Autocatalytic hydrothermal pretreatment by recycling byproduct organic acids to directionally depolymerize cassava straw

A two-stage autocatalytic hydrothermal pretreatment was proposed to improve the cassava straw utilization. The two-stage hydrothermal pretreatment was a process of which the first stage adopted lower-severity conditions (temperature and time) to improve the C-5 sugar yields and the second stage employed more severities to enhance C-6 sugar yield during enzyme hydrolysis. After employing this process, the maximum yields of C-5 and C-6 sugars were 68.49% and 81.02% when treating at 180 °C for 60 min for the first stage and 200 °C for 20 min for the second stage. Based on this, the autocatalytic pretreatment was investigated, which was a method to further enhance the pretreatment intensity by recycling pretreated liquid rich in byproduct organic acids (acetic acid, lactic acid and formic acid) during two-stage hydrothermal pretreatment. The results showed that the C-5 sugar yields of the first stage increased to 81.12% when recycled pretreated liquid twice, which led to 0.93 wt% byproduct organic acid. After the second stage, the C-6 sugar yield increased to 88.60% during enzymatic hydrolysis. Besides, mass balance and development potentials were analyzed. The results revealed that two-stage autocatalytic hydrothermal pretreatment could effectively enhance pretreatment intensity and provide promising methods of directionally depolymerizing cassava straws.     

Chemoenzymatic synthesis and antileukemic activity of novel C9- and C14-functionalized parthenolide analogs

Parthenolide is a naturally occurring terpene with promising anticancer properties, particularly in the context of acute myeloid leukemia (AML). Optimization of this natural product has been challenged by limited opportunities for the late-stage functionalization of this molecule without affecting the pharmacologically important α-methylene-γ-lactone moiety. Here, we report the further development and application of a chemoenzymatic strategy to afford a series of new analogs of parthenolide functionalized at the aliphatic positions C9 and C14. Several of these compounds were determined to be able to kill leukemia cells and patient-derived primary AML specimens with improved activity compared to parthenolide, exhibiting LC₅₀ values in the low micromolar range. These studies demonstrate that different O–H functionalization chemistries can be applied to elaborate the parthenolide scaffold and that modifications at the C9 or C14 position can effectively enhance the antileukemic properties of this natural product. The C9-functionalized analogs 22a and 25b were identified as the most interesting compounds in terms of antileukemic potency and selectivity toward AML versus healthy blood cells.     

Selective immobilization of Bacillus subtilis lipase A from cell culture supernatant: Improving catalytic performance and thermal resistance

Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; however, its immobilization and behavior as an insoluble biocatalyst have not been extensively explored. In this work, for the first time, a direct immobilization of recombinant BSLA from microbial culture supernatant was reported, using chemically modified porous with different electrostatic, hydrophobic, hydrophilic, and hydrophilic−hydrophobic enzyme-support interactions. The resulting biocatalysts were evaluated based on their immobilization kinetics, activity expression (pH 7.4), thermal stability (50 °C), solvent resistance and substrate preference. Biocatalysts obtained using glyoxyl silica support resulted in the selective immobilization of BSLA, resulting in an activity recovery of 50 % and an outstanding aqueous stabilization factor of 436, and 9.5 in isopropyl alcohol, compared to the free enzyme. This selective immobilization methodology of BSLA allows to efficiently generate immobilized biocatalysts, thus avoiding laborious purification steps from cell culture supernatant, which is usually a limiting step when large amounts of enzyme variants or candidates are assessed as immobilized biocatalysts. Direct enzyme immobilization from cell supernatant provides an interesting tool which can be used to facilitate the development and assessment of immobilized biocatalysts from engineered enzyme variants and mutant libraries, especially in harsh conditions, such as high temperatures or non-aqueous solvents, or against non-water-soluble substrates. Furthermore, selective immobilization approaches from cell culture supernatant or clarified lysates could help bridging the gap between protein engineering and enzyme immobilization, allowing for the implementation of immobilization steps in high throughput enzyme screening platforms for their potential use in directed evolution campaigns.     

Design, synthesis and catalytic activity of a serine protease synthetic model

Summary The design, synthesis and catalytic properties of a cyclic branched peptide carrier that possesses the catalytic triad residues of the serine proteases is reported. The synthesis of the peptide model was totally completed on solid support using three different orthogonal amino protecting groups. Hydrolytic activity measurements against Suc-Ala-Ala-Ala-pNA substrate showed that it is hydrolysed by the peptide model to a small extent. Despite this small hydrolytic activity, it is the first time, to our knowledge, that hydrolysis of such a substrate is reported by an enzyme model compound. Contrary, this enzyme model peptide showed considerable activity against the Boc-Ala-pNP substrate (k _cat =0.414 min⁻¹ andK _m =0.228 mm). These results suggest that the designed carrier brings in appropriate contact the catalytic triad residues (Ser, His, Asp) resulting in the obtained hydrolytic activity.     

Minimization of intermediate concentrations as a suggested optimality principle for biochemical networks

The multiobjective problem of minimizing all intermediate concentrations is solved for a model of glycolysis, the pentose monophosphate shunt and the glutathione system in human erythrocytes. It turns out that one solution out of four obtained corresponds qualitatively to the real system. Furthermore, it is shown that for any reaction system, the mentioned optimality principle implies distinct time hierarchy in that some reactions are infinitely fast and subsist in quasi-equilibrium. Finally, the relationships to the standard method of deriving enzymatic rate laws are discussed.     

Catalytic resolution of (RS)-HMPC acetate by immobilized cells of Acinetobacter sp. CGMCC 0789 in a medium with organic cosolvent

Kinetic resolution of a chiral alcohol, 4-hydroxy-3-methyl-2-(2′-propenyl)-2-cyclopentenone (HMPC), a key intermediate for the production of prallethrin insecticides, was successfully carried out by enantioselective hydrolysis of (RS)-HMPC acetate using calcium alginate gel-entrapped cells of a newly isolated esterase-producing bacterium Acinetobacter sp. CGMCC 0789. When the effect of different cosolvents was investigated, it was found that isopropanol could markedly enhance the activity and enantioselectivity of the immobilized cells. The optimum concentration of isopropanol was 10% (v/v) where immobilized cells still showed good operational stability. After 10 cycles of reaction, no significant decrease in the enzyme activity was observed. The catalytic specificity constants (V_max/K_m) for both enantiomers of the substrate were determined with partially purified enzyme, giving 0.0184 and 0.671 h⁻¹ for the (S)- and (R)-ester, respectively.