Screening And Enzymology Properties Of An Thermo-stable Xylanase Producing Strain From JiMo Hot Spring

Thermo-stable xylanase can hydrolyze xylans into xylo-oligosaccharides and xylose, and has great prospect for applications in feed industry , paper and pulp industry, food industry and environment science. With the development and application of the new technologies , such as molecular biology, structural biology and protein engineering, many progresses have been made in the research of structures and functions of xylanase. The research will make a basis to its further structure-functional study, its industrialization and application.Six xylanase producing strains(L₁, L₂, L₃, L₄, L₅, L₆) which could produce obvious transparent zones on selective medium were obtained from JiMo Hot Spring and the xylanase activity could be identified by these transparent zones. By analyzing the enzymatic characteristics,we find L₂ was the best strain among them .Then the effects of following factors : the addition method and dose of hemicellulose,temperature ,pH, carbon sources, nitrogen sources, surfactant Tween-80, mental iron and ferment time on L₂ enzyme production were studied . In this way, the efficiency of primary screening were effectively improved. The strain of L₂, which was isolated from related JiMo hot spring samples, showed the highest xylanase-producing ability (720.6IU/mL). The results showed that the optimum temperature of this xylanase was 75°C and its optimum reaction pH was 10.0. The effects of metal ions on xylanase showed that Li⁺, Mg²⁺, Ca²⁺ could increase its activity. On the other hand, Ba²⁺, Fe³⁺, Ag⁺, Sr⁺ inhibited the activity of xylanase. The inhibition of EDTA on xylanase was remarkable, which suggested there were metal ions in the active centre of xylanase.The orthogonal design has applied extensively to optimize xylanase fermentation of combination factors. In this paper, an orthogonal design of L9(4)3 was designed to arrange four factors in 3 levels experiments—fermentation temperature (28°C, 37°C, 50°C),fermentation time (48h, 96h, 144h) , pH of fermentation broth (6.09, 7.54, 8.69) and strains to optimize the xylanse fermentation . A simple fermentation device was designed and fermentations were done according to the orthogonal design. The activity of xylanase produced in fermentation process was determined by DNS (3, 5-nitro-2 salicylate) colorimetric method according to a xylose standard curve .The results of orthogonal experiment were evaluated by range analysis. The factors impacting xylanase production were: strains > time > pH > temperature. The optimal ferment temperature was 50°C, the optimal fermentation time was 144 hours, the optimal pH for the broth was 8.69, and the optimal strain was L₂. The fermentation conditions have been optimized, and xylanase production has been raised.This paper also focused on some factors of solid-state fermention about L₂,such as solid-state ferment time, temperature,thermostability, pH, PHstability,bran content and storage time. The optimal solid-state conditions were as follows: solid-state ferment time:144h, ferment temperature: 70°C, ferment pH: 7.54, bran content :2/3, storage time :more than 120d.By analysis, solid-state fermention can raise the xylanase activity.