Molecular Characterization And Functional Analysis Of Novel Amylase From Fungi

Starch hydrolyzing enzyme(i.e. amylase) is crucial in the starch industry. According to the application pattern, amylase is divided into liquefying amylase and saccharifying amylase.Glucoamylase(EC 3.2.1.3) and maltotriose-forming α-amylase(EC 3.2.1.116) are important saccharifying amylases. Glucoamylase is becoming attractive because the glucose released can be further processed to ethanol, amino acids, organic acids, etc. The maltotriose released by maltotriose-forming α-amylase is widely used in baking, sports drinks and medicine industry. This thesis aims to obtain novel acidic maltotriose-forming α-amylase and thermophilic glucoamylase and provide excellent candidate amylases for the starch industry.A maltotriose-forming α-amylase gene(Teamy13) of glycoside hydrolase(GH) family 13, 1488 bp in length, was cloned from Talaromyces emersonii JCM23024 and successfully expressed in Pichia pastoris GS115 with the yield of 5.6 U/m L. In comparison with the commercial fungal amylase(optimal temperature and p H at 55°C and p H 5.0- 6.0, respectively), recombinant Te Amy13 showed some distinct characteristics. It exhibited the maximum activity at 60°C and remaining 90% of the activity at 70°C. The optimum p H(4.5) of Te Amy13 made it compatible with thermophilic amylase,thus simplyfing starch processing. Te Amy13 had capacity to hydrolyze maltooligosaccharide and soluble starch. TLC analysis indicated the main product by Te Amy13 hydrolysis was maltotriose. Thus Te Amy13 is a novel maltotriose-forming α-amylase and has the potential to be used for mass-production of maltotriose for foods, sports drinks and medical tests.A glucoamylase gene(gla15) of GH15 was cloned from Bispora sp. MEY-1 and heterologously expressed in P. pastoris GS115. The c DNA of gla15 consists of 1827 bp and codes for a catalytic domain, a linker and a starch-binding domain of family 20. Purified recombinant GLA15 is a typical thermostable acidic enzyme. When using maltose as the substrate, GLA15 had p H and temperature optima at 4.0 and 70°C, respectively. The enzyme was stable at 2.0-11.0 and 70°C. GLA15 had ability to hydrolyze both gelatinized starch and raw starch. It cleaved the terminal α-1,4-linkage successively from the non-reducing ends and released β-D-glucose, and had the ability to break the α-1,6-glycosidic linkage of isomaltose. Compared with commercial glucoamylase, GLA15 had higher saccharification effeciency(9%) and glucose conversion rate under similar conditions.In summary, a maotriose-forming α-amylase gene of GH13 and a glucoamylase gene of GH15 were cloned from T. emersonii and Bispora sp., and achieved expression in P. pastoris GS115. The purified recombinant enzymes have distinct properties, and have application potentials in the production of gucose and maltotriose, respectively. This study not only enriches the genetic resources of amylase,but also prodive excellent enzymes ready for mass-production of glucose and maltotriose.