Expression Of A Cold-adapted A-amylase From Psychrotolerant Fungi In Aspergillus Oryzae And Its Immobilizion

The filamentous fungus Aspergillus oryzae,for which the complete genome sequence is known,is used extensively in the manufacture of fermented foods because of its proven safety record in the food industry and being generally recognized as safe(GRAS).A.oryzae has also been considered as a favourable host for enzyme production(e.g.amylases,proteases,phytases,lipases,etc.)because of its ability to secrete proteins into the extracellular medium and eukaryotic post-translational modifications.a-Amylase,also known as a-1,4-D-glucan-glucanhydrolase,is endo-acting enzyme catalyzing hydrolysis in a random manner in the interior of the starch molecule,producing linear and branched oligosaccharides of various chain length.a-Amylase has many applications in various industries including the paper,textile,pharmaceutical,detergent,and of course,the food industry,especially in relation to syrup industry,bread and baking.Therefore,the isolation of novel a-amylase that possess special properties is of great potential economic value and importance.In this study,pyrG gene was disrupted by gene recombination in RIB40 first.Then A.oryzae ligD gene involved in nonhomologous chromosomal integration was disrupted by the same way,followed by disruption of the pyrG gene for uridine/uracil auxotroph.The mutant named ?pyrG?ligD was picked out for further study.Expression plasmid pSKNHG was constructed by inserting promoter and terminator of A.oryzae amyB,signal peptide,pyrG and His-tag label.The sequence of Geomyces pannorum R1-2 a-amylase was digged out by NCBI.The cDNA is composed of 1482 nucleotides,corresponding to a protein of 492 amino acid residues.The cloned gene was defined as AmyA1.It shares the highest amino acid identity with the a-amylase from Geomyces pannorum(66%,GenBank Accession No.AHN65136.1),and shares only 54%and 44%identity with the a-amylase from Lipomyces kononenkoae(GenBank Accession No.AAO 12212.1)and the a-amylase from Aspergillus kawachii IFO 4308(GenBank Accession XP₀07584707.1),respectively.The recombinant a-amylase was purified by affinity chromatography and a single protein band of approximately 52 kDa was observed when subjected to SDS-PAGE.The specific activity of purified AmyAl was 12.8×103 U/ml.The purified AmyA1 had Km and Vmax values of 25.1 mg/ml and 0.824 mg/(ml·min)towards soluble starch,respectively.The optimum temperature and pH of recombinant a-amylase was 50? and 5.0,respectively.Catalytic efficiency of the purified AiyAl was increased by Mg2+ and Ca2+,while it was obviously inhibited by Ni2+,surfactants(Tween-80,Tween-20 and Triton X-100)and methanol.The purified AmyAl exhibited broad substrate specificity that could efficiently hydrolyze all the substrates tested except pullulan.The main hydrolysis products of soluble starch catalyzed by the purified AmyAl were glucose,maltose,maltotriose,maltotetraose and maltopentaose.The recombinant a-amylase was immobilized by IONPs.The specific activity of IONPs immobilized a-amylase is not strongly affected.The optimum temperature of immobilized a-amylase is changed from 50? to 60? and the optimum pH is changed from 5.0 to 6.0.The immobilized a-amylase was found to be active during reuse and 60%of its initial hydrolytic activity was obtained even after sixth cycle.