Pub Date : 2019-08-13DOI: 10.1080/10826068.2019.1650373
Ganesh T. Sivanathan, H. Mallubhotla, Satyanarayana V. Suggala
Abstract Proteolytic degradation is a serious problem that complicates downstream processing during production of recombinant therapeutic proteins. It can lead to decreased product yield, diminished biological activity, and suboptimal product quality. Proteolytic degradation or protein truncation is observed in various expression hosts and is mostly attributed to the activity of proteases released by host cells. Since these clipped proteins can impact pharmacokinetics and immunogenicity in addition to potency, they need to be appropriately controlled to ensure consistency of product quality and patient safety. A chromatography step for the selective removal of clipped proteins from an intact protein was developed in this study. Poly(ethylenimine)-grafted anion- exchange resins (PolyQUAT and PolyPEI) were evaluated and compared to traditional macroporous anion-exchange and tentacled anion-exchange resins. Isocratic retention experiments were conducted to determine the retention factors (k′) and charge factors (Z) were determined through the classical stoichiometric displacement model. High selectivity in separation of closely related clipped proteins was obtained with the PolyQUAT resin. A robust design space was established for the PolyQUAT chromatography through Design-Of-Experiments (DoE) based process optimization. Results showed a product recovery of up to 63% with purity levels >99.0%. Approximately, one-log clearance of host cell protein and two-logs clearance of host cell DNA were also obtained. The newly developed PolyQUAT process was compared with an existing process and shown to be superior with respect to the number of process steps, process time, process yield, and product quality.
摘要蛋白水解降解是一个严重的问题，使重组治疗蛋白生产过程中的下游加工复杂化。它会导致产品产量下降，生物活性降低，产品质量次优。蛋白水解降解或蛋白截断在不同的表达宿主中都有发生，这主要归因于宿主细胞释放的蛋白酶的活性。由于这些剪切蛋白除了效力外还会影响药代动力学和免疫原性，因此需要对其进行适当控制，以确保产品质量和患者安全的一致性。本研究开发了一种从完整蛋白中选择性去除剪切蛋白的色谱步骤。对聚乙亚胺接枝阴离子交换树脂(PolyQUAT和PolyPEI)进行了评价，并与传统的大孔阴离子交换树脂和触角阴离子交换树脂进行了比较。通过等压保留实验确定了保留因子k′，并通过经典的化学计量位移模型确定了电荷因子Z。PolyQUAT树脂对密切相关的剪切蛋白具有很高的选择性。通过基于实验设计(design - of - experiments, DoE)的工艺优化，为PolyQUAT色谱建立了稳健的设计空间。结果表明，产品回收率可达63%，纯度可达99.0%。大约，宿主细胞蛋白的一对数清除率和宿主细胞DNA的两对数清除率也得到了。新开发的PolyQUAT工艺与现有工艺进行了比较，结果表明，在工艺步骤数量、工艺时间、工艺收率和产品质量方面，PolyQUAT工艺具有优越性。
{"title":"Selective removal of closely related clipped protein impurities using poly(ethylenimine)- grafted anion-exchange chromatography resin","authors":"Ganesh T. Sivanathan, H. Mallubhotla, Satyanarayana V. Suggala","doi":"10.1080/10826068.2019.1650373","DOIUrl":"https://doi.org/10.1080/10826068.2019.1650373","url":null,"abstract":"Abstract Proteolytic degradation is a serious problem that complicates downstream processing during production of recombinant therapeutic proteins. It can lead to decreased product yield, diminished biological activity, and suboptimal product quality. Proteolytic degradation or protein truncation is observed in various expression hosts and is mostly attributed to the activity of proteases released by host cells. Since these clipped proteins can impact pharmacokinetics and immunogenicity in addition to potency, they need to be appropriately controlled to ensure consistency of product quality and patient safety. A chromatography step for the selective removal of clipped proteins from an intact protein was developed in this study. Poly(ethylenimine)-grafted anion- exchange resins (PolyQUAT and PolyPEI) were evaluated and compared to traditional macroporous anion-exchange and tentacled anion-exchange resins. Isocratic retention experiments were conducted to determine the retention factors (k′) and charge factors (Z) were determined through the classical stoichiometric displacement model. High selectivity in separation of closely related clipped proteins was obtained with the PolyQUAT resin. A robust design space was established for the PolyQUAT chromatography through Design-Of-Experiments (DoE) based process optimization. Results showed a product recovery of up to 63% with purity levels >99.0%. Approximately, one-log clearance of host cell protein and two-logs clearance of host cell DNA were also obtained. The newly developed PolyQUAT process was compared with an existing process and shown to be superior with respect to the number of process steps, process time, process yield, and product quality.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77027627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-08-13DOI: 10.1080/10826068.2019.1650374
S. Majumdar, B. Goswami, Ankita Chakraborty, D. Bhattacharyya, J. Bhowal
Abstract The present study investigated the operational conditions for different pretreatment approaches and subsequent enzymatic hydrolysis of cauliflower wastes (stalk and leaf) for better release of fermentable sugars. The structural analysis of raw and pretreated lignocellulosic biomasses was investigated using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transforms infrared (FTIR) analysis. Results demonstrated that the highest cellulose conversion rate and removal of most of the hemicellulose and lignin were obtained with organosolvent pretreatment. Using methanol in presence of sodium (Na) acetate was most effective in delignification of cauliflower wastes. In the present study, methanol (100% v/v) in presence of 0.1 M Na-acetate at 121 °C for 45 and 60 min for stalk and leaf, respectively, gave maximum reducing sugar yield. Response surface methodology was used to optimize different process parameters for enzymatic saccharification using microbial cellulase and xylanase. The optimum operation condition of enzymatic hydrolysis of organosolvent pretreated cauliflower wastes were substrate loading (2.5% w/v for both stalk and leaf), enzyme loading (15 and 10 U/g for stalk and leaf, respectively), pH (4.46 and 5.48 for stalk and leaf, respectively), at 60 °C and for 180 min.
摘要本研究研究了不同预处理方法的操作条件，并对花椰菜废弃物(茎叶)进行酶解，以更好地释放可发酵糖。采用扫描电镜(SEM)、x射线衍射(XRD)和傅里叶变换红外(FTIR)分析对原料和预处理木质纤维素生物质进行了结构分析。结果表明，有机溶剂预处理能获得最高的纤维素转化率和大部分半纤维素和木质素的去除率。甲醇在乙酸钠存在下对花椰菜废渣进行脱木质素处理是最有效的。在本研究中，甲醇(100% v/v)在0.1 M Na-acetate的存在下，在121°C下，对茎和叶分别处理45和60 min，可获得最大的还原糖产量。采用响应面法对微生物纤维素酶和木聚糖酶的糖化工艺参数进行优化。有机溶剂预处理菜花废弃物酶解的最佳操作条件为底物量(茎叶均为2.5% w/v)、酶量(茎叶分别为15和10 U/g)、pH(茎叶分别为4.46和5.48)、60℃、180 min。
{"title":"Effect of pretreatment with organic solvent on enzymatic digestibility of cauliflower wastes","authors":"S. Majumdar, B. Goswami, Ankita Chakraborty, D. Bhattacharyya, J. Bhowal","doi":"10.1080/10826068.2019.1650374","DOIUrl":"https://doi.org/10.1080/10826068.2019.1650374","url":null,"abstract":"Abstract The present study investigated the operational conditions for different pretreatment approaches and subsequent enzymatic hydrolysis of cauliflower wastes (stalk and leaf) for better release of fermentable sugars. The structural analysis of raw and pretreated lignocellulosic biomasses was investigated using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transforms infrared (FTIR) analysis. Results demonstrated that the highest cellulose conversion rate and removal of most of the hemicellulose and lignin were obtained with organosolvent pretreatment. Using methanol in presence of sodium (Na) acetate was most effective in delignification of cauliflower wastes. In the present study, methanol (100% v/v) in presence of 0.1 M Na-acetate at 121 °C for 45 and 60 min for stalk and leaf, respectively, gave maximum reducing sugar yield. Response surface methodology was used to optimize different process parameters for enzymatic saccharification using microbial cellulase and xylanase. The optimum operation condition of enzymatic hydrolysis of organosolvent pretreated cauliflower wastes were substrate loading (2.5% w/v for both stalk and leaf), enzyme loading (15 and 10 U/g for stalk and leaf, respectively), pH (4.46 and 5.48 for stalk and leaf, respectively), at 60 °C and for 180 min.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76333574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-08-13DOI: 10.1080/10826068.2019.1650378
F. R. Marín, J. Hernández-Ruiz, M. B. Arnao
Abstract In many occasions it is necessary to use fast and simple methods, different to the chromatographic techniques, for the quantification of biomolecules such as flavonoids. Also, the flavonoid levels in some foodstuffs can be influenced by industrial extraction processes such as pressing and squeezing, resulting in modification of their functional value. For this purpose, we have developed a rapid method to analyze flavonoids, based on a coupling reaction between ABTS and flavonoid mediated by peroxidase. The present method can be used to detect and measure flavonoids with hydroxyl moieties on A- or B-rings, not adjacent to methoxy or oxo substitutions. The visible spectrum of the ABTS-flavonoid complex, the calibration curve (within the range 5-50 μM) and the molar absorption coefficients for isosakuranetin, isonaringin, rhoifolin, hyperoside, rutin, hesperetin, quercetin, kaempherol and naringenin are given. The method has been applied to complex culture media and is sensitive, accurate, quick and easy to apply. This method can be used in laboratories that do not have sophisticated and expensive techniques such as liquid chromatography and also as a quick, simple and inexpensive technique for student practice laboratories.
{"title":"A colorimetric method for the determination of different functional flavonoids using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and peroxidase","authors":"F. R. Marín, J. Hernández-Ruiz, M. B. Arnao","doi":"10.1080/10826068.2019.1650378","DOIUrl":"https://doi.org/10.1080/10826068.2019.1650378","url":null,"abstract":"Abstract In many occasions it is necessary to use fast and simple methods, different to the chromatographic techniques, for the quantification of biomolecules such as flavonoids. Also, the flavonoid levels in some foodstuffs can be influenced by industrial extraction processes such as pressing and squeezing, resulting in modification of their functional value. For this purpose, we have developed a rapid method to analyze flavonoids, based on a coupling reaction between ABTS and flavonoid mediated by peroxidase. The present method can be used to detect and measure flavonoids with hydroxyl moieties on A- or B-rings, not adjacent to methoxy or oxo substitutions. The visible spectrum of the ABTS-flavonoid complex, the calibration curve (within the range 5-50 μM) and the molar absorption coefficients for isosakuranetin, isonaringin, rhoifolin, hyperoside, rutin, hesperetin, quercetin, kaempherol and naringenin are given. The method has been applied to complex culture media and is sensitive, accurate, quick and easy to apply. This method can be used in laboratories that do not have sophisticated and expensive techniques such as liquid chromatography and also as a quick, simple and inexpensive technique for student practice laboratories.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87131363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-08-12DOI: 10.1080/10826068.2019.1650376
Kamil Piwowarek, E. Lipińska, E. Hać-Szymańczuk, Anna Rudziak, M. Kieliszek
Abstract Sequential optimization of propionate production using apple pomace was studied. All experiments were performed in a static flask in anaerobic conditions. Effect of apple pomace as nitrogen source against conventional N sources (yeast extract, peptone) was studied. The double increase was observed in propionic acid production while using yeast extract and peptone (0.29 ± 0.01 g/g), as against the use of only apple pomace extract (APE) (0.14 ± 0.01 g/g). Intensification of propionic acid fermentation was also achieved by increasing the pH control frequency of the culture medium from 24-(0.29 ± 0.01 g/g) to 12-hour intervals (30 °C) (0.30 ± 0.02 g/g) and by increasing the temperature of the culture from 30 to 37 °C (12-hour intervals of pH control) (0.32 ± 0.01 g/g). An important factor in improving the parameters of fermentation was the addition of biotin to the medium. The 0.2 mg/L dose of biotin allowed to attain 7.66 g/L propionate with a yield of 0.38 ± 0.03 g/g (12-hour intervals of pH control, 37 °C).
{"title":"Optimization of propionic acid production in apple pomace extract with Propionibacterium freudenreichii","authors":"Kamil Piwowarek, E. Lipińska, E. Hać-Szymańczuk, Anna Rudziak, M. Kieliszek","doi":"10.1080/10826068.2019.1650376","DOIUrl":"https://doi.org/10.1080/10826068.2019.1650376","url":null,"abstract":"Abstract Sequential optimization of propionate production using apple pomace was studied. All experiments were performed in a static flask in anaerobic conditions. Effect of apple pomace as nitrogen source against conventional N sources (yeast extract, peptone) was studied. The double increase was observed in propionic acid production while using yeast extract and peptone (0.29 ± 0.01 g/g), as against the use of only apple pomace extract (APE) (0.14 ± 0.01 g/g). Intensification of propionic acid fermentation was also achieved by increasing the pH control frequency of the culture medium from 24-(0.29 ± 0.01 g/g) to 12-hour intervals (30 °C) (0.30 ± 0.02 g/g) and by increasing the temperature of the culture from 30 to 37 °C (12-hour intervals of pH control) (0.32 ± 0.01 g/g). An important factor in improving the parameters of fermentation was the addition of biotin to the medium. The 0.2 mg/L dose of biotin allowed to attain 7.66 g/L propionate with a yield of 0.38 ± 0.03 g/g (12-hour intervals of pH control, 37 °C).","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79500289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-08-12DOI: 10.1080/10826068.2019.1650377
Arthitaya Kawee‐ai, Phisit Seesuriyachan
Abstract Select LAB, including Lactobacillus fermentum TISTR 950, Lactobacillus plantarum TISTR 2265 and Lactobacillus casei TISTR 1500 were investigated for their ability to enhance GABA, TPC and the antioxidant activity of perilla seed juice. L. casei TISTR 1500 produced higher GABA and TPC contents and presented higher antioxidant activity than other strains. Furthermore, the optimal fermentation condition to perilla seeds inoculated with L. casei TISTR 1500 to improve the GABA, TPC and antioxidant activity was performed using 33 full factorial design. The final optimal values for perilla fermentation was found at fermentation time of 4.82 days (4 days 19 h 40 min), initial substrate of 5% (w/v) and fermentation temperature of 30.07 °C. Under the optimal fermentation condition, an observed values of GABA, TPC, ABTS, DPPH and FRAP were 71.46 µg/g, 3175.00 µg GAE/g, 1991.40 µg TEAC/g, 9178.29 µg TEAC/g and 7753.34 µg TEAC/g, respectively, which was 3.3, 0.9, 2.9, 10.8 and 10.2 times higher than that of unfermented perilla seeds, and 2.1, 0.8, 0.9, 10 and 9.2 times of fermented perilla seeds before the optimization. These results may provide the foundation to further target in industrial application for the production of plant-based and develop functional perilla seed products containing GABA. Highlights Improved GABA, TPC and antioxidant contents were found using Lactobacillus casei TISTR 1500 Full factorial design applied to optimize fermented perilla seeds by lactic acid fermentation The optimized conditions dramatically increased GABA and TPC contents
{"title":"Optimization of fermented Perilla frutescens seeds for enhancement of gamma-aminobutyric acid and bioactive compounds by Lactobacillus casei TISTR 1500","authors":"Arthitaya Kawee‐ai, Phisit Seesuriyachan","doi":"10.1080/10826068.2019.1650377","DOIUrl":"https://doi.org/10.1080/10826068.2019.1650377","url":null,"abstract":"Abstract Select LAB, including Lactobacillus fermentum TISTR 950, Lactobacillus plantarum TISTR 2265 and Lactobacillus casei TISTR 1500 were investigated for their ability to enhance GABA, TPC and the antioxidant activity of perilla seed juice. L. casei TISTR 1500 produced higher GABA and TPC contents and presented higher antioxidant activity than other strains. Furthermore, the optimal fermentation condition to perilla seeds inoculated with L. casei TISTR 1500 to improve the GABA, TPC and antioxidant activity was performed using 33 full factorial design. The final optimal values for perilla fermentation was found at fermentation time of 4.82 days (4 days 19 h 40 min), initial substrate of 5% (w/v) and fermentation temperature of 30.07 °C. Under the optimal fermentation condition, an observed values of GABA, TPC, ABTS, DPPH and FRAP were 71.46 µg/g, 3175.00 µg GAE/g, 1991.40 µg TEAC/g, 9178.29 µg TEAC/g and 7753.34 µg TEAC/g, respectively, which was 3.3, 0.9, 2.9, 10.8 and 10.2 times higher than that of unfermented perilla seeds, and 2.1, 0.8, 0.9, 10 and 9.2 times of fermented perilla seeds before the optimization. These results may provide the foundation to further target in industrial application for the production of plant-based and develop functional perilla seed products containing GABA. Highlights Improved GABA, TPC and antioxidant contents were found using Lactobacillus casei TISTR 1500 Full factorial design applied to optimize fermented perilla seeds by lactic acid fermentation The optimized conditions dramatically increased GABA and TPC contents","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87214882","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-08-09DOI: 10.1080/10826068.2019.1650375
A. Ademakinwa, F. Agboola
Abstract The production of cell-wall degrading enzymes (CWDE) such as cellulase and pectinase by Aureobasidium pullulans NAC8 through induction using orange peels was investigated for the potential application of these enzymes in the extraction of lycopene from tomato skin, waste, and paste (SWP). The CWDE was then immobilized via entrapment in alginate beads for lycopene extraction and the kinetic/thermodynamic properties of the free and immobilized CWDE investigated. The optimum production of CWDE occurred at pH, temperature, and orange peel concentration of 6.0, 50 °C, and 2.0% (w/v), respectively. The values obtained for some kinetic and thermodynamic parameters such as and indicate that both free and immobilized cellulase and pectinase were thermostable between 40 and 50 °C. Maximum lycopene extracted from the tomato SWP was 80 ± 2.4 mg/kg, 42 ± 1.3 mg/kg and 60 ± 1.2 mg/kg, respectively, using the immobilized CWDE. The entrapped CWDE was able to extract lycopene with yields of 58 ± 4.2, 51 ± 1.2 and 57 ± 4.2% for tomato SWP respectively after the fifth cycle. Using orange peels for the induction of CWDE by A. pullulans offers a unique and cheaper approach to obtaining thermostable multi-enzyme complexes employable for easy lycopene extraction from tomato SWP.
{"title":"Kinetic and thermodynamic investigations of cell-wall degrading enzymes produced by Aureobasidium pullulans via induction with orange peels: application in lycopene extraction","authors":"A. Ademakinwa, F. Agboola","doi":"10.1080/10826068.2019.1650375","DOIUrl":"https://doi.org/10.1080/10826068.2019.1650375","url":null,"abstract":"Abstract The production of cell-wall degrading enzymes (CWDE) such as cellulase and pectinase by Aureobasidium pullulans NAC8 through induction using orange peels was investigated for the potential application of these enzymes in the extraction of lycopene from tomato skin, waste, and paste (SWP). The CWDE was then immobilized via entrapment in alginate beads for lycopene extraction and the kinetic/thermodynamic properties of the free and immobilized CWDE investigated. The optimum production of CWDE occurred at pH, temperature, and orange peel concentration of 6.0, 50 °C, and 2.0% (w/v), respectively. The values obtained for some kinetic and thermodynamic parameters such as and indicate that both free and immobilized cellulase and pectinase were thermostable between 40 and 50 °C. Maximum lycopene extracted from the tomato SWP was 80 ± 2.4 mg/kg, 42 ± 1.3 mg/kg and 60 ± 1.2 mg/kg, respectively, using the immobilized CWDE. The entrapped CWDE was able to extract lycopene with yields of 58 ± 4.2, 51 ± 1.2 and 57 ± 4.2% for tomato SWP respectively after the fifth cycle. Using orange peels for the induction of CWDE by A. pullulans offers a unique and cheaper approach to obtaining thermostable multi-enzyme complexes employable for easy lycopene extraction from tomato SWP.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81965809","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-08-06DOI: 10.1080/10826068.2019.1650372
Razieh Jafari Hajati, V. Payamnoor, Najmeh Ahmadian Chashmi
Abstract Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem’s inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150 mg l−1 on the 8th day. The highest content of BA was 1.3 mg g−1 DW after treatment with 1 mg l−1CCC on the 4th and 6th days and 200 mg l−1CTS NF on the 10th day. The highest B content (0.94 mg g−1DW) was obtained in the treated hairy root by 2 mg l−1 CCC after 4 and 6 days.
摘要白桦素(Betulin, B)和白桦酸(betulinic acid, BA)是两种三萜类化合物，在白桦不同器官中具有广泛的生物活性和药用活性。本研究旨在通过7种生物和非生物诱导子，提高白桦毛状根培养中B和BA的积累。用C58C1菌株在WPM(木本植物培养基)培养基中诱导钟柏茎内皮产生毛状根。不同激发子浓度和不同采根时间对钟藻毛状根培养的影响表明，壳聚糖(CTS)、氯化氯胆碱(CCC)和壳聚糖纳米纤维(CTS - NF)处理的钟藻毛状根的生长指数(GI)、B和BA最高。CTS 150mg l−1处理后第8天，毛羽根的GI值最高，为13。第4、6天和第10天分别用1 mg l - 1CCC和200 mg l - 1CTS NF处理，BA含量最高为1.3 mg g - 1 DW。2 mg l - 1 CCC处理4和6天后毛状根的B含量最高，为0.94 mg g - 1DW。
{"title":"Effect of biotic and abiotic elicitors on production of betulin and betulinic acid in the hairy root culture of Betula pendula Roth","authors":"Razieh Jafari Hajati, V. Payamnoor, Najmeh Ahmadian Chashmi","doi":"10.1080/10826068.2019.1650372","DOIUrl":"https://doi.org/10.1080/10826068.2019.1650372","url":null,"abstract":"Abstract Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem’s inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150 mg l−1 on the 8th day. The highest content of BA was 1.3 mg g−1 DW after treatment with 1 mg l−1CCC on the 4th and 6th days and 200 mg l−1CTS NF on the 10th day. The highest B content (0.94 mg g−1DW) was obtained in the treated hairy root by 2 mg l−1 CCC after 4 and 6 days.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91059235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-07-30DOI: 10.1080/10826068.2019.1645695
N. Suryawanshi, Sweta H. Naik, J. Eswari
Abstract The microbial polysaccharides secreted and produced from various microbes into their extracellular environment is known as exopolysaccharide. These polysaccharides can be secreted from the microbes either in a soluble or insoluble form.Lactobacillus sp. is one of the organisms that have been found to produce exopolysaccharide. Exo-polysaccharides (EPS) have various applications such as drug delivery, antimicrobial activity, surgical implants and many more in different fields. Medium composition is one of the major aspects for the production of EPS from Lactobacillus sp., optimization of medium components can help to enhance the synthesis of EPS . In the present work, the production of exopolysaccharide with different medium composition was optimized by response surface methodology (RSM) followed by tested for fitting with artificial neural networks (ANN). Three algorithms of ANN were compared to investigate the highest yeild of EPS. The highest yeild of EPS production in RSM was achieved by the medium composition that consists of (g/L) dextrose 15, sodium dihydrogen phosphate 3, potassium dihydrogen phosphate 2.5, triammonium citrate 1.5, and, magnesium sulfate 0.25. The output of 32 sets of RSM experiments were tested for fitting with ANN with three algorithms viz. Levenberg–Marquardt Algorithm (LMA), Bayesian Regularization Algorithm (BRA) and Scaled Conjugate Gradient Algorithm (SCGA) among them LMA found to have best fit with the experiments as compared to the SCGA and BRA.
{"title":"Extraction and optimization of exopolysaccharide from Lactobacillus sp. using response surface methodology and artificial neural networks","authors":"N. Suryawanshi, Sweta H. Naik, J. Eswari","doi":"10.1080/10826068.2019.1645695","DOIUrl":"https://doi.org/10.1080/10826068.2019.1645695","url":null,"abstract":"Abstract The microbial polysaccharides secreted and produced from various microbes into their extracellular environment is known as exopolysaccharide. These polysaccharides can be secreted from the microbes either in a soluble or insoluble form.Lactobacillus sp. is one of the organisms that have been found to produce exopolysaccharide. Exo-polysaccharides (EPS) have various applications such as drug delivery, antimicrobial activity, surgical implants and many more in different fields. Medium composition is one of the major aspects for the production of EPS from Lactobacillus sp., optimization of medium components can help to enhance the synthesis of EPS . In the present work, the production of exopolysaccharide with different medium composition was optimized by response surface methodology (RSM) followed by tested for fitting with artificial neural networks (ANN). Three algorithms of ANN were compared to investigate the highest yeild of EPS. The highest yeild of EPS production in RSM was achieved by the medium composition that consists of (g/L) dextrose 15, sodium dihydrogen phosphate 3, potassium dihydrogen phosphate 2.5, triammonium citrate 1.5, and, magnesium sulfate 0.25. The output of 32 sets of RSM experiments were tested for fitting with ANN with three algorithms viz. Levenberg–Marquardt Algorithm (LMA), Bayesian Regularization Algorithm (BRA) and Scaled Conjugate Gradient Algorithm (SCGA) among them LMA found to have best fit with the experiments as compared to the SCGA and BRA.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81747230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
ABSTRACT Lincomycin is a lincosamide antibiotic produced by Streptomyces lincolnensis. Through mutagenesis by ethylmethansulfonate (EMS) and ultraviolet (UV) irradiation repeatedly, M2 was picked out in plate with glutamine and propylproline orderly. In 50-L stirred bioreactor, the production of lincomycin, fermented by M2, was increased to 8136 u/ml under the optimal condition as compared to original strain S. lincolnensis 07–5 (6634 u/ml). Two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS)-shown LmbG, LmbI, and acetohydroxy acid isomeroreductase were remarkably synthesized in M2. The gene lmbG and lmbI are responsible for methylation in the lincomycin biosynthetic cluster, while acetohydroxy acid isomeroreductase contributes to stronger metabolic capability. Finally, we obtained a better strain for industrial production.
{"title":"Improvement of lincomycin production by mutant selection and metabolic regulation","authors":"X. Pang, Yitao Zheng, Xianting Qiao, Quangui Mao, Q. Ma, Rui-fang Ye","doi":"10.1080/10826068.2016.1207083","DOIUrl":"https://doi.org/10.1080/10826068.2016.1207083","url":null,"abstract":"ABSTRACT Lincomycin is a lincosamide antibiotic produced by Streptomyces lincolnensis. Through mutagenesis by ethylmethansulfonate (EMS) and ultraviolet (UV) irradiation repeatedly, M2 was picked out in plate with glutamine and propylproline orderly. In 50-L stirred bioreactor, the production of lincomycin, fermented by M2, was increased to 8136 u/ml under the optimal condition as compared to original strain S. lincolnensis 07–5 (6634 u/ml). Two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS)-shown LmbG, LmbI, and acetohydroxy acid isomeroreductase were remarkably synthesized in M2. The gene lmbG and lmbI are responsible for methylation in the lincomycin biosynthetic cluster, while acetohydroxy acid isomeroreductase contributes to stronger metabolic capability. Finally, we obtained a better strain for industrial production.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83280686","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380 mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86 mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.
融合表达是在大肠杆菌中生物合成长肽的有效方法。然而，由于商业亲和树脂成本高昂，常用的融合标签主要适用于实验室规模的应用。在此，我们开发了一种新方法，利用水蛭素作为多用途融合标签，结合烟草蚀刻病毒（TEV）蛋白酶裂解，在大肠杆菌中高效、低成本地制备 43 氨基酸模型肽月桂苷。我们设计了一种融合基因，通过一个包含 TEV 蛋白酶裂解位点的柔性连接体，将月见草素的 N 端与水蛭素的 C 端融合，并将其克隆到分泌载体 pTASH 中。通过在 7 L 生物反应器中培养，融合蛋白以 ~380 mg/L 的高产率被排泄到培养液中，并通过廉价的 HP20 疏水层析进行回收和纯化，回收率高达 ~80%。经抛光和 TEV 蛋白酶裂解后，最终纯化的月季素纯度≥95%，产率约为 86 mg/L 培养液。总之，这种水蛭素标记策略在生产月季素方面非常有效，也可用于制备规模生产其他多肽。
{"title":"Hirudin as a novel fusion tag for efficient production of lunasin in Escherichia coli.","authors":"Qinghua Tian, Ping Zhang, Zhan Gao, Hengli Li, Zhengli Bai, Shuhua Tan","doi":"10.1080/10826068.2017.1286600","DOIUrl":"10.1080/10826068.2017.1286600","url":null,"abstract":"<p><p>Fusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380 mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86 mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.</p>","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/10826068.2017.1286600","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75226357","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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