BACKGROUND: As a low-cost alternative to the traditional ionic liquids (ILs), deep eutectic solvents (DESs) have currently attracted increasing attention for their various applications including biocatalysis. This study aimed at analyzing the effect of DESs on 1, 2-dehydrogeneration of cortisone acetate(CA) to prednisone acetate (PA) by Arthrobacter simplex.RESULTS: Of the three DESs examined, ChCl:U was successfully employed as co-solvent to improve the bioconversion efficiency. For substrate feeding concentration of 5 g L −1 , substrate conversion reached 93% by immobilized A. simplex cells (4 g DW L −1 ) with ChCl:U content of 6%, which is more efficient than conversions without DESs. Importantly, it has been shown that DESs and the immobilized cells can be easily recovered and then reused for five batches of bioconversion with a final conversion above 80%. CONCLUSION:Our results show the potential use of DESs-based systems in industrial steroid biodehydrogenation by A. simplex, and provide an environmentally benign means of steroid biotransformation.

BACKGROUND: The improved efficiency of steroid biotransformation using the biphasic system is generally attributed to the positive effect on the solubility of substrate in aqueous media. A promising alternative for the application of organic solvents in biphasic systems is the use of ionic liquids (ILs). This study aims to investigate the applicability of the biphasic ILs/water system for 11α hydroxylation of 16α, 17‐epoxyprogesterone (HEP) by Aspergillus ochraceus. RESULTS: Of the seven ILs tested, [C3mim][PF6] exhibited the best biocompatibility, with markedly improved biotransformation efficiency. In the [C3mim][PF6]‐based biphasic system, substrate conversion reached 90% under the condition in which buffer pH, volume ratio of buffer to ILs, cell concentration, and substrate concentration were 4.8, 10/1, 165 g L−1 and 20 g L−1, respectively. This is more efficient than that of the monophasic aqueous system. The effects of the cations and anions of these ILs on the 11α hydroxylation of 16α, 17‐epoxyprogesterone (HEP) by A. ochraceus is also discussed. CONCLUSION: The above results showed that IL/water biphasic system improved the efficiency of 11α hydroxylation of 16α, 17‐epoxyprogesterone (HEP) by A. ochraceus, thus suggesting the potential industrial application of ILs‐based biphasic systems for steroid biotransformation. © 2012 Society of Chemical Industry

d-Allulose is an attractive noncaloric sugar substitute with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability under harsh industrial reaction conditions hampered its practical applications. Here, we developed a continuous spectrophotometric assay (CSA) for the efficient analysis of d-allulose in a mixture. Furthermore, a high-throughput screening strategy for DAEases was developed using CSA by coupling DAEase with a NADH-dependent ribitol dehydrogenase, enabling high-throughput screening of DAEase variants with desired properties. The variant M15S/P40N/S209N exhibited a half-life of 22 h at 60 °C and an 8.7 °C increase of the T 50 60 value, with a 1.2-fold increase of activity. Structural modeling and molecular dynamics simulations indicated that the improvement of thermostability and activity was due to some new hydrogen bonds between chains at the dimer interface and between the residue and the substrate d-fructose. This work offers a robust tool and theoretical basis for the improvement of DAEases, which will benefit the enzymatic biosynthesis of d-allulose and promote its industrial application.

Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-△(1)-dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.

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BackgroundBiosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor.ResultsRecombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively).ConclusionsThe successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity. Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0981-0) contains supplementary material, which is available to authorized users.

Biphasic processes are used in whole-cell biotransformation to overcome the low water solubility of substrates and products as well as their inhibitory effects on the biocatalyst. Commercially available [NTf(2)]- and [PF(6)]-based ionic liquids (ILs) were used in a biphasic system for the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione by Penicillium raistrickii. With the substrate at 5 g l(-1) and a volume ratio of IL to buffer, buffer pH and cell density at, 1:9, 6.5, 16.8 g(DW) l(-1), respectively, the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione was achieved with a yield of 70 % after 72 h using [BMIm][NTf(2)] in a 50 ml biphasic system. This is compared to a 30 % yield in a monophasic aqueous system. This suggests the potential industrial application of ILs-based biphasic systems for steroid biotransformation.

BACKGROUND The discovery of deep eutectic solvents (DESs) has introduced a new class of promising green solvents to overcome many drawbacks associated with organic solvents. In this study, the synergistic effects of the components in the form of DESs on bacterial viability, membrane integrity, and retention of metabolic activity as well as the efficiency of Δ1,2‐dehydrogeneration of cortisone acetate (CA) using whole cells of Arthrobacter simplex were investigated. RESULTS The agar disc diffusion method, viability assays and the retention of sugar metabolic activity were used to investigate their toxicity to A. simplex. The results indicated that colinium salts were more toxic than the corresponding hydrogen bond donor (HBDs), but their deleterious effect could be weakened by incorporating them into DESs. Moreover, the best catalytic performance was found in the ChCl:U(DES)‐containing system when Δ1,2‐dehydrogeneration using A. simplex was carried out in three different systems followed by the addition of DES, their components and their mixtures, respectively. In addition, metabolites with significantly changed concentration were identified by means of gas chromatography–mass spectrometry (GC–MS) and principal component analysis, which revealed five compounds that may be used as potential indicators to discriminate the effects of different concentrations of DESs on A. simplex. CONCLUSION The cholinium salts used in this study were toxic to A. simplex to some extent, while synergistic effects were observed after DES formation. The efficient biotransformation in DESs especially in the ChCl:U(DES)‐containing system potentially offers a promising strategy for the production of many different steroids. © 2018 Society of Chemical Industry