DEVELOPMENT OF NEW BIOCATALYTIC PROCESSES FOR FRUCTOOLIGOSACCHARIDES (FOS) PREPARATION
Creators: name: ZAMBELLI, PAOLO
Publisher: name: Universit� degli Studi di Milano
Year: 2014

description: This PhD research project was aimed at the development of new biocatalytic processes to produce natural sugars by selection and characterisation of new enzymes able to produce fructooligosaccharides. Biochemical studies were performed to obtain information on the mechanism of action and understand the structural elements that define the activity. After the development of the biotransformation conditions, a continuous production of FOS was studied and a cheap separation method of the transformation products was also assessed, in order to obtain FOS in purified form. Mass spectrometry studies were performed on the purified enzymes after purification from wild-type strains.
After a screening for FOS production from sucrose two microorganisms were chosen for their activity up to 30 % (w/w) conversion and differences in FOS mixture production:
CF215 = Cladosporium cladosporioides 
CK1 = Penicilium sizovae
CF215 produce a mixture similar to the commercial product Actilight�, while CK1 produce for almost kestose (GF3). Under the optimised biotransformation conditions the maximum accumulation of FOS was 56 % (w/w) and 31 % (w/w) for CF215 and CK1 respectively. We were able to isolate and characterise seven different carbohydrates such as 1-kestose, 1-nystose, 1-fructofuranosylnystose, 6-kestose, neo-kestose and neo-nystose for CF215 while CK1 produce only 1-kestose, 1-nystose, 1-fructofuranosylnystose and 6-kestose. Another oligosaccharides was isolated and fully characterised from CF215 mixture, named blastose (Fru-?(2?6)-Glc). An immobilization study was carried using the DALGEEs (Dried Alginate Entrapped Enzymes) method on the mycelium of CF215 strain. The maximum accumulation of FOS using DALGEEs mycelium was 51 % (w/w), reached in common buffer and seawater. With this cheap technique we develop a continuous FOS production using the facilities of Flow chemistry. The reactor, filled with DALGEEs and celite, was stable for months and the maximum accumulation of FOS was 52% (w/w). At this flow stream of FOS mixture we added a batch step to purify the FOS from glucose that represent the 26 % (w/w) of entire mixture. Glucose Oxidase from Novozymes� named Glyzyme� MONO 10.000 BG was employed and the result was the reduction of glucose from 26 % (w/w) to 3% (w/w). This purification step was added for two reasons: to obtain a cheap and fast method for FOS purification from glucose and to simplify the blastose HPLC purification. 56 mg of purified blastose were obtained and utilised to perform a pioneer study of blastose prebiotic action. The growth of 5 different lactobacillus strains (Lactobacillus paracasei DG, Lactobacillus rhamnosus GG, Lactobacillus paracasei SHIROTA, Lactobacillus johnsonii LC1 and Lactobacillus reuteri ATCC55730) were followed with the addiction of different carbohydrates as only carbon source (glucose, Actilight�, inulin, blastose). The best results were reached with Lactobacillus johnsonii LC1 where the Vmax using 0.5 % (w/v) of blastose was higher than glucose 0.5 % (w/v) (1.125 � 0.023 1/h and 0.521 � 0.054 1/h respectively).
In the second part of this PhD project the purification of the enzymes involved in FOS formation was achieved after several chromatographic steps. The molecular weight (MW) of the two proteins was ?50 kDA for the enzyme from Cladosporium cladosporioides (monomeric) and ?75 kDa for the one from Penicilium sizovae (monomeric). The enzyme from C. cladosporioides was biochemically characterised and shown a Km of 129 � 6 mM, Vmax of 2.83 � 0.04 U/mL, Kcat of 2.88 � 0.04 1/s and a Kcat/Km of 22.3 � 1.4 1/M*s with sucrose and a Km of 268 � 6 mM, Vmax of 0.0328 � 0.003 U/mL, Kcat of 0.0334 � 0,003 1/s and Kcat/Km of 0.124 � 0.014 1/M*s with 1-kestose. A mass spectrometry MALDI-TOF analysis study was performed on the protein, showing a MW of 61178 Da. A trypsin digestion was performed and the fragments analysed but we didn?t found match in databases. The molecular study of the protein was stopped until protein sequence elucidation.