Optimization Of Strain For Trehalose Synthase Production And Analyzation Of Enzyme Properties

Trehalose, molecular formula C12H22O11?2H2O, is a non-reducing disaccharide widespread in many organism cells all over the world. It’s a biological metabolite in cells under hostile environment and has a high concentration in the organisms such as archaea,fugus and animal and plant cells living in extreme environment. Trehalose can protect cells from hostile conditions and as energy of cells. The protection aspects of trehalose include maintaining structure and function of biomembrane, protein and nucleic acid and holding the osmotic pressure inside and outside of cells and preventing the leaving of nutrients in cells. Trehalose can use in protecting medical biological products and strengthening the stress resistance of crops using the gene engineering to cultivate the crops with saline-alkaline tolerance and garden stuff with antifreeze ability because of the properties of trehalose. Trehalose also can use in food industry because of its non-reducing without maillard reaction. It’s meaningful to have a research in trehalose.The method for trehalose production is using enzyme in industry. This method is a good method with high production efficiency, saving time and labor and without pollution.The key to the method is obtaining the relative enzymes with high activity for trehalose production. There are 7 enzymes with 5 pathways for trehalose production such as Tre S pathway(trehalose synthase, Tre S), Tre YZ pathway(MTSase and MTHase), Ots AB(TPS,TPP), Tre T pathway(Tre T) and Tre P(Tre P). Many researches on trehalose focused on finding the strains with trehalose synthesis pathways and improving the strains to obtain the high yield of trehalose and then extract trehalose or enzymes for trehalose production.The main improving methods including traditional mutagenesis and gene engineering can obtain the mutants with high yield of trehalose or enzymes.This paper used the Corynebacterium glutamicum with 3 pathways(Tre S pathway、Tre YZ pathway and Ots AB pathway) as wild strain for trehalose production and obtained mutants with high yield of trehalose and trehalose synthase using traditional mutagenesis method. Then we extracted and purified the enzymes for trehalose production from cell and made an analysis for these enzymes. At last, we optimized the medium composition using response surface methodology for production of trehalose synthase and then optimized the fermentation conditions using “one at a time” method. The mutant with a good genetic stability has a high trehalose synthase with 621U/m L under optimum conditions 1.88 times than the former without optimum conditions.(1) The mutant obtained using traditional mutagenesis method has a raise substantially in yield with 1.28 g /100 g dry weight·h-1 and the wild strain with 0.45 g /100 g dry weight·h-1. The results of pass test shows that the mutant has a good genetic stability.(2)To use enzymes for trehlose production effectively, it’s necessary to assay theenzymes properties. We optimized the reaction conditions of trehalose synthse using orthogonal experiment because of the reversible activity. The conversion rate from maltose to trehalose reached 81.1% using trehalose synthase. The optimum p H value for trehalose synthase, MTSase, MTHase, TPS and TPP was 7.0, 7.5, 6.5, 6.5, 7.5 and the range of p H6.5-8.0, p H6.5-7.5, p H6.0-7.5, p H6.0-7.5, p H6.5-8 with over 80% of highest activity,respectively. The optimum reaction temperature for them was 35℃, 35℃, 40℃, 35℃,35℃ with maintaining higher reactivity at the range of 30-35℃, 35-40℃, 30-45℃,25-40℃, 30-35℃, respectively.(3)In this paper, we optimized the medium composition using fractional factoral design(FFD) and central composite design(CCD) and then optimized the fermentation conditions using one factor at a time method to obtain the maximum trehalose synthase production. The optimum medium composition was: maltose(22g/L), beef extract(7.49g/L), Na Cl(13.36g/L), K2HPO4(11.33g/L) and peptone(15.00g/L). The optimum fermentation conditions: The initial p H 7.0, incubated at 34℃ for 30 h, inoculated with1×108/m L and the velocity of rotary shaker was 200 rpm, the culture volume 70 m L/250 m L.The maximum trehalose synthase activity obtained finally was 621U/m L.