γ-聚谷氨酸发酵条件优化及结构表征分析
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摘要
γ-聚谷氨酸(γ-Polyglutamic acid,即γ-PGA)是一种具有水溶性、生物可降解性、无毒、对人体无害的高分子材料,它最初是从某些微生物的荚膜中提取出来的,是由D-谷氨酸和L-谷氨酸单体组成的聚合物。γ-PGA可以作为吸水保湿剂、药物缓释材料、防冻剂、生物絮凝剂、高吸水性树脂、可降解纤维、动物饲料添加剂和重金属吸附剂,广泛的应用于食品、农业、医药、化妆品和环境等领域。本文利用本实验室筛选的Bacillus subtills NOBEL-007摇瓶发酵合成γ-PGA,对γ-PGA的发酵条件、γ-PGA的分子结构和分子量等进行了研究,为工业化生产提供理论基础。主要研究内容如下:
(1)利用单因素实验对影响γ-PGA产量的碳源、氮源、谷氨酸钠和无机盐等因素进行研究,筛选出各因素大概的参考范围,为进一步优化做准备。经单因素实验得到的适宜初步发酵培养基配方为:葡萄糖60g/L,酵母粉6.0g/L,谷氨酸钠60g/L,NH_4Cl 3.0g/L,CaCl_2 0.2g/L、K_2HPO_4·3H_2O 3.0g/L,MgSO_4·7H_2O 0.75g/L,MnCl_2·4H_2O 0.04g/L。
(2)用SAS软件设计完成Plackett-Burman实验,实验结果表明酵母粉、谷氨酸钠和CaCl_2对γ-PGA的产量影响最为显著,因此重点对这三个因素进行优化分析。根据Plackett-Burman实验结果设计最陡爬坡实验,以便最大限度接近最高产γ-PGA的最优值区域,建立有效的拟合方程。
(3)根据最陡爬坡实验的结果,采用Box-Behnken实验研究结果完成系统分析后建立回归方程,结合Design-Expert软件绘出响应面分析图和等高线图,最终获得γ-PGA的最佳发酵培养基为:葡萄糖50g/L,酵母粉4.18g/L,谷氨酸钠76.89g/L,NH_4Cl 3g/L,CaCl_2 0.1422g/L、K_2HPO_4·3H_2O 3g/L,MgSO_4·7H_2O 0.75g/L,MnCl_2·4H_2O 0.04g/L,此时γ-PGA的理论产量为43.39g/L。对预测结果进行验证,得到γ-PGA的产量为43.26g/L,与理论预测值相近,说明模型的可信度较高。与优化前21.26g/L相比,γ-PGA的产量提高了1.035倍。
(4)应用红外光谱和核磁共振对发酵产物进行鉴定分析,结果证实为γ-PGA,并解析了它的分子结构式。纸层析和茚三酮比色法测定结果表明γ-PGA是由单一的谷氨酸组成,且发酵所得γ-PGA产品的纯度为85.13%。利用浊度法/分光光度计法测得γ-PGA的等电点为3.5。SDS-PAGE电泳测得γ-PGA的分子量,发现其同其他聚氨基酸一样,是多分子量聚合物,分子量范围在100-600kD之间。利用发酵所得γ-PGA产品进行吸水性实验、保水性实验和对种子发芽率影响的实验,发现γ-PGA土壤保水性与PAM相当,但其吸收水分的能力和提高种子发芽率的能力均优于PAM。此外,还发现γ-PGA的吸水能力在蒸馏水中最强,其次是生理盐水,井水和自来水最差,在土壤中的保水实验也得到类似结果,且质量浓度为12g/L的γ-PGA保水效果最好。
As a kind of macromolecular material,γ-polyglutamic acid is water soluble, biodegradable,nontoxic,harmless towards humans. It is a polymer materials which is made of D-glutamic acid and L-glutamic acid and extracted originally from the capsule of some microorganisms.γ-PGA can be used as absorbent moisturizer,drug delivery materials,antifreeze,flocculant,superabsorbent,biodegradable fiber,animal feed additives and heavy metal adsorbent,widely being used in food, agriculture, medicine,cosmetics and the environmental protection.This paper deals with Synthesis ofγ-PGA by fermentation with Bacillus subtills NOBEL-007 which was screened in our laboratory.Work have been carried out on the fermentation conditions, molecular structure and molecular weight ofγ-PGA,thus provided theoretical basis for the industrial production ofγ- PGA.
Single-factor design was used to study the effect of the carbon sources, nitrogen sources,sodium glutamat and inorganic salts onγ-poly glutamic acid production.Then selected the ranges of value of each related factor,making preparation for further optimization.From the single-factor test,we got appropriate fermentation medium: glucose 60g/L,yeast extract 6.0g/L,sodium glutamate 60g/L,NH_4Cl 3.0g/L, CaCl_2 0.2g/L,K_2HPO_4·3H_2O 3.0g/L,MgSO_4·7H_2O 0.75g/L,MnCl_2·4H_2O 0.04g/L.
Using SAS software to design Plackett-Burman experiment, the results showed that the effect of yeast extract,sodium glutamate and CaCl_2 on production ofγ-PGA was the most significant,so these three factors should be optimized emphatically.In order to approach the maximum production area ofγ-PGA and establish an effective fitting equation,we designed steepest ascent experiments according to the analysis of results of the Plackett-Burman experiment.
Design Box-Behnken experiment according to the results of steepest ascent. Analysized the results and established regression equation, and then combined the maps of response surface and contour which were droped with Design-Expert software,we get the optimal fermentation medium ofγ-PGA:glucose 50g/L,yeast extract 4.18g/L, sodium glutamate76.89g/L, NH_4Cl 3g/L, CaCl_2 0.1422g/L, K_2HPO_4·3H_2O 3g/L, MgSO_4·7H_2O 0.75g/L, MnCl_2·4H_2O 0.04g/L,and the theoretical yield ofγ-PGA was 43.39g/L. To verify the prediction results,we found that the Experimental values ofγ-PGA production was 43.26g/L which closed to the theoretical prediction, thus Demonstrated that the credibility of the model is high. Compared to the former 21.26g/L which was not optimized,γ-PGA yield increased by 1.035 times.
Identified and analysized fermentation products by using infrared spectroscopy and nuclear magnetic resonance,we comfined that the ermentation products wasγ-PGA actually, and also got its molecular structure.Using paper chromatography and ninhydrin colorimetry to assay the composition and the purity ofγ-PGA,we foundγ-PGA was made of pure glutamic acid,and the purity ofγ-PGA was. 85.13%.The isoelectric point ofγ-PGA was 3.5 measured by turbidimetry/spectro photometry. Finally,SDS-PAGE electrophoresis was used to measure molecular weight ofγ- PGA, the same like other amino acids,γ-PGA also had polymer molecular weight,and the molecular weight range from 100kDto 600kD.
From the study on water absorption and water holding capacity ofγ-PGA,and the effect ofγ-PGA on seed germination ratio in rats,we found thatγ-PGA had the same soil water holding capacity as PAM,and its ability to absorb water and increase seed germination rate was better than PAM.And WE also found that water absorption ofγ-PGA in distilled was the strongest,followed by saline, worst in wells and tap,and the similar results can be got by water retention experimentIn soil.The water holding capacity was hignest when the concentration ofγ-PGA was 12g/L.
(1)利用单因素实验对影响γ-PGA产量的碳源、氮源、谷氨酸钠和无机盐等因素进行研究,筛选出各因素大概的参考范围,为进一步优化做准备。经单因素实验得到的适宜初步发酵培养基配方为:葡萄糖60g/L,酵母粉6.0g/L,谷氨酸钠60g/L,NH_4Cl 3.0g/L,CaCl_2 0.2g/L、K_2HPO_4·3H_2O 3.0g/L,MgSO_4·7H_2O 0.75g/L,MnCl_2·4H_2O 0.04g/L。
(2)用SAS软件设计完成Plackett-Burman实验,实验结果表明酵母粉、谷氨酸钠和CaCl_2对γ-PGA的产量影响最为显著,因此重点对这三个因素进行优化分析。根据Plackett-Burman实验结果设计最陡爬坡实验,以便最大限度接近最高产γ-PGA的最优值区域,建立有效的拟合方程。
(3)根据最陡爬坡实验的结果,采用Box-Behnken实验研究结果完成系统分析后建立回归方程,结合Design-Expert软件绘出响应面分析图和等高线图,最终获得γ-PGA的最佳发酵培养基为:葡萄糖50g/L,酵母粉4.18g/L,谷氨酸钠76.89g/L,NH_4Cl 3g/L,CaCl_2 0.1422g/L、K_2HPO_4·3H_2O 3g/L,MgSO_4·7H_2O 0.75g/L,MnCl_2·4H_2O 0.04g/L,此时γ-PGA的理论产量为43.39g/L。对预测结果进行验证,得到γ-PGA的产量为43.26g/L,与理论预测值相近,说明模型的可信度较高。与优化前21.26g/L相比,γ-PGA的产量提高了1.035倍。
(4)应用红外光谱和核磁共振对发酵产物进行鉴定分析,结果证实为γ-PGA,并解析了它的分子结构式。纸层析和茚三酮比色法测定结果表明γ-PGA是由单一的谷氨酸组成,且发酵所得γ-PGA产品的纯度为85.13%。利用浊度法/分光光度计法测得γ-PGA的等电点为3.5。SDS-PAGE电泳测得γ-PGA的分子量,发现其同其他聚氨基酸一样,是多分子量聚合物,分子量范围在100-600kD之间。利用发酵所得γ-PGA产品进行吸水性实验、保水性实验和对种子发芽率影响的实验,发现γ-PGA土壤保水性与PAM相当,但其吸收水分的能力和提高种子发芽率的能力均优于PAM。此外,还发现γ-PGA的吸水能力在蒸馏水中最强,其次是生理盐水,井水和自来水最差,在土壤中的保水实验也得到类似结果,且质量浓度为12g/L的γ-PGA保水效果最好。
As a kind of macromolecular material,γ-polyglutamic acid is water soluble, biodegradable,nontoxic,harmless towards humans. It is a polymer materials which is made of D-glutamic acid and L-glutamic acid and extracted originally from the capsule of some microorganisms.γ-PGA can be used as absorbent moisturizer,drug delivery materials,antifreeze,flocculant,superabsorbent,biodegradable fiber,animal feed additives and heavy metal adsorbent,widely being used in food, agriculture, medicine,cosmetics and the environmental protection.This paper deals with Synthesis ofγ-PGA by fermentation with Bacillus subtills NOBEL-007 which was screened in our laboratory.Work have been carried out on the fermentation conditions, molecular structure and molecular weight ofγ-PGA,thus provided theoretical basis for the industrial production ofγ- PGA.
Single-factor design was used to study the effect of the carbon sources, nitrogen sources,sodium glutamat and inorganic salts onγ-poly glutamic acid production.Then selected the ranges of value of each related factor,making preparation for further optimization.From the single-factor test,we got appropriate fermentation medium: glucose 60g/L,yeast extract 6.0g/L,sodium glutamate 60g/L,NH_4Cl 3.0g/L, CaCl_2 0.2g/L,K_2HPO_4·3H_2O 3.0g/L,MgSO_4·7H_2O 0.75g/L,MnCl_2·4H_2O 0.04g/L.
Using SAS software to design Plackett-Burman experiment, the results showed that the effect of yeast extract,sodium glutamate and CaCl_2 on production ofγ-PGA was the most significant,so these three factors should be optimized emphatically.In order to approach the maximum production area ofγ-PGA and establish an effective fitting equation,we designed steepest ascent experiments according to the analysis of results of the Plackett-Burman experiment.
Design Box-Behnken experiment according to the results of steepest ascent. Analysized the results and established regression equation, and then combined the maps of response surface and contour which were droped with Design-Expert software,we get the optimal fermentation medium ofγ-PGA:glucose 50g/L,yeast extract 4.18g/L, sodium glutamate76.89g/L, NH_4Cl 3g/L, CaCl_2 0.1422g/L, K_2HPO_4·3H_2O 3g/L, MgSO_4·7H_2O 0.75g/L, MnCl_2·4H_2O 0.04g/L,and the theoretical yield ofγ-PGA was 43.39g/L. To verify the prediction results,we found that the Experimental values ofγ-PGA production was 43.26g/L which closed to the theoretical prediction, thus Demonstrated that the credibility of the model is high. Compared to the former 21.26g/L which was not optimized,γ-PGA yield increased by 1.035 times.
Identified and analysized fermentation products by using infrared spectroscopy and nuclear magnetic resonance,we comfined that the ermentation products wasγ-PGA actually, and also got its molecular structure.Using paper chromatography and ninhydrin colorimetry to assay the composition and the purity ofγ-PGA,we foundγ-PGA was made of pure glutamic acid,and the purity ofγ-PGA was. 85.13%.The isoelectric point ofγ-PGA was 3.5 measured by turbidimetry/spectro photometry. Finally,SDS-PAGE electrophoresis was used to measure molecular weight ofγ- PGA, the same like other amino acids,γ-PGA also had polymer molecular weight,and the molecular weight range from 100kDto 600kD.
From the study on water absorption and water holding capacity ofγ-PGA,and the effect ofγ-PGA on seed germination ratio in rats,we found thatγ-PGA had the same soil water holding capacity as PAM,and its ability to absorb water and increase seed germination rate was better than PAM.And WE also found that water absorption ofγ-PGA in distilled was the strongest,followed by saline, worst in wells and tap,and the similar results can be got by water retention experimentIn soil.The water holding capacity was hignest when the concentration ofγ-PGA was 12g/L.
引文
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