Production Of Thermophilic Xylanase By Thermotoga Maritima And Recombinant Pichia Pastoris And Modification Of Enzyme

In the paper, the culture condition of Thermotoga maritima and recombinant Pichi pastoris was considerated firstly, chemicial modification and active sites of thermophilic xylanase B was studied after purification of xylanase. The main content and result of the study were as following:1. The effects of different factors on thermophilic xylanase production by Thermotoga maritima were studied in hungate tube. Corncob xylan and yeast extract were the best carbon and nitrogen resources, respectively. The best initial pH value of growth medium for xylanase production was at pH 6.5, and the best concentration of Nad was 2.28%. The highest xylanase activity was 0.50 U/mL. The zymogram of the protein confirmed that the secretion of Thermotoga maritima is so complex, which explain that xylanase production by Thermotoga maritima was unreasonable.2. The fermentation result of Pichia pastoris in blask and 5L fermentor is that, the best originaland the best initial pH value of production medium were 8 and at pH 5.0, severally, moreover, the best concentration of methanol was 1.5%. The recombinant enzyme activity was increased to more than 2 times with the addition of 0.1% Triton-114 in blask, and was 104time of Thermtoga maritima. When the feed velocity of methanol was 3mL/(h.L), the OD600 of Pichia pastoris reached to 260 after 10 days, and the xylanase activity was 108 U/mL, the protein was 207mg/L.3. The recombinant thermophilic xylanase was separated and purified according to sephadex G-25, concentration and D380 ion exchange chromatography. The molecular weight of the recombinant xylanase was estimated to be 41.8 kDa by SDS-PAGE, which was different from the xylanase from E. coli4. The result of chemical modification indicated that tryptophan residues and carboxyl residues might be involved in the active site of the recombinant thermophilic xylanase. The thermol stability of xylanase was correlative with carboxyl residues. Chemical modification of xylanase with N-bromosuccinimide and WRK revealed that 1 mol each of trytophan and glutamate / aspartate were essential for the activity.