【作者】 段效辉；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2007， 博士

【摘要】 普鲁兰多糖(pullulan)是一种应用价值较高的微生物胞外多糖,除了普鲁兰多糖本身在农业、食品工业、制药工业和化妆品工业中具有重要用途外,经过化学改造的各种普鲁兰衍生物还是很重要的抗病毒、抗血栓、抗血凝固、抗肿瘤药物的工业原料。但由于现有工业化生产菌株产量低,实际应用就受到了很大的限制。根据国内外文献报道,现有的普鲁兰生产菌株主要是普鲁兰类酵母(Aureobasidium pullulans),其产量一般在3.0%-5.0%(w/v)左右,并且现已报导的产普鲁兰菌株发酵产生胞外多糖时容易产生色素,给产品的后处理带来极大的不便,随着发酵时间的进行,发酵液的pH不断下降,从而影响了胞外多糖的产生。因此,获得具有新特征和产量更高的普鲁兰生产菌株和研究这些菌株发酵最佳条件的工作就变得非常重要。目前有关A. pullulans产普鲁兰多糖的机理还了解很少,与普鲁兰合成相关的酶也鲜有报导。本研究主要利用生物化学方法测定了不同胞外多糖产量细胞中UDP-葡萄糖库的大小,有关的酶活性大小,以便了解UDP-葡萄糖库在真核微生物调节过量合成胞外普鲁兰中的作用,为进一步提高真核微生物胞外多糖的产量提供理论依据。这必将对普鲁兰多糖的工业化生产产生深远的意义。本实验室保存的菌种Y68在250mL摇瓶中发酵培养56h普鲁兰多糖产量可达52.47g/L。针对此菌株在发酵时的营养、初始pH、通气量和转速等条件的需求,我们在2L发酵罐中进行了发酵条件的研究,发现菌株Y68发酵产普鲁兰的最适条件为:葡萄糖碳源,初始pH7.0,最佳转速300rpm,最佳通气量为6.5 L/min。在此条件下,得到的普鲁兰最高产量为65 g/L。菌株Y68是目前所有酵母中普鲁兰产量最高的菌株。恒定pH对该菌株胞外普鲁兰多糖的生产同样有影响,最适生产恒定pH为6.0,但产量低于其在pH 7.0情况下的产量。在后面的研究中发现,胞内几种酶活力的差异可以解释该现象。经微生物常规方法鉴定以及分子生物学方法18S rDNA、ITS序列的克隆测序证实:Y68菌株应属Aureobasidium pullulans。为了确定胞外普鲁兰产量与胞内各酶活力水平间的关系,本文研究了不同培养条件、产不同量普鲁兰多糖情况下Y68菌株细胞中UDPG含量的变化以及在不同培养条件下细胞中普鲁兰多糖合成酶、葡萄糖基转移酶、UDPG焦磷酸化酶以及磷酸葡萄糖变位酶活力的差异。研究结果发现:1.研究Y68菌株在不同培养条件下(包括不同的pH、不同的碳源)和不同菌株在相同培养条件下胞外多糖产量以及其与细胞内UDPG含量的关系,结果发现:胞外普鲁兰多糖产量与细胞内UDPG含量成反比例关系,即胞外多糖量高的情况下胞内的UDPG含量反而低。反之多糖量高的情况下,胞内的UDPG含量反而较高。这一结果与乳酸菌中的情况恰好相反。2. Y68菌株在不同碳源培养基中培养,细胞内普鲁兰合成酶、葡萄糖基转移酶、UDPG焦磷酸化酶以及磷酸葡萄糖变位酶的酶活力都随胞外多糖产量的增加而升高,表明这些酶对胞外多糖生产有促进作用。Y68菌株在不同pH条件培养80小时结果发现,细胞中除普鲁兰合成酶活力有一个先升高后降低的过程外,其它酶都是逐渐升高后趋于稳定。总体看来这些胞内酶都对胞外普鲁兰多糖的累积有明显的促进作用。本研究纯化的Y68菌株细胞内葡萄糖基转移酶,其Native-PAGE表观分子量为350 kDa,而SDS-PAGE表观分子量为50.8 kDa。该酶的最适作用pH、温度分别为6.0、和40℃。化合物二硫苏糖醇(DTT)对酶有保护作用,而EDTA、碘乙酸和PMSF则对酶有明显的抑制作用。其对底物PNPG的Km值和Vmax分别为1.03 mmol/mL和1.107μg/min更多还原

【Abstract】 Pullulan is a linear homopolysaccharide of glucose that is often described as anα-（1, 6） linked polymer of maltotriose subunits produced by strains of Aureobasidium pullulans. It can be produced by A. pullulans from a variety of carbohydrate such as glucose, hydrolyzed amylum, sucrose and glucose and so on. The pullulan has the characteristics of water-soluble, no-gelation, no-aging and without any toxicity. Biodegradable properties of pullulan, production from starch or other sugars and the characteristics mentioned above make pullulan be a promising material and be applied in numerous fields such as biomedicine, chemical industries, food industries, biotechnology and so on. Many products consisting of pullulan function as films, binders, adhesives, thickeners, viscosity improvers and coating agentsStrain Y68, a high pullulan yield yeast strain kept in our laboratory, was identified using the routine method and molecular method. Results of 18S rDNA and ITS sequences showed that strain Y68 has 100% similarity to Aureobasidim pullulans and results of routine identification also indicate that physiological and biochemical characteristics of strain Y68 was identical with Aureobasidium pullulans. So strain Y68 was finally identified as Aureobasidium pullulans.To get the highest pullulan yield, it was very important to optimize the culture conditions for pullulan production by strain Y68. Effects of different initial pHs, constant pHs, carbon sources, agitation and aeration on pullulan production by strain Y68 were examined. It was found that the highest yield of pullulan was achieved when the yeast strain was grown in the medium with initial pH 7.0, agitation of 300 rpm and aeration of 6.5 L/min, containing glucose. Under these conditions, 65 g/L pullulan was yielded. Results of study on pullulan yield at constant pH showe that it was not necessary to keep broth pH at a constant level for pullulan yield.In order to better understand the correlation between some enzymes and pullulan yield in strain Y68, effects of different sugars and different pHs on UDP-glucose level, pullulan-related synthase,α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase activity in the cells of strain Y68 and UDP-glucose level in different pullulan-producing strains were examined. Our results demonstrate that the more pullulan was synthesized, the less UDP-glucose was left in the cells of Y68 and this phenomenon alsp happened in other pullulan-producing yeast strains which produced less pullulan. However, it was observed that the more pullulan was synthesized, the higher pullulan-related synthase activity was detected in the cells Y68. Therefore, high pullulan yield was related to low UDP-glucose level and high pullulan-related synthases activity in A. pullulans Y68. Furthermore, it was observed that more pullulan was synthesized, the higherα-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase activity were detected in the cells of Y68. Therefore, high pullulan yield is related to highα-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase activity in A. pullulans Y68 grown on different sugars.As a key enzyme in pullulan synthesis, glucosyltransferase in the cells of Y68 was purified and characterized. The results obtained in this study showed that the molecular mass of glucosyltransferase was 350 kDa on the Native-PAGE gel, but 50.8 kDa on the SDS-PAGE gel which indicated the enzyme was composed of several subunits. Study on the characteristic of enzyme showed activity of this enzyme was inhibited greatly by metal ion such as Hg²⁺, Mn²⁺, Ca²⁺ and so on. EDTA, PMSF, DTT and SDS also influenced the enzyme activity obviously,but only DTT can stimulate glucosyltransferase activity. The optimal pH for its activity was 6.0 and temperature was 40℃. Km and Vmax of this enzyme for PNPG were 1.03mmol/mL and 1.107μg/min, respectly.更多还原