Production,optimization, purification,characterization, and anti-cancer application of extracellular L-glutaminase produced from the marine bacterial isolate

Abstract

L-glutaminase from bacterial sources has been proven to be effective and economical agents in cancer therapy, food industry and high-value chemicals like threonine. In the present study, a newly isolated bacterial strain was potentially producing extracellular L-glutaminase, it identified as Bacillus subtilis OHEM11 (MK389501) using the 16S rRNA gene. L-glutaminase production optimized and the optimum factors for production under submerged fermentation were at pH 6.5–7.0 and 35?°C after 28?hr using rhamnose and glutamine as carbon and nitrogen sources, respectively, while bagasse was the best inducer for the production under solid-state fermentation. Ethanol precipitation and ion-exchange chromatography using QFF are the purification steps. L-glutaminase was purified to 2-fold with specific activity 89.78?U/mg and its molecular weight about 54.8?kDa with the alkaline property of the enzyme makes it clear having carcinostatic property; maximum enzyme activity at pH 8.2 and 40?°C and retained about 90% activity for 1?hr. The cytotoxicity effect of L-glutaminase indicated a significant safety on Vero cells with high anticancer activity against NFS-60, HepG-2, and MCF-7 cancer cell lines. The outcomes demonstrated that L-glutaminase could be applied in many biotechnological applications such as pharmaceutical and food processing.     

Optimization of parameters affecting on CHO cell culture producing recombinant erythropoietin

Abstract

Several factors may affect erythropoietin (EPO) sugar structures including designing cell culture procedure, pH, concentration of additives, dissolved oxygen, and other physicochemical parameters. In this study, we investigated the influence of changes in effective parameters and compounds on the growth rate of Chinese hamster ovary cell (CHO) cells producing recombinant EPO. Cell culture was performed at different temperature, buffering conditions, and varied concentrations of additives such as pyruvic acid, insulin, GlutaMAX, and sodium butyrate. Results indicated that the optimal temperature and pH were 37?°C and 7.2, respectively. Also, optimal concentrations for pyruvic acid, butyrate, glutamate, and insulin were obtained to be 20?mM, 1?mM, 2?mM, and 40?μg/mL, respectively. Then, cell culture was performed in microcarrier-coated spinner flasks under the optimized condition. The results showed recombinant human EPO (rhEPO) production with adequate purity. Optimization of physicochemical conditions and culture media are important factors to improve the quantity and quality of protein products. This study showed that cell growth and recombinant EPO protein production significantly increased under the optimized conditions. The results of this research can also be used in scale-up to increase the efficiency of EPO production.

Abbreviations: EPO: erythropoietin; CHO cell: Chinese hamster ovary cell; rhEPO: recombinant human EPO; DMEM: modified eagle’s medium; FBS: fetal bovine serum; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; IGF-1: insulin-like growth factor 1     

黑老虎叶总黄酮提取工艺及其抗氧化活性研究

为探讨超声波辅助提取黑老虎叶总黄酮的最佳提取工艺条件及其抗氧化活性,该文以黑老虎叶为研究对象,采用超声波提取法提取黑老虎叶总黄酮,通过单因素试验研究提取时间、乙醇浓度、提取温度、料液比对黑老虎叶总黄酮提取率的影响,在单因素试验的基础上,采用正交试验优化其提取工艺条件,测试了最优条件下提取的黑老虎叶总黄酮对DPPH自由基、·OH自由基及超氧阴离子的清除能力。结果表明:黑老虎叶总黄酮超声辅助提取最佳提取条件为提取时间 35 min、乙醇浓度80%、提取温度50 ℃、料液比1:20 g·mL^-1,最佳条件下提取率为4.83%。抗氧化活性测试结果显示,黑老虎叶总黄酮表现出较好的清除DPPH自由基、·OH自由基及超氧阴离子能力,其抗氧化能力为清除DPPH自由基能力>清除超氧阴离子能力>清除·OH自由基能力。在浓度为0.8 mg·mL^-1时,黑老虎叶总黄酮清除DPPH自由基、·OH自由基及超氧阴离子的能力相当于同浓度下Vc的97.6%、82.1%、95.5%,黑老虎叶总黄酮是天然抗氧化剂的良好来源。上述结果为黑老虎叶活性成分的提取及开发利用提供了理论基础。     

西南旱地油菜田土壤水分和作物光合特征对覆盖材料和垄沟比的响应

为了应对西南地区频发的季节性干旱,提高旱作农田水分利用效率和作物光能利用效率,设置大田试验,研究不同覆盖材料(普通白膜、普通黑膜、生物降解膜和不覆膜)和不同垄沟比(40 cm∶40 cm和40 cm∶80 cm)对土壤贮水量和油菜光合特性、荧光参数、叶绿素相对含量(SPAD)的影响。结果表明: 油菜生育期不同覆盖材料下的土壤贮水量为:普通黑膜垄作(BR)≈普通白膜垄作(WR)≈生物降解膜垄作(BDR)＞不覆膜垄作(NR)＞平作(FP);在同一覆盖材料下,垄沟比对土壤贮水量无显著影响。与平作相比,垄沟集雨处理的净光合速率、气孔导度、最大荧光、可变荧光、PSⅡ最大光化学量子产量、PSⅡ潜在活性、光化学猝灭系数和非光化学猝灭系数均有所提高;与平作相比,WR、BR、BDR和NR处理的SPAD值分别提高6.1%、8.6%、8.5%和3.6%,瞬时水分利用效率分别提高18.3%、11.4%、16.3%和10.4%。相比于平作,BR、WR和BDR处理可显著增加油菜产量,NR处理增产不显著,普通黑膜垄作+垄沟比40 cm∶40 cm处理的经济效益最高。可见,垄沟集雨种植可以改善西南旱地油菜田土壤水分,增强油菜叶片光合能力,提高油菜产量,增加农民收入。     

竹柏2种颜色叶片的光合特性研究

为了解竹柏(Podocarpus nagi)的光合特性,以3 a生全绿叶和花叶竹柏为材料,测定其光合色素含量和气体交换参数。结果表明,全绿叶竹柏叶片的叶绿素a、叶绿素b、类胡萝卜素、叶绿素a+b、叶绿素a/b和叶绿素a+b/类胡萝卜素均显著高于花叶竹柏;全绿叶竹柏叶片的初始量子效率、最大光合速率和暗呼吸速率均显著高于花叶,而光饱和点和光补偿点均显著低于花叶;全绿叶竹柏叶片的初始羧化效率、光合速率、CO2饱和点和光呼吸速率均高于花叶,而CO2补偿点低于花叶。2种颜色叶片的气孔导度、蒸腾速率和水分利用效率均随着光合有效辐射的增大而增大,且均表现为全绿叶花叶,而胞间CO2浓度则相反,表现为花叶全绿叶。因此,全绿叶竹柏利用弱光的能力强于花叶竹柏,而花叶竹柏利用强光的能力更强,在园林绿化配置中,可根据2种颜色叶片的光合特性合理配置。     

Optimization of culture media to enhance the growth of tissue engineered cartilage

Tissue engineering is a promising option for cartilage repair. However, several hurdles still need to be overcome to develop functional tissue constructs suitable for implantation. One of the most common challenges is the general low capacity of chondrocytes to synthesize cartilage-specific extracellular matrix (ECM). While different approaches have been explored to improve the biosynthetic response of chondrocytes, several studies have demonstrated that the nutritional environment (e.g., glucose concentration and media volume) can have a profound effect on ECM synthesis. Thus, the purpose of this study was to optimize the formulation of cell culture media to upregulate the accumulation of cartilaginous ECM constituents (i.e., proteoglycans and collagen) by chondrocytes in 3D culture. Using response surface methodology, four different media factors (basal media, media volume, glucose, and glutamine) were first screened to determine optimal media formulations. Constructs were then cultured under candidate optimal media formulations for 4 weeks and analyzed for their biochemical and structural properties. Interestingly, the maximal accumulation of proteoglycans and collagen appeared to be elicited by different media formulations. Most notably, proteoglycan accumulation was favored by high volume, low glucose-containing DMEM/F12 (1:1) media whereas collagen accumulation was favored by high volume, high glucose-containing F12 media. While high glutamine-containing media elicited increased DNA content, glutamine concentration had no apparent effect on ECM accumulation. Therefore, optimizing the nutritional environment during chondrocyte culture appears to be a promising, straight-forward approach to improve cartilaginous tissue formation. Future work will investigate the combined effects of the nutritional environment and external stimuli.     

Straightforward method for calibration of mechanistic cation exchange chromatography models for industrial applications

Mechanistic modeling of chromatography processes is one of the most promising techniques for the digitalization of biopharmaceutical process development. Possible applications of chromatography models range from in silico process optimization in early phase development to in silico root cause investigation during manufacturing. Nonetheless, the cumbersome and complex model calibration still decelerates the implementation of mechanistic modeling in industry. Therefore, the industry demands model calibration strategies that ensure adequate model certainty in a limited amount of time. This study introduces a directed and straightforward approach for the calibration of pH-dependent, multicomponent steric mass action (SMA) isotherm models for industrial applications. In the case investigated, the method was applied to a monoclonal antibody (mAb) polishing step including four protein species. The developed strategy combined well-established theories of preparative chromatography (e.g. Yamamoto method) and allowed a systematic reduction of unknown model parameters to 7 from initially 32. Model uncertainty was reduced by designing two representative calibration experiments for the inverse estimation of remaining model parameters. Dedicated experiments with aggregate-enriched load material led to a significant reduction of model uncertainty for the estimates of this low-concentrated product-related impurity. The model was validated beyond the operating ranges of the final unit operation, enabling its application to late-stage downstream process development. With the proposed model calibration strategy, a systematic experimental design is provided, calibration effort is strongly reduced, and local minima are avoided.     

Effects of stocking density during live transportation on haematological parameters of Siberian sturgeon (Acipenser baerii,Brandt, 1869)

In this study, we investigated the effects of different stocking densities water quality and blood parameters during the transportation of the Siberian sturgeon (Acipenser baerii). Experiments were carried out in high-density polyethylene tank with three different stocking densities (50, 100 and 150 kg/m³) for 20 h. Ammonium nitrogen (NH₄-N) and nitrite nitrogen (NO₂-N) were measured in the water samples. The erythrocyte, leucocyte, haematocrit (HCT), haemoglobin (Hb), cortisol, and sodium ion (Na⁺) were measured in blood samples. Ammonium nitrogen and nitrite nitrogen concentrations in the water have increased in parallel with the stocking density depending on the time. In the highest stocking density, the maximum levels of NH₄-N and NO₂-N at the 16^th h reached 2.64 mg/L and 4.74 mg/L, respectively. Erythrocyte, leucocyte, HCT, and Hb values did not differ significantly between the experimental groups (p > .05). The results showed that fish could be transported safely for 20 h in 15℃ water temperature at 50 kg/m³ stocking density; however, stocking density of 100 kg/m³ and over could threaten fish welfare and health as from 16^th h.     

利用基因组改组技术提高短杆菌素产量及其培养条件优化*

短杆菌素是一种广谱抗菌肽,对细菌和真菌均有较好的抑制作用,具有潜在的抗生素替代价值。通过对侧孢短芽孢杆菌fmb70进行紫外诱变、亚硝基胍诱变、常压室温等离子体诱变,获得3株短杆菌素产量提高的诱变菌株。随后以诱变菌株为亲本进行两轮基因组改组,获得融合子F₂-24,其短杆菌素产量为(340.5±16.35) μg/mL,是野生菌株fmb70短杆菌素产量的1.92倍。融合子传代5代后,该菌株短杆菌素产量无明显差异,说明菌株稳定性良好。最后对该菌株产短杆菌素的培养基和发酵条件进行优化,优化后的培养基为:4%蔗糖、2%牛肉膏、0.5%氯化镁,发酵温度30℃、培养24 h、培养基初始pH6.0。优化后的短杆菌素产量可达(442.45±9.58)μg/mL,是初始培养条件的2.50倍。     

Prediction of multi-criteria optimization (MCO) parameter efficiency in volumetric modulated arc therapy (VMAT) treatment planning using machine learning (ML)

PurposeTo predict the impact of optimization parameter changes on dosimetric plan quality criteria in multi-criteria optimized volumetric-modulated-arc therapy (VMAT) planning prior to optimization using machine learning (ML).MethodsA data base comprising a total of 21,266 VMAT treatment plans for 44 cranial and 18 spinal patient geometries was generated. The underlying optimization algorithm is governed by three highly composite parameters which model a combination of important aspects of the solution. Patient geometries were parametrized via volume- and shape properties of the voxel objects and overlap-volume histograms (OVH) of the planning-target-volume (PTV) and a relevant organ-at-risk (OAR). The impact of changes in one of the three optimization parameters on the maximally achievable value range of five dosimetric properties of the resulting dose distributions was studied. To predict the extent of this impact based on patient geometry, treatment site, and current parameter settings prior to optimization, three different ML-models were trained and tested. Precision-recall curves, as well as the area-under-curve (AUC) of the resulting receiver-operator-characteristic (ROC) curves were analyzed for model assessment.ResultsSuccessful identification of parameter regions resulting in a high variability of dosimetric plan properties depended on the choice of geometry features, the treatment indication and the plan property under investigation. AUC values between 0.82 and 0.99 could be achieved. The best average-precision (AP) values obtained from the corresponding precision/recall curves ranged from 0.71 to 0.99.ConclusionsMachine learning models trained on a database of pre-optimized treatment plans can help finding relevant optimization parameter ranges prior to optimization.