A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100°C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20–30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed–batch cultivation, glucose concentration was maintained at 2–3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.     

A recombinant lipoprotein antigen against Lyme disease expressed in E. coli: fermentor operating strategies for improved yield

Decorin-binding lipoprotein, lpp-DBP, a bacterial surface adhesin, shows promise as a vaccine against Lyme disease. It is expressed in recombinant E. coli as an undesirable 20.5 KDa apoprotein that is subsequently lipidated in vivo to the desired 22 KDa lpp-DBP form. This study defines fermentation conditions for maximizing lpp-DBP yield. Super broth medium, a low post-induction temperature (30 degrees C), and a glucose feed based on dissolved oxygen resulted in high lpp-DBP yield and minimized apoprotein formation. Since cells lysed within 2-3 h after induction, the cell yield was maximized by growing cells to high cell density prior to induction. Compared to a glucose feed based on maintaining a constant fermentor glucose concentration (Glucose-Stat), feeding based on maintaining a constant dissolved oxygen level (DO-Stat) improved yields. Also, a dissolved oxygen level of 60% (air saturation) was best, as no product degradation was detected by Western blotting and SDS-PAGE. Acetic acid levels under both modes of glucose feed were sufficiently low, and no adverse growth effects were observed.     

Batch and fed-batch cultivation for excretive production of human epidermal growth factor (hEGF) with recombinant E. Coli K12 system

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32 degrees C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.     

Lactobacillus amylovorus as a phytase producer in submerged culture

Nineteen strains of lactic acid producing bacteria of the genera Lactobacillus and Streptococcus collected from different culture collections were screened for the production of extracellular phytase. A number of them exhibited the enzyme activity in the fermentation medium but Lactobacillus amylovorus B4552 produced the maximum amounts of phytase ranging from 125 to 146 units ml^-1 extracellularly under the submerged cultivation conditions. The optimum glucose and inorganic phosphate levels for highest phytase production were found to be 1% and 24% mg, respectively. Lactobacillus amylovorus has potential in improving nutritional qualities of cereal and pulse-based food fermentations.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid

Escherichia coli harboring a recombinant plasmid was cultivated in fed-batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of beta-isopropylmalate dehydrogenase activity at 75 degrees C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH-stat was useful for feeding amino acids.     

抗菌肽Adenoregulin基因工程菌培养条件的优化及分批发酵研究

denoregulin（ADR）是来源于南美树蛙Phyllomedusa bicolor皮肤的含有33个氨基酸的抗菌肽,在非极性环境中形成α_螺旋型结构,具有抗菌活性强、抗菌谱广的特点。将ADR基因克隆于pET32a载体上,转化大肠杆菌BL21(DE3),对这一工程菌株的培养条件进行了优化。通过正交试验,考察诱导时机、诱导剂量和诱导时间三个因素的不同水平对蛋白表达的影响,结果发现诱导时机的影响尤为显著,考察了9种不同培养基对表达量的影响,发现培养基中加入葡萄糖对目标蛋白的稳定表达起了重要的作用,确定最佳培养条件为：培养基为2×YT+0.5%葡萄糖,诱导时机为OD600=0.9左右,诱导剂IPTG加入的终浓度为0.1mmol/L,诱导时间为4h。采用前期恒pH、后期指数流加的策略进行工程菌BL21(DE3)/pET32a-adr的高密度培养,在整个流加过程中,通过控制葡萄糖的加入量,将菌株的比生长速率控制在015h^-1,乙酸浓度也被控制在较低的水平(<2g/L),但是质粒丢失严重,在发酵结束时,约有40%的大肠杆菌中不带质粒,这导致了目标蛋白的表达量下降严重,但是表达的目标蛋白90%以上为可溶性形式。表达的融合蛋白无抑菌活性,而裂解后得到的ADR单体具有明显的抑菌活性。     

可溶性TRAIL蛋白的高密度培养及补料策略研究

采用分批补料的方法高密度培养重组大肠杆菌C600/PbvTRAIL制备人可溶性TRAIL蛋白,优化发酵工艺,探索简单高效的分离纯化方法并测定蛋白生物活性。通过比较几种不同的补料策略：间歇流加、Dostat、pHstat,摸索了一种流加策略,即DOstatpHstat组合流加,有效的避免了发酵过程中,尤其是诱导表达阶段乙酸积累的增加,使TRAIL蛋白在高密度培养条件下,得到高效表达。菌体密度最终达到300g/L（WCW）以上,可溶性TRAIL蛋白占菌体总蛋白的4.2%,含量为1.1g/L。在整个发酵过程中,乙酸浓度接近于0,且未使用任何特殊手段,如纯氧、加压等,简化了发酵工艺,降低了发酵成本,为TRAIL的工业化生产创造了条件。     

Protease production by Bacillus subtilis in oxygen-controlled,glucose fed-batch fermentations

Summary An open-loop, on-off control system using the dissolved oxygen level to control a glucose feed was used in a study of growth and production of protease by Bacillus subtilis CNIB 8054. With this system, both glucose and oxygen were controlled at low concentrations. In batch fermentations, protease activity in the fermentation broth was maximum when growth had stopped. During oxygen-controlled, glucose fed-batch fermentations, growth and the production of protease activity continued during glucose feeding. Oxygen-controlled, glucose fed-batch fermentations produced more protease activity than batch fermentations, depending upon the set point for dissolved oxygen. These results indicate that control of glucose and oxygen concentrations can result in improvements in protease production.     

Raman-based dynamic feeding strategies using real-time glucose concentration monitoring system during adalimumab producing CHO cell cultivation

The use of Process Analytical Technology tools coupled with chemometrics has been shown great potential for better understanding and control of mammalian cell cultivations through real-time process monitoring. In-line Raman spectroscopy was utilized to determine the glucose concentration of the complex bioreactor culture medium ensuring real-time information for our process control system. This work demonstrates a simple and fast method to achieve a robust partial least squares calibration model under laboratory conditions in an early phase of the development utilizing shake flask and bioreactor cultures. Two types of dynamic feeding strategies were accomplished where the multi-component feed medium additions were controlled manually and automatically based on the Raman monitored glucose concentration. The impact of these dynamic feedings was also investigated and compared to the traditional bolus feeding strategy on cellular metabolism, cell growth, productivity, and binding activity of the antibody product. Both manual and automated dynamic feeding strategies were successfully applied to maintain the glucose concentration within a narrower and lower concentration range. Thus, besides glucose, the glutamate was also limited at low level leading to reduced production of inhibitory metabolites, such as lactate and ammonia. Consequently, these feeding control strategies enabled to provide beneficial cultivation environment for the cells. In both experiments, higher cell growth and prolonged viable cell cultivation were achieved which in turn led to increased antibody product concentration compared to the reference bolus feeding cultivation.     

Online detection of feed demand in high cell density cultures of Escherichia coli by measurement of changes in dissolved oxygen transients in complex media

A starvation-based dissolved oxygen (DO) transient controller was developed to supply growth-limiting substrate to high cell density fed-batch cultures of recombinant Escherichia coli. The algorithm adjusted a preexisting feed rate in proportion to the culture's oxygen demand, which was estimated from transients in the DO concentration after short periods of feed interruption. In this manner, the addition of glucose feed was precisely controlled at a rate that did not exceed the acetate production threshold, thus preventing acetate accumulation. In comparison to exponential feed algorithms commonly used in industry, the implementation of the new feeding strategy increased the final cell density from 32 to 44 g (dry cell weight).L(-1), with less than 16 mM acetate accumulated, producing an ideal culture for subsequent induction. Despite a constant starvation level and relatively low levels of acetate, experimental cultivations still tended to produce acetate towards the end of the process. The use of a simple Monod model provided an explanation as to why this may occur in high cell density cultivations and suggests how it may be overcome.     

Simultaneous production of catalase, glucose oxidase and gluconic acid by Aspergillus niger mutant.

The production of gluconic acid, extracellular glucose oxidase and catalase in submerged culture by a number of biochemical mutants has been evaluated. Optimization of stirrer speed, time cultivation and buffering action of some chemicals on glucose oxidase, catalase and gluconic acid production by the most active mutant, AM-11, grown in a 3-L glass bioreactor was investigated. Three hundred rpm appeared to be optimum to ensure good growth and best glucose oxidase production, but gluconic acid or catalase activity obtained maximal value at 500 or 900 rpm, respectively. Significant increase of dissolved oxygen concentration in culture (16-21%) and extracellular catalase activity were obtained when the traditional aeration was employed together with automatic dosed hydrogen peroxide.     

Production of Escherichia coli Heat-labile Enterotoxin in Fermenter Dialysis Culture

Production of Escherichia coli heat-labile enterotoxin was investigated with one porcine and one human strain in three different media under different cultivation conditions. Cultivation in aerated fermenters at pH 7·0 yielded 10–20 times more enterotoxin/ml of culture fluid than cultivation in shake flasks. A trypton-yeast extract medium was optimal in fermenter cultures. Comparatively good yields of enterotoxin in fermenters were also obtained in a glucose-salts medium. Continuous feeding of glucose and salts during fermenter cultivation resulted in a lower production of enterotoxin/mg of bacterial cells. Since this decrease in specific yield could be reversed by using dialysis culture, it was concluded that inhibition of toxin formation was due to the accumulation of extracellular low molecular weight metabolites. The highest yield of enterotoxin in dialysis culture was 80 ED₅₀ ml⁻¹ (rabbit jejunal loop test) which is at least eight times more toxin than in ordinary fermenter culture and 80 times more toxin than in shake flask cultures.     

Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture

A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture. The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed-batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level.     

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

Fed-batch cultivation of Wautersia eutropha

Batch kinetics of polyhydroxybutyrate (PHB) synthesis in a bioreactor under controlled conditions of pH and dissolved oxygen gave a biomass of 14 g l(-1) with a PHB concentration of 6.1 g l(-1) in 60 h. The data of the batch kinetics was used to develop a mathematical model, which was then extrapolated to fed-batch by incorporating the dilution due to substrate feeding. Offline computer simulation of the fed-batch model was done to develop the nutrient feeding strategies in the fed-batch cultivation. Fed-batch strategies with constant feeding of only nitrogen and constant feeding of both nitrogen and fructose were tried. Constant feeding strategy for nitrogen and fructose gave a better PHB production rate of 0.56 g h(-1) over the value obtained in batch cultivation (PHB production rate - 0.4 g h(-1)).     

High-cell-density cultivation for co-production of ergosterol and reduced glutathione by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Two different high-cell-density cultivation processes based on the mutant Saccharomyces cerevisiae GE-2 for simultaneous production of glutathione and ergosterol were investigated. Compared with keeping the ethanol volumetric concentration at a constant low level, feedback control of glucose feeding rate (F) by keeping the descending rate of ethanol volumetric concentration (ΔE/Δt) between −0.1% and 0.15% per hour was much more efficient to achieve a high glutathione and ergosterol productivity. This bioprocess overcomes some disadvantages of traditional S. cerevisiae-based cultivation process, especially shortening cultivation period and making the cultivation process steady-going. A classical on or off controller was used to manipulate F to maintain ΔE/Δt at its set point. The dry cell weight, glutathione yield and ergosterol yield reached 110.0 ± 2.6 g/l, 2,280 ± 76 mg/l, and 1,510 ± 28 mg/l in 32 h, respectively.     

Induction of Cellulases in chaetomium cellulolyticum by cellobiose

The production of extracellular cellulases by Chaetomium cellulolyticum could be induced by slow feeding of cellobiose to the cultures. Both the rate of production and the amount of activity were comparable to that obtained in batch cultivation on cellulose. The specific filter paper activity of 2.06 U per mg protein was almost two times higher than that obtained in cellulose medium. Cellulases were not induced when glucose was slowly fed to the cultures. Changing the feed stream from glucose to cellobiose resulted in a rapid accumulation of cellulases. Thus cellobiose has a similar role in cellulase induction in C. cellulolyticum, as earlier shown for Trichoderma reesei.     

以葡萄糖为生长相基质的重组毕赤酵母表达植酸酶发酵

甲醇营养型毕赤酵母表达外源蛋白是在醇氧化酶（alcohol oxidase,AOX）启动子（PAOXI）严格调控下进行的,然而这种启动子在转录水平受到葡萄糖的阻遏。本文研究了毕赤酵母在葡萄糖替代甘油为生长相碳源时表达重组植酸酶蛋白的发酵特征。结果表明：初始葡萄糖浓度为20dL的细胞得率高,为0．39g[DCW]／g。通过基于实时参数（溶氧和呼吸商）调控的葡萄糖补料策略,生长相40h后细胞密度达到100g[DCW]／L,甲醇诱导100h后植酸酶产量达到2200FTUphytase／mL,甲醇得率系数为0．25FTU phytase／gmethnol。因此,在毕赤酵母高表达重组蛋白培养中葡萄糖能够用作生长相基质,并能实现重组蛋白的高效表达。     

Production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system E. coli[pIET98

A method for the production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale is described employing the temperature induced runaway replication vector pIET98 and the Escherichia coli host strain BL21. Fed-batch cultivation using a semi-synthetic high-cell densitiy medium was adjusted in 5-L scale to yield a constant growth rate of 0,17 h(-1) and a final cell concentration of 27 g dry weight/L by exponentially increasing the nutrient supply. Runaway replication and thus, LeuDH expression was induced during the feeding phase by increasing the cultivation temperature to 41 degrees C yielding a specific enzyme activity of 110 U/mg, which corresponds to 30% of the soluble cell protein. The cultivation was terminated when the dissolved oxygen content fell below 10% saturation. The final volume activity was 600,000 U/L cultivation. No change in growth, cell density, or expression activity was observed scaling up the cultivation volume to 200 L. Thus, 120,000,000 units L-leucine dehydrogenase were obtained from one cultivation. The purification of L-leucine dehydrogenase to homogeneity was carried out by heat denaturation, liquid-liquid extraction, gel filtration, and anion-exchange chromatography to give pure enzyme in 65% yield. The integrity of the recombinant enzyme was tested measuring the molecular weight and determining the N-terminal amino acid sequence.