Study On Tehalose Production Through Permeable Resting Cells

Tehalose is a secure non-reducing saccharide, moisturizing, resistant to frost and drying conditions, stabilized to heat and acid, and provides nonspecific protection for biologic molecule. It has a broad prospect of application for pharmaceuticals, agriculture, cosmetics and food industry, etc, but hasn't been widely used due to high cost and prepapation difficulty. This thesis mainly studied trehalose production through permeable resting cells from Pseudomonas putida. In this study, trehalose synthase is immobilized inside the cells, while the newly- synthetic trehalose is released across the cell membrane, which shows an innovative approach for trhalose production. The contents of this thesis include four parts: preparation for permeable resting cells, optimization for medium component and fermentation process, the impact of osmotic pressure on permeability and enzyme activity, and the study on enzymatic reaction process and enzyme characteristics.Trehalose synthase is intracellular, so enzyme purification after cell discruption would be difficult. Besides, the enzyme activity loss is huge, so permeable resting cell was adopted to catalyze the enzymatic reaction instead of pure enzymes after permeated by toluene of 2% for 2h. The treatment effects between toluene permeation and ultrasonication were compared to show the complete permeation ability of toluene.Medium components were optimized in this study to discuss their influences on enzyme activity and biomass, the optimum medium was then obtained: glucose 2%, beef extract 1%, K2HP04 0.1%,NaH2PO4 0.1%,MgSO4.7H2O 0.05%(w/v). The optimum fermentation process was confirmed after orthogonal test: temperature= 35℃, initial pH=7.0, rotatory speed=190r/min, volume of medium=90ml/500mL, inoculum size=10%(v/v), fermentation time=74h, seed culture time=10h. Trehalose yield had been increased by 10%.The impact of osmotic pressure on enzyme activity and permeability of resting cells was an innovation in this paper. The definition of cell's permeability and its fomular were proposed innovatively, while the influence of osmotic pressure on permeability, enzyme activity and cell morphology was revealed for the first time, mechanism was discussed then. Test showed that high osmotic pressure could improve permeability and enzyme activity by a wide margin, and cell volume had been increased by 10-20 times to adjust to the condition. Trehalose yield had been improved for 10 times when adding NaCl 2%(w/v) to the mudium at 24h after fermentation, which provided an innovative method for trehalose production and industrilization.The enzymatic process of trehalose synthase from permeable resting cells was optimized, and the characteristics of the enzyme were studied in this paper. The optimum enzymatic reaction process was: pH=7.8, rotatory speed=180r/min, temperature=40℃, reaction time=8h. Na+ and K+ could activate the enzyme activity while heavy metal ions could strongly inhibit both of the activity and cell growth. The substrate specificity was obvious, and the enzyme should be kept under low temperature no more than 7 days because of its unstability.