發(fā)布時(shí)間：2018-04-29 02:37

  本文選題：裂殖壺菌 + 酶法　； 參考：《中國(guó)海洋大學(xué)》2015年碩士論文

【摘要】：二十二碳六烯酸(Docosahexaenoic acid, DHA)屬于n-3多不飽和脂肪酸(PUFA),能夠促進(jìn)神經(jīng)系統(tǒng)的發(fā)育,對(duì)視覺和智力發(fā)育具有重要意義,此外還具有抑癌、抗炎、保護(hù)心腦血管等多方面的生物活性。裂殖壺菌(A urantiochytrium limacinum)是一種海洋真菌,其胞內(nèi)油脂含量超過細(xì)胞干重的50%,DHA含量豐富,兼具安全質(zhì)優(yōu)、無毒易培養(yǎng)等優(yōu)點(diǎn),在健康保健和養(yǎng)殖飼料領(lǐng)域日益受到關(guān)注。本文主要從酶法破壁提取裂殖壺菌胞內(nèi)油脂條件的優(yōu)化、裂殖壺菌脂肪酸合成相關(guān)基因的克隆及構(gòu)建超表達(dá)菌株等方面展開研究,為進(jìn)一步提高裂殖壺菌的油脂含量及工業(yè)提取裂殖壺菌胞內(nèi)油脂打下基礎(chǔ)。本文以裂殖壺菌Aurantiochytrium limacinum OUC168為研究對(duì)象,比較了酶法、超聲和索氏提取三種方法的油脂和DHA提取效率：其中主要研究了不同酶在不同條件下的破壁效率,結(jié)果顯示,破壁效率由高到低分別是木瓜蛋白酶中性蛋白酶堿性蛋白酶纖維素酶葡聚糖酶和蝸牛酶溶菌酶。選取木瓜蛋白酶,中性蛋白酶和纖維素酶,并結(jié)合各破壁條件進(jìn)行正交實(shí)驗(yàn),確定了單酶的最優(yōu)酶解條件為：木瓜蛋白酶,4000U/ml, pH6.5,45℃,4h,進(jìn)一步將中性蛋白酶添加到酶切體系中組成復(fù)合酶,可以使酶解后的油脂提取率進(jìn)一步提高,達(dá)56.37±1.41%,DHA提取率為12.82±0.66%；超聲破碎法油脂提取率為58.2±3.91%,DHA提取率為12.02±2.09%：索氏提取法油脂提取率為61.42±1.90%,DHA提取率為12.06±0.74%。在油脂提取方面,索氏提取法破壁效率最高,其次為超聲破碎法,最后為酶法；在DHA提取方面,酶法破壁效果最好,其次為索氏提取法,最后為超聲破碎法。�；�載體蛋白(ACP)是裂殖壺菌脂肪酸合成過程中的重要蛋白,本文克隆了裂殖壺菌的acp基因,分析發(fā)現(xiàn)acp基因有1個(gè)開放閱讀框,編碼由152個(gè)氨基酸組成的�；�載體蛋白(ACP),通過預(yù)測(cè),此蛋白分子質(zhì)量約為16.44kDa,等電點(diǎn)5.63。ACP在PKS途徑中是一種關(guān)鍵蛋白,主要作為脂肪酸合成過程中的酯酰基載體,蛋白的活性位點(diǎn)一般位于第37位的絲氨酸殘基,其上具有磷酸泛酰巰基乙胺基結(jié)合位點(diǎn)。在脂肪酸合成過程中,酰基載體蛋白與輔基結(jié)合,作為載體將輔基結(jié)合到新生成的脂肪酸上,接著在酮酯酰-ACP合成酶、酮酯酰-ACP還原酶、烯酯酰-ACP脫水異構(gòu)酶和烯酯酰-ACP還原酶等的催化作用下,合成多不飽和脂肪酸。酮酯酰-�；�載體蛋白還原酶(KR)、脫水異構(gòu)酶(DH)也是裂殖壺菌脂肪酸合成過程中的重要酶,由本實(shí)驗(yàn)室前期克隆獲得,并構(gòu)建了表達(dá)載體p18SZPC-KR-Cm、p18SZPC-DH-Cm。本研究構(gòu)建了含有acp基因的表達(dá)載體p18SZPC-ACP-Cm,分別將上述3個(gè)載體轉(zhuǎn)化入裂殖壺菌,通過篩選和檢測(cè),得到基因超表達(dá)菌株,生物性狀分析結(jié)果顯示,各轉(zhuǎn)化菌株的生物量大于出發(fā)菌株,KR+轉(zhuǎn)化株生物量為19.69±2.36g/l；其次依次為ACP+轉(zhuǎn)化株的19.02±2.27g/l；DH+轉(zhuǎn)化株的18.73±0.83g/l：未轉(zhuǎn)化菌株的生物量最低,為17.81±0.90g/l。3種轉(zhuǎn)化菌株的油脂含量和DHA積累量具有顯著提高,尤其是KR+和ACP+轉(zhuǎn)化菌株,KR+轉(zhuǎn)化株油脂含量達(dá)到48.07±4.31%：其次依次為ACP+轉(zhuǎn)化株的42.64±2.64%：DH+轉(zhuǎn)化株的39.00±5.00%；未轉(zhuǎn)化菌株的油脂含量最低,為36.24±3.76%。DHA結(jié)果顯示,KR+轉(zhuǎn)化菌株總DHA含量為8.47±2.26%,高于ACP+轉(zhuǎn)化株的7.98±0.39%,高于DH+轉(zhuǎn)化株的7.19±1.75%；而未轉(zhuǎn)化菌株的總DHA含量為6.71±1.60%；KR+, ACP+, DH+轉(zhuǎn)化菌株的總DHA含量分別比未轉(zhuǎn)化菌株高出26.23%,18.93%和7.15%。本研究?jī)?yōu)化了酶法破壁的條件為工業(yè)提取裂殖壺菌胞內(nèi)油脂和DHA提供了實(shí)驗(yàn)依據(jù)：通過克隆裂殖壺菌脂肪酸合成相關(guān)基因及構(gòu)建超表達(dá)菌株,為從分子水平提高裂殖壺菌的DHA含量奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：中國(guó)海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：Q939.9

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