•Design of a novel process for bio-synthesis of aviation fuel.•A plant origin FASs was introduced to improve the production of fatty acid.•A chain length specific thioesterase was used to control the chain length of alkane.•A heterologous ACP was used to improve the selectivity of introduced thioesterase.The aim of this work was to study the synthesis of medium-chain length alkanes (MCLA), as bio-aviation product. To control the chain length of alkanes and increase the production of MCLA, Escherichia coli cells were engineered by incorporating (i) a chain length specific thioesterase from Umbellularia californica (UC), (ii) a plant origin acyl carrier protein (ACP) gene and (iii) the whole fatty acid synthesis system (FASs) from Jatropha curcas (JC). The genetic combination was designed to control the product spectrum towards optimum MCLA. Decanoic, lauric and myristic acid were produced at concentrations of 0.011, 0.093 and 1.657 mg/g, respectively. The concentration of final products nonane, undecane and tridecane were 0.00062 mg/g, 0.0052 mg/g, and 0.249 mg/g respectively. Thioesterase from UC controlled the fatty acid chain length in a range of 10–14 carbons and the ACP gene with whole FASs from JC significantly increased the production of MCLA.Biosynthesis pathway for producing medium-chain alkanes.Download high-res image (83KB)Download full-size image

•A glucomannokinase from Mycobacterium phlei was characterized.•The glucomannokinase shows significant preference towards inorganic polyphosphate than ATP.•Enzymatic production of mannose-6-phosphate without using ATP was achieved.Mannose-6-phosphate is an important phosphor-sugar, which is involved in many physiological functions and it is used to treat many diseases. Its production is however expensive since it requires costly substrate ATP as phosphorylation agent. This study has focused upon the direct synthesis of M6P by glucomannokinase using inorganic polyphosphate without involvement of ATP. The gene cloned for glucomannokinase has been sequenced from Mycobacterium phlei and it is transformed into Escherichia coli for expression. After purification involving affinity chromatography, a band of 30 kDa corresponding to the enzyme has been isolated from induced crude supernatant. A total amount of 0.69 mg/ml of enzyme has been successively obtained and the purity exceeds 90%. The kinetic assay studies show that this enzyme has more affinity towards polyphosphate and glucose than ATP and mannose respectively. The KM values of the enzyme for glucose, mannose, ATP and hexametaphosphate derived from experiments are 9.5, 203.7, 4.6, 1.7 μM, respectively. The enzyme has shown a maximum production of mannose-6-phosphate at optimized conditions of pH 8.5, 25 °C, poly(P)/mannose ratio 3:1 and in the presence of bivalent ion Mg2+. The results reveal that the glucomannokinase from Mycobacterium phlei suitable for further production of mannose-6-phosphate.

MATTt (a thermostable methionine adenosyltransferase from Thermus thermophilus HB27) was overexpressed in Escherchia coli and purified using Ni-NTA affinity column. The enzymatic activity of MATTt was investigated in a temperature range from 30 °C to 90 °C, showing that MATTt exhibited a high enzymatic activity and good thermostability at 80 °C. Circular dichroism spectra reveals that MATTt contains high portion of β-sheet structures contributing to the thermostability of MATTt. The kinetic parameter, Km is 4.19 mmol/L and 1.2 mmol/L for ATP and methionine, respectively. MATTt exhibits the highest enzymatic activity at pH 8. Cobalt (Co2+) and zinc ion (Zn2+) enhances remarkably the activity of MATTt compared to the magnesium ion (Mg2+). All these results indicated that the thermostable MATTt has great potential for industry applications.

Non-edible oils are preferred raw materials for biodiesel production. However, the properties of raw materials significantly affect the synthesis process, leading to difficulties to design one process suitable for any kind of raw material. In this study, the composition of five typical non-edible oils was analyzed. The major difference was the content of free fatty acids, reflected from their acid values. The influence of different oils was investigated by using lipase from Candida sp. 99–125. At low lipase dosage and low water content, the conversion was found proportional to the acid value. However, by increasing the water content or lipase dosage, we observed that the conversions for all kinds of oils used in this study could exceed 80 %. Time course analysis indicates that the lipase used in this study catalyzed hydrolysis followed by esterification, rather than direct transesterification. Accumulation of free fatty acids at the very beginning was necessary. A high water content facilitated the hydrolysis of oils with low acid value. This lipase showed capability to transform all the oils by controlling the water content.

Currently, fumaric acid is produced by catalytic isomerization of maleic acid in aqueous solutions at low pH. Being petroleum based, requiring catalyst, and producing vast amounts of by-products and wastewater, the production of fumaric acid from renewable resources by a “green” process is increasingly attractive. In an aqueous solution, the reaction equilibrium constant of the fumarase-mediated conversion of l-malic acid to fumaric acid is 1:4.2 (fumaric acid to l-malic acid). To shift the reaction equilibrium to fumaric acid, solvent engineering was carried out by varying hydrophilic solvents and their concentrations. Generally, organic solvents may denature fumarase. Therefore, fumarase from Thermus thermophilus was employed to overcome this problem. Ethylene glycol was found more suitable than other solvents. This fumarase was shown to be more stable in 50 % than in 70 % ethylene glycol. Therefore, a preparation was carried out in 50 % ethylene glycol. Under this condition, 54.7 % conversion was observed using fumarase for transforming 1 mmol l-malic acid. After precipitation by adapting the pH, and washing to remove residual solvent and substrate, 27 % total yield was obtained with 99 % purity. The results demonstrated that the alternative green route to produce bio-based fumaric acid via l-malic acid is feasible and viable.

•Three-dimensional cicada wing substrate with decorated Au nanoparticle was synthesized.•The Au/CW substrate shows the SERS quantitative determinations of biomolecules, ATP and ADP.•The ATP percentage in a ATP:ADP mixed solution could be quantitatively determined.The ATP:ADP molar ratio is an important physiological factor. However, in previous literatures, ATP and ADP could not be distinguished by Raman spectroscopy due to the high similarity of molecular structure. To challenge this problem, also considering that the γ phosphate group may interact with adenine group and cause a different variation of the Raman spectrum than that of ADP, a highly sensitive, low-cost, environment protecting, flexible and super-hydrophobic Au nanoparticles/cicada wing (Au/CW) substrate with three-dimension structure was fabricated and employed as an active surface-enhanced Raman scattering (SERS) substrate to detect the ATP:ADP molar ratios. The concentration as low as 10−8 M for ATP and ADP was analyzed to determine the limit of detection. This SERS study on various ATP:ADP molar ratios demonstrates that ATP:ADP could be distinguished and the quantitative determination of ATP content was achieved. Moreover, a principle was speculated based on the molecular structures of ATP and ADP of the Raman peaks centered at ~685 and ~731 cm−1 to explain the linear relationship between the area ratio and the molar ratio. A new method has been developed for quantitative determination of ATP:ADP molar ratio based on Au/CW substrate by the SERS method.