Improving Thermostability And Catalytic Efficiency Of β-mannanase By Modifying Its Loop Structure

β-Mannanase(endo-β-1,4-D-mannanase, EC 3.2.1.78), which cleaves the internal β-1,4-D-mannosidic linkages of mannan backbone, is the key component in mannan-degrading enzyme system. It exists in various organisms especially in microorganisms, and can be broadly applied in industrial areas, such as food, feed, paper, textile, biofuel and so on. Although a number of different kinds of β-mannanases have been reported, only a few of them have excellent properties and industrial potential. A β-mannanase(AuMan5A) from Aspergillus usamii which belongs to glycoside hydrolase(GH) family 5 was used as the object in this study. We modified a loop structure of AuMan5 A by substitution in order to improve the enzyme’s thermostability and catalytic efficiency, and tried to explore the relationship between the structure and function of β-mannanase.A ligand of mannopentaose was docked in the substrate binding groove of AuMan5 A that was predicted by homology modeling. Based on structural bioinformatics and evolutionary analysis of this docked complex, an unconserved loop structure(Loop FG) was suggested to be involved in the interaction between enzyme and substrate, and thus Loop FG was chosen as the modifying place. According to the enzymatic properties and structural features of other fungal GH family 5 β-mannanases, three mutants, AuMan5A-Af, AuMan5A-An, and AuMan5A-Th, were designed by replacing Loop FG of AuMan5A(316KSPDGGN322) with the corresponding ones of other family 5 β-mannanases from Aspergillus fumigatus, Aspergillus nidulans, and Trichoderma harzianum, respectively. Three mutant-encoding genes were constructed by megaprimer PCR as designed theoretically and expressed in Pichia pastoris GS115 by using plasmid pPICZαA. The recombinant enzymes, re-AuMan5 A, re-AuMan5A-Af, re-AuMan5A-An and re-AuMan5A-Th, displayed the temperature optima of 65°C, 75°C, 65°C and 70°C, thermal inactivation half-lives of 10 min, 480 min, 5 min and 25 min at 70°C, melt temperatures of 64.5°C, 76.6°C, 63.2°C and 69.1°C, respectively; the kcat/Km values of re-AuMan5A-Af, re-AuMan5A-An and re-AuMan5A-Th were 12.7-, 6.0- and 11.0-fold that of re-AuMan5 A, respectively. re-AuMan5A-Af had the most excellent temperature characteristics and catalytic efficiency.To explore and explain the reason for AuMan5A-Af’s superior enzymatic properties, single-site mutants, AuMan5A-AfD320 G, AuMan5A-AfH321 G and AuMan5AG320 D, were designed and constructed by site-directed mutagenesis. re-AuMan5A-AfD320 G, re-AuMan5A-AfH321 G and re-AuMan5AG320 D displayed the temperature optima of 70°C, 75°C and 70°C, half-lives of 40 min, 300 min and 25 min at 70°C, and their kcat/Km values were 3.9-, 14.1- and 9.5-fold higher than that of the wild-type, respectively. Studying proteins’ intramolecular interactions and experimental results above, hydrogen bonds formed between G320/H321 and the surrounding residues were considered to be responsible for the high thermostability of AuMan5A-Af; the mutation G320 to D320(or D321) was the critical reason for the significant increase of all loop-substitution mutants’ catalytic efficiencies.By using the plasmid pKLAC1, Auman5 A and Auman5A-Af were successfully expressed in Kluyveromyces lactis GG799. The expressed recombinant enzymes, re-Kl/AuMan5 A and re-Kl/AuMan5A-Af, possessed an apparent molecular weight of 55.0 kDa; the expression supernatants had β-mannanase activities of 12.9 U?mL-1 and 74.0 U?m L-1, respectively; enzymatic properties of re-Kl/AuMan5 A and re-Kl/AuMan5A-Af were similar to those of proteins expressed in P. pastoris, re-AuMan5 A and re-AuMan5A-Af, respectively.