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摘 要: 聚苹果酸（ poly ( malic acid ) , PMA ）是以L-苹果酸为唯一单体通过酯键连接而成的高分子聚合物，它具有高度水溶性、生物相容性、生物可降解性及化学修饰性等优良性质，在药物载体、生物医学材料等领域具有广泛应用前景。 PMA可通过生物发酵法制备，它主要由短梗霉菌、绒泡黏菌等真核微生物合成，其中短梗霉属菌株具有优良的 PMA 合成能力。本研究拟筛选高产 PMA 的短梗霉属菌株，课题组从淮安市多地酸性丛林土壤中取样 70 份，根据短梗霉属的菌落形态特征进行初筛，筛选到 43 株具有酵母菌落形态的菌株，使用苹果酸脱氢酶催化法和高效液相色谱法复筛，筛选到 7 株可以合成 PMA 的菌株。选取产量最高的一株菌株进行鉴定， PCR 扩增其 ITS 序列后进行比对，确定该菌株为出芽短梗霉（ Aureobasidium pullulans ），其摇瓶发酵PMA产率为26.5 g/L。70299
毕业论文关键词：聚苹果酸，出牙短梗霉，菌株筛选与鉴定
Abstract：Poly ( malic acid ) is formed by L-malic acid as only monomer and linked each other with ester bond, PMA have lots of excellent characteristics, including highly water-soluble, biocompatible, biodegradable and can be chemically modified, etc. And PMA is being developed for pharmaceutical carrier, biomedical materials, etc based on these characteristics. PMA can be prepared by microbial fermentation, it is synthesized by Aureobasidium, Physarum polycephalum, etc eucaryotic organism, and Aureobasidium produced a high yield of PMA. This study aimed to screen Aureobasidium strains which can produce a high yield of PMA. We collect 70 samples from acidic forest soil. Based on the morphological features of Aureobasidium, 43 yeast-like strains were obtained, furthermore, with the use of catalysis of malate dehydrogenase and high performance liquid chromatography, 7 strains that can synthesize PMA were screened. The strain with highest yield of PMA was picked out and identified, its ITS sequence was amplified by PCR and aligned, and this strain was characterized as Aureobasidium pullulans, which produced 26.5 g/L PMA in shake flask scale.
Keywords：polymalic acid , Aureobasidium pullulans, strain screening and identification
目录
1.1 聚苹果酸的结构 5
1.2 聚苹果酸的性质 5
1.3 聚苹果酸的应用 6
1.4 聚苹果酸的生产方法 7
1.4.1 聚苹果酸的化学合成方法 7
1.4.2 聚苹果酸的生物合成法 7
第二章 材料与方法 9
2.1 材料 9
2.1.1 样本来源 9
2.1.2 主要试剂和仪器 9
2.1.3 培养基 10
2.2 产聚苹果酸菌株的筛选 11
2.2.1 土壤的选择 11
2.2.2 单菌落的分离 11
2.2.3 菌株的初筛 11
2.2.4 菌株的复筛 12
2.3 发酵产物的分析方法 12
2.3.1 发酵液的前处理 12
2.3.2 聚苹果酸的定量测定 12
2.4 菌种的鉴定 12
第三章 结果 14