Directed Modification Of β-mannanase Substrate Affinity Based On The Rational Design

β-Mannanases (endo-β-1,4-D-mannanases, EC3.2.1.78), which exist widely in variousorganisms especially in microorganisms, can randomly hydrolyze the internalβ-1,4-D-mannosidic linkages of mannan backbones to form mannooligosaccharides. Recently,they have attracted much attention due to their great potential applications in industrialprocesses, such as food, pharmaceutics, feed, paper and pulp, textile, biofuel and so on.The three-dimensional (3-D) structure of a docked complex of Aspergillus usamiiglycoside hydrolase5β-mannanase (AuMan5A) with mannobiose was predicted throughhomology modeling and molecular docking simulation. On the basis of this3-D structure,38amino acid sites in proximity to mannobiose within8were located by using the PyMolsoftware,21conserved sites were excluded by multiple alignment. According to theproperties of amino acids at17non-conserved sites and their locations on the3-D structure ofAuMan5A,5aromatic amino acids (Trp54, Tyr111, Tyr115, Phe206and Tyr243) were selected tobe substituted with the similar amino acids and/or high frequency ones in other β-mannanasesequences, respectively, forming a series of mutant enzymes. Binding free energies (ΔGbind)of various β-mannanases with mannobiose were calculated by using the molecular mechanicsPoisson-Boltzmann surface area (MM-PBSA) method. The ΔGbindof AuMan5AY111FandAuMan5AY115Fwere-56.8and-75.0kcal/mol, lower than those of AuMan5A and othercandidate mutant enzymes. AuMan5AY243Awith increased ΔGbindof-20.8kcal/mol wasselected as a negative control.Based on the rational design, the mutant genes, Y111F, Y115F and Y243A wereconstructed by megaprimer PCR, and the recombinant expression vectors pPIC9K-Y111F,pPIC9K-Y115F and pPIC9K-Y243A were constructed, respectively. Then pPIC9K-Auman5Aand these vectors were linearized with SacⅠ, then transformed into P. pastoris GS115byelectroporation, respectively. Recombinant β-mannanases WT (wild-type), Y111F and Y115F(mutants) showed the highest activity of44.8,53.0and50.4U/mL towards locust bean gum,respectively, after expressed by menthanol induction. However, Y243A exhibited littleβ-mannanase activity, which indicated that Tyr243played an important role in the maintainingof AuMan5A activity.The purification of recombinant β-mannanase was performed by a combination of(NH4)2SO4precipitation, DEAE Sepharose Fast Flow ion-exchange chromatography,ultrafiltration and Sephadex G-75gel filtration. WT, Y111F and Y115F were purified by1.9, 2.2,1.8folds with recoveries of48.4％,52.6％,56.8％, respectively. The specific activitiesof purified WT, Y111F and Y115F were209.5,293.0,284.1U/mg, respectively. SDS-PAGEanalysis of each purified recombinant β-mannanase displayed one single protein band with anapparent molecular weight of49.5kDa. The Kmvalues of WT, Y111F and Y115F towardsguar gum were (4.49±0.07),(2.95±0.22),(2.39±0.33) mg/mL, respectively. Compared withWT, the Kmvalues of Y111F and Y115F decreased34.3%and46.8%, the catalytic efficienciesof Y111F and Y115F increased0.5and0.7fold. However, other characteristics kept almostunchanged. Structural analysis showed that Y111F and Y115F increased their affinities bydecreasing the steric conflicts with those more complicated substrates.The double-mutant β-mannanase gene Y111F/Y115F was constructed by megaprimerPCR, and was expressed in Pichia pastoris GS115successfully. The activity of recombinantβ-mannanase Y111F/Y115F was45.7U/mL. After the same steps of purification, the enzymewas purified by2.4fold with a recovery of58.5%, the specific activity of the purifiedY111F/Y115F was327.5U/mg. The Kmvalue of Y111F/Y115F towards guar gum was(2.89±0.24) mg/mL. Compared with WT, this value decreased35.6%, and the catalyticefficiency increased1.5fold, however, other characteristics kept almost unchanged.The hydrolytic conditions of guar gum were investigated with the recombinantβ-mannanases constructed in this study. Under the condition of guar gum water solution20g/L, recombinant β-mannanase Y111F/Y115F dosage60U/g guar gum, hydrolytictemperature50°C and hydrolytic time6h, the hydrolytic rate can reach18.7%. As is known,this is the frst report about the directed modifcation of the A. usamii β-mannanase to improveits substrate affnity by in silico design and site-directed mutagenesis.