Structure-based substrate screening for an enzyme

<p>Abstract</p> <p>Background</p> <p>Nowadays, more and more novel enzymes can be easily found in the whole enzyme pool with the rapid development of genetic operation. However, experimental work for substrate screening of a new enzyme is laborious, time consuming and costly. On the other hand, many computational methods have been widely used in lead screening of drug design. Seeing that the ligand-target protein system in drug design and the substrate-enzyme system in enzyme applications share the similar molecular recognition mechanism, we aim to fulfill the goal of substrate screening by in silico means in the present study.</p> <p>Results</p> <p>A computer-aided substrate screening (CASS) system which was based on the enzyme structure was designed and employed successfully to help screen substrates of <it>Candida antarctica </it>lipase B (CALB). In this system, restricted molecular docking which was derived from the mechanism of the enzyme was applied to predict the energetically favorable poses of substrate-enzyme complexes. Thereafter, substrate conformation, distance between the oxygen atom of the alcohol part of the ester (in some compounds, this oxygen atom was replaced by nitrogen atom of the amine part of acid amine or sulfur atom of the thioester) and the hydrogen atom of imidazole of His224, distance between the carbon atom of the carbonyl group of the compound and the oxygen atom of hydroxyl group of Ser105 were used sequentially as the criteria to screen the binding poses. 223 out of 233 compounds were identified correctly for the enzyme by this screening system. Such high accuracy guaranteed the feasibility and reliability of the CASS system.</p> <p>Conclusion</p> <p>The idea of computer-aided substrate screening is a creative combination of computational skills and enzymology. Although the case studied in this paper is tentative, high accuracy of the CASS system sheds light on the field of computer-aided substrate screening.</p

Experimental study on stoichiometric laminar flame velocities and Markstein lengths of methane and PRF95 dual fuels

Natural gas is one of the most promising alternative fuels. The main constituent of natural gas is methane. The slow burning velocity of methane poses significant challenges for its utilization in future energy efficient combustion applications. Methane-gasoline dual fuelling has the potential to improve methane’s combustion. The fundamental combustion characteristics of a methane-gasoline Dual Fuel (DF) blend needs further investigation. In the current experimental study, the relationship between laminar flame velocity and Markstein length, with the ratio of gas to liquid in a DF blend has been investigated using spherical flames in a constant volume combustion vessel. A binary blend of primary reference fuels (PRF95) was used as the liquid fuel. Methane was added to PRF95 in three different energy ratios 25%, 50% and 75%. Values of the stoichiometric laminar flame velocities and Markstein lengths are measured at pressures of 2.5, 5, 10 Bar and a temperature of 373 K. It has been found that with a 25% increase in the DF ratio, the Markstein length is reduced by 15%, 21%, 32% at a pressure of 2.5, 5 and 10 Bar respectively whereas at the same pressures the laminar flame velocity is reduced by 2%, 3% and 5%. The flame evolution at the early stages of combustion is found to be faster with an increase in the DF ratio, and gradually as the flame develops it becomes slower

Efficient display of active lipase LipB52 with a Pichia pastoris cell surface display system and comparison with the LipB52 displayed on Saccharomyces cerevisiae cell surface

<p>Abstract</p> <p>Background</p> <p>For industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.</p> <p>Results</p> <p>Two <it>Pichia pastoris </it>cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the α-factor secretion signal sequence were constructed respectively. The lipase gene <it>lipB52 </it>fused with the <it>FLO </it>gene was successfully transformed into <it>Pichia pastoris </it>KM71. The lipase LipB52 was expressed under the control of the <it>AOX1 </it>promoter and displayed on <it>Pichia pastoris </it>KM71 cell surface with the two <it>Pichia pastoris </it>cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the <it>Pichia pastoris </it>cell surface exhibited activity toward <it>p</it>-nitrophenol ester with carbon chain length ranging from C₁₀to C₁₈, and the optimum substrate was <it>p</it>-nitrophenol-caprate (C₁₀), which was consistent with it displayed on the <it>Saccharomyces cerevisiae </it>EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on <it>Pichia pastoris </it>KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40°C at pH8.0, they retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.</p> <p>Conclusion</p> <p>The LipB52 displayed on the <it>Pichia pastoris </it>cell surface exhibited better stability than the lipase LipB52 displayed on <it>Saccharomyces cerevisiae </it>cell surface. The displayed lipases exhibited similar transesterification activity. But the <it>Pichia pastoris </it>dry cell weight per liter (DCW/L) ferment culture was about 5 times than <it>Saccharomyces cerevisiae</it>, the lipase displayed on <it>Pichia pastoris </it>are more suitable for whole-cell biocatalysts than that displayed on <it>Saccharomyces cerevisiae </it>cell surface.</p

Experimental investigation on the Laminar burning velocities and Markstein lengths of methane and PRF95 dual fuels

© 2016 American Chemical Society.Natural gas is a promising alternative fuel. The main constituent of natural gas is methane. The slow burning velocity of methane poses significant challenges for its utilization in future energy efficient combustion applications. The effects of methane addition to PRF95 on the fundamental combustion parameters, laminar burning velocity (Su0) and Markstein length (Lb), were experimentally investigated in a cylindrical combustion vessel at equivalence ratios of 0.8, 1, and 1.2, initial pressures of 2.5, 5, and 10 bar, and a constant temperature of 373 K. Methane was added to PRF95 in three different energy ratios 25%, 50%, and 75%. Spherically expanding flames were used to derive the flow-corrected flame velocities, from which the corresponding Lb and Su0 were obtained. The flame velocities were corrected for the motion of burned gas induced by the cylindrical confinement. It has been found that at stoichiometric conditions there is a linear decrease in Lb and Su0 with the dual fuel (DF) ratio in all investigated pressures. At rich conditions, all DFs resulted in having lower Su0 as compared to methane and to a larger extent PRF95. The values of Lb for all DFs were lower than methane and comparable to those of PRF95. At lean conditions, the values of Lb for all DFs are biased toward those of methane whereas the values of Su0 are found to be higher than those of PRF95 at pressures of 2.5 and 5 bar. At 10 bar both Lb and Su0 reduce with DF ratio although Su0 of all DFs converge to that of PRF95. The findings of the current study indicate a distinct synergy in the utilization of dual fueling in future lean burn energy efficient combustion applications

A screening analysis of the GJB2 c.176 del 16 mutation responsible for hereditary deafness in a Chinese family

AbstractObjectiveTo determine whether a new-born child from a family carrying a deafness gene needs cochlear implantation to avoid dysphonia by screening and sequencing a deafness-related gene.ResultsBoth screening and sequencing results confirmed that the new born child had a normal GJB2 gene despite the fact that she has a brother suffering from hearing loss triggered by an allelic GJB2 c.176 del 16 mutation. We cloned the GJB2 genes derived from their respective blood genomic DNA into GFP fused plasmids and transfected those plasmids into the 293T cell line to test for gene function. While the mutated GJB2 gene (GJB2 c.176 del 16) of her deaf brother was found to be unable to form the gap junction structure between two adjacent cells, the baby girl's GJB2 gene ran into no such problems.ConclusionThe screening and sequencing as well as the GJB2 gene function tests invariably showed results consistent with the ABR tested hearing phenotype, which means that the child, with a normal wild type GJB2 gene, does not need early intervention to prevent her from developing hearing loss and dysphonia at a later stage in life

Experimental study on the burning rate of Methane and PRF95 dual fuels

Natural gas as an alternative fuel offers the potential of clean combustion and emits relatively low CO2 emissions. The main constitute of natural gas is methane. Historically, the slow burning speed of methane has been a major concern for automotive applications. Literature on experimental methane–gasoline Dual Fuel (DF) studies on research engines showed that the DF strategy is improving methane combustion, leading to an enhanced initial establishment of burning speed even compared to that of gasoline. The mechanism of such an effect remains unclear. In the present study, pure methane (representing natural gas) and PRF95 (representing gasoline) were supplied to a constant volume combustion vessel to produce a DF air mixture. Methane was added to PRF95 in three different energy ratios 25%, 50% and 75%. Experiments have been conducted at equivalence ratios of 0.8, 1, 1.2, initial pressures of 2.5, 5 and 10 bar and a temperature of 373K. At stoichiometric conditions, experiments in an SI engine have been also performed. It has been found that methane and all DFs have their fastest burning rate at stoichiometric conditions whereas PRF95 at rich conditions (Φ=1.2). At lean conditions (Φ=0.8), all DFs resulted in faster combustion than PRF95, whereas methane is the slowest of all. At rich conditions, DF75 and DF50 are slower than methane. The transition mechanism between the constant volume combustion experiments and those in the engine environment resulted in a larger increase in the burning speed of methane and all DFs in comparison to that of the liquid fuel

Experimental Investigation on the Laminar Burning Velocities and Markstein Lengths of Methane and PRF95 Dual Fuels

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Energy and Fuels, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/acs.energyfuels.6b00644© 2016 American Chemical Society.Natural gas is a promising alternative fuel. The main constituent of natural gas is methane. The slow burning velocity of methane poses significant challenges for its utilization in future energy efficient combustion applications. The effects of methane addition to PRF95 on the fundamental combustion parameters, laminar burning velocity (Su0) and Markstein length (Lb), were experimentally investigated in a cylindrical combustion vessel at equivalence ratios of 0.8, 1, and 1.2, initial pressures of 2.5, 5, and 10 bar, and a constant temperature of 373 K. Methane was added to PRF95 in three different energy ratios 25%, 50%, and 75%. Spherically expanding flames were used to derive the flow-corrected flame velocities, from which the corresponding Lb and Su0 were obtained. The flame velocities were corrected for the motion of burned gas induced by the cylindrical confinement. It has been found that at stoichiometric conditions there is a linear decrease in Lb and Su0 with the dual fuel (DF) ratio in all investigated pressures. At rich conditions, all DFs resulted in having lower Su0 as compared to methane and to a larger extent PRF95. The values of Lb for all DFs were lower than methane and comparable to those of PRF95. At lean conditions, the values of Lb for all DFs are biased toward those of methane whereas the values of Su0 are found to be higher than those of PRF95 at pressures of 2.5 and 5 bar. At 10 bar both Lb and Su0 reduce with DF ratio although Su0 of all DFs converge to that of PRF95. The findings of the current study indicate a distinct synergy in the utilization of dual fueling in future lean burn energy efficient combustion applications

Poboljšanje enzimske proizvodnje cefaleksina upotrebom velikih koncentracija supstrata i uklanjanjem produkata reakcije kompleksiranjem in situ

Cephalexin (CEX) was synthesized with 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D(–)-phenylglycine methyl ester (PGME) using immobilized penicillin G acylase from Escherichia coli. It was found that substrate concentration and in situ product could remarkably influence the ratio of synthesis to hydrolysis (S/H) and the efficiency of CEX synthesis. The optimal ratio of enzyme to substrate was 65 IU/mM 7-ADCA. High substrate concentration improved the 7-ADCA conversion from 61 to 81 % in the process without in situ product removal (ISPR), while in the synthetic process with ISPR, high substrate concentration increased the 7-ADCA conversion from 88 to 98 %. CEX was easily separated from CEX/β-naphthol complex and its purity and overall yield were 99 and 70 %, respectively.Primjenom imobilizirane Penicilin-G-acilaze iz bakterije Escherichia coli sintetiziran je cefaleksin (CEX) iz 7-amino-3-deacetoksicefalosporanske kiseline (7-ADCA) i D(-)-fenilglicin-metilnog estera (PGME). Koncentracije supstrata i produkta in situ mogu znatno utjecati na omjer sinteze i hidrolize (S/H) i djelotvornost sinteze cefaleksina. Optimalni omjer enzima i supstrata iznosio je 65 IU/mM 7-ADCA. Velika koncentracija supstrata poboljšala je konverziju 7-ADCA sa 61 na 81 % u procesu bez uklanjanja produkta in situ (ISPR), a s 88 na 98 % u procesu sa ISPR. Cefaleksin je lako uklonjen iz kompleksa CEX/β-naftol, a dobiveni je proizvod imao čistoću od 99 % i ukupni prinos od 70 %

Turbulent flame propagation with pressure oscillation in the end gas region of confined combustion chamber equipped with different perforated plates

Experiments were conducted in a newly designed constant volume combustion chamber with a perforated plate by varying the initial conditions. Hydrogen-air mixtures were used and the turbulent flame, shock wave, and the processes of flame-shock interactions were tracked via high-speed Schlieren photography. The effects of hole size and porosities on flame and shock wave propagation, intensity of the shock wave and pressure oscillation in closed combustion chamber were analyzed in detail. The effect of interactions between the turbulent flame and reflected shock or acoustic wave on the turbulent flame propagation was comprehensively studied during the present experiment. The results demonstrated that flame front propagation velocity and pressure oscillation strongly depend on the hole size and porosities of the perforated plate. The flame front propagation velocity in the end gas region increases as hole size increases and porosity decreases. The flame front propagation intensity in the end region of a confined space is strongly relevant to two competing effects: the initial turbulent formation and turbulent flame development. The experimental results indicated that an oscillating flame is associated with both the reflected shock wave and the acoustic wave. Meanwhile, different turbulent flame propagations and combustion modes were observed