ABSTRACT
Virgibacillus sp. strain AGTR, which is a haloalkaliphilic microorganism, was isolated from a sediment sample collected in hypersaline Lake Acigöl in Turkey. It has the potential to produce biotechnologically essential proteases. Here, the whole-genome sequence and its annotations are reported.
ABSTRACT
We report on the fabrication of AqpZ immobilized flat sheet membranes. The effects of interfacial polymerization conditions as well as proteoliposome concentration were evaluated. Commercial AqpZ were used as positive control for cloned AqpZ. Specific permeate flux of membranes at higher proteoliposome concentrations increased up to 25 times higher than thin film composite membranes; however; MgSO4 rejection is lowered almost to 1.5%. FTIR and SEM confirm immobilization of proteoliposomes. Thermal analysis showed that increasing proteoliposome concentration has no positive effect on the incorporation of proteoliposomes into polyamide structures. On the contrary, at lower proteoliposome concentrations, incorporation of proteoliposomes was found better. When combined membrane performances were compared in terms of specific permeate flux; MgSO4 and humic rejection and flux recovery after humic acid filtration, the performance of cloned AqpZ incorporated membranes (having 0.1â mg/mL proteoliposome concentration and polyamide formed with 2â min piperazine reaction time) improved 1.7 times regarding TFC membranes. According to the results, increasing proteoliposome concentration did not improve nanofiltration membrane performance. On the contrary, lower proteoliposome concentrations were found to be more effective in increasing membrane performance.
Subject(s)
Aquaporins , Membranes, Artificial , Filtration , ProteolipidsABSTRACT
In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6 × 104-fold and 5 × 104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating "smarter libraries" for improving the properties of cmFDH.
ABSTRACT
Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self-assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site-specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N-terminus of AP (nGBP1-AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi-functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple-repeat constructs, 5GBP1-AP displayed the best bi-functional activity and, therefore, was chosen for self-immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1-AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self-immobilization of the bi-functional enzyme on micro-patterned substrates where genetically linked 5GBP1-AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site-specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic-binding peptides has a potential utility in a wide range of biosensing and bioconversion processes.
