Enabling biocatalysis in high-concentration organic cosolvent by enzyme gate engineering.

Biocatalysis in high-concentration organic solvents (OSs) offers many advantages, but realizing this process remains a huge challenge. An R-selective ω-amine transaminase variant (AcATAM2 ) exhibited high activity toward 50 g/L pro-sitagliptin ketone 1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione (PTfpB). However, AcATAM2 displayed unsatisfactory organic-cosolvent resistance against high-concentration dimethyl sulfoxide (DMSO), which is required to enhance the solubility of the hydrophobic substrate PTfpB. Located in the substrate-binding tunnel, enzyme gates are structural elements that undergo reversible conformational transitions, thus affecting the accessibility of the binding pocket to solvent molecules. Depending on the conformation of the enzyme gates, one can define an open or closed conformation on which the enzyme activity in OSs may depend. To enhance the DMSO resistance of AcATAM2 , we identified the beneficial residues at the "enzyme gate" region via computational analysis, alanine scanning, and site-saturation mutagenesis. Two beneficial variants, namely, AcATAM2F56D and AcATAM2F56V , not only displayed improved enzyme activity but also exhibited enhanced DMSO resistance (the half-life value increased from 25.71 to 42.49 h under 60% DMSO). Molecular dynamic simulations revealed that the increase in DMSO resistance was mainly caused by the decrease in the number of DMSO molecules in the substrate-binding pocket. Moreover, in the kilogram-scale experiment, the conversion of 80 g/L substrate was increased from 50% (AcATAM2 ) to 85% (M2F56D in 40% DMSO) with a high e.e. of >99% within 24 h.

Evolutionarily conserved vasa identifies embryonic and gonadal germ cells in spinyhead croaker Collichthys lucidus.

In this study, a 2198 bp full-length cDNA of spinyhead croaker Collichthys lucidus vasa gene encoding 616 amino-acid residues was obtained. Multiple alignment revealed that C. lucidus vasa has eight conserved characteristic motifs of the DEAD box protein family and has the highest identity to large yellow croaker Larimichthys croceas. Reverse-transcription (RT)-PCR and Western blot analyses indicated that the vasa messenger (m)RNA and Vasa protein are specifically expressed in the gonads in both sexes. In situ hybridisation (ISH) demonstrated that vasa RNA is exclusively detected in the germ cells in C. lucidus gonads and its temporospatial expression reveals a dynamic pattern during oogenesis. Surprisingly, C. lucidus vasa 3'UTR can direct stable and specific GFP expression in the primordial germ cells (PGC) of medaka Oryzias latipes embryos. Taken together, these results suggest that because C. lucidus vasa expression delineates critical stages of oogenesis, it may be a useful molecular marker for the identification of gonadal germ cells, facilitating the isolation and utilization of germ cells in future study.

Creation of a robust and R-selective ω-amine transaminase for the asymmetric synthesis of sitagliptin intermediate on a kilogram scale.

The creation of an R-selective ω-amine transaminase (ω-ATA) as biocatalyst is crucial for the asymmetric amination of prochiral ketones to produce sitagliptin intermediates because rare ω-ATAs are R-selective in nature and most of them suffer from poor stability and low activity toward bulky prochiral ketones. Here, the gene of an R-selective ω-ATA was cloned from Arthrobacter cumminsii ZJUT212 (AcATA) and expressed in Escherichia coli. The best variants (M1 + M122H and M1+T134 G) were obtained using a semi-rational protein design after screening. These variants not only exhibited improved activity and substrate affinity but also enhanced stability in aqueous phase containing 20 % dimethyl sulfoxide. The conversion of asymmetric amination on 50 g/L pro-sitagliptin ketone PTfpB (1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione) achieved 92 %, with an extremely high e.e. of >99 %, using 2 gDCW/L E. coli cells harboring M1 + M122H as biocatalyst. In the kilogram-scale experiment, approximately 40 kg of (R)-APTfpB (e.e. >99 %) was produced within 30 h when 50 kg PTfpB was used as the substrate. Furthermore, the space-time yield reached ≈32 g/(L·d).

Integral characterization of normal and alopecic hair at different degeneration stages by in-situ visible and chemical imaging.

Direct exploration to differences between normal hair (NH) and alopecic hair (AH) at different degeneration stages is still lacking. To reveal compositional and structural variation of AH with reference to NH internally and externally, infrared spectroscopic imaging combined with scanning electron microscopy was applied to investigate integral changes of hair chemical profiles and surface texture structures, and infrared macro-fingerprinting analysis revealed detailed chemical compositions of NH and AH. Results showed that AH had excessive irregular laminated structures compared to NH, leading to a lower weight bearing capacity. Spatial distributions of lipids, phosphates, lipoproteins and phospholipids in hair transverse sections showed that their infrared absorptions were intensified and gradually centralized to medulla with average variable-areas increasing upto 2.3 folds (lipoproteins area changed from 13% in NH to 30% in AH)as the alopecia progressed. Extracted pixel spectra from the chemical images showed different fingerprint characteristics in 1075-1120 cm-1. Specifically, compared to NH, AH showed red shift of phosphate peaks, indicating the occurrence of phosphates transformation. In this study, in-situ visible and infrared chemical imaging directly revealed more irregular laminated scalps with decreasing weight bearing capacity and increasing distributive areas expanding to medulla of key components (phosphates, phospholipids, etc.) that were relevant to alopecia development from NH to AH, and offered a fast, eco-friendly and effective method for hair research.

Identification of candidate piRNAs in the gonads of Paralichthys olivaceus (Japanese flounder).

Piwi-interacting RNA (piRNA) plays an important role in the gonadal development and maintenance of Teleostei. In this study, piRNA libraries derived from the adult gonads of Japanese flounder (Paralichthys olivaceus) were generated using next-generation sequencing technology. Using zebrafish piRNAs as a reference, 5 865 unique candidate piRNAs were identified; 289 candidate piRNA clusters (PRCs) were generated from the above piRNAs. Among the isolated candidate PRCs, a total of 38 ovary-specific, 45 ovary-bias, 24 testis-specific, and 131 testis-bias PRCs were found. The relative expression levels of seven PRCs were validated through quantitative reverse transcription-polymerase chain reaction. The results of this study will help facilitate exploration of the development and maintenance of the phenotypic sex mechanism in P. olivaceus.