利用嗜熱性Bacillus subtilis TF-141 發酵香菇柄所生產幾丁質脢之分離純化及其應用

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Title:	利用嗜熱性Bacillus subtilis TF-141 發酵香菇柄所生產幾丁質脢之分離純化及其應用 The Purification and Application of Chitinases Production from Shiitate Stipe by Thermophilie Bacillus subtilis TF-141
Authors:	張文智 Chang, Wen-Chih Chang, W. C. (行政院國家科學委員會) (東方技術學院食品科技系)
Keywords:	香菇柄;幾丁質脢;幾丁寡醣;Shiitate stipe;Chitinase;Chitooligosaccharide
Date:	2005
Issue Date:	2009-07-22 09:55:36 (UTC+8)
Abstract:	從台灣南部溫泉中篩選到能以香菇柄粉為主要營養源，且嗜熱性之菌株Bacillus subtilis TF-141，以2% 香菇柄粉、0.1% K2HPO4、0.05% MgSO4‧7H2O 為培養基，接種B. subtilis TF-141 之菌株，於 60°. 培養1 天，發現具有高耐熱性幾丁質. (熱安定為70-80°.) 活性。幾丁質.，一種能將幾丁質分解成低分子寡糖之酵素，廣存於微生物，被發表之幾丁質.生產菌係使用懸態幾丁質或幾丁質作為碳源。唯於商業化之立場而言，便宜且具耐熱性之幾丁質.將更受矚目於用來生產幾丁寡醣。若能利用廉價之香菇柄作為生產幾丁質.之碳源，則能增加此農業上副產物之經濟價值。所擬探討的因子包括最適反應酸鹼值、最適反應溫度、酸鹼安定性、熱安定性、半衰期等，並利用HPLC 分析所得寡醣產物之變化。簡而言之，本計畫之目的為： 1. 利用廉價之香菇柄 (不需滅菌) 經由B. subtilis TF-141 發酵生產耐熱性幾丁質.。 2. 建立最適生產幾丁寡醣之條件與方法。 3. 提升國內生物性農業技術，並提供產業界研發更經濟有效幾丁寡醣之製程。The thermophilie Bacillus subtilis TF-141 was isolated from the hot spring of southern Taiwan. B. subtilis TF-141 excreted thermostable chitinases when cultured in a medium containing 2% shiitate stipe powder, 0.1% K2HPO4 and 0.05% MgSO4?7H2O for 1 day at 45.degree.C. The chitinase, purified by sequential chromatography, had molecular weight of 31 kDa, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI of for the chitinase was 5.2. The optimum pH, optimum temperature, pH stability, and thermal stability of chitinase were pH 7, 50.degree.C, pH 6-8, and 70-80.degree.C. TF-141 chitinase showed higher hydrolyzing activity against ethylene glycol chitin and little activity against glycol chitosan. There was no enzymatic activity on p-NP-GlcNAc. According to this nomenclature system, TF-141 chitinase is not a beta-N-acetyl-hexosaminidases because it does not hydrolyze p-NP-GlcNAc. It could be an endochitinase since it catalyzes release of N-acetylchitotriose from colloidal chitin. Chitinases, a group of enzymes capable of degrading chitin directly to low molecular weight products, have been shown to be produced by a number of microorganisms. Chitin and its derivatives hold economic value because of their versatile biological and agrochemical application. Almost all of the reported chitinase-producing strains will use chitin or colloidal chitin as a carbon source. Commercial interest in the utilization of chitin and its derivatives has led to the need for inexpensive, reliable sources of active and thermostable chitinase preparation. The production of inexpensive chitinolytic enzymes is an important element in the utilization of shiitate stipe that promotes the economic value of the agricultural byproducts. The pH stability，thermal stability, optimum pH, optimum temperature, optimum pH, and half-life of the chitinases will be study. The compositions of the oligosaccharides obtained by enzymatic hydrolysis were analyzed by HPLC. In short, the objectives of this project are: 1. To investigate the microbial reclamation of inexpensive shiitate stipe for the production of thermostable chitinases. 2. To establish the conditions and method of producing chitooligosaccharides. 3. To help advance the technology of producing chitooligosaccharides. The results could be used to help our agriculture to develop more competitive process for chitooligosaccharides.
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