重组聚球藻固碳和生产海带多糖酶的研究

发布时间：2018-03-06 16:18

  本文选题：聚球藻7002　切入点：LamA基因　出处：《中国地质大学(北京)》2017年博士论文　论文类型：学位论文

【摘要】：矿石燃料燃烧排放到大气中的CO_2对全球气候的变化起了决定性作用。由于蓝藻具有生长速度快、CO_2固定速率高、其生物质和细胞产物具有较高的附加值等特点,利用蓝藻生物固碳具有突出的应用价值。本研究以蓝藻为受体,将海栖热胞菌Msb8中合成海带多糖酶的LamA基因转入聚球藻7002中。实现了利用密闭型光生物反应器培养蓝藻固碳和生产高附加值重组蛋白的双重目的。本文在成功构建了转LamA基因的重组聚球藻的基础上,对重组聚球藻生产的LamA编码的海带多糖酶的酶活性和理化特性进行了研究。结果表明,重组海带多糖酶的最大相对酶活性为4.325 U?mg-1 dry mass。其最适酶反应条件为pH=8.0,70℃。重组海带多糖酶在pH值为6-8的范围内具有较高的活性,同时表现出很强的耐热性。在80℃和90℃其酶活性的半衰期分别为40 min和20 min。根据密闭型光生物反应器的优缺点设计了改良型搅拌罐式光生物反应器。通过空载试验和完全析因设计,对反应器的动力学特性和重组聚球藻7002的生长特性进行分析,确定了对重组聚球藻Xmax的主要影响因素依次为:光照强度CO_2浓度温度*温度温度CO_2浓度*光照强度;对μmax的主要影响因素依次为:CO_2浓度光照强度温度。反应器的最佳运行条件为进气量为0.5 L·min-1,进气CO_2浓度为5%,光照强度为300μmol·m-2·s-1,温度为30℃。Xmax和μmax分别达到3.929 g·L-1和0.015 h-1。此外,当光照周期由连续光照变为间隙光照,重组聚球藻的比生长速率、产率、CO_2固定速率和最大生物质浓度均下降。高浓度CO_2(5%)对重组聚球藻7002的生长和海带多糖酶的相对活性均具抑制作用。虽然重组聚球藻在进气CO_2浓度为15%时仍可生长,但其Xmax、μmax和海带多糖酶相对酶活性分别为最适条件下的36.9%、40.7%和75.1%。通过pH控制和高浓度CO_2驯化培养的方法均可提高重组聚球藻7002的Xmax和μmax。结果显示,加入碳酸-碳酸氢钠缓冲液效果最佳,比未加入缓冲液的情况下(2.507 g·L-1)Xmax和μmax分别提高了33.3%和15.8%。但由于pH值的升高,进气CO_2的固定效率降低,高浓度CO_2驯化的重组聚球藻7002初代Xmax和μmax比控制组提高了24.4%和11.2%。并且数代培养后,其适应性稳中有升。
[Abstract]:The CO_2 released from the combustion of mineral fuels into the atmosphere plays a decisive role in global climate change. Because cyanobacteria have the characteristics of fast growth rate and high fixation rate of COSP, their biomass and cell products have higher added value, etc. Biological carbon sequestration by cyanobacteria is of great application value. In this study, cyanobacteria were used as receptors. The LamA gene of synthesizing kelp polysaccharide enzyme from Msb8 of Pyrocystis martensii was transferred into Chlorella 7002. The purpose of carbon sequestration of cyanobacteria and production of high value-added recombinant protein were achieved by using closed photobioreactor. On the basis of the recombinant Chlorella sp. LamA gene, The enzyme activity and physical and chemical properties of the LamA encoded kelp polysaccharide enzyme produced by recombinant Chlorella spp were studied. The results showed that the maximum relative enzyme activity of the recombinant kelp polysaccharide enzyme was 4.325 U? The optimum reaction conditions of mg-1 dry were as follows: pH = 8.0 ~ 70 鈩，

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