scholarly journals Techno-economic analysis of recombinant Endo-β-1,4-Glucanase production from Escherichia coli Eg-RK2 culture using oil palm empty fruit bunch

2021 ◽  
Vol 926 (1) ◽  
pp. 012070
Author(s):  
S Z Amraini ◽  
E A Surya ◽  
S Limoes ◽  
S Setyahadi ◽  
S Abd-Aziz ◽  
...  
Keyword(s):  
Economic Analysis ◽  
Steam Explosion ◽  
Cellulase Production ◽  
Alkaline Pretreatment ◽  
Market Demand ◽  
Fermentation Inhibitors ◽  
E Coli ◽  
Hemicellulose Removal ◽  
Recombinant Cellulase ◽  
Primary Substrate

Abstract A techno-economic analysis of recombinant cellulase production from E. coli Eg-RK2 was conducted to support the fulfilling of Indonesia’s energy roadmap for ethanol production. The plant utilizes OPEFB as a primary substrate in cellulase production, with an expected lifetime of 12 years. The plant is assumed to be built in Indonesia and it will fulfill 1% of the total market demand. The effect of different pretreatment processes (alkaline, steam explosion, and sequential acid-alkaline) on the profitability parameter was also studied. A simulation using SuperPro Designer was used to calculate the mass and energy balance based on the kinetic parameters of E. coli EgRK2. A technology evaluation showed that alkaline pretreatment provides the highest yield with no known inhibitors formed. The steam explosion pretreatment offers the lowest rate of lignin and hemicellulose removal, and it is understood to form known fermentation inhibitors. The NPVs of the alkaline, steam explosion and sequential acid-alkaline pretreatments are USD 32,121,000, USD -36,841,000, and USD 384,000, respectively, which means the alkaline pretreatment is economically very feasible for the production of cellulase.

Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

2019 ◽  
Vol 20 (6) ◽  
pp. 497-505 ◽  
Author(s):  
Abeer M. Abd El-Aziz ◽  
Mohamed A. Shaker ◽  
Mona I. Shaaban
Keyword(s):  
Gold Nanoparticles ◽  
Lipolytic Activity ◽  
Market Demand ◽  
Biotechnological Applications ◽  
Lipase Gene ◽  
Expression Plasmid ◽  
E Coli ◽  
Nickel Affinity Chromatography ◽  
Recombinant Production ◽  
Sds Page

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

scholarly journals Detection and Quantification of Superoxide Formed within the Periplasm of Escherichia coli

2006 ◽  
Vol 188 (17) ◽  
pp. 6326-6334 ◽  
Author(s):  
Sergei Korshunov ◽  
James A. Imlay
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Phagocytic Cells ◽  
Free Living ◽  
E Coli ◽  
Superoxide Formation ◽  
Genes Encoding ◽  
Copper Zinc ◽  
Primary Substrate

ABSTRACT Many gram-negative bacteria harbor a copper/zinc-containing superoxide dismutase (CuZnSOD) in their periplasms. In pathogenic bacteria, one role of this enzyme may be to protect periplasmic biomolecules from superoxide that is released by host phagocytic cells. However, the enzyme is also present in many nonpathogens and/or free-living bacteria, including Escherichia coli. In this study we were able to detect superoxide being released into the medium from growing cultures of E. coli. Exponential-phase cells do not normally synthesize CuZnSOD, which is specifically induced in stationary phase. However, the engineered expression of CuZnSOD in growing cells eliminated superoxide release, confirming that this superoxide was formed within the periplasm. The rate of periplasmic superoxide production was surprisingly high and approximated the estimated rate of cytoplasmic superoxide formation when both were normalized to the volume of the compartment. The rate increased in proportion to oxygen concentration, suggesting that the superoxide is generated by the adventitious oxidation of an electron carrier. Mutations that eliminated menaquinone synthesis eradicated the superoxide formation, while mutations in genes encoding respiratory complexes affected it only insofar as they are likely to affect the redox state of menaquinone. We infer that the adventitious autoxidation of dihydromenaquinone in the cytoplasmic membrane releases a steady flux of superoxide into the periplasm of E. coli. This endogenous superoxide may create oxidative stress in that compartment and be a primary substrate of CuZnSOD.

scholarly journals Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

2021 ◽  
Vol 12 ◽  
Author(s):  
Almaz Nigatu Tesfahun ◽  
Marina Alexeeva ◽  
Miglė Tomkuvienė ◽  
Aysha Arshad ◽  
Prashanna Guragain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Excision Repair ◽  
Dna Lesions ◽  
Base Pairs ◽  
E Coli ◽  
Thymine Glycol ◽  
Base Excision ◽  
The Cost ◽  
Primary Substrate

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

scholarly journals Economic analysis of mushroom enterprise in Chitwan district, Nepal

2021 ◽  
Vol 6 (4) ◽  
pp. 408-415
Author(s):  
Ranju Acharya ◽  
Ujjwal Tiwari
Keyword(s):  
Economic Analysis ◽  
Group Discussion ◽  
Secondary Data ◽  
Regression Function ◽  
Climatic Conditions ◽  
Primary Data ◽  
Cost Ratio ◽  
Market Demand ◽  
Gross Margin ◽  
Benefit Cost Ratio

The majority of the population (66%) in-country “Nepal” are engaged in agriculture. However, domestic production finds it difficult to meet the annual demand of the people. Hence, people are moving from subsistence agriculture to embrace mushroom farming. This study focuses on economic analysis and analysis of the present status of mushroom farming and enterprise in this country. The study was conducted in the land area of Kalika Municipality and Bharatpur Metropolitan City. 30 mushroom farmers with two huts and at least three years of experience were selected from the study area. The primary data were collected through face-to-face interviews with the farmers, focus group discussion (FGD) and key informant interviews (KII). The secondary data was collected through various published articles and documents. The data analysis was done using basic statistics and a regression function. The benefit-cost ratio is 2.54 and a high gross margin is NRs.490,876.65 per kattha per year. The return to scale (RTS) is 0.80. Five marketing channels are present among which wholesalers and local collectors contributed the highest percentage of the share. However, the dominance of the intermediaries, timely unavailability of inputs, price fluctuation, disease and pest infestation were the major constraints. Disease and pest control, formation of the producer organization, improvised cultivation practices, timely and affordable availability of quality can be the major solution measures. Whereas, suitable climatic conditions, high productivity and growing market demand are the strengths of mushroom production in this study area. Mushroom farming is found to be a profitable business concerning competitive and comparative markets. 

scholarly journals Efficient Biosynthesis of R-(-)-linalool through Adjusting Expression Strategy and Increasing GPP Supply in Escherichia coli

2019 ◽  
Author(s):  
Xun Wang ◽  
Jing Wu ◽  
Jiaming Chen ◽  
Longjie Xiao ◽  
Xun Li
Keyword(s):  
Shake Flask ◽  
Streptomyces Clavuligerus ◽  
Batch Cultivation ◽  
Market Demand ◽  
Protein Fusion ◽  
Fed Batch ◽  
Geranyl Diphosphate ◽  
E Coli ◽  
Key Enzyme ◽  
Linalool Synthase

Abstract Background: R-(-)-linalool is a versatile acyclic monoterpene alcohol with applications in the flavor and fragrance, pharmaceutical, and agrochemical industries. However, plant extraction furnishes only limited and unstable R-(-)-linalool yields that do not satisfy market demand. Therefore, a sustainable yet efficient and productive method of R-(-)-linalool synthesis is urgently needed. Results: To induce the R-(-)-linalool biosynthesis pathway in E. coli , we expressed heterologous (3R)-linalool synthase (LIS) from Lavandula angustifolia (laLIS). We then enhanced R-(-)-linalool production in the cells using a suitable LIS from Streptomyces clavuligerus (bLIS). The bLIS expression was markedly elevated by using optimized ribosomal binding sites (RBSs) and protein fusion tags. R-(-)-linalool output rose from 4.8 mg L -1 to 33.4 mg L -1 . To increase the geranyl diphosphate (GPP) content in E. coli , we tested various alterations in geranyl diphosphate synthases (GPPSs) and their mutants. The final E. coli strain harboring GPPS from Abies grandis ( Ag GPPS) accumulated ≤ 100.1 mg L -1 R-(-)-linalool after 72 h shake-flask fermentation. This yield gain constitutes a 60.7-fold improvement in R-(-)-linalool biosynthesis over the parent strain. Fed-batch cultivation of the engineered strain in a 1.3-L fermenter yielded 1,027.3 mg L -1 R-(-)-linalool. Conclusions: In this study, an efficient R-(-)-linalool production pathway was induced in E. coli via the heterologous MVA pathway, AgGPPS, and (3R)-linalool synthase (bLIS). By overexpressing the key enzyme in the engineered E. coli strain, R-(-)-linalool production reached 100.1 mg L -1 and 1,027.3 mg L -1 under shake flask- and fed-batch fermentation conditions, respectively. The latter is the highest reported R-(-)-linalool yield to date using an engineered E. coli strain. The strategies of key enzyme overexpression and mutation could lay theoretical and empirical foundations for engineering terpenoid pathways and optimizing other metabolic pathways.

scholarly journals Towards creating an extended metabolic model (EMM) for E. coli using enzyme promiscuity prediction and metabolomics data

2019 ◽  
Author(s):  
Sara A. Amin ◽  
Elizabeth Chavez ◽  
Nikhil U. Nair ◽  
Soha Hassoun
Keyword(s):  
Metabolic Model ◽  
Enzyme Promiscuity ◽  
Metabolomics Data ◽  
Structural Modifications ◽  
E Coli ◽  
Cellular Metabolic Activity ◽  
Metabolic Models ◽  
Genome Scale ◽  
Primary Substrate ◽  
Model Annotation

AbstractBackgroundMetabolic models are indispensable in guiding cellular engineering and in advancing our understanding of systems biology. As not all enzymatic activities are fully known and/or annotated, metabolic models remain incomplete, resulting in suboptimal computational analysis and leading to unexpected experimental results. We posit that one major source of unaccounted metabolism is promiscuous enzymatic activity. It is now well-accepted that most, if not all, enzymes are promiscuous – i.e., they transform substrates other than their primary substrate. However, there have been no systematic analyses of genome-scale metabolic models to predict putative reactions and/or metabolites that arise from enzyme promiscuity.ResultsOur workflow utilizes PROXIMAL – a tool that uses reactant-product transformation patterns from the KEGG database – to predict putative structural modifications due to promiscuous enzymes. Using iML1515 as a model system, we first utilized a computational workflow, referred to as Extended Metabolite Model Annotation (EMMA), to predict promiscuous reactions catalyzed, and metabolites produced, by natively encoded enzymes in E. coli. We predict hundreds of new metabolites that can be used to augment iML1515. We then validated our method by comparing predicted metabolites with the Escherichia coli Metabolome Database (ECMDB).ConclusionsWe utilized EMMA to augment the iML1515 metabolic model to more fully reflect cellular metabolic activity. This workflow uses enzyme promiscuity as basis to predict hundreds of reactions and metabolites that may exist in E. coli but have not been documented in iML1515 or other databases. Among these, we found that 17 metabolites have previously been documented in E. coli metabolomics studies. Further, 6 of these metabolites are not documented for any other E. coli metabolic model (e.g. KEGG, EcoCyc). The corresponding reactions should be added to iML1515 to create an Extended Metabolic Model (EMM). Other predicted metabolites and reactions can guide future experimental metabolomics studies. Further, our workflow can easily be applied to other organisms for which comprehensive genome-scale metabolic models are desirable.

scholarly journals Steam Explosion Impact to Technical Hemp Fiber Diameter

2015 ◽  
Vol 1 ◽  
pp. 112
Author(s):  
Laima Grāve ◽  
Anna Putniņa ◽  
Silvija Kukle ◽  
Veneranda Stramkale
Keyword(s):  
Mechanical Properties ◽  
Natural Products ◽  
Steam Explosion ◽  
Fine Particles ◽  
Fiber Diameter ◽  
Natural Fibers ◽  
Optical Microscope ◽  
Alkaline Pretreatment ◽  
Diameter Measurement ◽  
Hemp Fiber

Microfibers and nanofibers from natural products have high mechanical properties. For this reason researchers pay particular attention to the natural fibers and to the method how they are obtained. In the research samples with different treatments (alkaline pretreatment, steam explosion and water extraction) were investigated to evaluate treatment influence on fiber diameters. Fractionation does not provide actual results of fibers diameter as thinner fibers agglomerate to each other and around thicker fibers and cannot be sieved. Fiber diameter measurement with an optical microscope shows that pretreatment and treatment reduced the thickest fiber percentage and increase the number of fine particles. Dew-retted hemp gives 16% of thicker fibers and only 39% fibers with a diameters less than 63µm. 50% of alkaline pretreated and steam explosion treated fibers diameters are less than 63 µm and only 8% of diameters fall in range 160 - 630 µm.

scholarly journals Characterization of cellulase from E. coli BPPTCC-EGRK2

2018 ◽  
Vol 52 ◽  
pp. 00024
Author(s):  
Mas Gunawan Haryanto ◽  
Siswa Setyahadi ◽  
Muhammad Sahlan ◽  
Masafumi Yohda ◽  
Yosuke Fukutani ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Animal Feed ◽  
Maximum Velocity ◽  
Cellulase Production ◽  
Luria Bertani ◽  
E Coli ◽  
Sds Page ◽  
Weight Analysis

Cellulase enzymes are widely used in various industries such as detergent industry, bioethanol, animal feed, textile and paper. This research focused on characterization of cellulase produced from Eschericia coli BPPT-CC EgRK2, which is a recombinant that can produce protein enzymes endo- β-1,4-glucanase. Eschericia coli BPPT-CC EgRK2 was cultured in 1 litre liquid medium Luria Bertani. Because the bacteria is intracellular, sonication is needed for cell disruption to get the cellulase enzyme. The enzyme activity was then analyzed by CMC substrate at different concentration. The protein content analysis was carried out using Bradford method; the molecular weight analysis was done using SDS-PAGE; while the enzyme kinetics was plotted using Michaelis-Menten model. Our results showed the highest enzyme activity was 2.403 U/ml and the protein concentration was 5.352 mg/ml. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for CMC substrate hydrolysis were 0.07 μmol/ml and 2.49 μmol/ml/sec, respectively. The cellulase molecular weight was 58 kDa using SDS-PAGE with 7.5% stacking gel. The results indicated that Eschericia coli BPPT-CC EgRK2 is a promising renewable source for cellulase production for industrial application.

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