Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Kinetic Characterization of β-xylosidase (GbtXyl43A) from Wild-type Geobacillus thermoleovorans IT-08 and The Variant GbtXyl43A-D121N

Version 1 : Received: 23 September 2020 / Approved: 24 September 2020 / Online: 24 September 2020 (14:15:50 CEST)

How to cite: Palupi, I.F.J.; Putri, K.D.A.; Purwani, N.N.; Sumarsih, S.; Puspaningsih, N.N.T. Kinetic Characterization of β-xylosidase (GbtXyl43A) from Wild-type Geobacillus thermoleovorans IT-08 and The Variant GbtXyl43A-D121N. Preprints 2020, 2020090585 (doi: 10.20944/preprints202009.0585.v1). Palupi, I.F.J.; Putri, K.D.A.; Purwani, N.N.; Sumarsih, S.; Puspaningsih, N.N.T. Kinetic Characterization of β-xylosidase (GbtXyl43A) from Wild-type Geobacillus thermoleovorans IT-08 and The Variant GbtXyl43A-D121N. Preprints 2020, 2020090585 (doi: 10.20944/preprints202009.0585.v1).

Abstract

GbtXyl43A, a β-xylosidase that is isolated from Geobacillus thermoleovorans IT-08 and grouped in GH43 family. The substitution of 121Asp residue with Asn in GbtXyl43A caused decrease the enzyme activity. The aim of this study, determine the kinetic characteristics of wild-type GbtXyl43A and D121N variant using Vmax, KM, kcat, and kcat/KM. These parameters indicated catalytic mechanism of GbtXyl43A and its derivative. All of them were produced in Escherichia coli BL21 star. The purification of wild-type GbtXyl43A using affinity chromatography, but D121N variant also required anion-exchange chromatography. The specific activity of wild-type GbtXyl43A and D121N variant were 0.471 U mg-1 in purity level 55,44 and 0.012 U mg-1 in purity level 2,407, respectively. Both enzymes had same molecular weight, ~58 kDa. The kinetic parameters of wild-type GbtXyl43A were KM: 2.845 mM, kcat: 0.033 s-1, Vmax: 0.0033 mM min-1and kcat/KM: 0.0115 s-1mM-1. Furthermore, the KM, kcat, Vmax, and kcat/KM values of D121N variant were 4.565 mM, 1.01 × 10-4 mM min-1, 0.140 × 10-4 s-1, and 0.0307 s-1mM-1, respectively. The KM value of the D121N variant was higher than its wild type and showed the affinity of D121N variant was lower than GbtXyl43A

Subject Areas

Geobaccilus thermoleovorans IT-08; β-D-xylosidase; GbtXyl43A; kinetic characteristics

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