ABSTRACT
Abstract Rhamnolipid is a potent biodegradable surfactant, which frequently used in pharmaceutical and environmental industries, such as enhanced oil recovery and bioremediation. This study aims to engineer Escherichia coli for the heterologous host production of rhamnolipid, to characterize the rhamnolipid product, and to optimize the production using autoinduction medium and POME (palm oil mill effluent). The construction of genes involved in rhamnolipid biosynthesis was designed in two plasmids, pPM RHLAB (mono-rhamnolipid production plasmid) and pPM RHLABC (di-rhamnolipid production plasmid). The characterization of rhamnolipid congeners and activity using high-resolution mass spectrometry (HRMS) and critical micelle concentration (CMC). In order to estimate rhamnolipid yield, an oil spreading test was performed. HRMS and CMC result show E. coli pPM RHLAB mainly produced mono-rhamnolipid (Rha-C14:2) with 900 mg/L and 35.4 mN/m of CMC and surface tension value, whereas E. coli pPM RHLABC mainly produced di-rhamnolipid (Rha-Rha-C10) with 300 mg/L and 34.3 mN/m of CMC and surface tension value, respectively. The optimum condition to produce rhamnolipid was at 20 h cultivation time, 37 oC, and pH 7. In this condition, the maximum rhamnolipid yield of 1245.68 mg/L using autoinduction medium and 318.42 mg/L using 20% (v/v) of POME. In conclusion, the characteristics of the rhamnolipid by recombinant E. coli is very promising to be used in industries as the most economical way of producing rhamnolipid.
Subject(s)
Palm Oil , Escherichia coli , Electromagnetic Phenomena , GlycosylationABSTRACT
Objective To construct the recombinant streptolysin O antigen(SLO) prokaryotic expression plasmid and establish its best expression condition in Escherichia coli .Methods The DNA fragment encoding SLO was amplified from streptococcal ge-nomic DNA template by PCR ,and then incorporated into pET-32a(+ ) vector to construct pET-32a(+ )-SLO recombinant plas-mid .pET-32a(+ )-SLO was transformed into Escherichia coli BL21(DE3) and SLO protein was expressed and purified by isopro-pyl-β-D-thiogalactoside(IPTG)-induction and auto-induction ,respectively .Results The results of DNA electrophoresis and DNA sequencing showed that pET-32a(+ )-SLO recombinant plasmid was constructed successfully .When IPTG at different concentra-tion was used ,SLO expressed as inclusion body and its expression efficiency was low .Under auto-induction condition ,SLO ex-pressed as partly soluble manner ,and its expression efficiency increased .Conclusion The prokaryotic expression plasmid pET-32a (+ )-SLO is constructed successfully and the best condition for SLO expression and purification from Escherichia coli culture is es-tablished ,which lay the foundation for further basic and clinical application research with SLO .
ABSTRACT
Objective To construct recombinant plasmids containing HPV18E7 gene ,and explore the optimization condition of its expression in Escherichia coli .Methods The genomic DNA extracted from HeLa cell line which served as a template to the HPV18 E7 gene was amplified using PCR method ;and the amplified product of HPV18E7 gene was connected to the pET-32a(+ ) vector ,which composed the pET-32a(+ )-HPV18E7 recombinant plasmid ;the positive recombinant plasmids were transformed into BL21-DE3-pLysS competent cells and the optimized expression condition was explored in order to obtain a large amount of HPV18E7 oncogenic protein .Results The fragment length of PCR products of HeLa cell genomic DNA was consistent with that of HPV18 E7 gene .In LB medium ,the expression level of the target protein was not high under such conditions as different concentra-tion of IPTG and lactose ,different temperatures and different induction starting amount .Therefore the ZYM-5052 auto-induction medium was tried in this experiment ,and the expression amount of the fusion protein was much higher than that induced with IPTG and lactose .Conclusion The amount of HPV18E7 fusion protein in ZYM-5052 automatic induction medium is much higher than that induced with IPTG and lactose .
ABSTRACT
Signaling mechanisms that govern physiological and morphological responses to change the cell density are common in bacteria. Quorum sensing is signal transduction processes which involves the production and release of and response to hormone-like molecules (auto-inducers) that accumulate in the external environment as the cell population grows. Quorum sensing is found in a wide variety of bacteria, both Gram-positive and Gram-negative and the spectrum of physiological functions that can be regulated is impressive. Variation in the nature of the extra-cellular signal in the signal detection machinery and in the mechanisms of signal transmission demonstrates the evolutionary adaptability of quorum sensing systems for multiple uses.
Subject(s)
Quorum Sensing/physiologyABSTRACT
PcoI-PcoR is a quorum-sensing (QS) system influencing the biofilm formation and rhizosphere colonization in Pseudomonas fluorescens 2P24. The expression of the pcoI, a N-acyl-homoserne lactone (AHL) biosynthase gene, is under the regulation of a number of chromosomal factors, such as the GacS-GacA two-component system. In this paper, we investigated the upstream regulators that influence the transcription of pcoI gene using a chromosomal pcoI-lacZ fusion reporter strain PM203. Cosmids containing genomic DNA of the wild-type strain 2P24 were introduced into the reporter strain PM203 (gacA—, pcoI-lacZ) to screen positive transcriptional regulators of pcoI gene. One of them named pP32-24, which contained a 5-kb Pst I functional fragment was selected. Further analysis identified that the pcoI was the gene responsible for the increase of the pcoI-lacZ expression. The expression of pcoI-lacZ reporter was alsoimproved in both PM101 (pcoI-lacZ) and its gacAmutant PM203 after addition of exogenous AHL, indicating that the expression of pcoI is positively regulated by AHL (autoinduction) in strain 2P24. In addition, deletion mutagenesis and complementation experiments demonstrated that the transcriptional regulator PcoR positively controlled the expression of pcoI and the formation of biofilm. These results suggest that, in strain 2P24, the expression of PcoI-PcoR QS system is auto-inducted, and the transcriptional factor PcoR is involved in the regulation of pcoI transcription and the biofilm formation.
ABSTRACT
INTRODUCTION: Carbamazepine (CBZ) undergoes biotransformation, being CYP3A4 the major cytocrome P450 (CYP) enzyme catalyzing the carbamazepine-10,11-epoxide (EPOX) formation, which is quantitatively the most important pathway in CBZ metabolism. There is evidence of dose-dependent elimination of this drug due to its autoinduction capacity. Moreover, published data showed an incomplete bioavailability of CBZ since its absorption increases when grapefruit juice was administered. Both CYP3A4 and MRP2 (located in the enterocyte) are autoinduced during long term use of CBZ. As the other enzymes involved in CBZ metabolism are negligible in the gut, presystemic biotransformation through CYP3A4 could be responsible for the bioavailability of the drug as well as EPOX formation. OBJECTIVE: The purpose of our study was to assess the importance of presystemic formation of EPOX during the autoinduction of CBZ versus the daily administered dose. PATIENTS AND METHODS: 40 adults (average age: 28 years) and 29 children (average age: 9 years) receiving CBZ as monotherapy were included in the study. CBZ and EPOX plasma concentrations were analyzed by a previous validated HPLC method. RESULTS AND CONCLUSION: The results obtained confirmed the metabolic induction after chronic administration and provided new elements to suggest a strong contribution of dose-dependent bioavailability in the non linear kinetics of CBZ.