新型微生物多糖WL-26的结构与特性研究
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摘要
从实验室保藏的菌株中筛选出一株Sphingomonas sp. ZJ01发酵生产微生物多糖WL-26，以发酵液粘度和多糖产量为目的，优化并获得了最佳培养基组成和发酵条件；在摇床和3L发酵罐水平，从合成的代谢特点、培养方式和培养条件等多角度出发，实施一系列过程强化和调控策略，实现了微生物多糖WL-26的高水平发酵；并对分离纯化得到的多糖WL-26的高级结构进行了分析和结构鉴定。主要研究内容如下：
(1)研究确定发酵培养基组成中，蔗糖为碳源，硝酸钠和牛肉膏为氮源，在此基础上对发酵条件进行优化，确定培养基组成和发酵条件为：50g/L蔗糖，4g/L复合氮源（20%硝酸钠和80%牛肉膏），2g/L KH2PO4，0.1g/L MgSO4，初始pH7.0，接种量5%，装液量50mL/250mL三角瓶，转速200r/min，30℃恒温培养58h，微生物多糖WL-26产量达到22g/L。
(2)在摇瓶和3L发酵罐水平上分别考察了转速和溶氧浓度对微生物多糖WL-26发酵的影响，发现微生物多糖WL-26的合成和代谢适宜在较高的溶氧条件下；通过考察不同转速对微生物多糖WL-26合成的影响，提出了多阶段搅拌的策略；通过降低磷酸盐浓度，使用0.2mmol/L氢氧化钠调控pH的策略，将初始磷酸盐的浓度降低到0.8g/L，大大降低了后续分离纯化以及废水处理的难度和成本。在3L发酵罐中，通过多阶段搅拌转速控制、碳质元素的补料分批控制和氢氧化钠控制pH等策略逐步优化，使微生物多糖WL-26的产量达到27g/L，比摇床水平提高了23%。
(3)利用静态和动态流变学方法研究WL-26溶液的粘度与剪切速率、溶液浓度、温度、酸碱度、冻融、冷藏和盐离子等因素的关系，并和工业中常用的黄原胶的流变特性进行比较。结果证明WL-26溶液的流体特征表现为高假塑性，适用于高温、高盐和宽pH范围的复杂环境，具有很好的应用前景。
(4)对微生物多糖WL-26部分酸水解，Sephacryl S100凝胶柱分离纯化，获得一个不含蛋白质和核酸的均一组分F21。对该组分进行了单糖组成分析、甲基化分析、电喷雾质谱分析和核磁共振（一维，二维）分析，基本确定了F21的单糖组成、结构单元中所含糖残基的构型和个数、糖残基之间的链接方式和连接顺序。结果表明，寡糖组分F21中含有鼠李糖、葡萄糖、甘露糖、半乳糖和葡萄糖醛酸，摩尔比为3:1.7:1:1:0.7；甲基化分析结果表明，半乳糖以端基形式存在，葡萄糖主要以1,4,6和1,3连接形式存在，鼠李糖则以1,4连接方式存在，甘露糖以1,4连接方式存在，并存在少量1,3鼠李糖残基、鼠李糖和葡萄糖末端，结合电喷雾质谱和核磁共振技术确定其为分子量1280Da的酸性寡糖，结构为：
以上结果为开发新型微生物多糖WL-26提供了可靠的科学依据，并为其在工业上的应用打下了良好的基础。
Microbial polysaccharide WL-26was produced by submerged fermentation of strainsSphingomonas sp. ZJ01and the fermentation condition was optimized on the basis of solutionviscosity and yield of WL-26. The effect of rotational speed and dissolved oxygenconcentration on fermentation were evaluated in shake flasks and3L bioreactor respectively.In order to release the high efficiency production of microbial polysaccharide WL-26, a seriesof strategies was carried out including culture conditions, culture methods and metaboliccharacteristics. After purification the structure and characteristics of WL-26was analyzed.
The main contents in this study are as follows:
(1) Sucrose, NaNO3and beef extract were the optimal carbon and complex nitrogen sourcesrespectively and the optimal medium consisted of50g/L sucrose,4g/L complex nitrogen(20%NaNO3and80%beef extract),2g/L KH2PO4and0.1g/L MgSO4. Yield ofmicrobial polysaccharide WL-26reached22g/L under the following conditions: mediuminitial pH7.0,5%inoculation volume,1:5aeration ratio (medium volume: flask volume,50mL medium in250mL flask), cultured at200r/min,30oC for58h.
(2) Base on the characteristics of Sphingomonas sp. fermentation processes in shake flask and3-L B. Braun Biostat C stainless steel reactor, through optimization of pH control strategy,the concentration of KH2PO4is reduced from2g/L to0.8g/L. This result is significantlyreduced the problem faced on the follow-up step of isolation and purification of WL-26and wastewater treatment, WL-26yield reached27g/L which was increased23%compared with yield in shake flask through multi-stage agitation speed control, carbonfed-batch fermentation, and low concentration of phosphate by adjusting the buffer pH7.0using0.2mmol NaOH.
(3) The relations between viscosity and shear rate, concentration, temperature, pH, highpressure, freezing-thawing, cold stored, and salt were evaluated by determining thedynamic and static viscosity. Results showed that the water solution of microbialpolysaccharide WL-26is a typical pseudoplastic liquid, having high viscosity at lowconcentrations. Novel Microbial exopolysaccharides WL-26has been shown attractiveapplication prospect due to its high temperature stability, pH tolerance, and environmentfriendly properties. More and more researches have been focused on production andapplication of it.
(4) A new oligosaccharide F21was obtained by microbial polysaccharide WL-26hydrolyzedby0.2M trifuluoroacetic acid and applied on gel-filtration chromatography column(Sephacryl S100). UV scan showed no proteins and nucleic acids and detection of infrared spectra indicated sugar characteristic group in F21.
The molecular weight of F21was1280Da evaluated by Esi and F21was composed ofL-rhamnose, D-glucose, D-mannose, D-galactose，and glucuronic acid in a molar ratio of3:1.7:1:1:0.7determined by HPAEC-PAD. Methylation analysis of F21showed that the1,4,6-tri-substituted glucose,1,4-di-substituted rhamnose,1,3-di-substituted glucose and1,4-di-substituted mannose are the main chain, with minor amounts of1,3-di-substitutedrhamnose, terminal glucose and rhamnose residues. The galactose residues arecompletely distributed at non-reducing terminals, and possibly with a glucuronic acidresidue at the other non-reducing terminal by NMR analysis. The proposal structure asfollows:
These results provide new soliol scientific data and for further develpment ofpolysaccharide WL-26from Sphingomonas sp.ZJ01and laid a good foundation for the futuredevelpment and application based on the polysaccharide.
(1)研究确定发酵培养基组成中，蔗糖为碳源，硝酸钠和牛肉膏为氮源，在此基础上对发酵条件进行优化，确定培养基组成和发酵条件为：50g/L蔗糖，4g/L复合氮源（20%硝酸钠和80%牛肉膏），2g/L KH2PO4，0.1g/L MgSO4，初始pH7.0，接种量5%，装液量50mL/250mL三角瓶，转速200r/min，30℃恒温培养58h，微生物多糖WL-26产量达到22g/L。
(2)在摇瓶和3L发酵罐水平上分别考察了转速和溶氧浓度对微生物多糖WL-26发酵的影响，发现微生物多糖WL-26的合成和代谢适宜在较高的溶氧条件下；通过考察不同转速对微生物多糖WL-26合成的影响，提出了多阶段搅拌的策略；通过降低磷酸盐浓度，使用0.2mmol/L氢氧化钠调控pH的策略，将初始磷酸盐的浓度降低到0.8g/L，大大降低了后续分离纯化以及废水处理的难度和成本。在3L发酵罐中，通过多阶段搅拌转速控制、碳质元素的补料分批控制和氢氧化钠控制pH等策略逐步优化，使微生物多糖WL-26的产量达到27g/L，比摇床水平提高了23%。
(3)利用静态和动态流变学方法研究WL-26溶液的粘度与剪切速率、溶液浓度、温度、酸碱度、冻融、冷藏和盐离子等因素的关系，并和工业中常用的黄原胶的流变特性进行比较。结果证明WL-26溶液的流体特征表现为高假塑性，适用于高温、高盐和宽pH范围的复杂环境，具有很好的应用前景。
(4)对微生物多糖WL-26部分酸水解，Sephacryl S100凝胶柱分离纯化，获得一个不含蛋白质和核酸的均一组分F21。对该组分进行了单糖组成分析、甲基化分析、电喷雾质谱分析和核磁共振（一维，二维）分析，基本确定了F21的单糖组成、结构单元中所含糖残基的构型和个数、糖残基之间的链接方式和连接顺序。结果表明，寡糖组分F21中含有鼠李糖、葡萄糖、甘露糖、半乳糖和葡萄糖醛酸，摩尔比为3:1.7:1:1:0.7；甲基化分析结果表明，半乳糖以端基形式存在，葡萄糖主要以1,4,6和1,3连接形式存在，鼠李糖则以1,4连接方式存在，甘露糖以1,4连接方式存在，并存在少量1,3鼠李糖残基、鼠李糖和葡萄糖末端，结合电喷雾质谱和核磁共振技术确定其为分子量1280Da的酸性寡糖，结构为：
以上结果为开发新型微生物多糖WL-26提供了可靠的科学依据，并为其在工业上的应用打下了良好的基础。
Microbial polysaccharide WL-26was produced by submerged fermentation of strainsSphingomonas sp. ZJ01and the fermentation condition was optimized on the basis of solutionviscosity and yield of WL-26. The effect of rotational speed and dissolved oxygenconcentration on fermentation were evaluated in shake flasks and3L bioreactor respectively.In order to release the high efficiency production of microbial polysaccharide WL-26, a seriesof strategies was carried out including culture conditions, culture methods and metaboliccharacteristics. After purification the structure and characteristics of WL-26was analyzed.
The main contents in this study are as follows:
(1) Sucrose, NaNO3and beef extract were the optimal carbon and complex nitrogen sourcesrespectively and the optimal medium consisted of50g/L sucrose,4g/L complex nitrogen(20%NaNO3and80%beef extract),2g/L KH2PO4and0.1g/L MgSO4. Yield ofmicrobial polysaccharide WL-26reached22g/L under the following conditions: mediuminitial pH7.0,5%inoculation volume,1:5aeration ratio (medium volume: flask volume,50mL medium in250mL flask), cultured at200r/min,30oC for58h.
(2) Base on the characteristics of Sphingomonas sp. fermentation processes in shake flask and3-L B. Braun Biostat C stainless steel reactor, through optimization of pH control strategy,the concentration of KH2PO4is reduced from2g/L to0.8g/L. This result is significantlyreduced the problem faced on the follow-up step of isolation and purification of WL-26and wastewater treatment, WL-26yield reached27g/L which was increased23%compared with yield in shake flask through multi-stage agitation speed control, carbonfed-batch fermentation, and low concentration of phosphate by adjusting the buffer pH7.0using0.2mmol NaOH.
(3) The relations between viscosity and shear rate, concentration, temperature, pH, highpressure, freezing-thawing, cold stored, and salt were evaluated by determining thedynamic and static viscosity. Results showed that the water solution of microbialpolysaccharide WL-26is a typical pseudoplastic liquid, having high viscosity at lowconcentrations. Novel Microbial exopolysaccharides WL-26has been shown attractiveapplication prospect due to its high temperature stability, pH tolerance, and environmentfriendly properties. More and more researches have been focused on production andapplication of it.
(4) A new oligosaccharide F21was obtained by microbial polysaccharide WL-26hydrolyzedby0.2M trifuluoroacetic acid and applied on gel-filtration chromatography column(Sephacryl S100). UV scan showed no proteins and nucleic acids and detection of infrared spectra indicated sugar characteristic group in F21.
The molecular weight of F21was1280Da evaluated by Esi and F21was composed ofL-rhamnose, D-glucose, D-mannose, D-galactose，and glucuronic acid in a molar ratio of3:1.7:1:1:0.7determined by HPAEC-PAD. Methylation analysis of F21showed that the1,4,6-tri-substituted glucose,1,4-di-substituted rhamnose,1,3-di-substituted glucose and1,4-di-substituted mannose are the main chain, with minor amounts of1,3-di-substitutedrhamnose, terminal glucose and rhamnose residues. The galactose residues arecompletely distributed at non-reducing terminals, and possibly with a glucuronic acidresidue at the other non-reducing terminal by NMR analysis. The proposal structure asfollows:
These results provide new soliol scientific data and for further develpment ofpolysaccharide WL-26from Sphingomonas sp.ZJ01and laid a good foundation for the futuredevelpment and application based on the polysaccharide.
引文
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