| Mannanase is one of the most important hemicellulases and is widely used in animal feed, food, oil exploration and detergent. To obtain more mannanases with favorable properties, two strategies were employed in this study. One is to explore the genetic resources of mannanase in fungi, the other is to study thermostability mechanism with protein engineering techniques to meet the demand of industries.In this study, five endo-b-1,4-mannanase genes of glycoside hydrolase(GH) family 5(man5A, man5 B, man5 D, man5 DB and man5DW) and one GH26 mannanase gene(man26DW) were cloned from Talaromyces leycettanus JCM12802, Gloeophyllum trabeum CBS900.73, Staphylotrichum coccosporum NBRC 31817 and Alternaria sp., and successfully expressed in Pichia pastoris GS115, respectively. The optimal temperature of Man5 A from T. leycettanus JCM12802 was 90 °C, higher than that of all known fungal counterparts. It is highly stable at 70 °C. It had optimal p H at 4.5, was highly stable over the broad p H range of 3.0-10.0, and had capacity to degrade mannan with different side chains. Man5 B from G. trabeum CBS900.73 was acidophilic, exhibiting high activity(>80 %) at a broad p H range(p H 2.0-6.0) with an optimum of 2.5 and excellent stability over a broad p H range(p H 2.0-10.0). The enzyme showed strong resistance to pepsin and trypsin digestion and had higher dry matter digestibility than commercial counterpart. The superior properties as well as its great substrate degrading ability and transglycosylation capacity make Man5 B prospective for application in the feed and food industries. The other four mannanases had a similar p H optimum of 5.0-6.0, but varied in temperature optimum. The optimal temperature of Man5 D and Man5 DB from S. coccosporum NBRC 31817 and Man5 DW from Alternaria sp. was 65-70 °C, and that of Man26 DW from Alternaria sp. was 50 °C. These four mannanases showed resistance against most metal ions and chemical reagents and had the capacity to degrade mannan with different side chains and catalyze the transglycosylation reaction.To undermine the thermostability mechanism of mannanase, multiple sequence alignment of thermophilic Man5 A from T. leycettanus JCM12802 with mannanases of different temperature optima and structural simulation were conducted, and one CBM and two unique amino acid residues(Asp191 and Arg286) were identified to be probably related to thermostability. Truncation mutant without the CBM, Man5A-CBD, and double substitution mutant D191G/R286 H were constructed and expressed in P. pastoris. Both mutant enzymes showed reduced thermal stability, confirming their roles in mannanase thermostability. In addition, the effect of glycosylation on mannanase thermostability was also studied in the mannanase Bman from Bispora sp. MEY-1. Random combinations of these mutants(15) were constructed based on the seven putative glycosylation sites, expressed in P. pastoris, and all the mutant enzymes showed improved thermostability. A tentative conclusion was drawn that it’s the location instead of the number of glycosylation sites that play the key role in thermostability. Further experiment is required to verify it.In summary, six fungal mannanase genes were cloned and heterologously expressed in P. pastoris at high levels, and factors influencing thermostability was identified by site-directed/truncation mutagenesis and homologous simulation. This study not only enriches the genetic resources of mannanases and provides six excellent candidate enzymes for industrial use, but also identifies two key factors related to thermostability and provides the theoretical basis for the improvement of mannanases thermostability. |
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