| Amylase can degrade ordinary starch, but only10percent of starch-degrading enzymes can bind and degrade the raw starch. Glucoamylases(GA) possess specific raw starch binding ability through their starch binding domain(SBD). SBDs have important industrial applications, such as in food processing, starch liquefaction, selective removal of starch from textiles, affinity purification of recombinant proteins and improving efficiencies of non-amylolytic enzymes. While in the process of indus-trial application, the purification is very difficult because of low expression of GA a-nd presence of other proteins. What’s more, because of the insufficiency of raw-starch degradation enzyme, the raw-starch should be destroyed by cooking them at high te-mperature and then we can make use of them through gelation and saccharification, which needs both extreme energy and money. So it is more urgent for us to construct recombinant strains with higher expression, easier purification, lower energy consu-mption and higher enzyme activity.The circulation permutation of a protein is to connect the amino termini and ca-rboxyl termini via a covalent linker and produce new terminals through the cleavage of an existing peptide bond. After circulation permutation, the protein gains higher ca-talytic ability, ligand binding affinity and stability, alters the oligomerization state and the substrate selectivity. The study was to investigate the raw starch adsorbility of SBD of recombinat strains with different termini SBD after circulation permutation. What’s more, we studied the changes of the affinity of the purpose protein-amylase with raw starch and soluble starch and the activity of the amylase after the circular permutation of SBD.We copied the gene of GA of Aspergillus niger and gene of SBD domain. We ga- ined six kinds of circulation permutation(CP) SBD genes:ANGCP527、ANGCP546、 ANGCP559、ANGCP585、ANGCP593and ANGCP605. The six CP SBD genes and the original SBD gene were cloned into pet28a to construct the recombinant plasmid. We studied the changes of the raw starch adsorbility of SBD of the six CP recombin-ant ANGCP-pet28a against the wild type ANG-pet28a. In order to discuss the enzyme properties and the affinity of the GA, the gene of GA was cloned into pPIC9K to con-struct the recombinat ANG-pPIC9K in Pichia pastoris GS115. Last, the six CP SBD genes replaced the original SBD gene of recombinant ANG-pPIC9K via overlapping PCR and created six recombinant ANGCP-pPIC9K in Pichia pastoris GS115:CP527、CP546、CP559、CP585、CP593and CP605.The study indicated that, the combining capacity and the hydrolysis activity with raw starch of the CP546, CP559and CP593had improved to a large extent. The com-bining capacity of CP546was1.5times of the control group, CP559and CP593had increased0.27and0.33times, respectively. CP546had the highest combining capaci-ty with raw starch and its purified glucoamylase’s hydrolysis activity of raw starch w-as the best:5.59IU/mg, while the control group was4.51IU/mg. The combining cap-acity and the hydrolysis activity with soluble starch of CP527and CP585had reduced a lot, while CP605remained the same as the control group.The combining capacity a-nd the hydrolysis activity with soluble starch of the six circulation permutation reco-mbinant hadn’t changed much and remained about the same with the control group: ANG. Our research had indicated that, after the circulation permutation of the SBD, the higher the combining activity with the raw-starch is, the greater the degradation activity of gluco-amylase gained. While the circulation permutation didn’t change the hydrolysis activity of GA with soluble starch. |
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