Gene Cloning And Expression, Thermal Stability And Catalytic Mechanism Of The Mannanase From The Thermophiles

With the development of biotechnology, mannanase, one of the main hemicellulose-degradingenzymes, is widely used in animal feed, paper, detergent, food, oil exploration and other fields.Thermophilic mannanase has a high reaction temperature （60℃） and good thermal stability, and isattracting much more attention in industrial and scientific researches. The objective of this study is todiscover more thermophilic mannanses with application potentials and undermine their thermophilicmechanisms.In this study, five thermophilic fungal strains XZ3, XZ7, XZ8, YH3and P1were isolated from thedesert and salt lake samples and showed ability to degrade mannan. These strains were identified to beAspergillus nidulans, Thielavia, Achaetomium sp, Aspergillus flavus and Neosartorya sp, respectively,based on morphological characteristics and phylogenetic analysis of the ITS rDNA sequences. All thesestrains showed optimal growth at or above45°C that is typical of thermophilic fungi and thus representexcellent microbial sources of thermophilic enyzmes.A total of five mannanase genes of glycoside hydrolase （GH） family5, man5XZ3, man5XZ7,man5XZ8, man5YH3and man5P1, were cloned from strain XZ3, XZ7, XZ8, YH3and P1, respectively.The gene products of man5XZ7, man5YH3and man5P1were heterologously expressed in Pichiapastoris GS115, purified and characterized. The optimal pH of Man5XZ7for enzyme activity was5.0,and it remained>20%activity at pH9.0. Man5XZ7was stable at pH3.0–12.0and50℃. It is a typicalthermophilic enzyme that showed optimal growth at75°C and retained>50%activity after incubation at70℃for10min. The Km and Vmax values of Man5XZ7were6.72mg/mL and909.09mol/min mg,respectively. The optimal temperature and pH of Man5P1for activity were80℃and pH4.0, and itremained>50%activity at pH3.0. Man5P1was stable, retaining85%activity at pH3.0–12.0whenincubated at60℃for1h. The Km and Vmax values of Man5P1were0.83mg/mL and192.31mol/min mg, respectively. The pH optimum of Man5YH3was5.0, and the temperatureoptimum was60℃. Man5YH3was highly resistant to most metal ions and chemical reagents,especially to SDS. It maintained60%of the enzyme activity in the presence of high concentrations ofSDS, and thus had application potentials in the detergent industry.In recent years, factors contributing to the thermostability of proteins have been concerned. In thisstudy, Man5XZ3and Man5XZ8were selected and their mechanisms of thermal stability wereundermined. Structure analysis of Man5XZ3indicated that it was mainly composed of three modularcomponents, an N-terminal carbohydrate-binding module of family1（CBM1）, a serine/threonine-richlinker region and a catalytic domain of GH5. In order to study the effect of each component on enzymethermostability, mutants Man5XZ3△CBM with removal of CBM1and Man5XZ3△LR with removalof both CBM1and the linker region were constructed and expressed in P. pastoris. These threeenzymes had similar pH and temperature optima, but varied in thermal stability after incubation at60℃for2h. Man5XZ3retained48%of its activity, Man5XZ3△CBM maintained57%of the activity, and Man5XZ3△LR only maintained30%of the enzyme activity. The results suggested that the deletion ofthe linker region greatly weakened the thermal stability of Man5XZ3. The Km and Vmax values ofMan5XZ3, Man5XZ3△CBM, and Man5XZ3△LR were0.92mg/mL and270.27mol/min mg,0.79mg/mL and227.27mol/min mg, and1.08mg/mL and277.79mol/min·mg, respectively.The amino acid sequence of Man5XZ8shared⁹⁰.1%identity with that of the thermophilic Man5Afrom Humicola sp. Y1, but these two enzymes had distinct enzymatic properties. Based onbioinformatics analysis, we constructed seven mutants with replacement of V124I, T224S, A251S,E263D, S264T, L298A and deletion of SG. The wide-type and mutant enzymes had no difference in pHand temperature optima, but mutants V124I, A251S and SG showed enhanced thermostability byretaining76%,32%, and74%of the activity, respectively.Moreover, a new mannanase gene of GH1¹³, man1¹³, was cloned from the thermophilicbacterium Alicyclobacillus sp. A4. The deduced amino acid sequence showed55%identity with knownsequences. The optimal pH and temperature for Man1¹³ activity was6.0and55℃. It remained>60%activity between pH4.5and pH10.0when incubated at40°C for1h and retained20%activity at60℃for10min. Homology modeling, molecular simulation and molecular docking analysis of thesubstrate-recognition mode of Man1¹³ indicated that seven amino acid residues, including Tyr¹³,Asn⁹⁰, Tyr⁹⁶, Arg⁹⁷, Cys¹⁴³, Tyr1⁹⁶, and Trp²⁷⁴, were necessary for substrate binding ofmannanases.In summary, five GH5mannanase genes and one GH1¹³ mannanase gene were identified inthermophilic microorganisms in this study. These genes were heterologously expressed andcharacterized. Of them, four mannanases had excellent enzyme properties. Moreover, factors related tothermostability were identified through analysis of the modular components and active sites ofmannanases, and residues involving substrate binding were also identified in GH1¹³ mannanses bymolecular docking method. This study not only enriches the microbial source of mannanase, but alsoprovides great information for scientific researches and industrial applications.