出芽短梗霉G16酸性多糖的分离纯化、结构及性质研究
发布时间：2019-03-08 19:23
【摘要】：微生物多糖具备良好的物理性质及生物活性,且生产周期短,能利用规模化深层发酵实现工业化生产,具有广阔的工业用途。因此开发性质优良的新型微生物胞外多糖具有重要意义。出芽短梗霉(Aureobasidium pullulans)合成的中性普鲁兰多糖性质优良,能在多领域广泛应用。除此之外,出芽短梗霉还能合成酸性多糖。本实验室选育保藏有一株A.pullulans G16,能同时合成普鲁兰多糖和酸性多糖。本课题从A.pullulans G16的发酵液中分离出酸性多糖,对其纯化后进行结构和性质分析,并且研究了三种氮源对A.pullulans G16合成胞外多糖的影响。首先经过乙醇沉淀、酶法-Sevag法结合脱蛋白、十六烷基三甲基溴化铵沉淀等步骤从A.pullulans G16发酵液中分离酸性多糖。再采用乙醇分级沉淀、DEAE-52纤维素柱层析等纯化方法得到A.pullulans G16酸性多糖的主要组分。通过Sephadex G-200葡聚糖凝胶柱层析、紫外全波段扫描对纯化后的A.pullulans G16酸性多糖进行纯度鉴定,结果表明,经过纯化的酸性多糖为均一组分。通过高效凝胶渗透色谱法得到A.pullulans G16酸性多糖的重均相对分子质量为495450。综合化学分析法、气相色谱法、红外光谱法、高碘酸氧化、Smith降解、部分酸水解法、糖苷酶酶解法及液质联用等分析结果可得,该酸性多糖是一种含有12.76%甘露糖醛酸的吡喃杂多糖,主要由甘露糖、葡萄糖、半乳糖按摩尔比2.52:27.21:0.74组成。该酸性多糖的主链由甘露糖、葡萄糖、半乳糖组成,摩尔比为2.48:15.92:0.72,其中以β-(1→3)糖苷键连接的甘露糖及葡萄糖摩尔比为1.56:12.80,其余甘露糖、葡萄糖及半乳糖以α-(1→4)键连接。该酸性多糖的侧链由β-(1→6)糖苷键连接的葡萄糖组成。多糖的性质研究表明,A.pullulans G16酸性多糖溶解性较差,微溶于去离子水、NaOH与HCl溶液,不溶于DMSO、乙醚与丙酮。该酸性多糖水溶液表观粘度较大,且随溶液浓度增大而增加;在30-80℃以及pH 4.0-10.0范围内,溶液表观粘度稳定。该酸性多糖溶液表现出一定耐盐性,K+和Ca2+对其溶液表观粘度无明显影响。在0-1mg/m L浓度范围内,A.pullulans G16酸性多糖的还原能力以及对羟基自由基、超氧阴离子自由基、DPPH自由基的清除率随溶液浓度增大而增加,该酸性多糖对上述自由基清除率的IC50值分别为1.45 mg/mL、3.62 mg/mL及1.11 mg/mL。研究三种氮源对A.pullulans G16合成胞外多糖的影响,结果表明,(NH4)2SO4和高浓度(≥1.8 g/L)酵母粉有利于A.pullulans G16合成普鲁兰多糖,而NaNO3和低浓度(1.8 g/L)酵母粉则更有利于A.pullulans G16合成酸性多糖。
[Abstract]:Microbial polysaccharide has good physical properties and biological activity, and its production cycle is short. It can be used in large-scale submerged fermentation to achieve industrial production, and has broad industrial use. Therefore, it is of great significance to develop a new type of microbe exopolysaccharide with excellent properties. The neutral pullulan synthesized by (Aureobasidium pullulans) has good properties and can be widely used in many fields. In addition, it is also able to synthesize acidic polysaccharides by Phyllostachys communis. A strain of A.pullulans G16 was selected and preserved in our laboratory, which can synthesize both pullulan polysaccharide and acid polysaccharide simultaneously. In this paper, acidic polysaccharides were isolated from the fermentation broth of A.pullulans G16, and their structures and properties were analyzed after purification. The effects of three nitrogen sources on the synthesis of extracellular polysaccharides from A.pullulans G16 were studied. At first, acidic polysaccharides were separated from A.pullulans G16 fermentation broth by ethanol precipitation, enzymatic-Sevag deproteinization and cetyltrimethylammonium bromide precipitation. The main components of A.pullulans G16 acid polysaccharide were obtained by ethanol fractionation, DEAE-52 cellulose column chromatography and other purification methods. The purified acidic polysaccharide of A.pullulans G16 was identified by Sephadex-G _ (200) dextran gel column chromatography and UV-full-band scanning. The results showed that the purified acid polysaccharide was homogeneous. The average molecular weight of A.pullulans G16 acid polysaccharide was 495450 by high performance gel permeation chromatography. The results of chemical analysis, gas chromatography, infrared spectroscopy, periodate oxidation, Smith degradation, partial acid hydrolysis, glucosidase hydrolysis and liquid mass spectrometry were obtained. The acidic polysaccharide, which contains 12.76% mannitonic acid, is mainly composed of mannose, glucose and galactose in the molar ratio of 2.52 to 27.21 脳 0.74. The main chain of the acidic polysaccharide was composed of mannose, glucose and galactose, and the molar ratio of mannose to glucose was 2.48 脳 15.92 and 0.72, in which the molar ratio of mannose to glucose connected with 尾-(1) glycoside bond was 1.56% and 12.80, respectively. The remaining mannose, glucose and galactose were linked by a-(1)-4 bond. The side chain of the acid polysaccharide consists of 尾-(1)-glucoside-bonded glucose. The results showed that A.pullulans G16 acid polysaccharide had poor solubility and was slightly soluble in deionized water, NaOH and HCl solution, and insoluble in DMSO, ether and acetone. The apparent viscosity of the acidic polysaccharide aqueous solution is larger and increases with the increase of the concentration of the solution, and the apparent viscosity of the solution is stable in the range of 30? 80 鈩，
本文编号：2437129
[Abstract]:Microbial polysaccharide has good physical properties and biological activity, and its production cycle is short. It can be used in large-scale submerged fermentation to achieve industrial production, and has broad industrial use. Therefore, it is of great significance to develop a new type of microbe exopolysaccharide with excellent properties. The neutral pullulan synthesized by (Aureobasidium pullulans) has good properties and can be widely used in many fields. In addition, it is also able to synthesize acidic polysaccharides by Phyllostachys communis. A strain of A.pullulans G16 was selected and preserved in our laboratory, which can synthesize both pullulan polysaccharide and acid polysaccharide simultaneously. In this paper, acidic polysaccharides were isolated from the fermentation broth of A.pullulans G16, and their structures and properties were analyzed after purification. The effects of three nitrogen sources on the synthesis of extracellular polysaccharides from A.pullulans G16 were studied. At first, acidic polysaccharides were separated from A.pullulans G16 fermentation broth by ethanol precipitation, enzymatic-Sevag deproteinization and cetyltrimethylammonium bromide precipitation. The main components of A.pullulans G16 acid polysaccharide were obtained by ethanol fractionation, DEAE-52 cellulose column chromatography and other purification methods. The purified acidic polysaccharide of A.pullulans G16 was identified by Sephadex-G _ (200) dextran gel column chromatography and UV-full-band scanning. The results showed that the purified acid polysaccharide was homogeneous. The average molecular weight of A.pullulans G16 acid polysaccharide was 495450 by high performance gel permeation chromatography. The results of chemical analysis, gas chromatography, infrared spectroscopy, periodate oxidation, Smith degradation, partial acid hydrolysis, glucosidase hydrolysis and liquid mass spectrometry were obtained. The acidic polysaccharide, which contains 12.76% mannitonic acid, is mainly composed of mannose, glucose and galactose in the molar ratio of 2.52 to 27.21 脳 0.74. The main chain of the acidic polysaccharide was composed of mannose, glucose and galactose, and the molar ratio of mannose to glucose was 2.48 脳 15.92 and 0.72, in which the molar ratio of mannose to glucose connected with 尾-(1) glycoside bond was 1.56% and 12.80, respectively. The remaining mannose, glucose and galactose were linked by a-(1)-4 bond. The side chain of the acid polysaccharide consists of 尾-(1)-glucoside-bonded glucose. The results showed that A.pullulans G16 acid polysaccharide had poor solubility and was slightly soluble in deionized water, NaOH and HCl solution, and insoluble in DMSO, ether and acetone. The apparent viscosity of the acidic polysaccharide aqueous solution is larger and increases with the increase of the concentration of the solution, and the apparent viscosity of the solution is stable in the range of 30? 80 鈩，
本文编号：2437129
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