Study On The Affinity Of Glucoamylase Starch Binding Domain In Aspergillus Niger And Its Application In Potato Starch Quality Improvement

Potato starch possess many excellent properties and it has been widely used in paper,food,adhesive and pharmacy industries.But due to some limitations in terms of physical and chemical properties,natural starches do not properly play its role in industrial applications.Therefore,natural starches are often modified by chemical or physical methods before being used.Chemical or physical modifications of natural starches are costly and chemicals used are often harmful.The composition and structure of starch can be modified in planta by genetic engineering and tailoring starches with new functionalities may be obtained.Previous studies showed that SBD(starch-binding domain)-platform technology could be effectively to target engineering artificial granule-bound recombinant proteins into granules during the starch biosynthesis for modification of the composition and structure of starch,in which SBD of cyclodextrin glycosyltransferase from Bacillus circulans was used as an anchor.Starch binding domain of glucoamylase(GASBD)from Aspergillus niger and starch binding domain(SBD)of cyclodextrin glycosyltransferase from Bacillus circulans belong to members of the CBM20 family,and their three-dimensional structure and starch binding sites are similar.Related researches were carried out using starch binding domain of glucoamylase from Aspergillus niger as materials in this study.The main results obtained are as follows:1.Affinities of tandem repeat GASBDs and starchA starch-binding domain encoding region of glucoamylase(GASBD)was amplified by PCR from Aspergillus niger 2316 and sequenced.The result(GenBank JQ814697)revealed that it consisted of 324bp nucleotides and encodes a peptide of 108 amino acids.Sequence analysis showed that GASBD was a member of the CBM20 family.To investigate the affinities of artificial tandem repeat GASBDs for insoluble starch in vitro,the pET28a plasmids harbouring,respectively,single GASBD and multiple-repeat GASBDs genes from two to four were constructed and expressed in Escherichia coli BL21(DE3).The GASBD(s)recombinant proteins were purified to homogeneity by Ni2+-NTA affinity chromatography and the affinities of the purified recombinant proteins for insoluble starches were determined.The results revealed that GASBD2,GASBD3,and GASBD4 had,respectively,approximately 5,8 and 10-fold higher affinities for starch than single GASBD,indicating that the multiple-repeat GASBDs act in a synergistic way when binding to starches.2.Expression of GASBD and GASBD2 genes in potatoThe encoding sequence of single GASBD or artificial tandem repeat GASBD2 gene was fused to the sequence encoding the transit peptide of potato granule-bound starch synthaseⅠ(GBSSI).The GASBD and GASBD2 genes were introduced into the potato cultivars Desiree using Agrobacterium-mediated method,respectively.They could be expressed in the tubers of potato under the control of potato GBSSI promoter.To determin whether GASBD and GASBD2 proteins are capable of granule-targeting during starch biosynthesis,the accumulation of GASBD and GASBD2 into starch granules of transgenic lines was analyzed by Western dot blot.The results demonstrated that the GASBD and GASBD2 proteins could be accumulated in starch granules and the highest accumulation of GASBD2 into starch granules was approximately 3-fold higher than that of GASBD.The starch contents of tubers of transgenic lines had no change,indicating the GASBD and GASBD2 expression have no effect on the starch biosynthesis.Furthermore,the impact of GASBD and GASBD2 accumulation in starch granules on the physicochemical properties of starches of transgenic lines was investigated.The results revealed that the accumulation of GASBD in starch granules had no significant effect on the granule morphology of starches of transgenic lines,but the accumulation of GASBD2 in starch granules had significant effect on the granule morphology of starches of transgenic lines.Other physicochemical properties of starch had no change between the starches of transgenic GASBD or GASBD2 lines and their corresponding controls.These results suggested that GASBD or GASBD2 could be used in SBD-platform technology for starch modification.3.Expression of glgB/GASBD fusion genes in potatoPrevious study has shown that the expression of the glycogen branching enzyme(glgB)gene of Escherichia coli in the tubers of potato resulted in an increased degree of branching of starches of transgenic lines.In the present study,in order to obtain transgenic potato starches with a higher degree of branching than before,the Escherichia coli glgB gene was fused to either the 5’-or 3’-terminus of the GASBD gene.The glgB/GASBD fusion genes and glgB gene using as the control were introduced into the potato cultivars Desiree using Agrobacterium-mediated method,respectively.They could be expressed in the tubers of potato under the control of potato GBSSI promoter.The accumulation of glgB/GASBD fusion proteins into starch granules of transgenic lines was analyzed by Western dot blot.The results revealed that a large amount of glgB/SBD fusion proteins could be accumulated inside starch granules and the GASBD positions in the fusion proteins(the N-terminus or C-terminus)had no significant effect on the accumulation of fusion proteins in starch granules.The expression of glgB/GASBD fusion genes in the tubers of potato resulted in the starch branching degree increasing up to 35%in some transgenic lines compared with the non-transgenic control starch,but compared with the starches of transgenic glgB lines,there was no significant increase in the branching degree of the starches of transgenic glgB/GASBD lines.The results of the starch contents of transgenic tubers showed that the expression of glgB/GASBD fusion proteins or glgB protein had no effect on the starch biosynthesis.However,the accumulation of glgB/GASBD fusion proteins in starch granules could alter the starch granule morphology of transgenic lines.The measurement results of the amylopectin chain length distribution showed that compared with the non-transgenic control starch,amylopectin molecules of starches with an increased degree of branching contained more short chains,the socalled A chains.This may be the main reason of the increase of branching degree of starches of transgenic lines.The influence of the increased degree of branching on the physico-chemical properties and functional characteristics of starches of transgenic lines was investigated.The results revealed that the increase of starch branching degree leaded to a lower peak viscosity and set back viscosity of starches of transgenic lines.Compared with the non-transgenic control starch,the pasting temperature of starches of transgenic lines was decreased by 2-3 ℃ and the starch gelatinization enthalpy change was also decreased.The transparency of starches of transgenic lines was decreased,whereas the freeze-thaw stability of starches of transgenic lines was increased.These results showed that increasing the starch branching degree leaded to specific changes in the physico-chemical properties and functional characteristics of the starch.