• Archives of Pharmacal Research
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• v.14 no.3
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• pp.261-265
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• 1991

In a microbial cell-free extract system, side chain cleavage on various sterols and steroids was tested. The cell-free extracts of Mycobacterium sp. NRRL B-3805 showed the side chain cleavage activity on lithocholic acid to form 5$\beta$-androstan-3.17-dione. The properties of the activity were examined.

• Journal of Industrial and Engineering Chemistry
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• v.67
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• pp.231-235
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• 2018

In this study, we demonstrate the conversion of starch to (R,R)-2,3-butanediol (2,3-BD) in a hybrid cell-free synthesis system containing a mixture of lysates derived from Escherichia coli (E. coli) and cyanobacteria. A sufficient pool of pyruvate required for the synthesis of 2,3-BD was generated by combining metabolic pathways of cyanobacteria and E. coli. Successful synthesis of 2,3-BD was achieved by additional modifications of the hybrid cell-free system with the enzymes required to convert pyruvate to 2,3-BD. The results demonstrate a new approach to harness biological pathways to expand the scope of cell-free metabolic engineering by cross-species combinations of cell lysates.

• Molecules and Cells
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• v.37 no.6
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• pp.473-479
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• 2014

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost-effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.3
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• pp.258-261
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• 2006

The addition of zeolite particles enhances the stability of mRNA molecules in a cell-free protein synthesis system. When $20{\mu}g/{\mu}L$ of zeolite (Y5.4) is added to a reaction mixture of cell-free protein synthesis, a substantial increase in protein synthesis is observed. The stabilizing effect of zeolite is most dearly observed in an in vitro translation reaction directed by purified mRNA, as opposed to a coupled transcription and translation reaction. Upon the addition of zeolite in the in vitro translation reaction, the life span of the mRNA molecules is substantially extended, leading to an 80% increase in protein synthesis. The effect of zeolite upon the mRNA stability appears be strongly related to the cation exchange (potassium to sodium) reaction. Our results demonstrate the possibility of modifying this biological process using heterogeneous, non-biological substances in a cell-free protein synthesis system.

• Korean Journal of Microbiology
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• v.8 no.1
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• pp.35-40
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• 1970

In the cell-free systems of Dunaliella tertiolecta, fructosediphosphate aldolase hardly contribute to synthesize hexosephosphate from triosephosphate derived from pentosphosphate pathway, and it could be considered that glycerol synthesized from added glucose was synthesized but via 3-phosphoglyceraldehyde as an intermediate not hydroxypyruvate.

• Microbiology and Biotechnology Letters
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• v.26 no.3
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• pp.195-199
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• 1998

AH alkalophilic Bacillus SP. AIk-7, isolated from soil, produced ethylene. The characteristics of this microorganism is the ability to grow well under the alkaline condition, at pH 10.3. This strain is similar to Bacillus alkalophilus in terms of morphological, physiological and biological characteristics. In observation of relationship of cell growth and ethylene production according to incubation times, the ethylene synthesis mostly occur from the late exponential phase to the death phase of growth. The purpose of this paper is to study the effects of various substrates on the biosynthesis of ethylene in the intact cell and the cell-free system by the Bacillus sp. AIk-7. In both intact cell and cell-free extract, optimum conditions for ethylene production was achieved at pH 10.3 and 3$0^{\circ}C$. Ethylene was effectively produced from L-Met and 1-aminocyclopropane-1-carboxylic acid (ACC). In this case, ACC as the substrate on ethylene production were two fold higher than L-met at each concentration of substrates. On the other hand, the cell-free ethylene-forming system was used as a tool for the elucidation of the biochemical reaction involved in the formation of ethylene by Bacillus sp. AIk-7. Ethylene production in the cell-free system required the presence of manganese and cobalt ion to be stimulated a little. The result obtained in this work suggests that L-met and ACC may be a precursor more directly related to bacterial ethylene production than any other substrates tested.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.3
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• pp.235-239
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• 2006

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast to in vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately $650{\mu}g/mL$ of protein was produced after 2h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.

• Korean Chemical Engineering Research
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• v.54 no.5
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• pp.665-670
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• 2016

This study deals with the possible degradation of toluene and acetic acid when subjected to cell-free enzyme system from the toluene degrading bacteria Pseudomonas putida and acetic acid degrading bacteria Cupriavidus necator. P. putida produces toluene dioxygenase only under the existence of toluene in culture medium and toluene is degraded to cis-toluene dihydrodiol by this enzyme. C. necator produces acetyl coenzyme A synthetase-1 and converts acetic acid to acetyl CoA in order to synthesize ATP to need for growth or PHA which is biodegradable polymer. In case of toluene degradation, the experiment was conducted before and after production of toluene dioxygenase as this enzyme, produced by P. putida, is an inducible enzyme. Toluene was detected using gas chromatography (GC). Similar amount of toluene was found in control group and before production of toluene dioxygenase (experimental group 1). However, reduction in toluene was detected after the production of toluene dioxygenase (experimental group 2). Acetic acid was detected through application of gas chromatography-mass spectrometer (GC-MS). The results showed the acetic acid peak was not detected in the experimental group to apply cell-free enzyme system. These results show that the cell-free enzyme system obtained from P. putida and C. necator retained the ability to degrade toluene and acetic acid. However, P. putida needs to produce the inducible enzyme before preparation of the cell-free enzyme system.

• Journal of Life Science
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• v.15 no.5 s.72
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• pp.779-782
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• 2005

Cell-free expression is a powerful tool for rapid protein analysis, enabling an efficient identification of gene without cumbersome procedure of transformation and cell culture. Epoxide hydrolase (EH) gene of Rhodotorula glutinis was simply amplified by PCR, and the resultant gene was expressed in vitro using a coupled Transcription/translation system. The cell-free expressed EH protein mixture exhibited the enantioselective hydrolysis activity toward (R)-styrene oxide, representing that cell-free protein synthesis system can be used for the rapid expression of an enantioselective enzyme for an efficient identification of the chiral activity.

• Journal of Plant Biology
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• v.12 no.2
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• pp.7-14
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• 1969

Dunaliella tertiolecta did not show any increase in respiration rate when supplied with glucose, glycerol, sucrose, L-alanine, acetate, pyruvate and succinate. This was in contrast to Chlorella pyrenoidosa, which, under identical conditions, showed significant increase when supplied with glucose or acetate but not with the other compounds. Production of 14CO2 from added 14C-glucose in D. tertiolecta was lower than the other 14C-labelled substrates: L-alinine, glycerol, succinate, but higher than 14C-sucrose addition. And it was also lower than C. pyrenoidosa experiments which was added 14C-glucose as a substrate. Light reduced amounts of labelled carbon dioxide from 14C-glucose or 14C-acetate and increased incorporation of 14C from the substrates to cell materials in either D. tertiolecta or C. pyrenoidosa. The contribution of 14C from 14C-glucose to 14CO2 in cell-free system of D. tertiolecta were much higher than in whole cell suspension. It was contrast to C. pyrenoidosa which were showed reduction of 14CO2 production in cell-free systems than whole cell suspensions. When cell-free systems of D. tertiolecta and C. pyrenoidosa were supplied with ATP, NAD, NADP or/and hexokinase, it was remarkably increased production of 14CO2 from the substrates than the control. It was concluded that the low ability of D. tertiolecta to metabolize glucose were caused by the impermeability of the cell membrane to glucose and were not due to deficiencies of enzyme systems concerning glucose metabolism. In the cell-free systems, it seemed to be more active pentose phosphate pathway than glycolytic pathway in D. tertiolecta.