scholarly journals Scale-up fermentation of Escherichia coli for the production of recombinant endoglucanase from Clostridium thermocellum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Iram Shahzadi ◽  
Maryam A. Al-Ghamdi ◽  
Muhammad Shahid Nadeem ◽  
Muhammad Sajjad ◽  
Asif Ali ◽  
...  
Keyword(s):  
Scale Up ◽  
Quadratic Model ◽  
Agitation Rate ◽  
Recombinant Enzymes ◽  
E Coli ◽  
Predicted Model ◽  
Hydrolysis Of ◽  
Positive Effect ◽  
Central Composite ◽  
Volumetric Yield

AbstractEndoglucanase (EC 3.2.1.4) catalysing the hydrolysis of β-1.4-glycosidic linkage of cellulose molecules is an enzyme of tremendous industrial importance. The present study describes a response surface methodology based predicted model to deduce a set of fermentation conditions for optimum growth and activity of recombinant endoglucanase in E. coli BL21 (DE3). Numerous significant parameters including fermentation media composition, temperature (Celsius), pH and agitation rate (rpm) were analysed systemically by employing central composite design. This effort reports highly efficient recombinant endoglucanase overproduction (6.9 gl−1 of biomass) with 30% expression by E. coli in modified M9NG media incubated at 37 °C and pH 7 agitated at 200 rpm. Addition of 3 mM glucose and 24 mM glycerol in the M9NG media has shown positive effect on the enzyme yield and activity. The CMCase activity experimentally estimated was found to be 1185 U/mg with the optimized parameters. The outcomes of both the responses by the predicted quadratic model were found in consensus with the obtained values. Our results well depicted the favourable conditions to further scale-up the volumetric yield of other relevant recombinant enzymes and proteins.

scholarly journals The Optimised Statistical Model for Enzymatic Hydrolysis of Tapioca by Glucoamylase Immobilised on Mesostructured Cellular Foam Silica

2019 ◽  
Vol 14 (2) ◽  
pp. 380
Author(s):  
Joni Agustian ◽  
Lilis Hermida
Keyword(s):  
Enzymatic Hydrolysis ◽  
Large Scale ◽  
Specific Activity ◽  
Agitation Speed ◽  
Quadratic Model ◽  
Lack Of Fit ◽  
Km Value ◽  
Hydrolysis Of ◽  
Central Composite ◽  
Better Than

Enzymatic hydrolysis of starches using free glucoamylase to reducing sugars have difficulties in recovering and recycling of the enzyme, hence immobilisation on inert supports were widely studied. However, effectiveness of the immobilised glucoamylase were merely observed only on soluble starches. It was considered a valuable thing to know performance of glucoamylase on Mesostructured Cellular Foam (MCF) silica in hydrolysing of tapioca. An optimised study on enzymatic hydrolysis of tapioca using glucoamylase on MCF silica (9.2T-3D) and its kinetics were described including justification of the predicted model as it was required to develop in large scale operations. Central Composite Design was used to model the process by studying effects of three factors on DE values after enzyme immobilisation.  Immobilisation of glucoamylase on this support gave up to 82% efficiency with the specific activity of 1,856.78 U.g-1. Its used to hydrolysis of tapioca resulted DE values of 1.740-76.303% (w/w) where the highest DE was obtained at pH of 4.1, temperature of 70 ℃ and agitation speed of 140 rpm. The optimisation produced a polynomial quadratic model having insignificant lack-of-fit and low standard deviation, so that it was applicable and reliable in simulating the DE with only 0.80% of data were not described. Temperature affected the process highly, but the buffer pH, agitation speed and factorial interactions were considered not important. KM value for immobilised enzyme was better than the free glucoamylase, however, its reaction rate was slower than the free glucoamylase catalysis. Copyright © 2019 BCREC Group. All rights reserved 

Enzyme-assisted Extraction for Optimized Recovery of Phenolic Bioactives from Peganum hermala Leaves Using Response Surface Methodology

2018 ◽  
Vol 17 (4) ◽  
pp. 349-354
Author(s):  
Qadir Rahman ◽  
Anwar Farooq ◽  
Amjad Gilani Mazhar ◽  
Nadeem Yaqoob Muhammad ◽  
Ahmad Mukhtar
Keyword(s):  
Response Surface Methodology ◽  
Response Surface ◽  
Enzyme Concentration ◽  
Coumaric Acid ◽  
Assisted Extraction ◽  
Pre Treatment ◽  
Optimized Conditions ◽  
Enzyme Complexes ◽  
Hydrolysis Of ◽  
Central Composite

This study investigates the effect of enzyme formulations (Zympex-014, Kemzyme dry-plus and Natuzyme) on recovery of phenolics from Peganum hermala (harmal) leaves, under optimized conditions using response surface methodology. As compared to the other enzyme complexes, the yield (34 g/100g) obtained through Zympex-014-assisted extraction was higher under optimized conditions such as time (75 min), temperature (70°C), pH (6.5) and enzyme concentration (5 g/100 g) using central composite design (CCD). Effectiveness of Zympex-014 towards hydrolysis of P. hermala leaves cell wall was examined by analyzing the control and enzyme-treated leave residues using scanning electron microscope (SEM). GC/MS characterization authenticated the presence of quercetin (1.44), gallic acid (0.23), caffeic acid (0.04), cinnamic acid (0.05), m-coumaric acid (0.23) and p-coumaric acid (0.37 μg/g) as the potent phenolics in Zympex-014 based extract. It can be concluded from the findings of the current work that pre-treatment of P. hermala leaves with Zympex-014 significantly enhanced the recovery of phenolics that supports its potential uses in the nutra-pharamaceutical industry.

scholarly journals Complete biosynthesis of a sulfated chondroitin in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Abinaya Badri ◽  
Asher Williams ◽  
Adeola Awofiranye ◽  
Payel Datta ◽  
Ke Xia ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Scale Up ◽  
Production Methods ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Microbial Product ◽  
Dry Cell Weight ◽  
One Step ◽  
Dry Cell

AbstractSulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

Kinetics and Equilibrium of Radioactive Metal Adsorption onto Sugarcane Bagasse Waste: Comparison of Batch and Column Adsorption Modes

2021 ◽  
Vol 235 (3) ◽  
pp. 281-294
Author(s):  
Abida Kausar ◽  
Haq Nawaz Bhatti ◽  
Munawar Iqbal
Keyword(s):  
Sugarcane Bagasse ◽  
Flow Rate ◽  
Breakthrough Curves ◽  
Quadratic Model ◽  
Waste Biomass ◽  
Initial Concentration ◽  
Bed Height ◽  
Column Adsorption ◽  
Central Composite ◽  
Bagasse Waste

Abstract Sugarcane bagasse waste biomass (SBWB) efficacy for the adsorption of Zr(IV) was investigated in batch and column modes. The process variables i.e. pH 1–4 (A), adsorbent dosage 0.0–0.3 g (B), and Zr(IV) ions initial concentration 25–200 mg/L (C) were studied. The experiments were run under central composite design (CCD) and data was analysed by response surface methodology (RSM) methodology. The factor A, B, C, AB interaction and square factor A2, C2 affected the Zr(IV) ions adsorption onto SBWB. The quadratic model fitted well to the adsorption data with high R2 values. The effect of bed height, flow rate and Zr(IV) ions initial concentration was also studied for column mode adsorption and efficiency was evaluated by breakthrough curves as well as Bed Depth Service and Thomas models. Bed height and Zr(IV) ions initial concentration enhanced the adsorption of capacity of Zr(IV) ions, whereas flow rate reduced the column efficiency.

scholarly journals Optimization of Supercritical Extraction Conditions of Senna Alata and Evaluation of Biological Activity

2018 ◽  
Vol 7 (3.11) ◽  
pp. 94
Author(s):  
Istikamah Subuki ◽  
Aiman Nabilah Abdul Malek ◽  
Saidatul Husni Saidin ◽  
Mazura Md. Pisar
Keyword(s):  
Crude Extract ◽  
Extraction Process ◽  
Quadratic Model ◽  
Total Flavonoids ◽  
Inhibition Activity ◽  
Backward Elimination ◽  
Total Flavonoids Content ◽  
Senna Alata ◽  
Central Composite ◽  
Experimental Runs

Supercritical fluid extraction (SFE) offer faster extraction process, decreased solvent usage and more selectivity on desired compounds. In this present study, the influence of pressure (100, 200 and 300 bar) and temperature (40, 50 and 60˚C) on the Senna alata crude yield were investigated with fixed supercritical carbon dioxide (SC-CO2) at the flow rate of 35 g/min. The parameters were optimised and modelled using response surface methodology (RSM) and central composite design (CCD). The analysis of variance (ANOVA) experimental design consists of 13 experimental runs with 5 replicates at the central points. Well-fitting quadratic model were successfully established for crude extract through backward elimination. The optimum crude extract yield pointed out by RSM was at the pressure of 300 bar and temperature 40˚C respectively. Extraction yields based on SC-CO2 varied in the range of 0.28 to 3.62%. The highest hyaluronidase inhibition activity and total flavonoids content obtained by S.alata crude extracts were 41.19% and 52.53% w/w, respectively. SC-CO2 proves to have great potential for extraction of yield, hyaluronidase inhibition activity and total flavonoids content for S.alata.  

Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2257-2266 ◽  
Author(s):  
Helmuth Adelsberger ◽  
Christian Hertel ◽  
Erich Glawischnig ◽  
Vladimir V. Zverlov ◽  
Wolfgang H. Schwarz
Keyword(s):  
Extracellular Enzymes ◽  
Enzyme System ◽  
Thermophilic Bacterium ◽  
Recombinant Enzymes ◽  
Type Mode ◽  
Complete Set ◽  
First Time ◽  
Hydrolysis Of

Four extracellular enzymes of the thermophilic bacterium Clostridium stercorarium are involved in the depolymerization of de-esterified arabinoxylan: Xyn11A, Xyn10C, Bxl3B, and Arf51B. They were identified in a collection of eight clones producing enzymes hydrolysing xylan (xynA, xynB, xynC), β-xyloside (bxlA, bxlB, bglZ) and α-arabinofuranoside (arfA, arfB). The modular enzymes Xyn11A and Xyn10C represent the major xylanases in the culture supernatant of C. stercorarium. Both hydrolyse arabinoxylan in an endo-type mode, but differ in the pattern of the oligosaccharides produced. Of the glycosidases, Bxl3B degrades xylobiose and xylooligosaccharides to xylose, and Arf51B is able to release arabinose residues from de-esterified arabinoxylan and from the oligosaccharides generated. The other glycosidases either did not attack or only marginally attacked these oligosaccharides. Significantly more xylanase and xylosidase activity was produced during growth on xylose and xylan. This is believed to be the first time that, in a single thermophilic micro-organism, the complete set of enzymes (as well as the respective genes) to completely hydrolyse de-esterified arabinoxylan to its monomeric sugar constituents, xylose and arabinose, has been identified and the enzymes produced in vivo. The active enzyme system was reconstituted in vitro from recombinant enzymes.

Improved Enzymatic Depolymerization of Chitosan by Ionic Liquid Pretreatment and the Effect of Oligo-Chitosan on Intestinal Flora In Vitro

2011 ◽  
Vol 391-392 ◽  
pp. 1319-1323
Author(s):  
Cui Zheng ◽  
Lin Li ◽  
Hao Pang ◽  
Zhao Mei Wang ◽  
Na Li
Keyword(s):  
Ionic Liquid ◽  
Hydrogen Bonds ◽  
Molecular Hydrogen ◽  
Intestinal Flora ◽  
X Ray Diffraction ◽  
X Ray ◽  
Hydrolysis Of ◽  
Positive Effect ◽  
Enzymatic Depolymerization

It still remains challenging for effective hydrolysis of chitosan into chitosan oligomers. In this work, a pretreatment was conducted on chitosan by an ionic liquid 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), aiming at improving enzymatic depolymerization of chitosan. X-ray diffraction analysis indicated that the inter- and intra-molecular hydrogen bonds within chitosan molecules were broken by [C4mim]Cl and the crystalline was destroyed. The oligo-chitosan hydrolyzed from IL-pretreated chitosan, coded as COS-IL, showed a DP of 3~5, in contrast to DP 5~8 with oligo-chitosan obtained from untreated chitosan(coded as COS-UN). COS-IL was more effective than COS-UN in inhibiting intestinal spoilage bacterials growth and it has positive effect on the growth of intestinal probiotic bacterials.

scholarly journals Expression of Heterologous Cellulases inThermotogasp. Strain RQ2

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Hui Xu ◽  
Dongmei Han ◽  
Zhaohui Xu
Keyword(s):  
Enzyme Activities ◽  
Bacterial Cells ◽  
Recombinant Enzymes ◽  
Caldicellulosiruptor Saccharolyticus ◽  
Shuttle Vectors ◽  
E Coli ◽  
Chimeric Enzymes ◽  
Coding Regions ◽  
First Time ◽  
Culture Supernatants

The ability ofThermotogaspp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) fromCaldicellulosiruptor saccharolyticuswere cloned intoT.sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried byThermotoga-E. colishuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed inE. coliDH5αandT.sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. InE. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas inT.sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost inT.sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed inThermotoga, demonstrating the feasibility of using engineeredThermotogaspp. for efficient cellulose utilization.

scholarly journals Enhanced production of taxadiene in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Behnaz Nowrouzi ◽  
Rachel Li ◽  
Laura E. Walls ◽  
Leopold d’Espaux ◽  
Koray Malci ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Shake Flask ◽  
Scale Up ◽  
Cost Effective ◽  
Cancer Drug ◽  
List Type ◽  
Taxadiene Synthase ◽  
Enhanced Production ◽  
Anti Cancer ◽  
Positive Effect

AbstractCost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at shake flask scale. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively. Maximum taxadiene titres of 129 ± 15 mg/L and 119 mg/L were achieved through shake flask and bioreactor cultivation, respectively, of the optimal strain at a reduced temperature of 20 °C. The results highlight the positive effect of coupling molecular biology tools with bioprocess variable optimisation on synthetic pathway development.HighlightsMaximum taxadiene titre of 129 ± 15 mg/L in Saccharomyces cerevisiae at 20 °CIntegrating fusion protein tagged-taxadiene synthase improved taxadiene titre.Consistent taxadiene titres were achieved at the micro-and mini-bioreactor scales.

scholarly journals Public-good driven release of heterogeneous resources leads to genotypic diversification of an isogenic yeast population in melibiose.

2021 ◽  
Author(s):  
Anjali Mahilkar ◽  
Phaniendra Alugoju ◽  
Vijendra Kavatalkar ◽  
Rajeshkannan E. ◽  
Jayadeva Bhat ◽  
...  
Keyword(s):  
Carbon Source ◽  
Public Good ◽  
Molecular Basis ◽  
Model System ◽  
Microbial Populations ◽  
Adaptive Diversification ◽  
E Coli ◽  
Hydrolysis Of ◽  
Convenient Model ◽  
Heterogeneous Resources

Adaptive diversification of an isogenic population, and its molecular basis has been a subject of a number of studies in the last few years. Microbial populations offer a relatively convenient model system to study this question. In this context, an isogenic population of bacteria (E. coli, B. subtilis, and Pseudomonas) has been shown to lead to genetic diversification in the population, when propagated for a number of generations. This diversification is known to occur when the individuals in the population have access to two or more resources/environments, which are separated either temporally or spatially. Here, we report adaptive diversification in an isogenic population of yeast, S. cerevisiae, when propagated in an environment containing melibiose as the carbon source. The diversification is driven due to a public good, enzyme α-galactosidase, leading to hydrolysis of melibiose into two distinct resources, glucose and galactose. The diversification is driven by a mutations at a single locus, in the GAL3 gene in the GAL/MEL regulon in the yeast.

Sign in / Sign up

Export Citation Format

Share Document