Escherichia coli is the most prevalent host organism for the production of recombinant en-zymes. This was feasible due to the possibility of genetic modification and the availability of multiple E. coli strains as recombinant systems. The primary disadvantage of using E. coli as a host, however, is bacterial cell lysis due to tension build-up in the periplasmic space caused by the overexpression of the recombinant enzyme. Therefore, immobilization is preferable to cytoplasmic excretion for directing the expression of recombinant enzymes into the culture medium. This research investigated the effect of graphene oxide (GO) on the xylanase and β-galactosidase activity of immobilized recombinant E. coli. The effect of culture conditions (expression medium, IPTG, post induction temperature, post induction duration, agitation rate, and pH) on xylanase excretion and cell survival of an immobilized cell was studied using the one factor at a time (OFAT) method. After 24 hours of induction, using terrific broth (TB) as a medium increased xylanase excretion to 0.060 U/ml and resulted in decreased β-galactosidase activity (1.218 U/ml). Apart from that, a lower concentration of isopropyl -D-1-thiogalactopyranoside (IPTG) at 0.01 mM, a lower post-induction temperature (25°C), a 5-hour post-induction time, neutral pH, and 150 rpm significantly increased the xylanase excretion of immobilized cells with low β-galactosidase activity. This study established that immobilizing recombinant E. coli on GO may be advantageous for the excretion of recombinant proteins with a high cell viability.
Glucose is a cheap and readily available substrate for production of large-scale chemicals. Synthesis of xylitol, a high demand chemical in global market is currently done by using xylose, which contributes to its high operational cost. Studies on production of xylitol from glucose have explored several approaches, from sequential fermentation to multiple and single gene expression. Xylitol-5-phosphate dehydrogenase (XPDH), is an enzyme that enables conversion of glucose to xylitol in a single step fermentation. This study explores conversion of xylitol from glucose in E. coli by the expression of xpdh from Clostridium difficile with modifications in metabolic pathways to enhance xylitol production. The xpdh gene was carried by pACYC-Duet-1 expression vector and induced by the addition of IPTG. Initial screening of E. coli expressing xpdh (NA116) was done by shake-flask fermentation for 24 hours and its metabolites were analyzed by HPLC. NA116 was able to produce 0.273 g/L xylitol from 4.33 g/L consumed glucose in 24 hours. Further metabolic pathway modification to eliminate competing pathways yielded four mutants, NA207 (∆rpiA), NA208 (∆rpiB), NA209 (∆pgi) and NA211 (∆rpi∆Apgi). Screening of mutants for xylitol production showed that highest xylitol production from glucose was achieved by NA211 with almost double the amount of the original strain, 0.585 g/L. This showed successful xylitol conversion from glucose in a single fermentation in E. coli with improved yield through metabolic pathway modification.
Aim: This paper reviews the different in vitro models of human intestinal epithelium that have been utilized for studying the adhesion and invasion properties. Problem Statement: The cell adhesion and invasion are the key mechanisms of bacterial pathogenicity that determines their possible routes of transmission. Numerous investigations related to the adhesion and invasion ability of bacterial isolates have been reported on monoculture human intestinal cells. However, the use of monoculture cells has several major disadvantages, such as the inability to reproduce the complex structure that defines the intestine and the inability to accurately predict the mechanism of bacterial adhesion and invasion. Approach: Co-culture models of human intestine have been developed as an alternative to improve the monoculture epithelial cell for adhesion and invasion studies, which provide more flexibility and overcome some of the limitations Conclusion: With the use of diverse in vitro approach, it could provide thorough information on different ability of bacterial adhesion and invasion and it could help to clarify the intricacy of host-pathogen interactions that underpin bacterial pathogenesis.
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