本文選題：南極菌 + 卡拉膠酶　； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：本論文從85株南極細(xì)菌中篩選獲得12株具有產(chǎn)卡拉膠酶活性的菌株,并對(duì)高活性菌株R11-5進(jìn)行種屬鑒定,優(yōu)化其發(fā)酵條件;通過(guò)對(duì)該菌全基因組測(cè)序分析,克隆獲得卡拉膠酶基因Car1853,并實(shí)現(xiàn)其異源高效表達(dá);純化重組卡拉膠酶,系統(tǒng)研究其酶學(xué)性質(zhì),分析該酶的降解特性,闡明其降解產(chǎn)物及其類(lèi)型,以期為卡拉膠酶和卡拉寡糖的工業(yè)化生產(chǎn)提供理論和技術(shù)支持。本實(shí)驗(yàn)首先對(duì)篩選出的產(chǎn)高活性卡拉膠酶菌株R11-5進(jìn)行種屬鑒定,根據(jù)形態(tài)學(xué)和16S rDNA法的種屬鑒定結(jié)果表明該南極菌屬于交替單胞菌屬(Alteromonas)。通過(guò)響應(yīng)面分析法對(duì)菌株優(yōu)化培養(yǎng)單因素實(shí)驗(yàn)的結(jié)果最佳培養(yǎng)條件為溫度15.7℃、p H7.0、酵母粉0.6%、牛肉膏1.17%、卡拉膠1.05‰、CaCl25.78 mmol/L,接種量為2%,在此培養(yǎng)條件下,能夠得到理論上最大酶活值為56.972 U·mL-1,經(jīng)過(guò)優(yōu)化培養(yǎng)后酶活提高了1.7倍。通過(guò)對(duì)該菌全基因組測(cè)序分析,克隆獲得了卡拉膠酶基因Car1853,采用基因工程手段構(gòu)建重組質(zhì)粒pET30a-Car1853,,并成功轉(zhuǎn)化大腸桿菌BL-21。采用Ni柱技術(shù)純化獲得單一條帶的重組卡拉膠酶,SDS-PAGE顯示其分子量為42 kDa;酶學(xué)性質(zhì)研究表明,該酶最佳反應(yīng)溫度為55℃,最佳pH為7.0,且多種金屬離子對(duì)其活性具有抑制作用。薄層層析法初步表明該卡拉膠酶裂解卡拉膠的終產(chǎn)物主要是卡拉膠二糖。
[Abstract]:In this paper, 12 strains with carrageenase-producing activity were obtained from 85 Antarctic bacteria, and the strain R11-5 with high activity was identified to optimize the fermentation conditions, and the whole genome of R11-5 was sequenced. The carrageenase gene Car1853 was cloned, and its heterologous expression was achieved. The recombinant carrageenase was purified, its enzymatic properties were systematically studied, the degradation characteristics of the enzyme were analyzed, and its degradation products and types were elucidated. In order to provide theoretical and technical support for the industrial production of carrageenase and carrageenase. In this experiment, the strain R11-5 producing high activity carrageenase was first identified. According to the morphological and 16s rDNA identification results, the Antarctic strain belongs to Alteromonas alternata. The results of single factor experiment with response surface analysis were as follows: temperature 15.7 鈩，

本文編號(hào)：1882436