Data on gene cloning, expression, purification, and characterization of the glycoside hydrolase family 11 from Bacillus velezensis

Bacillus velezensis RB.IBE29 is a chitinolytic bacterium originally isolated from the rhizospheric soil of black pepper grown in Vietnam. This bacterium is a strong biocontrol agent against plant pathogens and possesses a novel chitinase system. Genome sequences available in CAZy database revealed B. velezensis possesses one gene encoding xylanase belonging to glycoside hydrolase family 11; however, this enzyme has yet to be un-experimentally characterized. In this work, xyA gene was isolated from the genomic DNA of strain RB.IBE29 and cloned in Escherichia coli DH5α cells using the pUC19 vector. Sequencing analysis showed that the ORF of xyA contains 642 bp and encodes the deduced xylanase with 213 aa and 23.27 kDa. The domain structure of the enzyme has a signal peptide and a family 11 catalytic domain. xyA (without peptide sequence) was successfully expressed in E. coli BL21-CodonPlus (DE3)-RIPL cells using the pColdII vector and purified using the HisTrap FF column. Purified recombinant xylanase degraded xylan substrates, had the highest hydrolytic activity at 55°C in 20 mM sodium phosphate buffer (pH 6.0), and MgCl2, CoCl2, and MnCl2 enhanced the enzymatic activity. Nucleotide sequence of xyA was submitted to the DDBJ/GenBank/EMBL under accession number LC779040. This is the first data on the gene cloning, expression, purification, and characterization of the glycoside hydrolase family 11 from B. velezensis.

a b s t r a c t Bacillus velezensis RB.IBE29 is a chitinolytic bacterium originally isolated from the rhizospheric soil of black pepper grown in Vietnam.This bacterium is a strong biocontrol agent against plant pathogens and possesses a novel chitinase system.Genome sequences available in CAZy database revealed B. velezensis possesses one gene encoding xylanase belonging to glycoside hydrolase family 11; however, this enzyme has yet to be un-experimentally characterized.In this work, xyA gene was isolated from the genomic DNA of strain RB.IBE29 and cloned in Escherichia coli DH5 α cells using the pUC19 vector.Sequencing analysis showed that the ORF of xyA contains 642 bp and encodes the deduced xylanase with 213 aa and 23.27 kDa.The domain structure of the enzyme has a signal peptide and a family 11 catalytic domain.xyA (without peptide sequence) was successfully expressed in E. coli BL21-CodonPlus (DE3)-RIPL cells using the pColdII vector and purified using the HisTrap FF column.Purified recombinant xylanase degraded xylan substrates, had the highest hydrolytic activity at 55 °C in 20 mM sodium phosphate buffer (pH 6.0), and MgCl 2 , CoCl 2 , and MnCl 2 enhanced the enzymatic activity.Nucleotide sequence of xyA was submitted to the DDBJ/GenBank/EMBL under accession number LC779040.This is the first data on the gene cloning, expression, purifi-

Background
B. velezensis RB.IBE29 is a chitinolytic bacterium isolated from the rhizospheric soil of black pepper in Vietnam.According to experimental findings, strain RB.IBE29 had a high chitindegrading activity and reduced the growth of Phytophthora , a fungal pathogen of black pepper [1] .According to field tests, strain RB.IBE29 was a suitable choice for producing black pepper sustainably [2] .Additionally, strain RB.IBE29 possessed a novel chitinase system with two family 18 chitinases and an AA10 protein.These enzymes have been expressed in Escherichia coli cells.Purified recombinant chitinases strongly affected the germination of fungal spores and the hatching of nematode eggs [3][4] .Genome sequences available in the CAZy database showed that B. velezensis possesses one gene encoding xylanase belonging to glycoside hydrolase family 11; however, this enzyme has been un-experimentally characterized.This work aimed to establish data on the gene cloning, expression, purification, and characterization of the glycoside hydrolase family 11 from B. velezensis RB.IBE29.

Data Description
As shown in Fig. 1 , the xyA encoding xylanase was identified and successfully isolated from the genomic DNA of the strain RB.IBE29 by PCR.The purified PCR product was inserted into the pUC19 vector previously digested with SmaI, following which the nucleotide sequence of the gene was sequenced and analyzed.The ORF of xyA contains 642 base pairs (bp) and encodes a deduced protein of 213 amino acids (aa).The primary structure of the deduced protein was analyzed by using the SMART program.The result showed that the deduced protein contains a signal peptide sequence (28 aa, residues 1-28) at the N-terminus and a catalytic domain of family 11 xylanase (183 aa, residues 29-211) at the C-terminus ( Fig. 2 ).The molecular size of the predicted enzyme is 23.27 kDa with signal peptide and 22.23 kDa without the signal peptide, respectively.The catalytic domain of deduced xylanase showed 100 % identity to that of characterized endo-1,4-beta-xylanase XynA (WP_003151206) of B. subtilis 168 [5] , 100 % to uncharacterized glycosyl hydrolases family 11 protein (GAD94166) of Paecilomyces variotii No. 5 [6] , and 96.15 % to xylanase (AFV73208) of Bacillus subtilis ABGx [7] .An alignment analysis showed that amino acids in the catalytic domain of the B. velezensis RB.IBE29 xylanase have a high sequence similarity to those of xylanases from other bacteria ( Fig. 3 ).In addition, two glutamic acids, which were reported to be catalytic residues of family 11 xylanase [8] , were found in the xylanase of strain RB.IBE29 (corresponding to Glu-106 and Glu-200) ( Fig. 2 and 3 ).Nucleotide and protein sequences of the xylanase in this work were submitted to the DDBJ/GenBank/EMBL under accessions LC779040 and BES79751 and can be downloaded at https://www.ncbi.nlm.nih.gov/nuccore/LC779040 and https://www.ncbi.nlm.nih.gov/protein/2581250181, respectively.Fig. 5A shows that the purified recombinant xylanase showed the highest substrate specificity toward xylan with 100 % relative activity (112.51 μmol reducing sugars), and very low activity toward CMC, chitin, and starch, respectively.As shown in Fig. 5B , temperatures ranging from 45 to 60 °C were suitable for xylanase activity; among them, 55 °C was the optimum temperature with 100 % relative activity (119.75 μmol reducing sugars).pH values ranging from 5.0 to 7.0 were suitable conditions for the hydrolysis of xylan by the purified xylanase, and pH 6.0 was shown to be the optimal pH of xylanase activity with 100 % relative activity (125.46 μmol reducing sugars) ( Fig. 5C ).Among the tested metal ions in this work, MgCl 2 , CoCl 2 , and MnCl 2 enhanced enzymatic activity of the purified xylanase by 109.12,103.56, and 119.75 % relative activity (135.6,128.69, and 148.81 μmol reducing sugars), respectively ( Fig. 5D ).

Gene cloning and sequencing analysis
Gene cloning and analysis were conducted as described by Tran et al. [3][4] .Briefly, the open reading frame of the xyA , which encodes the family 11 xylanase, was iso-lated from the genomic DNA of B. velezensis RB.IBE29 by PCR using forward primer 5'-ATGTTTAAGTTTAAAAAGAAATTCTTAG-3', reverse primer 5'-TTACCACACTGTTACATTAGAACTTC-3', and Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, USA) per the supplier's instructions.Electrophoresis was used to separate the amplified products on a 1.2 % agarose gel (w/v).Using the Wizard SV Gel and PCR Clean-Up System (Promega Co., USA), the target band was purified.The vector pUC19 was incubated with the FastDigest SmaI (Thermo Fisher Scientific, USA), and the purified fragment was inserted into the SmaI site of the treated vector using the Mighty Mix (Takara, Japan) to create the recombinant vector pUC19-xyA.The recombinant vector pUC19-xyA was introduced into E. coli DH5 α by heat shock.Transformants were cultured at 37 °C on LB agar plates containing X-Gal (0.04 mg/mL), ampicillin (100 μg/mL), and IPTG (Isopropyl β-D-thiogalactopyranoside, 0.1 mM).The recombinant vector pUC19-xyA from the positive colonies was examined by colony PCR, isolated, and purified using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, USA).Inserts from purified recombinant vectors were sequenced at the 1 st BASE Company (Selangor, Malaysia).The signal peptide sequence of the deduced enzyme was predicted using SignalP 5.0 [9] .The domain structure was analyzed using the SMART database [10] .Molecular weight was calculated using the Compute pI/Mw tool [11] .The BLASTp program [12] was used to analyze the homology of the predicted enzyme and reported bacterial xylanases.

Expression and purification of recombinant xylanase
Expression and purification of recombinant xylanase were performed by Tran et al. [3][4] .Briefly, the ORF (without signal peptide sequence, starting from Ala-29) of xyA was amplified by PCR using primers (forward primer 5'-GGTGGTGGATCCGCTGGCACAGATTACTG-3' and reverse primer 5'-GGTGGTAAGCTTCCACACTGTTACATTAGAACTTCCGCTG-3', underlines indicate cleavage sites of BamHI and HindIII, respectively), the pUC19-xyA , and the Phusion High-Fidelity DNA polymerase.After analyzing the amplified product by electrophoresis on a 1.2 % agarose gel (w/v), the target band was recovered and purified using the Wizard SV Gel and PCR Clean-Up System.After incubating the purified fragment with the FastDigest BamHI-HindIII (Thermo Fisher Scientific, USA), the Mighty Mix was used to ligate the treated insert into the BamHI-HindIII site of the vector pColdII, creating the recombinant plasmid pCold-xyA.The plasmid pCold-xyA was introduced into E. coli BL21-CodonPlus (DE3)-RIPL cells by heat shock.Transformants were grown at 33 °C on LB agar plates containing ampicillin (100 μg/mL).Recombinant vectors from the positive colonies were examined by colony PCR and then verified by sequencing.Cells from a positive colony were grown in 1.0 L of LB containing ampicillin at 33 °C with shaking until an OD600 nm of 0.6 was reached.The cells were induced by addition of IPTG to a final concentration 0.5 mM, and the culture was further incubated for an additional 33 h at 15 °C.Cultured cells were collected by centrifugation, resuspended in 125 mL of a buffer (20 mM sodium phosphate buffer (pH 7.4), 40 mM imidazole, and 0.5 M NaCl), and then sonicated using a Q700 sonicator (Qsonica, USA).The remaining cell debris was discarded by centrifugation, and a 1 mL HisTrap FF column (Cytiva, Sweden) was used to purify the recombinant xylanase present in the supernatant.Finally, SDS-PAGE was used to examine the eluted proteins.

Substrate Specificity of Purified Recombinant Protein
Xylanase activity was examined using various substrates, including xylan from beech wood, carboxymethyl cellulose (CMC), rice straw, sugarcane bagasse, α-chitin, β-chitin, and starch.A reaction mixture (900 μL) contained one of the substrates (1 % each, w/v) and 5 μg purified xylanase in 20 mM phosphate buffer (pH 6.0).In control, inactivated xylanase by incubating purified enzyme at 100 °C for 10 min was applied.The reaction mixture was incubated at 50 °C for 30 min, and then stopped with 500 μL of 3,5-dinitrosalicylic acid reagent.The reaction mixture was boiled at 100 °C for 5 min and reducing sugars generated from the reaction were measured at 540 nm with D-xylose used as the standard [13][14][15] .

Effect of temperature on hydrolytic activity of purified recombinant xylanase
To examine the optimal temperature, the reaction mixture containing 1 % xylan from beech wood (w/v), 5 μg purified recombinant xylanase in 20 mM phosphate buffer (pH 6.0) was incubated at temperatures ranging from 30 to 80 °C for 30 min [13][14][15] .

Limitations
Not applicable.

Ethics Statement
The current work does not involve human subjects, animal experiments, or any data collected from social media platforms.

Fig. 2 .
Fig. 2. Nucleotide sequence of xyA gene and primary structure of xylanase of Bacillus velezensis RB.IBE29.Note: An asterisk indicates the stop codon, and closed circles indicate two catalytic residues.

Fig. 5 .
Fig. 5. Bio-properties of purified recombinant xylanase of Bacillus velezensis RB.IBE29.Note: All results were the mean of triplicates and standard deviations.

1. Value of the Data
cation, and characterization of the glycoside hydrolase family 11 from B. velezensis .© 2023 The Author(s).Published by Elsevier Inc.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) -Expression of xyA in E. coli BL21-CodonPlus (DE3)-RIPL cells using the pColdII expression vector -Purification of recombinant xylanase using the HisTrap FF column -Characterization of purified recombinant xylanase Data source location • Institution: Institute of Biotechnology and Environment, Tay Nguyen University • City/Province/Region: Buon Ma Thuot/Dak Lak/The Central Highlands • Country: Vietnam Data accessibility 1.Nucleotide sequence Repository name: DDBJ/GenBank/EMBL Data identification number: Accession number LC779040 Direct URL to data: https://www.ncbi.nlm.nih.gov/nuccore/