| Author | Wang, Xiao-dong |
| Call Number | AIT Diss. no.BP-98-01 |
| Subject(s) | Oligosaccharides
|
| Note | A dissertation in partial fulfillment of the requirements for the degree of Doctor of Technical Science, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Abstract | Iso-oligosaccharides have a number of health and nutraceutical applications. The overall aim of this project was to study the production, characterizations and application of transferase enzyme used in the biosynthesis of beneficial iso-oligosaccharides. The fungus Aspergillusfoetidus NRRL 33 7', one of the best known producers of the transferase enzyme, was used in the study. These enzymes can be utilised for the synthesis of fructose~ oligosaccharides and isomalto-oligosaccharides. Maltose and corn steep liquor were found to be the best carbon and nitrogen substrates at optimum concentration of 1% and 3% respectively in shake flask studies. The surfactant Tween 80 and metal salts of Fe , Zn , Mg and Cu were found to have no obvious effect on the enzyme productivity. Bioreactor studies with 3% maltose as carbon source indicated that uncontrolled pH starting with a pH value of 5.0 gave the highest transferase production compared with pH controlled at constant levels and control after natural fall. The optimum temperature was found to be 30 0C. Enzyme activity increased 8 fold in reactors, with better aeration and agitation condition, as compared to shake flask. The morphology of the fungal mycelium was highly dependent on the nitrogen source and had great influence on enzyme production. Four major protein fractions having transferase enzyme activity were obtained from the broth of Aspergillus foetidus NRRL 337 by ammonium sulphate precipitation and DEAE-cellulose chromatography. The optimum temperature (60 OC) and pH stability (in the range 4 to 6) of all fractions were nearly the same. The heat sensitivity and pH optima for the four fractions were however different. While EDTA and phenylmethylsulfonyl fluoride had no obvious effect on the enzyme activity of all four transferase enzyme fractions they showed different action patterns in the presence of metal salts. All four enzyme fractions could not utilise lactose and cellobiose to synthesise iso—oligosaccharide, but the I-IPLC iso-oligosaccharides product analysis of these transferase enzymes on maltose and SucrOSe reveal that the four forms of enzymes are indeed distinct. These multiple forms of enzymes for iso-oligosaccharides synthesis include glucosyltransferase, fructosyltransferase and glucosidase enzymes. A crude enzyme sample which was a mixture of these enzymes was extracted and concentrated by ammonium sulphate precipitation and used in the synthesis isomalto- oligosaccharide from maltose and fi-ucto-oligosaccharide from sucrose. The iso- oligosaccharide concentration increased with the increasing reaction time. However, the formed iso-oligosaccharides were hydrolysed to monosaccharides after extended reaction times. This was also dependent on the enzyme quantity used in the reaction and On the substrate concentration. For isomalto-oligosaccharide and fi'ucto-oligosaccharide synthesis, 30% maltose and 40% sucrose were optimum leading to a productivity of 0.22 and 0.16 glunithour respectively under the best studied conditions. Initial studies on the immobilisation of the crude transferase enzyme using sodium alginate was done for isomalto-oligosaccharide synthesis. The immobilized transferase enzyme showed improved kinetics, thermal and pH stability as compared to free enzyme. The productivity for somalto-oligosaccharide synthesis was about 0.44 g/unithour which is much higher than that of free enzyme (0.16 g/unit.hour). The results obtained from this work indicate that Aspergillus foeridus could indeed be used in the hyper production of the transferase enzymes which in turn can be applied in a practical process for the production of valuable iso-oligosaccharides like panose and kestose. |
| Year | 1998 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Bioprocess Technology (BP) |
| Chairperson(s) | Rakshit, Sudip K.; |
| Examination Committee(s) | Stevens, Willem F.;Athapol Noomhorm;Montct, Didier; |
| Scholarship Donor(s) | Employee Student; |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology |