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

Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Kampus C-UNAIR, Surabaya, 60115, Indonesia; kartika.dwi.asni-2019@fst.unair.ac.id (K.D.A.P.); sri-s@fst.unair.ac.id (S.S.). Department of Health, Faculty of Vocational Studies, Universitas Airlangga, Kampus B-UNAIR, Surabaya 60286, Indonesia; nyoman.purwani@vokasi.unair.ac.id (N.P.). Department of Mineral Chemical Engineering, Polytechnic of Metal Industry, Morowali, 94974, Indonesia; palupi.ikafj@gmail.com (I.F.J.P.). Laboratory of Proteomics, University CoE-Research Centre for Bio-Molecule Engineering, Universitas Airlangga, Surabaya, Indonesia * Correspondence: ni-nyoman-t-p@fst.unair.ac.id; Tel.: +62-0811-345-2009

Greater catalytic activities were observed for variant enzymes compare to the wild-type enzyme for the β-Dxylosidase of S. ruminantium, at 46%, 71%, and 48%, respectively [8]. A study by Wagschal et [15]. Huang reported that for β-xylosidase from G. stearothermophilus, substitution of tyrosine 509 into glutamic acid leads to a new exoxylanase activity. The KM and kcat values of the Y509E variant xylanase was determined using beechwood xylan as a substrate were 5.10 mg/mL and 22.53 s −1 , respectively [9].
Kinetic parameters that used were derived from the Michaelis-Menten equation, including the maximum enzyme activity (Vmax) and Michaelis constant (KM), which has a role to measure the reflecting of the enzyme affinity [1].
The importance of Asp residue as a pKa modulator was described by Brux (2006). A similar study was conducted by Hartanti (2016). The substitution of the Asp121 residue from GbtXyl43A with glutamic acid (Glu), asparagine (Asn) and valine (Val) reduced the enzyme activity dramatically. The decrease in activity in the three variants showed a change of conformation due to the mutation. Thus, the substitution of Asp with Glu (D121E), Asn (D121N), and Val (D121V) caused some changes in the characteristics of GbtXyl43A, with respect to its pH and temperature optima [6]. Based on that analysis, it could be predicted that the GbtXyl43A variant had a lower affinity value against the substrate compared with its wild type. Enzymes with good catalytic properties (e.g., a high rate of substrate degradation) and acid-base-tolerance are needed in industrial process to reduce the production cost. Therefore, it is necessary to carry out advanced characterization of GbtXyl43A

Materials and methods
The materials that used in this study were Escherichia coli BL21 containing a wild-type GbtXyl43A gene and the D121N variant, were belong to Proteomic Laboratory, University CoE-Research Center for Bio-Molecule Engineering, Institute of Tropical Diseases, Universitas Airlangga, Surabaya, East Java, Indonesia.

Expression and production of proteins
The protein of GbtXyl43A was expressed in E. coli BL21 (DE3) which contained of pET-xyl plasmid and its purification had established [6]. First, pre-culture was prepared and incubated in 37°C for overnight. This pre-culture was used Luria-Bertani (LB) medium that supplemented with 100 µg/mL of ampicillin and grown by the recombinant plasmid E. coli BL21 (DE3)-pET-xyl. It was used 1% for inoculating in 1 L of fresh LB medium contained 100 μg/mL of ampicillin. The production was shaked until OD600nm value between 0.7 and 0.8 (±2.5 h) and induced by 1 mM isopropyl-β-D-thiogalactosidase for 3 h (wild-type GbtXyl43A) and 6 h (GbtXyl43A-D121N). The harvesting of cells was used centrifugation (3,500 rpm) for 30 min at 4°C. The pellet was resuspended by 10 mM phosphate buffer (pH 7.5). The phosphate buffer also contained of 10 mM imidazole and 50 mM NaCl. This solution of cells was sonicated. The supernatants of recombinant protein were taken after centrifugation (10,000 rpm) for 15 min at 4°C.

Purification of GbtXyl43A
The protein of GbtXYl43A was purified using affinity chromatography while the column containing nickelnitrilotriacetic acid (Ni-NTA) resin. First, the 1 mL column of Ni-NTA was washed by five column volumes of sterile aquabidest and buffer phosphate pH 8 for equilibration. The buffer phosphate pH 8 that used was Wild-type GbtXyl43A was eluted using increasing imidazole concentration (60, 100, and 300 mM). Moreover,

Enzymatic activity
The purified enzyme was analyzed about the activity using 2.0 M of pNP-X (pNP-β-D-xylopyranoside) as a substrate. This analysis was incubated at 50°C. The mixture solution for assay was contained of 450 μL substrate in phosphate buffer and 50 μL enzyme. The enzyme had pre-treatment by incubating at 50°C for 30 min. The analysis was continued by adding 50 μL of 0,4 M Na2CO3 and measured at 400 nm. The measurement was calculated in duplicate. the activity of recombinant GbtXyl43A was defined in one unit of activity (IU).
This analysis enzyme was required 1 μmol pNP per minute under reaction conditions.

Enzyme kinetic assays
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 24 September 2020 doi:10.20944/preprints202009.0585.v1 The enzyme kinetic parameters that contained of KM, Vmax, kcat, and kcat/KM were measured by observing the absorbance changes with a UV-Vis spectrophotometer at 405 nm. The hydrolysis reaction was determined by incubating the pNP-X substrate with a variety of concentrations (0.5, 2, 4, 6, and 10 mM), as adapted from Wagschal (2009). The substrate was dissolved in phosphate buffer of pH 6. The reaction was initiated by adding an enzyme solution to each substrate, which was incubated at 50°C. Each reaction was observed at 0, 6, 14, 22, 30, 38, 46, and 54 min. The initial velocity (V0) of each substrate concentration was obtained from the linearity of each curve by plotting the incubation time on an axis and forming the product as an ordinate. Next, KM, Vmax, kcat, and kcat/KM were determined using the Lineweaver-Burk plot.

Expression and purification of the wild-type GbtXyl43A and the D121N variant
The β-D-xylosidase of wild-type GbtXyl43A and the D121N variant were expressed in E. coli BL21 and purified. To elucidate the xylanolytic activity on synthetic substrates, the GbtXyl43A properties were explored.
Purified wild-type GbtXyl43A was recovered after an elution of buffer with 100 mM imidazole concentration. was showed that change in its optimum pH of enzyme activity and was significantly higher than the wild-type enzyme [6].

The activity of GbtXyl43A and the D121N variant
The enzyme of β-xylosidase that isolated from G. thermoleovorans IT-08 (Xyl) was grouped to the GH43 family. It has a three-dimensional structure that consist of a five-bladed β-propeller fold [13]. β-Xylosidase hydrolyzes the substrate with the use of a mechanism that leads to inversion the structure by the anomeric configuration. It was caused by the displacement of nucleophilic. There are three important residues that have role in catalytic site, they are Glu177, Asp14, and Asp121. The role of Glu177, Asp14, and Asp121 are as general acid, general base, and modulate the pKa of the acid and keeping all of them in the correct orientation toward to the substrate [6,3,13].
In addition, to modulate the pKa of the catalytic acid, the residue had a role to guide the catalytic acid to the substrate. It was shown in the binding of 2-O of the sugar bond and subsite-1. It was influenced in the stabilization by the substrate's transition state. There was a mutation by changing the Asp into Asn (D121N) was decreased the active enzyme than the wild type did [6].
Wild-type GbtXyl43A showed the specific activity of 0.47110 (U mg -1 ), or 55.44-fold over that of the activity of supernatant, when pNP-X was used as a substrate. By contrast, the D121N variant exhibited a specific activity of 0.01241 (U mg -1 ), or 2.407-fold over that of the activity of its supernatant, when the same substrate was used ( Table 1).
The activity of the β-D-xylosidase D121N variant was considerably lower than that of its wild type. The low activity value could have been due to the Asp121 substitution being Asn in the GbtXyl43A-D121N variant.
This substitution caused a change in the nature of the enzyme. The GbtXyl43A-D121N variant had an optimal temperature and pH of 90°C and 9, respectively, whereas the optimal temperature and pH of wild-type GbtXyl43A were 50°C and 6, respectively. Asp121 mutation also caused changes in enzyme conformation.
Asp121 plays a role as the pKa modulator and also keep it in the correct orientation to the substrate in the Asn is a non-charged amino acid. Deep View analysis of the substitution that produce the variant was predicted that it changed into carboxamide function from the carboxylate function and it introduce a new hydrogen bond that showed by the O of Glu177 to HN of Asn121. The new hydrogen bonding had an interaction between these two residues because of the location was closer and also the differences of electronegativity N and O (N<O).
It's caused the ability of N δ2 in Asn121 to donate the proton to O ε1 in Glu177. Thus, the Glu177 also needs a donation of proton. Besides, the optimal pH of the enzyme was decreased and changed to be alkaline. Otherwise, changing Asp into Asn increased the interaction of hydrophobic that happen on the catalytic area, and thus it also influenced the protein's stability [6].

Enzyme kinetic assays
The parameters of enzyme activity were obtained from the Michaelis-Menten equation as follows: where V0 is the velocity in the substrate reaction, Vmax is the maximum rate of enzyme activity at a reaction that given temperature, whereas the KM is the half-saturation constant (Michaelis constant), and [S] is the concentration of the substrate [7].
When the substrate concentration is greater than KM, the rate of catalysis is equal to kcat. The kinetic constant kcat is the number of substrate molecules that converted into product per unit of time at a single catalytic site when the enzyme is fully saturated with the substrate. The ratio of kcat/KM provides a penetrating probe into enzyme efficiency. To identify the Asp121 as the residue that had an important role to modulate the pKa of the general acid and also guide it in the correct orientation to the substrate, an extensive study was performed by mutating this residue into Asn, Val, and Glu [6]. In this work, we determined the kinetic parameters of the D121N variant by using pNP-X as the substrate. We examined the D121N variant because it has the highest activity among the three variants.
The kinetic reaction of the p-nitrophenyl-β-D-xylopyranoside substrate by wild-type GbtXyl43A and the D121N variant was performed with a variety of substrate concentrations. An increased rate of product formation occurs with an increase in substrate concentration. In a relatively high substrate concentration, the reaction speed reaches a maximum. Therefore, a continuous monitoring method was applied to ascertain the initial-rate reactions for determining the kinetic parameters of the GbtXyl43A acting on the pNP-X substrate.
The kinetic constants for the synthetic substrates were determined using Lineweaver-Burk plots (Figures 2 and   3). The wild-type enzyme exhibited kinetic constants for the hydrolysis of pNP-X, of 2.845 mM and 0.0033 mM min -1 for KM and Vmax, respectively. Furthermore, the D121N variant exhibited kinetic constants for the hydrolysis of pNP-X, of 4.565 mM and 0.101 × 10 -3 mM min -1 for KM and Vmax, respectively (   Asn was thermolabile amino acid; however, the substitution was performed at the important residue at the catalytic site, a decrease in the bonding power of the substrate ensues. The Vmax value expressed the velocity of the product formation of p-nitrophenol. The Vmax value of the D121N variant was lower than that of its wild type, demonstrating that the speed of enzymatic reactions to produce the p-nitrophenol of the variant D121N is slower than that of the wild type. The substitution of aspartate (Asp121) with asparagine (Asn121) in β-D-xylosidase GbtXyl43A increased the optimal temperature and pH. These changes were supported by their kinetic properties.