RESUMO
The rice xylanase inhibitor gene, rixi, was cloned from rice genome. The open reading frame of rixi was 915â¯bp and encoded 304 amino acids with the theoretical molecular mass of 33.9â¯kDa. The rixi was inserted into the new-type expression vector pCold TF, and was high-level expressed in Escherichia coli BL21 (DE3). SDS-PAGE and Western blot analysis revealed that the molecular weight of the recombinant rice xylanase inhibitor, namely reERIXI, was approximately 89.8â¯kDa. The reERIXI exhibited significant inhibitory activities against several family GH11 xylanases. After interaction with reERIXI, the residual activity of reBaxA50 and TfxA_CD214 were 59.24% and 44.41%, respectively. The optimal temperature of reERIXI inhibitory activity to reBaxA50 and TfxA_CD214 were 60⯰C and 50⯰C, respectively. The thermostability assay revealed that reERIXI was stable below 60⯰C. reERIXI showed high inhibitory when interacting with reBaxA50 and TfxA_CD214 for 30-60â¯min. The intrinsic fluorescence spectroscopy of reBaxA50 and TfxA_CD214 was quenched with increasing reERIXI concentration. Circular dichroism measurement revealed that ratio of helix of reBaxA50 and ratio of beta of TfxA_CD214 significantly decreased when interacting with reERIXI. The total concentration of hydrolytic products from beechwood xylan decreased when reERIXI was added.
Assuntos
Endo-1,4-beta-Xilanases/antagonistas & inibidores , Endo-1,4-beta-Xilanases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oryza/enzimologia , Oryza/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Relação Dose-Resposta a Droga , Endo-1,4-beta-Xilanases/genética , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hidrólise , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Análise Espectral , Fatores de TempoRESUMO
In this study, BaxA (GenBank: KM624029), which encodes the Bacillus amyloliquefaciens xylanase A (BaxA), was highly expressed in Pichia pastoris GS115 under the control of the AOX1 promoter. The recombinant xylanase, namely rePBaxA, was purified to homogeneity by using Ni-affinity resin and its molecular weight was 35.0kDa. The optimum temperature and pH of rePBaxA were 50°C and 5.0, respectively. The kinetic parameters Michaelis-Menten constant (Km) and maximum reaction rate (Vmax) of rePBaxA were 5.41mg/mL and 22.42µmol/min/mL, respectively. High-performance liquid chromatography results showed that after 6h of hydrolysis, rePBaxA released xylose-xylohexaose (X1-X6) mixture from beechwood and birchwood xylan, with xylobiose (X2) and xylotriose (X3) as the major products, respectively. The hydrolyates from oat spelt, wheat bran insoluble xylan and pretreated wheat bran by rePBaxA included X2-X6, with X6 having the highest concentration. The mode of action analysis revealed that rePBaxA was an endo-acting xylanase with transglycosylation activity. X2 might be the minimum oligomer hydrolyzed by rePBaxA. The pretreated wheat bran and wheat bran insoluble xylan could be directly hydrolyzed by rePBaxA. This study provided a basis for using agricultural waste by-products as substrates for manufacting value-added probiotics, namely, xylooligosaccharides.
Assuntos
Bacillus amyloliquefaciens/enzimologia , Fibras na Dieta , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Oligossacarídeos/metabolismo , Pichia/genética , Xilanos/química , Bacillus amyloliquefaciens/genética , Endo-1,4-beta-Xilanases/isolamento & purificação , Estabilidade Enzimática , Expressão Gênica , Hidrólise , Oligossacarídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , TemperaturaRESUMO
This study aimed to obtain xylanase exhibiting improved enzyme properties to satisfy the requirements for industrial applications. The baxA gene encoding Bacillus amyloliquefaciens xylanase A was mutated by error-prone touchdown PCR. The mutant, pCbaxA50, was screened from the mutant library by using the 96-well plate high-throughput screening method. Sequence alignment revealed the identical mutation point S138T in xylanase (reBaxA50) produced by the pCbaxA50. The specific activity of the purified reBaxA50 was 9.38U/mg, which was 3.5 times higher than that of its parent expressed in Escherichia coli BL21 (DE3), named reBaxA. The optimum temperature of reBaxA and reBaxA50 were 55°C and 50°C, respectively. The optimum pH of reBaxA and reBaxA50 were pH 6 and pH 5, respectively. Moreover, reBaxA50 was more stable than reBaxA under thermal and extreme pH treatment. The half-life at 60°C and apparent melting temperature of reBaxA50 were 9.74min and 89.15°C, respectively. High-performance liquid chromatography showed that reBaxA50 released xylooligosaccharides from oat spelt, birchwood, and beechwood xylans, with xylotriose as the major product; beechwood xylan was also the most thoroughly hydrolyzed. This study demonstrated that the S138T mutation possibly improved the catalytic activity and thermostability of reBaxA50.
