**5. Use of carbohydrate-binding modules to change amylose - amylopectin ratio and obtaining of modified starches.**

In the past few years the search for different strategies in order to produce starches with new properties was intensified. One of these strategies is to evaluate the possibility whether the microbial starch binding domains (SBDs) could be used as a universal tool for starch modification in plant biotechnology.

It has been reported that SBDs are also present in microbial starch degrading enzymes. As mentioned above, one of the functions of SBD is to attach amylolytic enzymes to the insoluble starch granule. The amino acid sequences of these modules are very well conserved among different enzymes (i.e. glucoamylase, α-amylase, β-amylase, etc.), as well as among different species such as *Clostridium thermosulfurogenes, Bacillus circulans, Aspergillus niger, Klebsiella pneumonia, Streptomyces limosus, Pseudomonas stutzeri,* etc. [50,71- 73]. Several studies have shown that these enzymes lose (most of) their catalytic activity towards raw starch granules upon removal of the SBD, whereas their activity on soluble substrates remains unaltered. Besides their affinity for starch granules, SBDs can also bind maltodextrins and cyclodextrins [71]. Ji et al [72], explored the possibility of engineering artificial granule-bound proteins, which can be incorporated in the granule during biosynthesis. The SBD-encoding region of cyclodextrin glycosyltransferase from *B. circulans* was fused to the sequence encoding the transit peptide (amyloplast entry) of potato GBSSI. The synthetic gene was expressed in the tubers of two potato cultivars and one amylose-free (amf) potato mutant. The results showed that SBDs are accumulated inside starch granules, not at the granule surface and amylose-free granules contained 8 times more SBD than the amylose-containing ones. However, no consistent differences in physicochemical properties between transgenic SBD starches and their corresponding controls were found, suggesting that SBD can be used as an anchor for effector proteins without having side-effects [72].

On the other hand it was also evaluated whether is it possible to produce an amylose-free potato starch by displacing GBSSI, from the starch granule by engineering multiple-repeat CBM20 SBD (two, three, four and five). The constructs were introduced in wild type potato cultivar, and the starches of the resulting transformants were compared with those expressing amf potato clones. The amount of SBDs accumulated in starch granules was increased progressively from SBD to SBD3 and not when were used SBD4 and SBD5; however, a reduction in amylose content was not achieved in any of the transformants. It was shown that SBDn expression can affect the physical process underlying granule assembly in both potato genetic backgrounds, without altering the primary structure of the constituent starch polymers and the granule melting temperature. Granule size distribution of the starches obtained from transgenic plants was similar to untransformed controls, irrespective of the amount of SBDn accumulated. In the amf background, granule size is severely affected [74].

In the case of starches which require chemical modifications to enhance their properties, such as the improved stability in solution by acetylation, a drawback is generated when pollutant chemicals are used. A biological alternative to the derivatization process was investigated by the expression of an amyloplast-targeted *Escherichia coli* maltose acetyltransferase (MAT) in tubers of wild-type and mutant amf potato plants. MAT was expressed alone, or fused in its N- or C-terminus to a SBD to be target to the starch granule. Starch granules derived from transgenic plants contained acetyl groups in low number. In addition, MAT protein on the starch granules present catalytic activity even after postharvesting, when supplied with glucose or maltose and acetyl-coenzyme A, but it was not able to acetylate starch polymers in vitro. Starch granules from transformants where MAT was expressed alone also showed MAT catalytic activity, indicating that MAT is accumulated in starch granules, and could bind to the polymer without the presence of any SBD. Furthermore, the fusion of MAT and SBD affects granule morphology: in potato transformants, the percentage of altered granules when the SBD was located at the Cterminal end correlated with the amount of fusion protein accumulated. When SBD was located at the N-terminus of MAT or it is absent, no differences were found respect to the untransformed controls, indicating that not only is the simultaneous presence of SBD and MAT important for altering granule morphology, but also their localization in the fusion protein [75].

502 The Complex World of Polysaccharides

severely affected [74].

It has been reported that SBDs are also present in microbial starch degrading enzymes. As mentioned above, one of the functions of SBD is to attach amylolytic enzymes to the insoluble starch granule. The amino acid sequences of these modules are very well conserved among different enzymes (i.e. glucoamylase, α-amylase, β-amylase, etc.), as well as among different species such as *Clostridium thermosulfurogenes, Bacillus circulans, Aspergillus niger, Klebsiella pneumonia, Streptomyces limosus, Pseudomonas stutzeri,* etc. [50,71- 73]. Several studies have shown that these enzymes lose (most of) their catalytic activity towards raw starch granules upon removal of the SBD, whereas their activity on soluble substrates remains unaltered. Besides their affinity for starch granules, SBDs can also bind maltodextrins and cyclodextrins [71]. Ji et al [72], explored the possibility of engineering artificial granule-bound proteins, which can be incorporated in the granule during biosynthesis. The SBD-encoding region of cyclodextrin glycosyltransferase from *B. circulans* was fused to the sequence encoding the transit peptide (amyloplast entry) of potato GBSSI. The synthetic gene was expressed in the tubers of two potato cultivars and one amylose-free (amf) potato mutant. The results showed that SBDs are accumulated inside starch granules, not at the granule surface and amylose-free granules contained 8 times more SBD than the amylose-containing ones. However, no consistent differences in physicochemical properties between transgenic SBD starches and their corresponding controls were found, suggesting that SBD can be used as an anchor for effector proteins without having side-effects [72].

On the other hand it was also evaluated whether is it possible to produce an amylose-free potato starch by displacing GBSSI, from the starch granule by engineering multiple-repeat CBM20 SBD (two, three, four and five). The constructs were introduced in wild type potato cultivar, and the starches of the resulting transformants were compared with those expressing amf potato clones. The amount of SBDs accumulated in starch granules was increased progressively from SBD to SBD3 and not when were used SBD4 and SBD5; however, a reduction in amylose content was not achieved in any of the transformants. It was shown that SBDn expression can affect the physical process underlying granule assembly in both potato genetic backgrounds, without altering the primary structure of the constituent starch polymers and the granule melting temperature. Granule size distribution of the starches obtained from transgenic plants was similar to untransformed controls, irrespective of the amount of SBDn accumulated. In the amf background, granule size is

In the case of starches which require chemical modifications to enhance their properties, such as the improved stability in solution by acetylation, a drawback is generated when pollutant chemicals are used. A biological alternative to the derivatization process was investigated by the expression of an amyloplast-targeted *Escherichia coli* maltose acetyltransferase (MAT) in tubers of wild-type and mutant amf potato plants. MAT was expressed alone, or fused in its N- or C-terminus to a SBD to be target to the starch granule. Starch granules derived from transgenic plants contained acetyl groups in low number. In addition, MAT protein on the starch granules present catalytic activity even after postharvesting, when supplied with glucose or maltose and acetyl-coenzyme A, but it was not able to acetylate starch polymers in vitro. Starch granules from transformants where MAT Another approach to obtain modified starches involves the bacterial glucansucrases [76]. Certain bacteria possess an array of enzymes, so-called glucansucrases, which can attach (contiguous) 1,6-linked or 1,3-linked glucosyl residues to maltodextrins. This, together with the presence of sucrose inside the potato tuber amyloplast [77], suggests that glucansucrases are of great interest for diversifying starch structure. With few exceptions, glucansucrases are extracellular enzymes, which are produced by lactic acid bacteria such as *Leuconostoc mesenteroides*, oral *Streptococci* and some species of *Lactococcus* and *Lactobacillus* [78]. The glucansucrases catalyze the polymerization of glucose residues from sucrose, which leads to the production of a large variety of α-glucans with different sizes and structures, and composed of diverse linkage types. Most glucansucrases share a common structure composed of four different regions: a signal peptide, a variable region, a catalytic domain, and a glucan-binding domain (GBD) [76].

Production of water-insoluble mutan polymers in wild type potato tubers was investigated by Kok-Jacon et al (2005) after expression of full-length GTFI (mutansucrase) and a truncated version without glucan-binding domain from *Streptococcus downei.* Mutan polymers are bacterial polysaccharides that are secreted by oral microorganisms and have adhesive properties and different degrees of water-solubility [81]. They account for about 70% of the carbohydrates present in dental plaque [79] in addition to dextrans and levans [80]. When the short form of the protein was expressed, low amounts of mutan polymer attached to the starch granules has been detected. Besides, these plants exhibited severely altered tuber phenotype and starch granule morphology in comparison to those expressing the full-length GTFI gene, whereas no changes at the starch level were observed. Finally, the rheological properties of the starch obtained from plants expressing the truncated protein were also altered, showing a higher retrogradation during cooling of the starch paste [80].

Subsequently, the same group of investigators fused the truncated form of a mutansucrase (without glucan binding domain) to an N- or C- terminal SBD. The different enzymes were introduced into two genetically different potato backgrounds (wild type and amf lines), in order to attach the enzyme to the growing starch granules, and to facilitate the incorporation of mutan polymers in starch. Starches from the chimeric transformants seemed to contain less amounts of mutan than those from plants expressing the mutansucrase alone, suggesting that SBD might inhibit the catalytic activity of the enzyme. Scanning electron microscopy showed that expression of SBD-mutansucrase fusion proteins resulted in alterations of granule morphology in both genetic backgrounds. Surprisingly, the amf starches containing the chimeric form had a spongeous appearance, as the granule surface contained many small holes and grooves, indicating that this fusion protein can interfere with the lateral interactions of amylopectin sidechains. No differences in physicochemical properties of the transgenic starches were observed [82].

Finally, all the knowledge gained about the characteristics, structure, function and occurrence of SBD and GBD will support current and future experimental research. Since SBD are domains which retain their structural fold and functional properties independently of the remaining parts of the protein molecule including the catalytic domain, they can be applied in various fields of biotechnology [48,83-86]. It is important to note that most of the applications have involved only the CBM20 SBD. One of the most attractive fields is represented by starch processing in the food industry, especially the hydrolysis of starch into maltodextrins and maltooligosaccharides [87]. Since conventional processes require starch gelatinization at elevated temperature and thus use of thermostable amylolytic enzymes [88], the possibility of carrying out the processes without gelatinization, by utilizing new enzymes with attached SBD is desirable [52,89,90].
