裂殖壶菌脂肪酸合成相关蛋白过表达菌株的构建和酶法破壁提取油脂工艺的研究

发布时间：2018-04-29 02:37

本文选题：裂殖壶菌 + 酶法　；参考：《中国海洋大学》2015年硕士论文

【摘要】：二十二碳六烯酸(Docosahexaenoic acid, DHA)属于n-3多不饱和脂肪酸(PUFA),能够促进神经系统的发育,对视觉和智力发育具有重要意义,此外还具有抑癌、抗炎、保护心脑血管等多方面的生物活性。裂殖壶菌(A urantiochytrium limacinum)是一种海洋真菌,其胞内油脂含量超过细胞干重的50%,DHA含量丰富,兼具安全质优、无毒易培养等优点,在健康保健和养殖饲料领域日益受到关注。本文主要从酶法破壁提取裂殖壶菌胞内油脂条件的优化、裂殖壶菌脂肪酸合成相关基因的克隆及构建超表达菌株等方面展开研究,为进一步提高裂殖壶菌的油脂含量及工业提取裂殖壶菌胞内油脂打下基础。本文以裂殖壶菌Aurantiochytrium limacinum OUC168为研究对象,比较了酶法、超声和索氏提取三种方法的油脂和DHA提取效率：其中主要研究了不同酶在不同条件下的破壁效率,结果显示,破壁效率由高到低分别是木瓜蛋白酶中性蛋白酶碱性蛋白酶纤维素酶葡聚糖酶和蜗牛酶溶菌酶。选取木瓜蛋白酶,中性蛋白酶和纤维素酶,并结合各破壁条件进行正交实验,确定了单酶的最优酶解条件为：木瓜蛋白酶,4000U/ml, pH6.5,45℃,4h,进一步将中性蛋白酶添加到酶切体系中组成复合酶,可以使酶解后的油脂提取率进一步提高,达56.37±1.41%,DHA提取率为12.82±0.66%；超声破碎法油脂提取率为58.2±3.91%,DHA提取率为12.02±2.09%：索氏提取法油脂提取率为61.42±1.90%,DHA提取率为12.06±0.74%。在油脂提取方面,索氏提取法破壁效率最高,其次为超声破碎法,最后为酶法；在DHA提取方面,酶法破壁效果最好,其次为索氏提取法,最后为超声破碎法。酰基载体蛋白(ACP)是裂殖壶菌脂肪酸合成过程中的重要蛋白,本文克隆了裂殖壶菌的acp基因,分析发现acp基因有1个开放阅读框,编码由152个氨基酸组成的酰基载体蛋白(ACP),通过预测,此蛋白分子质量约为16.44kDa,等电点5.63。ACP在PKS途径中是一种关键蛋白,主要作为脂肪酸合成过程中的酯酰基载体,蛋白的活性位点一般位于第37位的丝氨酸残基,其上具有磷酸泛酰巯基乙胺基结合位点。在脂肪酸合成过程中,酰基载体蛋白与辅基结合,作为载体将辅基结合到新生成的脂肪酸上,接着在酮酯酰-ACP合成酶、酮酯酰-ACP还原酶、烯酯酰-ACP脱水异构酶和烯酯酰-ACP还原酶等的催化作用下,合成多不饱和脂肪酸。酮酯酰-酰基载体蛋白还原酶(KR)、脱水异构酶(DH)也是裂殖壶菌脂肪酸合成过程中的重要酶,由本实验室前期克隆获得,并构建了表达载体p18SZPC-KR-Cm、p18SZPC-DH-Cm。本研究构建了含有acp基因的表达载体p18SZPC-ACP-Cm,分别将上述3个载体转化入裂殖壶菌,通过筛选和检测,得到基因超表达菌株,生物性状分析结果显示,各转化菌株的生物量大于出发菌株,KR+转化株生物量为19.69±2.36g/l；其次依次为ACP+转化株的19.02±2.27g/l；DH+转化株的18.73±0.83g/l：未转化菌株的生物量最低,为17.81±0.90g/l。3种转化菌株的油脂含量和DHA积累量具有显著提高,尤其是KR+和ACP+转化菌株,KR+转化株油脂含量达到48.07±4.31%：其次依次为ACP+转化株的42.64±2.64%：DH+转化株的39.00±5.00%；未转化菌株的油脂含量最低,为36.24±3.76%。DHA结果显示,KR+转化菌株总DHA含量为8.47±2.26%,高于ACP+转化株的7.98±0.39%,高于DH+转化株的7.19±1.75%；而未转化菌株的总DHA含量为6.71±1.60%；KR+, ACP+, DH+转化菌株的总DHA含量分别比未转化菌株高出26.23%,18.93%和7.15%。本研究优化了酶法破壁的条件为工业提取裂殖壶菌胞内油脂和DHA提供了实验依据：通过克隆裂殖壶菌脂肪酸合成相关基因及构建超表达菌株,为从分子水平提高裂殖壶菌的DHA含量奠定了基础。
[Abstract]:Twenty-two carbon and six enoic acids (Docosahexaenoic acid, DHA) belong to n-3 polyunsaturated fatty acids (PUFA), which can promote the development of the nervous system and have important significance to visual and intellectual development. In addition, it also has biological activity in many aspects, such as cancer suppression, anti-inflammatory and protecting the heart and brain vessels. The A urantiochytrium limacinum (A urantiochytrium limacinum) is a marine fungus. The intracellular oil content is more than 50% of the cell dry weight, the content of DHA is rich, and it has the advantages of high safety and innocuity. It has attracted more and more attention in the field of health care and feed. This paper mainly extracts the conditions of the intracellular grease from the enzyme broken wall, and the cloning of the related genes of fatty acid synthesis and the construction of the Super Table of the fatty acid synthesis of hyphis. In order to further improve the oil content of the strain and the base of the intracellular oil extraction of the strain of the strain of the strain of the strain of the strain of the strain of the strain of the fungus, Aurantiochytrium limacinum OUC168 was used as the research object. The oil and DHA extraction efficiency of the three methods: enzyme method, ultrasonic and Soxhlet extraction were compared. The wall breaking efficiency of different enzymes in different conditions showed that the wall breaking efficiency was from high to low was the papain neutral protease alkaline protease glucan enzyme glucan enzyme and snail enzyme lysozyme. Papain, neutral protease and cellulase were selected, and the orthogonal experiment was carried out in combination with the wall breaking conditions to determine the most single enzyme. The optimum enzymolysis conditions are: papain, 4000U/ml, pH6.5,45, 4h, and adding neutral protease to the enzyme cutting system to make a compound enzyme, which can further improve the extraction rate of oil after enzymatic hydrolysis, reach 56.37 + 1.41%, the extraction rate of DHA is 12.82 + 0.66%, the extraction rate of oil is 58.2 + 3.91%, and the extraction rate of DHA is 12.02 + 2.09%. The extraction efficiency of Soxhlet extraction was 61.42 + 1.90%, the extraction rate of DHA was 12.06 + 0.74%. in oil extraction. The Soxhlet extraction method had the highest wall breaking efficiency, followed by ultrasonic breakage, and finally the enzyme method. In the DHA extraction, the enzyme breaking effect was the best, followed by Soxhlet extraction, and finally the ultrasonic breakage method. The acyl carrier protein (ACP) was the crack. The important protein in the fatty acid synthesis of Paragonimus was cloned, and the ACP gene was cloned in this paper. It was found that the ACP gene has 1 open reading frames and encodes the acyl carrier protein (ACP), which is composed of 152 amino acids. It is predicted that the mass of the protein is about 16.44kDa, and the isoelectric point 5.63.ACP is a key protein in the PKS pathway. As the ester acyl carrier in the fatty acid synthesis process, the active site of the protein is generally located in the thirty-seventh bit serine residue, which has a phosphoryl acid sulfhydryl ethylamine binding site. In the process of fatty acid synthesis, the acyl carrier protein is combined with the cofactor to bind the cofactor to the newly formed fatty acid as a carrier and then in the ketoyl -AC. P synthetase, ketoacyl -ACP reductase, allyl -ACP dehydrating isomerase and allyl -ACP reductase, the synthesis of polyunsaturated fatty acids, ketoylacyl acyl carrier protein reductase (KR) and dehydrated isomerase (DH) are also important enzymes in the fatty acid synthesis of fission bacteria, obtained by early cloning and constructed in our laboratory. The expression vector p18SZPC-KR-Cm, p18SZPC-DH-Cm., which constructed the expression vector p18SZPC-ACP-Cm containing ACP gene, transformed the 3 vectors into the strains of hypo hypomiapus respectively. Through screening and detection, the gene overexpression strains were obtained. The biotrait analysis results showed that the biomass of all the transformed strains was larger than the starting strain and the biomass of the KR+ transformed strain. It was 19.69 + 2.36g/l, followed by 19.02 + 2.27g/l of ACP+ transformation plant and 18.73 + 0.83g/l of DH+ transformation strain: the biomass of untransformed strain was the lowest. The oil content and DHA accumulation of 17.81 + 0.90g/l.3 strains were significantly improved, especially KR+ and ACP+ transformation strains, and the oil content of KR+ transformants reached 48.07 + 4.31%: The 42.64 + 2.64%:DH+ transformation strain of the ACP+ transformation strain was 39 + 5%, and the oil content of the unconverted strain was the lowest, which was 36.24 + 3.76%.DHA. The total DHA content of the KR+ transformation strain was 8.47 + 2.26%, higher than 7.98 + 0.39% of the ACP+ transformation strain and 7.19 + 1.75% of the DH+ transformation strain, while the total DHA content of the unconverted strain was 6.71 + 1.60. The total DHA content of KR+, ACP+, DH+ transformation strains was 26.23% higher than that of the untransformed strain, 18.93% and 7.15%., which optimized the condition of enzyme breaking to provide experimental basis for the industrial extraction of intracellular oil and DHA of hyphimyphus. It laid the foundation for improving the DHA content of the bacteria.

【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q939.9

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