添加剂对大孔吸附树脂固定化脂肪酶的影响

利用大孔吸附树脂DA-201为载体对海洋脂肪酶固定化,并探寻添加剂对固定化过程的影响。分别以NH_4Cl、甘露糖和甘氨酸为添加剂,采用单因素和正交实验相结合的方法优化条件。结果显示,以NH_4Cl为添加剂的最优条件:柠檬酸-柠檬酸钠缓冲液pH 6. 0,固定化温度30℃,载体投放量0. 5g,NH_4Cl浓度25mmol/L,固定化时间3. 0h,酶活力达到115. 27U/g;比不含有添加剂的固定化酶固定化效率提高47. 42%。以甘露糖为添加剂最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度35℃,载体投放量0. 5g,甘露糖浓度10mmol/L,固定化时间4. 5h;酶活力达到122. 75U/g,比不含有添加剂的固定化酶固定化效率提高6. 50%。以甘氨酸为添加剂的最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度20℃,载体投放量0. 5g,甘氨酸浓度为25mmol/L,固定化时间7. 5h;酶活力达到141. 69U/g,比不含有添加剂的固定化酶固定化效率提高26. 12%。采用不同添加剂对大孔吸附树脂DA-201的吸附固定化过程有较大影响,可以极大地提高吸附效率;同时发现缓冲液类型、pH、温度、添加剂浓度和固定化时间等对DA-201树脂吸附脂肪酶有很大影响,对后续吸附固定化工业酶研究有较好的参考价值。     

10升气升环流式生物反应器培养紫草细胞

本文采用自行设计研制的10升气升环流式生物反应器培养紫草细胞,培养周期34d．前14d为细胞生长培养,细胞生长呈正常的S型曲线,细胞增长到原细胞接人量的4倍．后20d为紫草色素生产培养,细胞增长到32倍。整个周期每升培养液可生产紫草色素0．6g,在反应器中,培养液pH值的变化与细胞生长呈正相关,与紫草色素的形成呈负相关,pH值变化规律可用于监测紫草细胞在生物反应器的生长和色素形成．     

原位吸附浸没式膜生物反应器催化制备茚二醇

在浸没式膜反应器(IMB)中利用恶臭假单胞菌ATCC 55687催化茚制备顺式茚二醇。采用25根膜丝制作的膜组件,在茚为3 g/L时,经过24 h培养,IMB中顺式茚二醇产量达到悬浮细胞反应器(SCB)的4倍多;进一步,在培养开始阶段加入10 g/L sp-207树脂进行原位吸附,IMB中顺式茚二醇产量最高达709 mg/L,为SCB产量的660%,容积产率也从SCB中的6 mg/(L·h)提高到30 mg/(L·h)。在原位吸附IMB中,中空纤维膜既可作为固定化细胞载体,同时又可作为第二相吸附不溶性底物,降低底物和产物抑制,兼有固定化反应器和两相反应器的优点。     

紫草组织培养研究概况

本文简要综述硬紫草、滇紫草和新疆紫草在利用愈伤组织培养、细胞悬浮培养和固定化培养生产紫草及其衍生物方面的研究进展。     

聚乙二醇对东北红豆杉培养细胞的生长及紫杉醇生产的影响

在改良的B5培养基中加入不同浓度的聚乙二醇对东北红豆杉培养细胞进行摇瓶培养,通过不同时期取样并测定细胞鲜,干重及用HPLC测定紫杉醇的含量,发现聚乙二醇对东北红豆杉培养细胞的生长及紫杉醇生产均有明显的促进作用,聚乙二醇为10g/L时,对细胞生长最为有利,细胞培养16d可达到最大生物量,其平均鲜重为28．73g/瓶,增重3．8倍,平均干重为2．14g/瓶,增重3．1倍,聚乙二醇为20g/L,对紫杉醇的生产最有利;细胞培养25d时,培养基中紫杉醇的含量达到最高水平,其含量为2350ug/L,是不加聚乙二醇的11倍。     

水-有机溶剂介质中固定化不动杆菌细胞催化拆分(RS)-环戊烯酮乙酸酯的研究

不动杆菌CGMCC 0789的海藻酸凝胶包埋固定化细胞可高对映选择性地水解拆分环戊烯酮(简称HMPC)乙酸酯.异丙醇对固定化细胞的活力和对映选择性有显著提高.反应体系中异丙醇浓度为10%(体积分数,全文同)时,固定化细胞的活力最高,为6.22 mmol/(L·min·g)细胞干重,是未添加异丙醇的对照组的150%.此时E值为94±6,是对照组的1.7倍.以光学纯环戊烯酮乙酸酯为底物,对部分纯化的不动杆菌酯酶进行了动力学考察.通过动力学参数推算,10%异丙醇存在时,酯酶的对映选择性(E值)为36.5,是空白的2.3倍.10%异丙醇存在下,固定化细胞仍具有良好的操作稳定性.连续反应10批,固定化细胞的活力保持良好.     

固定化桔青霉产生核酸酶P_1的研究

利用吸附固定在多孔聚酯载体上的桔青霉(Penicillium citrinum)菌丝细胞,在摇瓶中以分批发酵方式合成核酸酶P_1.试验结果表明:在固定化细胞产酶的条件下,培养液中葡萄糖和蛋白胨的最适浓度分别为10g/L和1g/L,摇瓶转速以180~200r/min为宜.固定化细胞经过48h的产酶周期,培养液中的核酸酶P_1活力可高达513.3U/ml,其产酶效率是游离菌丝的3.6倍,而葡萄糖和蛋白胨的用量仅为游离菌丝产酶的五分之一.在重复分批发酵试验中,固定化菌丝细胞形状稳定,连续28批(共56d)的产酶结果基本一致,每批的平均酶活力为507.4U/ml,在经济上和工艺上均显示了明显的优越性.     

固定化桔青霉产生核酸酶P_1的研究

利用吸附固定在多孔聚酯载体上的桔青霉(Penicillium citrinum)菌丝细胞,在摇瓶中以分批发酵方式合成核酸酶P_1.试验结果表明:在固定化细胞产酶的条件下,培养液中葡萄糖和蛋白胨的最适浓度分别为10g/L和1g/L,摇瓶转速以180~200r/min为宜.固定化细胞经过48h的产酶周期,培养液中的核酸酶P_1活力可高达513.3U/ml,其产酶效率是游离菌丝的3.6倍,而葡萄糖和蛋白胨的用量仅为游离菌丝产酶的五分之一.在重复分批发酵试验中,固定化菌丝细胞形状稳定,连续28批(共56d)的产酶结果基本一致,每批的平均酶活力为507.4U/ml,在经济上和工艺上均显示了明显的优越性.     

DA-F127水凝胶包埋固定化含腈水合酶细胞

腈水合酶是一类可催化腈类化合物转化生成相应酰胺类物质的酶。含腈水合酶的游离细胞催化水合反应存在酶容易失活、细胞无法重复利用、分离纯化困难等缺陷,细胞固定化技术可有效解决这些问题。为探索合适的固定化方法,以含腈水合酶的重组E. coli细胞为研究对象,以固定化酶活回收率和批次反应情况为评价指标,筛选比较了几种常用的包埋固定化方法。结果表明,DA-F127水凝胶包埋固定化细胞不仅具有较高的酶活回收率,而且稳定性也很好。对该方法进行了固定化条件和操作稳定性优化,当DA-F127浓度为15%、UV光源距离为20cm、光照时间为6min、菌体含量为20mg/g固定化细胞时,酶活回收率为89. 74%,并且可以催化9批次150g/L的3-氰基吡啶完成转化,第九批次转化率可达98. 26%。与游离细胞催化过程相比,单位质量游离细胞的烟酰胺产量提高了12倍,具有良好的工业应用前景。     

DA-F127水凝胶包埋固定化含腈水合酶细胞

腈水合酶是一类可催化腈类化合物转化生成相应酰胺类物质的酶。含腈水合酶的游离细胞催化水合反应存在酶容易失活、细胞无法重复利用、分离纯化困难等缺陷,细胞固定化技术可有效解决这些问题。为探索合适的固定化方法,以含腈水合酶的重组E.coli细胞为研究对象,以固定化酶活回收率和批次反应情况为评价指标,筛选比较了几种常用的包埋固定化方法。结果表明,DA-F127水凝胶包埋固定化细胞不仅具有较高的酶活回收率,而且稳定性也很好。对该方法进行了固定化条件和操作稳定性优化,当DA-F127浓度为15%、UV光源距离为20cm、光照时间为6min、菌体含量为20mg/g 固定化细胞时,酶活回收率为89.74%,并且可以催化9批次150g/L的3-氰基吡啶完成转化,第九批次转化率可达98.26%。与游离细胞催化过程相比,单位质量游离细胞的烟酰胺产量提高了12倍,具有良好的工业应用前景。     

Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization

Plant cell cultures of Lithospermum erythrorhizon were carried out to produce shikonin by in situ extraction and cell immobilization in calcium alginate bead in shake flask cultures. In situ product extraction and cell immobilization enhanced shikonin production and facilitated product recovery. In situ extraction by n-hexadecane and cell immobilization by calcium alginate gave higher specific shikonin productivities of 7.4 and 2.5 times, respectively, than those from the cultures of free cells without extraction. Simultaneous use of both techniques increased specific and volumetric productivities of shikonin 25- and 15-fold, respectively. In calcium alginate immobilized cell cultures, n-hexadecane addition at an early stage (before 15 days) was effective for shikonin production, and solvent addition after 15 days of the culture significantly reduced shikonin production. Higher numbers of plant cell immobilized bead inoculation did not increase shikonin production and sucrose consumption. Most of the produced shikonin was dissolved in the solvent layer.     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

Studies on Cell Suspension Culture and Fermentation Culture in Arnebia euchroma

This paper reports some characteristics of cell suspension and fermentation culture in Arnebia euchroma (Royle) Johnst. The yield of suspension culture reached 22.0g dry wt/L per month when inoculum quantity was 2.50 g dry wt/L. Time-course study showed that cell growith lagged in 0–3 days and enhanced greatly in 3–12 days, and almost ceased after 12 days of culture, pH value changed during the culture period and peaked on the 12th day after inoculation. When cells were cultured in liquid production medium, the contents of shikonin derivatives increased quickly and reached to the maximum about the 25th day. The cell yield of 9.47 and 9.34 g dry wt/L per month was obtained in fermentation culture. Timecourse of cell growth in fermentation culture was similar to that in suspension culture. The total content of shikonin derivatives in fermentation culture was 14.26% dry weight from 10 L bioreactor. The yield of shikonin derivatives was 1.93 g/L.     

Cell Growth and Shikonin Production of Arnebia euchroma in a Periodically Submerged Airlift Bioreactor

Arnebia euchroma was grown in a 2-l periodically submerged, airlift bioreactor (PSAB) in which the non-submerged (immobilization culture) and submerged (suspension culture) operations were controlled automatically. PSAB had advantages in improving cell growth, shikonin content, shikonin production and cell aggregation compared with suspension culture. Under the optimal submerged/non-submerged period of 10 min/15 h, the shikonin content (4.6%, w/w) and, cell dry mass (16.8 g/l) were 229 and 26% higher than those in suspension culture. Revisions requested 31 October 2005 and 6 December 2005; Revisions received 2 December 2005 and 13 January 2006     

An novel immobilization method of Saccharomyces cerevisiae to sorghum bagasse for ethanol production

Natural sorghum bagasse without any treatment was used to immobilize Saccharomyces cerevisiae at 0.6+/-0.2g dry cell weight (DCW)/g dry sorghum bagasse weight (DSW) through solid-state or semi-solid state incubation. The scanning electron microscopy (SEM) of the carriers revealed the friendship between yeast cells and sorghum bagasse are adsorption and embedding. The ethanol productivity of the immobilized cells was 2.24 times higher than the free cells. In repeated batch fermentation with an initial sugar concentration of 200g/L, nearly 100% total sugar was consumed after 16 h. The ethanol yield and productivity were 4.9 g/g consumed sugar on average and 5.72 g/(Lh), respectively. The immobilized cell reactor was operated over a period of 20 days without breakage of the carriers, while the free cell concentration in the effluent remained less than 5 g/L thoughout the fermentation. The maximum ethanol productivity of 16.68 g/(Lh) appeared at the dilution rate of 0.3h(-1).     

Selection of Cell Lines with High Productivity of Shikonin Derivatives by Protoplast Culture of Lithospermum erythrorhizon Cells

We selected high-yield cell lines, using protoplast culture of Lithospermum erythrorhizon cells. Three cell lines having different shikonin productivities were used as parent cells for the selection, and cell lines with high productivity were obtained efficiently in every case. The best cell line had 6.45 g shikonin/g inoculum/23 days of production which was almost 1.5 times higher than that of the original cell line. The productivities of protoplast-derived cell lines were distributed widely and their average productivity was similar to the original one. The subculture of such a protoplast-derived cell line for eight months showed that its shikonin productivity was stabler than the original cell line.     

新疆紫草细胞生产阶段培养中细胞生长及产物合成的研究Ⅱ:发酵培养

用内循环气升式反应器进行新疆紫草细胞生产阶段培养,当培养基浓度增加时,色素产率也随之提高,其数量及增长的幅度远大于悬浮培养。当接种量为180gFW／1,培养基为3倍的M9时能获得最大的色素产率：2．28g／l,是培养基非加倍时的5．4倍,太高的接种量不利于色素的合成。为降低成本而减少培养基中某些成份的用量时,要得到较高的色素产量,需同时调整其它成份的浓度。     

γ—射线对滇紫草细胞产生色素的影响

应用５００～２５００伦琴（Ｒ）的６０钴γ射线照射滇紫草（ＯｎｏｓｍａｐａｎｉｃｕｌａｔｕｍＢｕｒ．ｅｔＦｒａｎｃｈ．）细胞系,确定了应用γ射线处理促进紫草素合成的最适辐照剂量为１５００Ｒ,处理后的细胞系在生产培养基上培养２１ｄ后紫草素含量（以干重计）达到９４．７９ｍｇ／ｇ,较对照提高了１４４６％,通过小团块选种法从中筛选到了色素含量（以干重计）高达１０３．４２ｍｇ／ｇ的高产细胞系Ｍｕｔ１,其营养生长与对照相比没有差别。     

The Effective Production of Shikonin by Cultures with an Increased Cell Population

We have studied the efficient production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon with an increased cell population. The yield of shikonin derivatives was highest (800 mg/liter) when 2.8 g dry wt/liter of the cells was inoculated into the M-2 medium which we had developed for the production, but the excess inoculum lowered the yield.

We investigated suitable conditions for production with the increased cell population. The optimum amount of inoculum rose to 4.9 g dry wt/liter when the concentrations of all the components contained in the M-8 medium, which we developed for increasing the productivity by modification of the M-2 medium, were increased in proportion to the amount of inoculum, and consequently we could increase the yield of the shikonin derivatives from 1400 mg/liter to 1900 mg/liter. Moreover, the increased rate of oxygen supply in addition to the enrichment of the medium made it possible to produce 2300 mg/liter of the shikonin derivatives from a culture for which 5.6 g dry wt/liter of the cells was inoculated.     

Critical factors affecting ethanol production by immobilized Pichia stipitis using corn cob hemicellulosic hydrolysate

Fermentation of xylose from hydrolysate of acid-treated corn cob by Pichia stipitis is inhibited by acetic acid and lignin derivatives. In the present study, we have designed and implemented an immobilized cell culture for xylose to ethanol conversion from acid-treated corn cob hydrolysate without the removal of fermentation inhibitors. In this study, cultivations of suspended and immobilized Pichia were compared in terms of ethanol yield and productivity to investigate whether the cell immobilization could improve resistance to inhibitors. Cell immobilization clearly favored the fermentative metabolism in nondetoxified corn cob hydrolysate leading to an improvement of twofold ethanol productivity as compared to that achieved with suspension culture. Calcium alginate as an immobilization matrix was selected to immobilize Pichia cells. Concentrations of sodium alginate, calcium chloride, and fermentor agitation speed were optimized for ethanol production using statistical method. Statistical analysis showed that agitation speed had maximum influence on ethanol production by immobilized Pichia cells. In comparison to suspension culture, immobilization had a positive impact on the fermentative metabolism of Pichia, improving the ethanol yield from 0.40 to 0.43?g/g and productivity from 0.31 to 0.51?g/L/h for acid-treated corn cob hydrolysate.