Improving hydrophobicity of N-terminal increased molecular stability of mannanase Man1312
Abstract
Protein terminals have important roles in molecular structural stability and in some occasions they have regulatory roles in catalytic reaction. To expand our understanding on the influences of distal residues mutation, we explored the molecular stability and kinetics of mannanase Man1312 mutants. For Man1312, the N-terminal loop was more disordered and changeable; therefore the mutations on N-terminal should have significant effects on enzymic properties. The experiment was investigated by spectrophotometer, circular dichroism and differential scanning calorimetry assays. As a result, positive mutations were found from sites of T2 and Q9 and double-site mutant ManY9G2 showed increased catalytic activity by 7.7% higher than Man1312. Meanwhile, ManY9G2 had significantly increased thermostability with promoted Topt by 6 oC and elongated t1⁄2 by 7 min, which was resulted from the optimized intermolecular forces and a newly-built hydrogen bond in ManY9G2. In our experiment, the specific residues were mutated from hydrophilic to hydrophobic and the improved hydrophobicity on N-terminal had a positive impact on the properties of mannanase Man1312.
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Introduction
Hemicelluloses are a series of polysaccharides consisting of linear chain and branches in the plants cell walls which are associated to the cellulose and lignin forming lignocelluloses biomass (1). Mannan is the most abundant of polysaccharide present in softwood hemicelluloses and targeted by mannanase to degrade into oligosaccharides. β-Mannanases catalyze random hydrolysis of beta-1,4-mannosidic bonds by releasing a single β-D-mannose unit from the nonreducing end of manno-oligo saccharides (2).
In the past few decades, various mannanases were from isolated from plants (3), marine mollusk (4), and a body of bacteria and fungi (5, 6), but in general, production of β-mannanase by microorganisms is more promising because of its low cost, high production rate and readily controlled conditions (7).
Mannanases play important roles in fundamental biological processes and also have potential applications in various industries. They are widely used by industries including food processing, feed, oil mining, paper making, pharmaceutical, and second generation biofuel (8, 9) as well as manufacture of oligosaccharide (7).Meanwhile, mannanases could be used in prebiotic preparation which is expected to improve the growth performance of animal.
Mannanase Man1312 screened from Bacillus subtilis (B. subtilis) B23 has the mechanism of Glycoside Hydrolase Family 26 (GH26). Although the members of GH26 family display great diversity in their sequence and function, all of them have a canonical α/β hydrolase fold, which consists of eight β-sheets flanked by α-helices. This core architecture is the structural foundation as a stable scaffold to accommodate the catalytic residues and tolerate mutations at some degree without losing its molecular stability or catalytic machinery. The loop which connects α-helices and β-strand is important to molecular structural stability and activity as well. Pleiss team studied the sequence and structure of epoxide hydrolases and proposed that NC-loop might interact with the substrate by defining the substrate-binding pocket and regulating the accessibility of active site (10, 11). The residue Tyr on the NC-loop of epoxide hydrolases is involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen (12, 13).
Conclusion
According to the hydrophobic cluster analysis, Mannanase Man1312 has been classified into GH26 family. The (α/β)8 barrel is one of GH26 hydrolase features and enzymes in this family display a broad variety of activity, including endo-β-1,4- mannanases, exo-acting β-mannanase (28), β-1,3:1,4-glucanase (29) and β-1,3-xylanase activities (30). For this reason, they are one of the most widely used groups of biocatalysts in industry.
In our previous work, the active sites were clarified and the expression system of mannanase Man1312 decoded gene was optimized. Furthermore, rational evolution strategy was performed on α/β fold of Man1312 to improve the molecular stability. The present study focused on the relationship of mutations on the N-terminal loop and molecular stability and activity basing on the structural analysis.
Our results suggest that the N-terminal loop could modulate both catalytic activity and stability of Man1312 and the mutations increased Man1312 thermostability and activity. The double mutant ManY9G2 increased catalytic activity by 7.7% higher than Man1312 and meanwhile, it increased Topt by 6oC and t1⁄2 elongated by 7 min in compare with Man1312.
From the spectra and modeling, ManY9G2 increased contents of the helix and strand and thus, led to a slow decreased △G 0 tendency in the thermal denaturation process, which indicated ManY9G2 had more mechanical stabilities and higher stiffness. Moreover, around the mutated positions 9, ManY9G2 formed stronger van der waals force due to its greater electron cloud overlap and formed a new hydrogen bond between Tyr9 and Asn7, indicating that substitution on position 9 had more important effects on thermal stability.
Similar to our work, Zhai constructed a loop-replacement mutant (L6RM) and solution nuclear magnetic resonance data show that the L6RM results in significant chemical shift changes in the loop and surrounding regions. The interactions with the L6RM loop stabilize the enediolate intermediate toward the elimination reaction catalyzed by the LDM (31). Results from Pertusa suggested the N-terminal domain of TRPM8 had a critical contribution to thermal and chemical sensitivity. Single point mutations caused a comparable increase in the responses to cold and menthol (32). The five N-terminal mutations might confer structural stability, and hence, prevent the overall thermal unfolding of the mesophilic Streptomyces olivaceovirdis xylanase (33). Dumon researched on the 15 most thermostable xylanases, the results showed that the Nterminal region was more susceptible to thermal unfolding (34).
Our results demonstrate that the specific mutations on N-terminus can have a significant impact on chemical properties of mannanase Man1312. With the better understanding of sequence-structure- function relationship of the second structure in different α/β hydrolases, the more benefit for industrial applications would be achieved through engineering this considerable group of enzymes.