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γ-聚谷氨酸在食品、化妆品、生物医药等领域具有广泛的应用,目前主要的生产菌株是谷氨酸依赖型菌株,在生产过程中需要添加谷氨酸作为前体,因而生产γ-聚谷氨酸的成本较高。文中主要研究从糖质原料一步法发酵合成γ-聚谷氨酸的生产工艺。首先,从产γ-聚谷氨酸的菌株枯草芽孢杆菌中克隆γ-聚谷氨酸合成酶的基因簇pgs BCA,在谷氨酸棒杆菌模式菌株ATCC13032中进行诱导型和组成型表达,结果显示,仅诱导型表达菌株可以积累γ-聚谷氨酸,产量为1.43 g/L。进一步对诱导条件进行优化,确定诱导时间为2 h,IPTG浓度为0.8 mmol/L,γ-聚谷氨酸产量为1.98g/L。在此基础上,在一株高产谷氨酸的谷氨酸棒杆菌F343中外源表达pgs BCA,对重组菌进行发酵,结果表明,在摇瓶发酵中γ-聚谷氨酸产量达到10.23g/L,在5L发酵罐中产量达到20.08g/L;继而对γ-聚谷氨酸进行分子量测定,结果显示,产自F343重组菌的γ-聚谷氨酸的重均分子量比产自枯草芽孢杆菌的提高34.77%。文中构建了一步法发酵糖质原料生产γ-聚谷氨酸的新途径,同时为开发其潜在应用奠定了基础。 相似文献
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Jun Yao Jin Jing Hong Xu Jinfeng Liang Qun Wu Xiaohai Feng Pingkai Ouyang 《Journal of Molecular Catalysis .B, Enzymatic》2009,56(2-3):158-164
The preparation of γ-polyglutamic acid (γ-PGA) from Bacillus subtilis NX-2 has been previously investigated, and its depolymerization during the batch culture was studied in this paper. The results suggested that the γ-PGA depolymerase was present and active extracellularly in the culture. The ywtD gene from B. subtilis NX-2, encoding the γ-PGA depolymerase was cloned and expressed in Escherichia coli. The YwtD protein was purified by metal-chelating affinity chromatography. YwtD was proved to be an endo-hydrolase enzyme and exhibited a remarkable activity in γ-PGA degradation at a wide range of temperature (30–40 °C) and pH (5.0–8.0). On an optimal condition of 30 °C and pH 5.0, an efficient γ-PGA enzymatic degradation was achieved. The molecular weight of γ-PGA could be reduced within the range of 1000–20 kDa and the polydispersity also decreased as a function of depolymerization time. Therefore, a controllable degradation of γ-PGA could be available by enzymatic depolymerization. 相似文献
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In this study, γ-polyglutamic acid (γ-PGA) and chitosan (CS) nanoparticles were characterized as a carrier for the anti-cancer drug doxorubicin (DOX). Using ionic complexation between the positively charged DOX and the negatively charged polyelectrolyte γ-PGA, DOX:γ-PGA complexes were produced with an efficiency of approximately 99%. SEM micrographs demonstrated that the complexation of γ-PGA and DOX alone does not lead to the formation of nanoparticles and that the addition of a third component, chitosan, is required. Drug-loaded DOX:γ-PGA:CS nanoparticles were produced with particle sizes ranging from ~150 to ~630 nm. The stability of the DOX:γ-PGA:CS nanoparticles was examined by suspending the nanoparticles in different kinds of aqueous media. For the first time, in vitro studies with DOX-loaded nanoparticles demonstrated the cytotoxicity of the nanoparticles against a human oral squamous cell carcinoma cell line (HN-5a). Non-drug-loaded γ-PGA:CS nanoparticles did not display cytotoxic effects. It was shown that the encapsulated or surface-bound DOX did not lose its bioactivity and the prepared drug-loaded particles exhibited a considerable anti-proliferative activity against the human cancer cell line. 相似文献
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Kyra Hoffmann Birthe Halmschlag Simon Briel Michaela Sieben Sastia Putri Eiichiro Fukusaki Lars M. Blank Jochen Büchs 《Biotechnology progress》2023,39(1):e3293
Poly-γ-glutamic acid (γ-PGA) is a biopolymer with a wide range of applications, mainly produced using Bacillus strains. The formation and concomitant secretion of γ-PGA increases the culture broth viscosity, while enzymatic depolymerisation and degradation of γ-PGA decreases the culture broth viscosity. In this study, the recently published ViMOS (V iscosity M onitoring O nline S ystem) is applied for optical online measurements of broth viscosity in eight parallel shake flasks. It is shown that the ViMOS is suitable to monitor γ-PGA production and degradation online in shake flasks. This online monitoring enables the detailed analysis of the Ppst promoter and γ-PGA depolymerase knockout mutants in genetically modified Bacillus subtilis 168. The Ppst promoter becomes active under phosphate starvation. The different single depolymerase knockout mutants are ∆ggt, ∆pgdS, ∆cwlO and a triple knockout mutant. An increase in γ-PGA yield in gγ-PGA/gglucose of 190% could be achieved with the triple knockout mutant compared to the Ppst reference strain. The single cwlO knockout also increased γ-PGA production, while the other single knockouts of ggt and pgdS showed no impact. Partial depolymerisation of γ-PGA occurred despite the triple knockout. The online measured data are confirmed with offline measurements. The online viscosity system directly reflects γ-PGA synthesis, γ-PGA depolymerisation, and changes in the molecular weight. Thus, the ViMOS has great potential to rapidly gain detailed and reliable information about new strains and cultivation conditions. The broadened knowledge will facilitate the further optimization of γ-PGA production. 相似文献
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分别采用海藻酸钠、明胶和壳聚糖为载体,并以戊二醛为交联剂,通过包埋-交联和吸附-交联两种耦合固定化方法制备固定化锰过氧化物酶。探讨了酶的不同固定化条件和固定化酶的部分性能。与游离酶相比,制备的3种固定化酶最适反应pH分别由7·0降低到5·0、5·0和3·0,最适反应温度分别由35℃升高到75℃、55℃和75℃。3种固定化酶的耐热性都显著提高,其中用壳聚糖制成的固定化酶在pH2·2~11的宽范围内表现出很好的酸碱耐受性。30℃连续测定6~9次酶活力,重复使用的3种固定化酶显示出良好的稳定性。将固定化酶应用在偶氮染料的脱色中,用明胶制成的固定化酶在静置和摇床条件下,以及用海藻酸钠制成的固定化酶在摇床条件下,均表现出与游离酶相近的脱色能力,并且在重复进行的摇床实验中,脱色能力未降低,反应前后的酶活力均没有损失。 相似文献
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Ching Ting Tsao Chih Hao Chang Yu Yung Lin Jaw-Lin Wang Kuo Huang Hsieh 《Carbohydrate research》2010,345(12):1774-413
In this study, we prepared a polyelectrolyte complex (PEC) hydrogel comprising chitosan as the cationic polyelectrolyte and γ-poly(glutamic acid) (γ-PGA) as the anionic polyelectrolyte. Fourier transform infrared spectroscopy revealed that ionic complex interactions existed in the chitosan-γ-PGA PEC hydrogels. The compressive modulus increased upon increasing the degree of complex formation in the chitosan-γ-PGA PEC hydrogel; the water uptake decreased upon increasing the degree of complex formation. At the same degree of complex formation, the compressive modulus was larger for the chitosan-dominated PEC hydrogels; the water uptake was larger for the γ-PGA-dominated ones. Scanning electron microscopy images revealed the existence of interconnected porous structures (pore size: 30-100 μm) in all of the chitosan-γ-PGA PEC hydrogels. The chitosan-γ-PGA PEC hydrogels also exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, in vitro cell culturing of 3T3 fibroblasts revealed that all the chitosan-γ-PGA PEC hydrogels were effective in promoting cell proliferation, especially the positively charged ones (chitosan-dominated). Therefore, the chitosan-γ-PGA polyelectrolyte hydrogel appears to have potential as a new material for biomedical applications. 相似文献
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Jun Feng Weixia Gao Yanyan Gu Wei Zhang Mingfeng Cao Cunjiang Song Peng Zhang Min Sun Chao Yang Shufang Wang 《Applied microbiology and biotechnology》2014,98(14):6397-6407
Poly-γ-glutamic acid (γ-PGA) is an important biopolymer with greatly potential in industrial and medical applications. In the present study, we constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens LL3 strain with considerable γ-PGA production, which was carried out by single, double, and triple markerless deletions of three degradation genes pgdS, ggt, and cwlO. The highest γ-PGA production (7.12 g/L) was obtained from the pgdS and cwlO double-deletion strain NK-pc, which was 93 % higher than that of wild-type LL3 strain (3.69 g/L). The triple-gene-deletion strain NK-pgc showed a 28 % decrease in γ-PGA production, leading to a yield of 2.69 g/L. Furthermore, the cell morphologies of the mutant strains were also characterized. The cell length of cwlO deletion strains NK-c and NK-pc was shorter than that of the wild-type strain, while the ggt deletion strains NK-g, NK-pg, NK-gc, and NK-pgc showed longer cell lengths. This is the first report concerning the markerless deletion of γ-PGA degradation genes to improve γ-PGA production in a glutamate-independent strain and the first observation that γ-glutamyltranspeptidase (encoded by ggt) could be involved in the inhibition of cell elongation. 相似文献
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采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%. 相似文献
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目的:验证在地衣芽胞杆菌ATCC9945A中存在着γ-聚谷氨酸降解酶基因(ywtD),为下一步解决在γ-聚谷氨酸微生物发酵合成过程中产物γ-PGA降解的技术性难题提供依据。方法:通过在pET-28b(+)大肠杆菌表达系统克隆表达地衣芽孢杆菌ATCC9945A中的ywtD基因,对诱导表达条件进行优化,采用SDS-PAGE和Western Blot方法检测目的蛋白的表达,并体外酶解实验验证其活性。结果:PCR扩增得到了一个1 245bp的基因片段,预期编码414个氨基酸,诱导表达后得到一个分子量大小约为45.6 kDa的表达产物。Western Blot分析结果表明ywtD基因得到了有效表达。体外酶解实验表明该表达产物具有降解γ-PGA的活性。结论:证明在地衣芽胞杆菌ATCC9945A中存在着γ-聚谷氨酸降解酶基因。 相似文献
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Xia Liu Fei Liu Shaoying Liu Haijun Li Peixue Ling Xiqiang Zhu 《Applied microbiology and biotechnology》2013,97(22):9801-9809
Poly-γ-glutamate (γ-PGA) has been considered as one of the most promising biomaterials with a wide range of applications, but there has been no report that directly shows the anti-tyrosinase and anti-melanogenesis properties of γ-PGA. In the present study, we investigated the inhibitory effects of γ-PGA with low molecular weight (Mw; lγ-PGA) and high Mw (hγ-PGA) on mushroom tyrosinase and murine tyrosinase activities and on melanogenesis in B16 melanoma cells. First, we showed that both lγ-PGA and hγ-PGA could effectively inhibit mushroom tyrosinase activities including monophenolase and diphenolase activities in a dose-dependent manner. Second, both lγ-PGA and hγ-PGA showed strong anti-tyrosinase activity and anti-melanogenesis in B16 melanoma cells. Third, both lγ-PGA and hγ-PGA inhibited forskolin-induced tyrosinase activity and melanogenesis by decreasing the levels of intracellular reactive oxygen species and nitric oxide while increasing the catalase activity in B16 cells. This is the first report on the anti-melanogenesis effect of γ-PGA, which suggests that γ-PGA could have a potential in the cosmetic skin whitening business, therapeutic applications and the food industry. 相似文献
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Poly-γ-glutamic acid (γ-PGA) is a retaining agent; it has applications in the food, medicine, agriculture, cosmetics and wastewater treatment industries. Most of the γ-PGA producing strains belong to the genus Bacillus. This study reports on a novel γ-PGA producing species. Bacillus siamensis SB1001 was screened and isolated from organically cultivated soybeans exhibiting a high γ-PGA producing ability. The fermentation medium and culture parameters for γ-PGA production by Bacillus siamensis SB1001 were optimized by statistical methods. The sucrose, l-glutamic acid and dipotassium phosphate in the medium were shown to be the significant factors of the γ-PGA production, and the optimum medium obtained consisted of the following: 106.86 g/L sucrose, 69.84 g/L l-glutamic acid and 2.39 g/L dipotassium phosphate. Using the optimized medium, 25.22 g/L γ-PGA were produced with a productivity of 1.05 g/L/h. The γ-PGA obtained had a molecular weight of 7.9 × 105 Da and a polydispersity index of 2.34, and the ratio of d-/L-glutamic acid was 89.71%:10.29%. To the best of our knowledge, this is the first report of γ-PGA production by B. siamensis strain. B. siamensis SB1001 has great potential as an industrial γ-PGA producer. 相似文献
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Jang-Ho Kim Jong Min Lee Won Je Jang Hae Dae Park Young-Ok Kim Chang-Hoon Kim In-Soo Kong 《Biotechnology and bioengineering》2019,116(7):1594-1603
Numerous studies on poly γ-d -glutamicacid (γ-PGA) production have investigated terrestrial renewable sources for reducing production costs, but there are no studies using waste marine resources so far. We aimed to develop a cost-effective production method of γ-d -PGA by Bacillus sp. SJ-10 using green macroalgae (Ulva sp.) as a major substrate without hydrolysis pretreatment. The SJ-10 was shown to not only cause immediate tissue degradation of the Ulva membrane but also grew well as a sole substrate. The γ-d -PGA yield was 6.29 ± 0.34 g/L under optimized conditions via the response surface method, and the produced γ-d -PGA had a thermal decomposition temperature of 310°C and molecular weight of 250–1780 kDa. The calculated cost efficiency for the final yield was 32% when compared with complex media. Therefore, the present study provided a strategy for promoting an ecofriendly and cost-effective means to produce γ-d -PGA via a marine renewable resource. 相似文献
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漆酶在磁性壳聚糖微球上的固定及其酶学性质研究 总被引:5,自引:0,他引:5
以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为:50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液(0.1mol/L,pH 7.0),在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。 相似文献
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Mingfeng Cao Jun Feng Sarote Sirisansaneeyakul Cunjiang Song Yusuf Chisti 《Biotechnology advances》2018,36(5):1424-1433
Poly-γ-glutamic acid (γ-PGA) is a natural biopolymer of glutamic acid. The repeating units of γ-PGA may be derived exclusively from d-glutamic acid, or l-glutamic acid, or both. The monomer units are linked by amide bonds between the α-amino group and the γ-carboxylic acid group. γ-PGA is biodegradable, edible and water-soluble. It has numerous existing and emerging applications in processing of foods, medicines and cosmetics. This review focuses on microbial production of γ-PGA via genetically and metabolically engineered recombinant bacteria. Strategies for improving production of γ-PGA include modification of its biosynthesis pathway, enhancing the production of its precursor (glutamic acid), and preventing loss of the precursor to competing byproducts. These and other strategies are discussed. Heterologous synthesis of γ-PGA in industrial bacterial hosts that do not naturally produce γ-PGA is discussed. Emerging trends and the challenges affecting the production of γ-PGA are reviewed. 相似文献
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CaCl(2) was used as a novel additive to enhance poly-(γ-glutamic acid) (γ-PGA) production by Bacillus subtilis strain CGMCC 2108. Addition of CaCl(2) to medium effectively reduced viscosity of culture broth, and increased consumption of extracellular glutamate by 11.4%, leading to γ-PGA yield of 9.07 g/l, compared to 7.88 g/l in control. CaCl(2) also increased activities of three key enzymes around the crucial 2-oxoglutarate branch of the γ-PGA biosynthesis pathway: isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), and 2-oxoglutarate dehydrogenase complex (ODHC). In particular, GDH activity was increased more than 8-fold, indicating that more 2-oxoglutarate was directed to synthesis of glutamate, the substrate of γ-PGA. Interestingly, the molecular weight of γ-PGA remained constant regardless of CaCl(2) addition. 相似文献