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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.126, #2)
Surface plasmon resonance by Priyabrata Pattnaik (pp. 79-92).
During last decade there has been significant progress in the development of analytical techniques for evaluation of receptor-ligand iteraction. Surface plasmon resonance (SPR)-based optical biosensors are now being used extensively to defined the kinetics of wide variety of macromolecular interactions and high- and low-affinity small molecule interactions. The experimental design data analysis methods are evolving along with widespread applications in ligand fishing, microbiology, virology, host-pathogen interaction, epitope mapping and protein-, cell-, membrane-, nucleic acid-protein interactions. SPR based biosensors have strong impact on basic and applied research significantly. This brief review describes the SPR technology and few of its applications in relation to receptor-ligand interaction that has brought significant change in the methodology, analysis, interpretation, and application of the SPR technology.
Keywords: Surface plasmon resonance (SPR); optical biosensor; receptor-ligand interaction; Biacore; ligand fishing
Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system by Shamik S. Sharma; Shaorong Chong; Sarah W. Harcum (pp. 93-117).
Inteins are self-cleavalbe proteins that under reducing conditions can be cleaved from a recombinant target protein. Industrially, an intein-based system could potentially reduce production costs of recombinant proteins by facilitating a highly selective affinity purification using an inexpensive substrate such as chitin. In this study, SuperPro® Designer was used to simulate the large-scale recovery of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification process based on the commercially available IMPACT® system. The intein process was also compared with a conventional process simulated by SuperPro. The intein purification process initially simulated was significantly more expensive than the conventional process, primarily owing to the properties of the chitin resin and high reducing-agent (dithiothreitol [DTT]) raw material cost. The intein process was sensitive to the chitin resin binding capacity, cleavage efficiency of the intein fusion protein, the size of the target protein relative to the intein tag, and DTT costs. An optimized intein purification process considerably reduced costs by simulating an improved chitin resin and alternative reduced agents. Thus, to realize the full potential of intein purification processes, research is needed to improve the properties of chitin resin and to find alternative, inexpensive raw materials.
Keywords: Chitin; process simulation; recombinant protein production; intein; economic analysis
Optimization of culture medium and conditions for penicillin acylase production by streptomyces lavendulae ATCC 13664 by Jesús Torres-Bacete; Miguel Arroyo; Raquel Torres-Guzmán; Isabel de La Mata; Carmen Acebal; M. Pilar Castillón (pp. 119-131).
The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO4·5H2O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.
Keywords: Streptomyces lavendulae ; penicillin acylase; fermentation; medium optimization; factorial design
Effect of pH and phosphate on trapping capacity of various heavy metal ions with ferritin reactor in flowing seawater by Bo Kong; He-Qing Huang; Qing-Mei Lin; Zong-Wei Cai; Ping Chen (pp. 133-148).
We describe a protein reactor consisting of native liver ferritin of Dasyatis akajei (DALF) and a dialysis bag. Our goal was to study a ferritin reactor for its capacity to trap various heavy metal ions (M2+) in flowing seawater. The reactor is sensitive and inexpensive and can be operated by nonprofessional technicians. A positive relationship between the number of trapped M2+ with the DALF reactor and its concentration in the flowing seawater was observed. Both the pH in the medium and the phosphate content within the ferritin cavity strongly affected trapping capacity. It was found that the ferritin released its phosphate compound directly with a shift in pH without the need for releasing reagent, which differs from the phosphate release characteristics of horse spleen ferritin, as previously described. This behavior evidently makes the trapping capacity with the ferritin reactor weaken, indicating that this trapping capacity is tightly connected to its phosphate compound. Our study shows that a self-regulation ability of the ferritin shell rather than its phosphate compound plays an important role in controlling the rate and capacity of trapping M2+. The ferritin reactor was constructed to monitor the contamination level of M2+ in flowing seawater. Our preliminary data along with fieldwork indicate that the DALF reactor is an analytical means for effectively monitoring the contamination level of M2+ in flowing seawater.
Keywords: Dasyatis akajei ; heavy metalions; ferritin reactor; phosphate effect; seawater; contamination level
Effect of pH on thermal- and chemical-induced denaturation of GFP by Klaithem M. Alkaabi; Abeer Yafea; S. Salman Ashraf (pp. 149-156).
Green fluorescent protein (GFP) is an unusually stable autofluorescent protein that is increasingly being exploited for many applications. In this report, we have used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with sodium dodecyl sulfate (SDS), urea, and heat. Surprisingly, SDS (up to 0.5%) did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5 buffers; however, at pH 6.5, the protein lost all fluorescence within 1 min of incubation. Similarly, incubation of GFP with 8 M urea at 50°C resulted in time dependent denaturation of GFP, but only in pH 6.5 buffer. At higher pH values (pH 7.5 and pH 8.5), the GFP was quite stable in 8 M urea at 50°C, showing only a slight decrease in fluorescence. Heat denaturation of GFP was found to be pH dependent as well, with the denaturation being fastest at pH 6.5 as compared to pH 7.5 or pH 8.5. Like the denaturation studies, renaturation of heat-denatured GFP was most efficient at pH 8.5, followed by pH 7.5, and then pH 6.5. These results suggests that GFP undergoes a structural/stability shift between pH 6.5 and pH 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat.
Keywords: E. coli ; GFP; SDS; urea; thermal denaturation; renaturation