| Trehalose is a non-reducing disaccharide consisting of two a-1,1bond-linked glucose molecules. It’s an unusual sugar as compared with other disaccharides, and widely distributed in various organisms such as bacteria, yeast, fungies and insects. It can be used as a component of sweeteners, seasonings, preserved and frozen foods, and as a moisture retainer in cosmetics and the preservatives in pharmaceuticals. Until now, several biosynthesis methods of trehalose have been developed. Among these methods, the enzymatic synthesis of trehalose is considered as a valuable method for the production of trehalose.In this study, a procedure of enzymatic synthesis of trehalose was developed which can apply to produce trehalose on a large scale. QS412was obtained by screening and mutation breeding, which could produce maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase). Fermentation of QS412, enzyme reaction and purification of trehalose were investigated carefully in this paper. The research was comprised of five sections as follow.The first section was about mutation breeding of strains. After Q186was treated with UV、acridine、manganese chloride、hydroxylamine and DES, two strains, QS412and QF611, were obtained, and enzyme activity of them increased more than20%. Activity of QS412obtained from UV mutation was65%higher than the wild strain. Enzyme activity increased only by13%after the strain was treated with DES. The strain which had as high as120%activity of wild strain was obtained after treatment in combination with UV and DES, but the strain was instable. The stability of strain QS412was very good. MTSase and MTHase were located in the cells and could convert starch into trehalose. In the further experiment, QS412was used to produce MTSase and MTHase and synthesize trehalose.Fermentation condition and kinesics of QS412were investigated in the second section. The optimized medium contained20g/L glucose,20g/L yeast extract,10g/L peptone,1g/L K2HPO4-3H2O and0.5g/L MgSO4·7H2O. After QS412was cultured with this medium, the wet weight of biomass was about17.3g/1, and enzyme activity of trehalose synthesis was up to12.32Upc.0.2%soybean flour and5%corn steep liquor was used to replace peptone in the fermentation, that made biomass improve to19.5g wet weight/L. Enzyme activity of trehalose synthesis was almost the same as those cultured with peptone. The fermentation condition was as follow:30℃culture temperature,72h culture time,50ml medium in250ml conical flask (34Upc)oFermentation in20L fermentor was performed under30℃. The inoculation ratio was5%, and the medium contained20g/L glucose,0.2%soybean flour,5%corn steep liquor, lg/L K2HPO4·3H2O and0.5g/L MgSO4·7H2O. The fermentation cycle was about48h. The biomass was up to61.2g/L. Kinetic analysis of batch culture indicated that the production of MTSase and MTHase was partial correlated with the growth of QS412.The third section was about study on the production characteristics of the two enzyme. In this section, the releasing condition of the enzymes from cells was determined. The optimal activity of two enzyme system was achieved under condition of pH8%100mM、40℃. But40℃could inactivate the enzymes. The activity was lost about50%after5h static incubation or3.5h incubation with stirring. The optimized reaction condition of free MTSase and MTHase was as follow:100mM pH8buffer,30℃,10%starch(10%DE),25h reaction time. After reaction, the obtained solution contained31.73mg/mL trehalose, and the yield from starch was63.5%.The substrate and product had no inhibitory action on the enzyme reaction. When concentration of trehalose was more than0.3mol/1, the enzyme activity increased. Mg2+could enhance enzyme activity, while Zn+, Mn2+,Cu+, Fe+, Hg2+could inhibit enzyme activity obviously. The inhibitory effect of Hg2+was most obvious. Among them. Zn2+above40mM could inhibit activity of MTHase, and this inhibitory effect could be eliminated by EDTA. So activity assay of MTSase could be performed under existence of two enzyme by adding Zn2+inhibiting MTHase, and the substrate of MTHase also could be prepared by the same method.Permeabilized Micrococcus QS412cells were used to produce trehalose from starch in the fourth section. The reagent, reagent dosage and time of cell permeabilization treatment were determined. The maximum trehalose biosynthesis activity was obtained after the cells were treated with5%(w/v) of toluene at30℃for40min. Reaction conditions of trehalose synthesis of permeabilized cells were optimized. The yield of trehalose was up to369mg/g wet permeabilized cells in pH8.0,100mmol/1phosphate buffer at30℃after12h reaction, and the transformation efficiency of starch was73.8%. Batch reactions showed that the permeabilized cells could be reused for16cycles in the biosynthesis reaction. The total trehalose yield was up to5.05g/g wet permeabilized cells. The eigen kinetic equations (rp=27.78Cs/(29.96+Cs)) and apparent kinetic equations (Rp=24.63Cs/(133.78+Cs)) were obtained. Kinetic analysis showed that inner diffusion had prominent effects on this process. Development of permeabilized cells provide a new cheaply alternative technology for trehalose production. The fifth section was about purification of trehalose. The trehalose crystal with95%purity was obtained through amylases treatment, removal protein by membranes and adsorption by the absorbite column.Total yield of trehalose was95%. The crystal was proved to be trehalose through detection by HPLC and MRI. |
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