Directed Evolution Of Endo-β-1,4-mannanase From Pantoea Agglomerans

After proteases, cellulases and hemicellulases are widely applied in the industrial production. As one of hemicellulases, β-mannanase (EC3.2.1.78) hydrolyzes hemicellulose in the mannan-based chains and completely hydrolyzes mannan by the synergistic action with β-mannosidase (E.C.3.2.1.25), β-glucosidase (E.C.3.2.1.21), a-galactosidase (E.C.3.2.1.22) and acetyl mannan esterase (E.C.3.1.1.6). P-mannanase has a wide variety of sources and commonly exists in microorganisms, plant and animal species. Furthermore, mannanases are widely used as industrial enzymes in food processing, coffee extraction, bioethanol production, pharmaceutical manufacturing, and pulp and paper industry.In a previous study of our lab, Man26P, a member of GH26family, was screened from the deep-sea bacterium Pantoea agglomerans, whose gene (GenBank accession no. FJ648764) consisted of1047bp and encoded348amino acids with a molecular mass of approximately38.51kDa. Its Km value toward locust bean gum (LBG) was5.78mg/mL, and its catalytic efficiency (kcat/Km) was106.65mL/mg-s. And its optimum temperature and pH were55℃and6.0, respectively. Besides, Man26P actived well and stably at30～70℃or in buffer (pH4.0～8.0). In order to obtain improved enzymes and understand the structure-function relationship better, Man26P was engineered using a modified DNA shuffling method with200μmol/L Mn2+and site-directed mutagenesis technology.Congo red plate assay combined with a96-well plate high-throughput screening technology was used to screen the mutant library. From the mutant library containing19,700clones, two mutants G267S and H134R/F141L were obtained. Compared to the wild-type enzyme (Man26P), G267S and H134R/F141L exhibited3.78-and2.77-fold decreased Km values toward locust bean gum (LBG) and exhibited1.14-and3.30-fold increased catalytic efficiency (kcat/Km). Combined with site-directed mutagenesis, more substitutions were introduced into the134site, among which, H134R and H134K exhibited2.32-and1.56-fold decreased Km values toward locust bean gum (LBG) and exhibited2.81-and2.75-fold increased catalytic efficiency versus the wild-type enzyme. And with an increase in the pKa values of the side chains of the mutated residues at the134site, a decrease was observed in their Km values against LBG, which was speculated to facilitate the enhancement of polar contacts or positive charges at the134site. In addition, the molecular modeling analysis suggested that the nature of the residue at the267site could significantly affect the enzyme activity by regulating substrate binding affinity and product release.Due to their activity improvement and structural characteristics, H134K and G267S were chosen to study the effects of temperature and pH on enzyme activity. The result showed that the temperature optima for H134K and G267S were55℃and50℃, respectively. In the thermal inactivation study, they lost their activity quickly after10min60℃, and they retained less than50%of the original activity after30min incubation at60℃. For the pH optima, both H134K and G267S had the same optimal activity at pH6.0and similar pH stability profiles with Man26P, exhibiting significant stability over a broad pH range from pH4.0to10.0.The study provides useful information for the directed evolution of mannanases.