Cloning, Protein Expression And Mutagenesis Of Thermophilic Xylanase

Xylan is the main constituent components of the hemicellulose of the plant cell wall, and the complete degradation of xylan requires the coordinate action of a variety of enzymes. Among them, endo-β-1,4-xylanase is the crucial enzyme to randomly cleave internal linkages on the β-1,4-xylose backbone of xylan to produce xylooligosaccharides and xylose. Xylanases have a wide range of commercial applications. In this study, xylanase gene was obtained from Geobacillus stearothermophilus, successfully cloned and expressed in Escherichia coli, and its enzymatic properties were analyzed. At the same time, the study improved the catalytic efficiency of xylanase using random mutagenesis. The obtained results were as follows:1. Endo-P-1,4-xylanase gene was cloned from Geobacillus stearothermophilus1A05583by PCR. XynA gene consisted of996bp, the overall GC content was49.5%, and encoded331amino-acid peptide with a molecular weight38.63kDa. XynA belonged to family10of the glycosyl hydrolases. Then xynA gene was cloned into the vector pGEX-6p-1and expressed in E. coli BL21(DE3). The enzymatic properties of the purified XynA were analyzed. XynA exhibited the optimal activity at pH6.5. XynA retained more than60%of the original activity after overnight incubation over a pH range from4.5to8.0. XynA was a partial neutral xylanase. The temperature optimum for XynA was65℃, and it had a good thermal stability. XynA still had80%activity when incubated at60℃for1h. Cu2+, Mg2+, Ba2+, Zn2+and Mn2+had a promote effect on the activity of the enzyme, however, Hg2+, Pb2+, SDS and beta-mercaptoethanol had a strong inhibitory effect on the activity of the enzyme. The Km, Vmax, kcat and kcat/Km values of XynA for beechwood xylan were1.655mg/mL,765.0μmol/mg-min,492.5s-1and297.6mL·mg-1s-1, respectively.2. Enzymes with improved catalytic efficiency were obtained using error-prone PCR and the96-well plate high-throughout screening system. Two variants1-B8and2-H6were screened from the mutant library containing9,000colonies.1-B8and2-H6exhibited the optimal activity at pH7.0and at pH7.5, respectively. Two mutants were stable over a broad pH range, and retained more than60%of the original activity after overnight incubation over a pH range from5.0to10.5. The temperature optima for mutant1-B8and mutant2-H6were60℃and65℃, respectively. And the thermal stability of them decreased.1-B8still had80%activity after1h incubation at50℃, but showed a sharp decrease in its activity above50℃, and lost activity after incubation at60℃for1h.2-H6retained60%activity when incubated at60℃for1h. The Km, Vmax, kcat and kcat/Km values of1-B8for beechwood xylan were2.0mg/mL,1158.0μmol/mg-min,745.5s-1, and372.8mL·mg-1s-1, respectively. Compared with the wild-type enzyme,1-B8gained a25%increase in the catalytic efficiency (kcat/Km). The Km, Vmax, kcat and kcat/Km values of2-H6for beechwood xylan were1.302mg/mL,1137.6μmol/mg-min,732.3s-1,and562.4mL·mg-1s-1, respectively. Compared with the wild-type enzyme,2-H6gained an89%increase in the catalytic efficiency (kcat/Km).3. By sequencing1-B8and2-H6, an identical mutation point histidine179was detected. Following the introduction of the remaining19amino acids into position179by site-saturation mutagenesis, found the Km, Vmax, kcat and kcat/Km values for beechwood xylan were1.084mg/mL,1734.5μmol/mg·min,1116.6s-1and1030.1mL·mg-1s-1when histidine was substituted by phenylalanine. And the catalytic efficiency was found to be2.46-fold higher than that of the wild-type. When histidine was substituted by tryptophan, arginine, methionine and proline, respectively, the enzyme lost activity. In contrast, when histidine was substituted by other amino acids, the enzyme showed slight changes in catalytic efficiency. The site179of XynA may play a crucial role in regulating substrate specificity and product dissociation. In the future, these experimental results provide useful information to probe important features of protein structure and function.