Abstract
BackgroundStarch-binding domains from carbohydrate binding module family 20 have been used as a tool for starch engineering. Previous studies showed that expression of starch binding domain fusion proteinsin planta resulted in modified starch granule structures and physicochemical properties. However, although 13 carbohydrate binding module families have been reported to contain starch-binding domains, only starch-binding domains from carbohydrate binding module family 20 have been well studied and introduced into plants successfully.In this study, two fragments, the tandem CBM25 domain and the tandem CBM25 with multiple fibronectin type III (FN3) domains of the α-amylase enzyme from Microbacterium aurum, were expressed in the tubers of a wild type potato cultivar (cv. Kardal) and an amylose-free (amf) potato mutant.ResultsThe (CBM25)2 and FN3 protein were successfully accumulated in the starch granules of both Kardal and amf transformants. The accumulation of (CBM25)2 protein did not result in starch morphological alterations in Kardal but gave rise to rough starch granules in amf, while the FN3 resulted in morphological changes of starch granules (helical starch granules in Kardal and rough surface granules in amf) but only at a very low frequency. The starches of the different transformants did not show significant differences in starch size distribution, apparent amylose content, and physico-chemical properties in comparison to that of untransformed controls.ConclusionThese results suggest that the starch-binding domains from carbohydrate binding module family 25 can be used as a novel tool for targeting proteins to starch granules during starch biosynthesis without side-effects on starch morphology, composition and properties.
Highlights
Starch-binding domains from carbohydrate binding module family 20 have been used as a tool for starch engineering
It is known that Starch-Binding Domains (SBDs) play the following three roles: they allow the interaction between the insoluble substrate and the enzyme in solution; they help the catalytic domain of enzymes to catch the substrate; and in some cases they have a disruptive function on the starch granule surface [11]
MRNA transcript analysis of (CBM25)2 and FN3 transformants Expression of the two gene fragments, (CBM25)2 and FN3, in potato tubers was determined by semi-quantitative RTPCR
Summary
Starch-binding domains from carbohydrate binding module family 20 have been used as a tool for starch engineering. Previous studies showed that expression of starch binding domain fusion proteins in planta resulted in modified starch granule structures and physicochemical properties. 13 carbohydrate binding module families have been reported to contain starch-binding domains, only starch-binding domains from carbohydrate binding module family 20 have been well studied and introduced into plants successfully. Microbial extracellular hydrolytic enzymes that catalyze the degradation of insoluble polysaccharides, such as glycogen, starch, and cellulose, typically possess a modular architecture referred to as carbohydrate-binding modules (CBMs) which target cognate catalytic modules to specific polysaccharide structure [5, 6]. CBMs which show affinity to starch granules are called Starch-Binding Domains (SBDs). It is known that SBDs play the following three roles: they allow the interaction between the insoluble substrate and the enzyme in solution; they help the catalytic domain of enzymes to catch the substrate; and in some cases they have a disruptive function on the starch granule surface [11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
