| Glycoconjugates are very important compounds in many biological processes,and they are widely used in food and pharmaceutical industries.Glycoconjugates are formed in processes termed glycosylation in which glycosidases play an important role.In this thesis,we used some thermophic glycosidases from Thermotoga naphthophila RUK-10,?-glucosidase(TN0602),?-galactosidase(TN1577),?-galactosidase(TN0949),and TN0602 mutants,to explore their role in the synthesis of different glycoconjugates,such as galacto-oligosaccharides,alkyl galactopyranosides and L-ascorbic galactopyranoside.For the galacto-oligosaccharides(GOS)sythesis,TN0602 demonstrated exceptionally high catalytic selectivity for synthesis of GOS with lactose as both galactosyl donor and acceptor.A high volumetric productivity of galactotrisaccharide(GOS3)up to 23.28 g L-1 h-1was achieved at p H 6.5 and 75°C with 100% selectivity.A comparative study with ?-galactosidase from Aspergillus oryzae(AO)with respect to reaction kinetics,enzyme-substrate thermodynamic binding and molecular docking simulation elucidated that,TN0602 has a deep catalytic pocket with a narrow entrance which prevents a simultaneous access of lactose and GOS3 to the catalytic site,dedicating its distinct difference in catalytic specificity and reaction kinetics from AO.To comfirm this opinion,and to decrease the hydrolytic activity of TN0602,some residues were selected to mutant in TN0602.The effect of the mutants on kinetic,enzyme activity,and substrate specificity were then determined.The results showed some mutants enhanced the synthetic properties of TN0602.The replacement of phenylalanine to serine(F414S)increased the GOS production(207.63 m M)compared with the wild-type TN0602(138.58 m M).The improvement of production was due to an increase in the ratio of transgalactosylation to hydrolysis.The results of molecular docking showed that the mutanted sites and the catalytic pocket shape of TN0602 are the main reasons why it could not produce GOS4.L-ascorbic acid(L-AA)is an essential nutrient that cannot be synthesized by the human body.Due to its extreme instability,various L-AA derivatives have been developed in an attempt to improve stability while retaining the same biological activity,a new method of efficient enzymatic transgalactosylation from lactose to L-ascorbic acid with TN0602 was developed.TN0602 was observed to have a high transgalactosylation activity for the synthesis of galactosylated L-ascorbic acid.The optimal temperature and p H for the enzymatic L-ascorbic galactopyranoside production were 75°C and 5.0,respectively,and the optimal TN0602 concentration and substrate mole ratio were 20 mg m L-1 and 2:1(L-ascorbic acid to lactose),respectively.In these optimal process conditions,maximal L-ascorbic galactopyranoside production reached 138.88 m M.Thus a highly scalable and facile procedure for the enzymatic transgalactosylation of L-ascorbic acid was developed compared to previous reports in literature.Moreover,compared to free L-ascorbic acid,L-ascorbic galactopyranoside still possessed antioxidant capability and exhibited improved oxidative stability in the presence of Cu2+.This implies that it can be used in applications as L-ascorbic acid.The results may be useful for the industrial scale production of L-ascorbic galactopyranoside.Green synthesis is of pivotal importance for environmental sustainability.In this study,we found a novel approach to synthesize an array of alkyl galactopyranosides using TN1577 as biocatalyst and lactose as galactosyl donor.Ammoeng 102(only 2.5% addition of total reaction volume),a functionalized ionic liquid(IL)containing tetraaminum cation with C18 acyl and oligoethyleneglycol,was identified as the most promising IL from a variety of structurally diverse ionic liquids,affording 2.37-fold increase in octyl galactopyranoside yield compared to a buffer system.Up to 18.2 g L-1 octyl galactopyranoside could be produced in 7 h,which is significantly higher(p<0.05)than any previous report in terms of time-space efficiency.Kinetic studies and Conductor-like Screening Model for Real Solvents(COSMO-RS)in silico predictions elucidated that the thermophilic nature of TN1577,increased solubility of substrate,suppression of hydrolysis,and excellent biocompatibility of Ammoeng 102 with enzyme(allowing TN1577 ?-galactosidase to perform optimal catalysis up to 95°C)are the main reasons.A series of alkyl galactopyranosides were successfully synthesized with n-butanol to n-tetradecanol as alkyl acceptors and lactose as galactosyl donor with in Ammoeng 102 system.Microwave irradiation shortened reaction times,increased the productivity of the alkyl galactopyranoside reaction,and decreased the amount of enzyme required for the reaction.The optimal conditions for the enzymatic synthesis of alkyl galactopyranoside from lactose and n-butanol by TN1577 under a microwave heating system were determined.The influences of the molar ratio between lactose and n-butanol(1:200–1:40)and temperature(65–85°C)on the transgalactosylation yield were investigated.The output power of the microwave ranged from 80 W to 800 W.Under optimal conditions(i.e.,a lactose and n-butanol molar ratio of 1:40 and a reaction temperature of 75°C),a yield of 17.07 mg m L-1 was obtained after a 3.5 h reaction incubation period.Compared with conventional heating,the microwave-assisted method improved the yield 11-fold.Microwave irradiation significantly increased the kcat of TN1577,but had little effect on Km compared with conventional heating.Based on these findings,the microwave-assisted enzymatic synthesis method enables the rapid and more economical production of butyl galactopyranoside with lower time and energy costs.Therefore,this work not only provides some new choices for industrial production of L-ascorbyl glycosides,alkyl glycosides,and high-purity galacto-oligosaccharides,but also screened some thermophic glycosidases for industry,which could provide potent biosynthesis approaches to produce glycocongates. |
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