121 to Catalytic Activity of b-D-Xylosidase. Disertasi thesis, UNIVERSITAS AIRLANGGA.

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Abstract

To understand the important role of Asp121 in catalytic activity of b-Dxylosidase from Geobacillus thermoleovorans IT-08 (GbtXyl43A), a series of separate site-directed mutagenesis of this residue had been done. Asp121 was mutated into Glu (D121E), Asn (D121N), or Val (D121V) by PCR-site directed mutagenesis. The recombinant plasmids obtained were moved into E. coli TOP 10 and expressed into E. coli BL21 (DE3 Star). Mutations were checked by restriction analysis and sequencing of variants xyl43A harboured in each of recombinant plasmids. To increase the expression level, cells were induced by IPTG 1.5 mM in various inducing time. Inducing for 6 hours was able to increase the specific activity of GbtXyl43A D121E and D121N variants. All variants of GbtXyl43A were purified in three consecutively purification steps, started with heating at 50 °C for 30 min, followed by affinity chromatography using Ni-NTA column and anion-exchange chromatography using ResourceTM Q column. Pure enzymes were characterized its activities in various pHs and temperatures, and enzymes were crystallized to determine its structure by X-ray crystallography. The three variants of GbtXyl43A showed very low activities compared to wild type’s activities, but two variants, i.e. D121V and D121N, showed optimum pH shifts towards pH 9. All variants of GbtXyl43A showed optimum temperature shifts into 90 °C, but their activities decreased drastically compared to that of the wild types. The highest activity of GbtXyl43A was 0.342 IU/mg or 0.338 IU/mg at pH 6 and 50 °C, while the highest activities of GbtXyl43A D121E, D121V, and D121N variants at 90 °C were 4.7, 12.4 and 13.3% from the wild type’s maximum activity, respectively. All variants of GbtXyl43A showed significant difference in its activities compared to that of the wild type’s in 99% level of confidence (ANOVA). Hence, Asp121 plays a crucial role in GbtXyl43A and is proposed as the third catalytic residue in b-D-xylosidase in GH43 family with triad-catalytic mechanism involving Asp I – Glu – Asp II, where Asp I function as proton donor to Glu (not directly involved in catalytic reaction), Glu as general acid or electrophile, and Asp II as general base or nucleophile.

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