一株海洋細(xì)菌renibacteriumsp.qd1中的殼聚糖酶及其產(chǎn)酶條件研究

1、 1 一株海洋細(xì)菌 Renibacterium sp. QD1 中的殼聚糖酶及其產(chǎn)酶條件研究 摘 要 殼聚糖(chitosan) 也叫甲殼胺，它是由氨基葡萄糖及少量乙酰氨基葡萄糖殘基通過(guò) β-1、4 糖苷鍵連結(jié)而成的高聚物，是幾丁質(zhì)進(jìn)一步脫乙?；纬傻漠a(chǎn)物。殼聚糖具有許多生理活性，但是由于多糖存在分子量大、不溶于水、生物利用度低等特點(diǎn)，其應(yīng)用受到很大限制。相比于多糖寡糖具有高效、安全、生物利用度高等優(yōu)點(diǎn)， 而且殼寡糖不但保留了殼聚糖所

2、具有的大部分性質(zhì)， 還具備獨(dú)特的生理活性，如：防治糖尿病、降低膽固醇、降血壓、消除脂肪肝等。傳統(tǒng)的化學(xué)法、物理法制備寡糖的方法存在著反應(yīng)條件難控制、 降解產(chǎn)物不均一、 環(huán)境污染嚴(yán)重等問(wèn)題，已成為制約殼寡糖應(yīng)用的瓶頸。相反，利用多糖降解酶制備殼寡糖，具有高效、特異、無(wú)污染、低成本等優(yōu)點(diǎn)，因此，對(duì)殼聚糖酶的研究具有重要的意義。目前市面上已經(jīng)商品化的殼聚糖酶非常少，而且存在活力低、生產(chǎn)成本高、價(jià)格昂貴等缺點(diǎn)，因此篩選具有工業(yè)化應(yīng)用潛力的新酶十

3、分必要。 我們利用殼聚糖選擇性培養(yǎng)基從青島海域篩選到一株高產(chǎn)殼聚糖酶的菌株，對(duì)菌落的形態(tài)和 16srDNA 分析顯示該菌株屬于 Renibacterium sp.菌屬，命名為Renibacterium sp. QD1。 菌株 QD1 為革蘭氏陽(yáng)性菌， 它能夠在以膠體殼聚糖為碳源的固體平板上生長(zhǎng)并且產(chǎn)生透明圈。對(duì) QD1 的發(fā)酵液采用鹽析、疏水層析等方法分離得到一種電泳純的殼聚糖酶 Csn-A，其純化效率達(dá) 67%以上，純化倍數(shù)為 7.8

4、9 倍。對(duì) Csn-A 的基本酶學(xué)性質(zhì)進(jìn)行了研究，表明該酶的分子量大小為26.1kDa， 酶的最適反應(yīng)溫度和 pH 分別為 55℃和 pH 5.3-6.5， 并且在<50℃、 pH4-8范圍內(nèi)比較穩(wěn)定。 該酶能夠被 Fe3+, Cu2+, Al3+, Zn2+ 和 SDS 強(qiáng)烈抑制， 但是 Mn2+能夠促進(jìn)該酶的活性（約提高 2 倍） 。對(duì)其降解產(chǎn)物分析發(fā)現(xiàn)其徹底降解產(chǎn)物為殼二糖和殼三糖。該酶的比活力可達(dá) 1574.5U/mg，經(jīng)

5、優(yōu)化發(fā)酵條件后粗酶液活力可達(dá) 300-400U/ml。 為了克隆得到 Csn-A 的編碼基因，本文首先嘗試了通過(guò)構(gòu)建基因組文庫(kù)的方法，用含有殼聚糖的 LB 培養(yǎng)基作為功能篩選的平板，結(jié)果顯示殼聚糖對(duì)宿主菌 DH5α的生長(zhǎng)抑制非常嚴(yán)重，平板無(wú)法發(fā)揮功能篩選的作用，因而不能夠篩選出陽(yáng)性克隆子。繼之，我們利用蛋白質(zhì) N 端測(cè)序技術(shù)對(duì)分離純化得3 Screening, identification and research on enzyme

6、production of the chitosanase produced from marine bacteria Renibacterium sp. QD1 Abstract Chitosan, an important marine polysaccharide, is limited to be used due to its low solubility, high molecular weight, and absorpt

7、ion rate. Chitooligosaccharides can be prepared through chemical method and physical method. However, the hydrolysis product is not homogenous and the hydrolyzing reaction is uncontrollable. Those methods also causing se

8、rious environmental pollution. Enzymatic degradation of chitosan should be a promising alternative to chemical method and physical method with high specificity and under mild conditions. Especially for it’s low cost and

9、no pollution, It is significante to do research of chitosanase . Currently, it was found a wide range of chitosanase. But because most of the chitosanase activity is low, and poor degradation efficiency, it is difficult

10、 to be applied to the industry for the preparation of chitooligosaccharides. Therefore, finding and screening yield novel and high activity chitosanase strains become a hot topic in the field of chitosan research.. Renib

11、acterium sp. QD1, a bacteria strain capable of hydrolyzing chitosan was isolated from the homogenate of small crustaceans in QingDao. Based on the 16srDNA sequence, QD1 was assessed to be Renibacterium sp., and is thus n

12、amed Renibacterium sp. QD1. Isolate QD1 is a gram-positive strain that formed a colorless colony on a screening plate. An extracellular chitosanase, Csn-A, was purified from the QD1 fermentation broth by (NH4)2SO4 saltin

13、g and Phenyl Sepharose HP. Csn-A is a monomeric enzyme and had a molecular weight of 26.1 kDa. The optimum pH and temperature of Csn-A were 5.3 to 6.5 and 55° C, respectively. The enzyme is stable under the conditio

14、ns of <50℃ and pH4-8. The enzyme was significantly activated by Mn2+ but was inhibited by Fe3+, Cu2+, Al3+, Zn2+, and SDS. The enzyme was purified 7.89-fold with a yield of 67%, and a specific activity of 1574.5 U/mg

15、proteins by performing two consecutive procedures. The strain QD1 which produced the largest clearing zone on the chitosan plate, show good signs of chitosanase production. Csn-A hydrolyzed N-deacetylated polymeric gluco