GABAA受体蛋白片段在大肠杆菌细胞表达条件研究

为了提高GABAA受体α1蛋白片段在大肠杆菌中表达量,研究了重组菌的发酵条件,包括培养基,接种量,温度,摇床转速,pH,诱导培养时间和诱导剂IPTG使用浓度等对GABAA受体蛋白片段表达的影响。结果表明重组菌以LB培养基为发酵基质,按3%接种量,37℃培养细胞3.5h后IPTG32℃诱导5h,菌体生物量为3.25g/L,目标蛋白表达量达95mg/L。用16L发酵罐进行放大培养,菌体生物量达4.95g/L,发酵周期5.5h。最高目标蛋白表达量达到136mg/L。     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

法夫酵母PLX-ll发酵纤维素酶水解物合成虾青素

法夫酵母（Phaffiarhodozyma)PLX朅ll菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素*

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

环境条件对丙酮酸分批发酵的影响

考察了搅拌转速、pH和温度对丙酮酸分批发酵的影响。高转速（500r／min）下,丙酮酸产率较高（71％）,但葡萄糖消耗速度较慢（1．23g／（L·h））;低转速（300r／min）下,细胞消耗葡萄糖的速度加快（1．95g/（L·h））,而丙酮酸产率（0．48％）却明显下降。将搅拌转速恒定在400r／min可在一定程度上获得较高的丙酮酸产率（0．62％）和葡萄糖消耗速度（1．66g／（L·h））。CaCO3调节pH时,较多碳流从丙酮酸节点转向α-酮戊二酸节点和细胞生长,最终丙酮酸产量比NaOH调节pH时的发酵结果低38．7％;NH3·H2O调节pH时最终细胞浓度和丙酮酸产量仅为NaOH调节时的77．8％和90．9％。pH5．5时最利于丙酮酸的合成。较高的发酵温度加速T.glabrata积累丙酮酸,但同时会导致α-酮戊二酸的提前积累;而较低的温度下甘油和α-酮戊二酸积累较少,丙酮酸发酵的最适温度为28～30℃。     

溶氧反馈分批补料高密度培养人骨形成蛋白-2工程菌

对表达人骨形成蛋白－2成熟肽的基因工程大肠杆菌E.coli DH5α/pDH-B2m在500mL摇瓶中进行了培养条件的摸索实验,并在此基础上扩大至NBS Bioflo IV20L发酵罐,利用溶氧反馈－分批补料培养技术：在培养过程中保持适当的溶解氧（40％）,以溶氧值在线反馈控制搅拌速度及流加补料培养基,使细菌保持适当的比生长率,成功地进行了工程菌的高密度培养,最终菌体密度达OD600＝57,每升干菌量22.8g,目的蛋白的表达量占细菌总蛋白的30％,人骨形成蛋白－2成熟肽的理论产率达到3.59g/L。     

代谢工程改造大肠杆菌合成丁二酸及发酵罐放大工艺

【背景】Escherichia coli AFP111发酵生产丁二酸时大量副产乙酸,丁二酸得率低。【目的】代谢工程改造EscherichiacoliAFP111,提高丁二酸得率,降低副产物乙酸的生成,建立100 L规模的丁二酸发酵工艺。【方法】一步同源重组敲除乙酸合成途径关键酶基因,改造丁二酸合成途径关键酶启动子实现过表达;单因素优化5L发酵罐培养条件。【结果】敲除乙酸产生途径编码乙酸激酶和磷酸转乙酰酶的基因ackA-pta、苏氨酸脱羧酶和2-酮丁酸甲酸裂解酶的基因tdcDE获得SX02菌株,摇瓶发酵条件下其乙酸产量下降了53.42%,丁二酸得率提高9.85%。在SX02菌株基础上,经启动子改造过表达编码葡萄糖激酶的基因glk后获得菌株SX03,其Glk酶活性提高3.66倍,乙酸产量下降了31.62%,丁二酸得率提高8.28%。SX03菌株发酵生产丁二酸在5 L发酵罐进行放大,其乙酸产量为3.97 g/L,丁二酸得率为1.62 mol/mol葡萄糖,相比出发菌株的乙酸产量下降了75.76%,丁二酸得率提高19.12%。在5L发酵罐上对比研究了中和剂Na2CO3和NaOH混合液替换碱式MgCO3的发酵效果,并优化了发酵pH、搅拌转速和葡萄糖浓度,获得如下最适发酵条件:pH6.8,搅拌转速250r/min,葡萄糖100g/L,发酵结束时乙酸产量为2.24 g/L,丁二酸得率为1.66 mol/mol葡萄糖。中和剂替换优化后乙酸产量下降了20.65%,丁二酸得率提高2.47%。菌株SX03发酵工艺进一步在100 L发酵罐上实现放大,其乙酸产量为1.91 g/L,丁二酸得率为1.30 mol/mol葡萄糖。【结论】通过代谢工程改造的大肠杆菌,其副产物乙酸含量显著下降,丁二酸得率提高,并在5 L和100 L发酵罐上实现了工艺放大,展现出较大的工业化利用潜力。     

弧菌外膜蛋白ompk工程菌规模化培养表达初探

目的:为Ompk亚单位疫苗生产提供参数。方法:利用振荡和发酵培养,测定不同培养时间的菌液浊度和蛋白诱导表达效果。结果:工程菌30℃摇瓶培养10h,种子罐培养8～10h,较为合适;摇瓶和5L发酵罐诱导表达培养,升温42℃诱导表达,7h效果较佳;经5批次50L、500L发酵罐诱导表达培养,均可得到重组蛋白表达的工程菌体,50L和500L发酵罐最高生物量分别为5.32g/L和6.38g/L,平均可达到3.75 g/L和4.74 g/L;适当延迟升温诱导前的培养时间,可提高工程菌的得率。结论:初步确定了重组蛋白工程菌规模化诱导表达培养方法,利用500L发酵罐培养可得到诱导表达的工程菌体。     

利用Pichia pastoris生产S-腺苷甲硫氨酸的发酵工艺

在摇瓶中考察了重组Pichia pastoris发酵的诱导剂量,L-甲硫氨酸,以及pH对腺苷甲硫氨酸产量的影响.放大到3.7 L发酵罐和30 L发酵罐后,研究了重组细胞的发酵过程变化,对S-腺苷甲硫氨酸初步纯化.摇瓶中优化后的发酵条件是:每天添加1%甲醇诱导,L-甲硫氨酸为50mmol/L,培养基pH 5.0.培养144 h后SAM产量达到2.32 g/L.3.7 L发酵罐中发酵251 h后细胞浓度为120 g/L,SAM总量为15.18 g.放大到30 L发酵罐中,发酵225.5 h后细胞浓度约为120 g/L,SAM总量为145.05 g.纯化后SAM的纯度为93.5%,回收率为84.5%.     

侧孢霉液体深层发酵生产木质纤维素分解酶

近年来,直接利用农业秸秆及其废弃物通过微生物转化生产纤维素酶和蛋白饲料引起了广大的关注（Buswelletal,1996,okekeetal,1993）。但目前对木质素分解酶的研究报导很少。本文采用7L搅拌式发酵罐,对侧抱霉（砂orotrichumsp．）直接利用甜菜渣液体深层发酵生产木质纤维素分解酶及酶反应条件进行了初步探讨。l材料与方法1．l菌种和培养条件侧抱震（》orotrichumsp）由中国农科院原子能利用研究所提供。菌株PDA斜面在4℃冰箱条件下保存,用于本项研究。液体种子培养基（g／L）：结晶纤维素10;酵母粉l;无机盐溶液10ml。发酵培养基（g…     

Investigation into the structural, morphological, mechanical and thermal behaviour of bacterial cellulose after a two-step purification process

Bacterial cellulose (BC) is a natural hydrogel, which is produced by Acetobacter xylinum (recently renamed Gluconacetobacter xylinum) in culture and constitutes of a three-dimensional network of ribbon-shaped bundles of cellulose microfibrils. Here, a two-step purification process is presented that significantly improves the structural, mechanical, thermal and morphological behaviour of BC sheet processed from these hydrogels produced in static culture. Alkalisation of BC using a single-step treatment of 2.5 wt.% NaOH solution produced a twofold increase in Young's modulus of processed BC sheet over untreated BC sheet. Further enhancements are achieved after a second treatment with 2.5 wt.% NaOCl (bleaching). These treatments were carefully designed in order to prevent any polymorphic crystal transformation from cellulose I to cellulose II, which can be detrimental for the mechanical properties. Scanning electron microscopy and thermogravimetric analysis reveals that with increasing chemical treatment, morphological and thermal stability of the processed films are also improved.     

Resistin and RELM-alpha gene expression in white adipose tissue of lactating mice

An ORF2 gene located upstream of the cellulose synthase (bcs) operon of Acetobacter xylinum BPR2001 was disrupted and a mutant (M2-2) was constructed. In static cultivation, the parent strain produced a tough, colorless, and insoluble cellulose pellicle, whereas M2-2 culture produced a thin, yellow, and fragile pellicle. The results of X-ray diffraction and 13C solid-state NMR indicated that the product of M2-2 is a mixture of cellulose I, cellulose II, and amorphous cellulose. The cellulose I to cellulose II ratio of the mixture was evaluated from the signal areas of C6 to be about 1:2. Electron microscopy revealed that the product of M2-2 included ribbon-like cellulose and irregularly shaped particles attached to the ribbons. On the other hand, the mutant complemented with plasmid pSA-ORF2/k containing the ORF2 gene and BPR2001 produced only cellulose I. These results indicate that the ORF2 gene is involved in the production and crystallization of cellulose I microfibrils by this microorganism.     

Bacterial cellulose production by Acetobacter xylinum in a 50-L internal-loop airlift reactor

Bacterial cellulose (BC) production was realized in a batch cultivation of Acetobacter xylinum subsp. sucrofermentans BPR2001 in a 50-L internal-loop airlift reactor. When the bacterium was cultivated with air supply, 3.8 g/L of BC was produced after 67 hours. When oxygen-enriched gas was supplied, the concentration of BC was doubled and the production rate of BC was 0.116 g/L. h, which was two times higher than that of air-supplied culture and comparable to that in a mechanically agitated stirred-tank fermentor. Bacterial cellulose produced by the airlift reactor formed a unique ellipse pellet (BC pellet), different from the fibrous form which was produced in an agitated stirred-tank fermentor. The BC-pellet suspension was demonstrated to have a higher volumetric oxygen transfer coefficient than the fibrous BC suspension in a 50-L internal-loop airlift reactor. The mixing time of BC-pellet suspension in the airlift reactor was also shorter than that in water.     

Effect of addition of sodium alginate on bacterial cellulose production by <Emphasis Type="Italic">Acetobacter xylinum</Emphasis>

Bacterial cellulose (BC) production by Acetobacter xylinum NUST4.1 was carried out in the shake flask and in a stirred-tank reactor by means of adding sodium alginate (NaAlg) into the medium. When 0.04% (w/v) NaAlg was added in the shake flask, BC production reached 6.0 g/l and the terminal yield of the cellulose was 27% of the total sugar initially added, compared with 3.7 g/l and 24% in the control, respectively. The variation between replicates in all determinations was less than 5%. During the cultivation in the stirred-tank reactor, the addition of NaAlg changed the morphology of cellulose from the irregular clumps and fibrous masses entangled in the internals to discrete masses dispersing into the broth, which indicates that NaAlg hinders formation of large clumps of BC, and enhances cellulose yield. Because the structure of cellulose is changed depending on the culture condition such as additives, structural characteristics of BC produced in the NaAlg-free and NaAlg medium are compared using scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD). SEM photographs show some differences in reticulated structures and ribbon width and FT-IR spectra indicate that there is the hydrogen bonding interaction between BC and NaAlg, then X-ray diffraction (XRD) analysis reveals that BC produced with NaAlg-added has a lower crystallinity and a smaller crystalline size. The results show that enhanced yields and modification of cellulose structure occur in the presence of NaAlg.     

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.     

Study of the Conditions for the Biosynthesis of Bacterial Cellulose by the Producer <Emphasis Type="Italic">Medusomyces gisevii</Emphasis> Sa-12

The effect of culture conditions on the bacterial cellulose (BC) yield synthesized by symbiotic culture Medusomyces gisevii Sa-12 grown in synthetic nutrient medium was studied for the first time. The conditions providing the maximum yield of bacterial cellulose were evaluated: the initial glucose concentration in the culture medium was 20–25 g/L, the temperature was 24–27°C, the ratio of the nutrient medium to the air volume was 1: 10, and the content of the black tea extracts comprised 1.6–4.8 g/L. A sample of chemically pure BC with the following characteristics was obtained under the selected culture conditions: moisture— 99.0%, degree of polymerization—4800, average width of microfibrillar ribbons—30.6 nm, degree of crystallinity— 86%, and the content of triclinic modification Iα—98%.     

Illustration of the development of bacterial cellulose bundles/ribbons by Gluconacetobacter xylinus via atomic force microscopy

The development of bacterial cellulose (BC) fibrils biosynthesized by Gluconacetobacter xylinus was investigated using atomic force microscopy (AFM). After various incubation times at 30 °C, both the length of BC fibrils and their average diameters increased significantly. After the first 2-h incubation, not only single BC microfibrils with an average diameter of 5.8?±?0.7 nm were biosynthesized but single microfibrils also began to bind with each other forming bundles. After longer incubation times of 6 h, 16 h, and 48 h, only BC bundles and ribbons or even only ribbons were detectable. The development of BC fibrils and the formation of BC bundles/ribbons along with the biosynthesis time were illustrated using AFM. Furthermore, single BC fibrils were twisted in a right-handed manner. The twisting of BC fibrils possibly promoted the formation of bigger ribbons.     

Schizophyllan production by newly isolated fungus <Emphasis Type="Italic">Schizophyllum commune</Emphasis> IBRC-M 30213: optimization of culture medium using response surface methodology

Schizophyllan (SPG) is a commercially attractive biopolymer produced by Schizophyllum commune. An investigation on the potential for SPG production by Iranian native S. commune was conducted based on culture medium, fermentation conditions and bioreactor type, . Nine native fungal strains were isolated from the northern forest of Iran at different times. Based on growth rate and SPG production, one strain was selected for further study. Optimal medium composition and inoculum size for maximizing SPG production and minimizing biomass were determined using central composite design by setting sucrose, yeast extract, inoculum size, carboxymethyl cellulose and oleic acid in the ranges of 50–200 g/L, 1–4 g/L, 2–10%, 2–12 g/L and 0.032–0.222%, respectively. The results showed that optimal results were obtained at 93.47 g/L sucrose, 1.87 g/L yeast extract, 7.68% inoculum size, 9.07 g/L carboxymethyl cellulose and 0.13% oleic acid, with maximum SPG production of 9.97 g/L and minimum biomass of 35.18 g/L. Under these optimal conditions, the production of SPG was studied in stirred tank and bubble column bioreactors. The results revealed greater production in the stirred tank because of better mixing of the culture medium. The SPG produced was characterized using rheometery, Fourier transform infrared spectroscopy, nuclear magnetic resonance), scanning electron microscopy and gel permeation chromatography. The results of these characterizations demonstrated the similarity of the SPG produced by S. commune IBRC-M 30213 to commercial SPG. Thus, the SPG produced shows good potential as a polysaccharide for use in various industries.     

Enhanced production of bacterial cellulose by using a biofilm reactor and its material property analysis

Bacterial cellulose has been used in the food industry for applications such as low-calorie desserts, salads, and fabricated foods. It has also been used in the paper manufacturing industry to enhance paper strength, the electronics industry in acoustic diaphragms for audio speakers, the pharmaceutical industry as filtration membranes, and in the medical field as wound dressing and artificial skin material. In this study, different types of plastic composite support (PCS) were implemented separately within a fermentation medium in order to enhance bacterial cellulose (BC) production by Acetobacter xylinum. The optimal composition of nutritious compounds in PCS was chosen based on the amount of BC produced. The selected PCS was implemented within a bioreactor to examine the effects on BC production in a batch fermentation. The produced BC was analyzed using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), thermogravimetric analysis (TGA), and dynamic mechanical analysis (DMA). Among thirteen types of PCS, the type SFYR+ was selected as solid support for BC production by A. xylinum in a batch biofilm reactor due to its high nitrogen content, moderate nitrogen leaching rate, and sufficient biomass attached on PCS. The PCS biofilm reactor yielded BC production (7.05 g/L) that was 2.5-fold greater than the control (2.82 g/L). The XRD results indicated that the PCS-grown BC exhibited higher crystallinity (93%) and similar crystal size (5.2 nm) to the control. FESEM results showed the attachment of A. xylinum on PCS, producing an interweaving BC product. TGA results demonstrated that PCS-grown BC had about 95% water retention ability, which was lower than BC produced within suspended-cell reactor. PCS-grown BC also exhibited higher T _max compared to the control. Finally, DMA results showed that BC from the PCS biofilm reactor increased its mechanical property values, i.e., stress at break and Young's modulus when compared to the control BC. The results clearly demonstrated that implementation of PCS within agitated fermentation enhanced BC production and improved its mechanical properties and thermal stability.     

Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor

Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.