scholarly journals Elucidating the potential of crude cell extracts for producing pyruvate from glucose

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
David C Garcia ◽  
Benjamin P Mohr ◽  
Jakob T Dovgan ◽  
Gregory B Hurst ◽  
Robert F Standaert ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Cell Growth ◽  
Enzymatic Activity ◽  
Crude Extract ◽  
Growth Conditions ◽  
Living Systems ◽  
Fine Chemicals ◽  
Pathway Optimization ◽  
Cell Extracts ◽  
Crude Extracts

Abstract Living systems possess a rich biochemistry that can be harnessed through metabolic engineering to produce valuable therapeutics, fuels and fine chemicals. In spite of the tools created for this purpose, many organisms tend to be recalcitrant to modification or difficult to optimize. Crude cellular extracts, made by lysis of cells, possess much of the same biochemical capability, but in an easier to manipulate context. Metabolic engineering in crude extracts, or cell-free metabolic engineering, can harness these capabilities to feed heterologous pathways for metabolite production and serve as a platform for pathway optimization. However, the inherent biochemical potential of a crude extract remains ill-defined, and consequently, the use of such extracts can result in inefficient processes and unintended side products. Herein, we show that changes in cell growth conditions lead to changes in the enzymatic activity of crude cell extracts and result in different abilities to produce the central biochemical precursor pyruvate when fed glucose. Proteomic analyses coupled with metabolite measurements uncover the diverse biochemical capabilities of these different crude extract preparations and provide a framework for how analytical measurements can be used to inform and improve crude extract performance. Such informed developments can allow enrichment of crude extracts with pathways that promote or deplete particular metabolic processes and aid in the metabolic engineering of defined products.

scholarly journals A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production

2020 ◽  
Author(s):  
David C. Garcia ◽  
Jaime Lorenzo N. Dinglasan ◽  
Him Shrestha ◽  
Paul E. Abraham ◽  
Robert L. Hettich ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Cell Growth ◽  
Metabolic Flux ◽  
Selective Removal ◽  
List Type ◽  
Metabolic Potential ◽  
Metabolite Production ◽  
Genomic Engineering ◽  
Crude Extracts ◽  
Engineering Strategy

AbstractCell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell’s capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is limited. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective removal from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes are demonstrated that result in engineered crude lysates that are capable of 10 to 20-fold increases of pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to develop cell-free metabolic engineering into a practical platform for both bioprototyping and bioproduction.HighlightsA novel method of engineering crude cell lysates for enhancing specific metabolic processes is described.Multiplex Automated Genomic Engineering (MAGE) can be used to engineer donor strains for improving cell-free metabolite production with minimal impact on cell-growth.The described lysate engineering strategy can specifically direct metabolic flux and create metabolic states not possible in living cells.Pooling of the central precursor pyruvate was significantly improved through use of this lysate proteome engineering strategy.

Metabolomics and Pharmacological Screening of Aspergillus versicolor Isolated from Hyrtios erectus Red Sea Sponge; Egypt

2020 ◽  
Vol 16 (7) ◽  
pp. 1083-1102
Author(s):  
Mohamed A. Shreadah ◽  
Nehad M.A. El Moneam ◽  
Samy A. El-Assar ◽  
Asmaa Nabil-Adam
Keyword(s):  
Oxidative Stress ◽  
Red Sea ◽  
Crude Extract ◽  
Quantitative Methods ◽  
Total Phenolic ◽  
Aspergillus Versicolor ◽  
Crude Extracts ◽  
Study Results ◽  
Anti Inflammatory ◽  
Pharmacological Screening

Background: Aspergillus Versicolor is a marine-derived fungus isolated from Hyrtios Erectus Red Sea sponge. Methods: The aim of this study was to carry out a pharmacological screening and investigation for the in vitro biological activity (antioxidant, cholinergic, antidiabetic and anticancer) of Aspergillus Versicolor crude extract’s active compounds by using different qualitative and quantitative methods. Results: The present study results showed that Aspergillus Versicolor crude extracts contain 0.6 mg total phenolic/mg crude extract. Aspergillus Versicolor also showed a potent antioxidative capacity by decreasing the oxidation of ABTS. The anticancer and inhibitory effects of Aspergillus Versicolor crude extracts on PTK and SHKI were found to be 75.29 % and 80.76%; respectively. The AChE inhibitory assay revealed that Aspergillus Versicolor extracts had an inhibitory percentage of 86.67%. Furthermore, the anti-inflammatory activity using COX1, COX2, TNF, and IL6 was 77.32, 85.21 %, 59.83%, and 56.15%; respectively. Additionally, the anti-viral effect using reverse transcriptase enzyme showed high antiviral activity with 92.10 %. Conclusion: The current study confirmed that the Aspergillus versicolor crude extract and its active constituents showed strong effects on diminishing the oxidative stress, neurodegenerative damage, antiinflammatory, anti-cancer and anti-viral, suggesting their beneficial role as a promising fermented product in the treatment of cancer, oxidative stress, Alzheimer's, anti-inflammatory and anti-viral diseases.

Enzymatic Activity Assays in Yeast Cell Extracts

BIO-PROTOCOL ◽  
2014 ◽  
Vol 4 (23) ◽  
Author(s):  
Melike Çağlayan ◽  
Samuel Wilson
Keyword(s):  
Enzymatic Activity ◽  
Yeast Cell ◽  
Cell Extracts

scholarly journals Longevity of Raw and Lyophilized Crude Urease Extracts

2021 ◽  
Vol 2 (2) ◽  
pp. 325-334
Author(s):  
Neda Javadi ◽  
Hamed Khodadadi Tirkolaei ◽  
Nasser Hamdan ◽  
Edward Kavazanjian
Keyword(s):  
Crude Extract ◽  
Room Temperature ◽  
Low Cost ◽  
Extraction Process ◽  
Crude Extracts ◽  
Simple Extraction ◽  
Commercial Applications ◽  
Stable Source ◽  
The Stability ◽  
The Cost

The stability (longevity of activity) of three crude urease extracts was evaluated in a laboratory study as part of an effort to reduce the cost of urease for applications that do not require high purity enzyme. A low-cost, stable source of urease will greatly facilitate engineering applications of urease such as biocementation of soil. Inexpensive crude extracts of urease have been shown to be effective at hydrolyzing urea for carbonate precipitation. However, some studies have suggested that the activity of a crude extract may decrease with time, limiting the potential for its mass production for commercial applications. The stability of crude urease extracts shown to be effective for biocementation was studied. The crude extracts were obtained from jack beans via a simple extraction process, stored at room temperature and at 4 ℃, and periodically tested to evaluate their stability. To facilitate storage and transportation of the extracted enzyme, the longevity of the enzyme following freeze drying (lyophilization) to reduce the crude extract to a powder and subsequent re-hydration into an aqueous solution was evaluated. In an attempt to improve the shelf life of the lyophilized extract, dextran and sucrose were added during lyophilization. The stability of purified commercial urease following rehydration was also investigated. Results of the laboratory tests showed that the lyophilized crude extract maintained its activity during storage more effectively than either the crude extract solution or the rehydrated commercial urease. While incorporating 2% dextran (w/v) prior to lyophilization of the crude extract increased the overall enzymatic activity, it did not enhance the stability of the urease during storage.

scholarly journals The global impact of bacterial processes on carbon mass

2019 ◽  
Author(s):  
Barbara Ervens ◽  
Pierre Amato
Keyword(s):  
Cell Growth ◽  
Organic Carbon ◽  
Growth Conditions ◽  
Water Soluble ◽  
Microbial Processes ◽  
Cloud Formation ◽  
Cloud Droplets ◽  
Spatial And Temporal Scales ◽  
Adverse Health Effects ◽  
Oh Reactions

Abstract. Many recent studies have identified biological material as a major fraction of ambient aerosol loading. A small fraction of these bioaerosols consist of bacteria that have attracted a lot of attention due to their role in cloud formation and adverse health effects. Current atmospheric models consider bacteria as inert quantities and neglect cell growth and multiplication. We provide here a framework to estimate the production of secondary biological aerosol (SBA) mass in clouds by microbial cell growth and multiplication. The best estimate of SBA formation rates of 3.7 Tg yr-1 are comparable to previous model estimates of the primary emission of bacteria into the atmosphere, and thus might represent a previously unrecognized source of biological aerosol material. We discuss in detail the large uncertainties associated with our estimates based on the rather sparse available data on bacteria abundance, growth conditions and properties. Additionally, the loss of water-soluble organic carbon (WSOC) due to microbial processes in cloud droplets has been suggested to compete under some conditions with WSOC loss by chemical (OH) reactions. Our estimates suggest that microbial and chemical processes might lead to a global loss of WSOC of 8–11 Tg yr-1 and 8–20 Tg yr-1, respectively. While also this estimate is very approximate, the analysis of the uncertainties and ranges of all parameters gives hints about the conditions under which microbial processes cannot be neglected as organic carbon sinks in clouds. Our estimates also highlight the urgent needs for more data concerning microbial concentrations, fluxes and activity in the atmosphere to evaluate the role of bacterial processes as net aerosol sink or source on various spatial and temporal scales.

scholarly journals GC-MS analyses of young and mature Archidium Ohioense Schimp Ex. C. Mull and Philonotis hastate (Duby) Wijk & Margad extracts

2021 ◽  
Vol 23 (1) ◽  
pp. 89-103
Author(s):  
M.O. Isa ◽  
B.A. Akinpelu ◽  
A.M. Makinde
Keyword(s):  
Ambient Temperature ◽  
Ethyl Ester ◽  
Crude Extract ◽  
Hexadecanoic Acid ◽  
Bioactive Constituents ◽  
Moss Species ◽  
Maturity Stages ◽  
Crude Extracts ◽  
Young Stage ◽  
Ethylhexyl Phthalate

Analyses of the constituents of the crude extracts obtained from young and mature moss species namely: Archidium ohioense and Philonotis hastata were conducted with a view to investigating the effects of maturity stages on their bioactive constituents. The mosses collected from their natural population were air dried at ambient temperature in the laboratory, extracted with methanol and the crude extracts subjected to gas chromatographymass spectrometry (GC-MS) analysis. The results of the analyses showed the presence of 20 compounds in young A. ohioense with n-hexadecanoic acid [26.60%], bis(2-ethylhexyl) phthalate [12.47%], bicyclo (3.1.1)heptane 2,6,6-trimethyl-[1r-(1.alpha.,2.beta.,5.alpha.)]- [11.59%] and phytol [9.69%] forming the prominent components while in the mature A. ohioense, 13 compounds were present, from which n-hexadecanoic acid [51.25%], hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)-ethyl ester [9.90%] and n-propyl 9-octadecenoate [7.47%] formed the prominent components. In P. hastata, 20 compounds were identified in the young stage sample with n-hexadecanoic acid [22.46%], bis(2-ethylhexyl) phthalate [20.95%] and phytol [18.14%] as the prominent components while 9 compounds were identified in the mature sample with n-hexadecanoic acid[51.84%], hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)ethyl ester [18.12%] and bis(2-ethylhexyl) phthalate [9.11%] which formed the prominent components. The study indicated that, maturity stages at collection of the mosses affected their bioactive compositions, with the young stage mosses showing more bioactive compounds than the mature ones. Keywords: A. ohioense, P. hastata, Mosses, Maturity stages, Crude extract, GC-MS.

scholarly journals Kinetic Behaviour of Pancreatic Lipase Inhibition by Ultrasonicated A. malaccensis and A. subintegra Leaves of Different Particle Sizes

2020 ◽  
Vol 15 (3) ◽  
pp. 818-828
Author(s):  
Miradatul Najwa Muhd Rodhi ◽  
Fazlena Hamzah ◽  
Ku Halim Ku Hamid
Keyword(s):  
Particle Size ◽  
Gallic Acid ◽  
Pancreatic Lipase ◽  
Crude Extract ◽  
Competitive Inhibition ◽  
Treatment Method ◽  
Crude Extracts ◽  
Pre Treatment ◽  
Pancreatic Lipase Inhibition ◽  
Lipase Inhibition

Gallic acid and quercetin equivalent were determined in the crude extract of matured leaves Aquilaria malaccensis and Aquilaria subintegra. The leaves of both Aquilaria species were dried at 60 °C for 24 hours, ground and sieved into particle size of 250, 300, 400, 500, and 1000 µm. Then, each particle size of leaves was soaked in distilled water with a ratio of 1:100 (w/v) for 24 hours and undergoes the pre-treatment method by using ultrasonicator (37 kHz), at the temperature of 60 °C for 30 minutes. The crude extracts were obtained after about 4 hours of hydrodistillation process. The highest concentration of gallic acid and quercetin equivalent was determined in the crude extract from the particle size of 250 µm. The kinetics of pancreatic lipase inhibition was further studied based using the Lineweaver-Burk plot, wherein the concentration of p-NPP as the substrate and pancreatic lipase were varied. Based on the formation of the lines in the plot, the crude leaves extract of both Aquilaria species exhibit the mixed-inhibition on pancreatic lipase, which indicates that in the reaction, the inhibitors were not only attached to the free pancreatic lipase, but also to the pancreatic lipase-(p-NPP) complex. The reaction mechanism was similar to non-competitive inhibition; however the value of dissociation constant, Ki, for both inhibition pathways was different. The inhibition shows an increment in Michaelis-Menten constant (Km) and a reduction in the maximum pancreatic lipase activity (Vm) compared to the reaction without Aquilaria spp. crude extracts (control). This proved that the inhibition occurred in this reaction. Copyright © 2020 BCREC Group. All rights reserved 

scholarly journals INHIBITION ACTIVITY OF CLEOME SPECIES EXTRACT ON ONION GUEST ROOT

2019 ◽  
pp. 1-2
Author(s):  
SUTTHIDUEAN CHUNHAKARN ◽  
PRAKAIDAO YINGSANGA ◽  
LADA MATHURASA
Keyword(s):  
Root Growth ◽  
Ethyl Acetate ◽  
Crude Extract ◽  
Inhibitory Activity ◽  
Crop Productivity ◽  
Distilled Water ◽  
Crude Extracts ◽  
Cleome Spinosa ◽  
Whole Plants ◽  
Potent Inhibitory Activity

Objective: Biological control is useful in agricultural. Allelopathy plays an important factor in crop productivity. The effect of allelopathy is able to produce and release allelochemicals or phytochemicals to inhibit or stimulate seed germination, seeding growth, shoot and root growth of other crops. The aim of this study was to evaluate the inhibitory activity of Cleome viscose and Cleome spinosa extracts on onion guest roots length.Methods: The whole plants of C. viscose and C. spinosa were extracted with dichloromethane, ethyl acetate, methanol and distilled water, respectively. Each group of onion guests was sprayed at various concentrations as 100, 200, 400, 800 and 1600 parts per million for the treatment. The root growth was measured every day for 1 month.Results: At concentration of 1600 ppm, methanol crude extract of C. spinose showed the highest percent inhibitory activity value of 91.68. Ethyl acetate and methanol crude extracts of C. viscose showed strong inhibitory activity with percentage values of 90.17 and 90.90, respectively, at concentration of 1600 ppm. Moreover, the methanol extract of C. viscose and C. spinosa evaluated higher inhibitory activity than other solvent. However, distilled water crude extract of C. spinose showed weak inhibitory activity with the percentage value of 40.05 at concentration of 1600 ppm.Conclusions: In this study, the methanol crude extract of C. spinose showed potent inhibitory activity on root growth. Moreover, ethyl acetate and methanol crude extracts of C. viscose evaluated strong inhibitory activity. C. viscose and C. spinosa extracts possessed allelochemicals for postharvest biology and technology in plants.

scholarly journals Selective inhibition of Ph-positive ALL cell growth through kinase-dependent and -independent effects by CDK6-specific PROTACs

Blood ◽  
2020 ◽  
Vol 135 (18) ◽  
pp. 1560-1573 ◽  
Author(s):  
Marco De Dominici ◽  
Patrizia Porazzi ◽  
Youcai Xiao ◽  
Allen Chao ◽  
Hsin-Yao Tang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Enzymatic Activity ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Regulatory Gene ◽  
Selective Inhibition ◽  
Hematologic Malignancies ◽  
Hematopoietic Progenitors

Abstract Expression of the cell cycle regulatory gene CDK6 is required for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cell growth, whereas expression of the closely related CDK4 protein is dispensable. Moreover, CDK6 silencing is more effective than treatment with the dual CDK4/6 inhibitor palbociclib in suppressing Ph+ ALL in mice, suggesting that the growth-promoting effects of CDK6 are, in part, kinase-independent in Ph+ ALL. Accordingly, we developed CDK4/6–targeted proteolysis-targeting chimeras (PROTACs) that inhibit CDK6 enzymatic activity in vitro, promote the rapid and preferential degradation of CDK6 over CDK4 in Ph+ ALL cells, and markedly suppress S-phase cells concomitant with inhibition of CDK6-regulated phospho-RB and FOXM1 expression. No such effects were observed in CD34+ normal hematopoietic progenitors, although CDK6 was efficiently degraded. Treatment with the CDK6-degrading PROTAC YX-2-107 markedly suppressed leukemia burden in mice injected with de novo or tyrosine kinase inhibitor–resistant primary Ph+ ALL cells, and this effect was comparable or superior to that of the CDK4/6 enzymatic inhibitor palbociclib. These studies provide “proof of principle” that targeting CDK6 with PROTACs that inhibit its enzymatic activity and promote its degradation represents an effective strategy to exploit the “CDK6 dependence” of Ph+ ALL and, perhaps, of other hematologic malignancies. Moreover, they suggest that treatment of Ph+ ALL with CDK6-selective PROTACs would spare a high proportion of normal hematopoietic progenitors, preventing the neutropenia induced by treatment with dual CDK4/6 inhibitors.

scholarly journals The global impact of bacterial processes on carbon mass

2020 ◽  
Vol 20 (3) ◽  
pp. 1777-1794
Author(s):  
Barbara Ervens ◽  
Pierre Amato
Keyword(s):  
Cell Growth ◽  
Growth Conditions ◽  
Water Soluble ◽  
Cloud Formation ◽  
Cloud Droplets ◽  
Spatial And Temporal Scales ◽  
Adverse Health Effects ◽  
Oh Reactions ◽  
Carbon Mass ◽  
High Concentrations

Abstract. Many recent studies have identified biological material as a major fraction of ambient aerosol loading. A small fraction of these bioaerosols consist of bacteria that have attracted a lot of attention due to their role in cloud formation and adverse health effects. Current atmospheric models consider bacteria as inert quantities and neglect cell growth and multiplication. We provide here a framework to estimate the production of secondary biological aerosol (SBA) mass in clouds by microbial cell growth and multiplication. The best estimate of SBA formation rates of 3.7 Tg yr−1 is comparable to previous model estimates of the primary emission of bacteria into the atmosphere, and thus this might represent a previously unrecognized source of biological aerosol material. We discuss in detail the large uncertainties associated with our estimates based on the rather sparse available data on bacteria abundance, growth conditions, and properties. Additionally, the loss of water-soluble organic carbon (WSOC) due to microbial processes in cloud droplets has been suggested to compete under some conditions with WSOC loss by chemical (OH) reactions. Our estimates suggest that microbial and chemical processes might lead to a global loss of WSOC of 8–11 and 8–20 Tg yr−1, respectively. While this estimate is very approximate, the analysis of the uncertainties and ranges of all parameters suggests that high concentrations of metabolically active bacteria in clouds might represent an efficient sink for organics. Our estimates also highlight the urgent need for more data concerning microbial concentrations, fluxes, and activity in the atmosphere to evaluate the role of bacterial processes as net aerosol sinks or sources on various spatial and temporal scales.

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