Study On Pullulan Production And Its Mechanism By A High Pulllulan Producing Yeast Strain Y68

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.