Rational redesign of the loop dynamics of carbonyl reductase LfSDR1 to improve the stereoselectivity for asymmetric synthesis of bulky chiral alcohols.

Engineering biocatalysts with enhanced stereoselectivity is highly desirable, and active-site loop dynamics play an important role in its regulation. However, knowledge of their precise roles in catalysis and evolution is limited. Here, we used the strategy of Rosetta enzyme design combined molecular dynamic simulations (MDs) to reprogram the landscapes of the key active-site loop dynamics of the carbonyl reductase LfSDR1 to improve stereoselectivity. The key flexible loop in the active site showed the potential to regulate the catalytic properties. A library of virtual variants was produced using the Rosetta design and assessed dynamic effect of the loop with the aid of MDs. A potential candidate was obtained with significant stereoselectivity (ee > 99 %) compared to the wild-type (ee = 42 %) without loss of catalytic activity or thermostability. The molecular basis of the catalytic property enhancement was flanked by MDs, which revealed the role of the G92L mutation in regulating loop dynamics to stabilize the environment of the active site. Finally, a series of the challenge bulky substrate derivatives were assessed using the G92L variant, and all showed improved stereoselectivity ee > 99 %. This study provides novel insights for improving stereoselectivity through rational engineering of the loop dynamics of biocatalysts.

Revealing the Mechanism of Aroma Production Driven by High Salt Stress in Trichomonascus ciferrii WLW.

Douchi is a Chinese traditional fermented food with a unique flavor. Methyl anthranilate (MA) plays an important role in formation of this flavor. However, the complicated relationship between the MA formation and the metabolic mechanism of the key functional microorganisms remains unclear. Here, we elucidated the response mechanism of aroma production driven by high salt stress in Trichomonascus ciferrii WLW (T. ciferrii WLW), which originates from the douchi fermentation process. The highest production of MA was obtained in a 10% NaCl environment. The enhanced expression of the key enzyme genes of the pentose phosphate pathway and shikimic acid pathway directed carbon flow toward aromatic amino acid synthesis and helped sustain an increased expression of metK to synthesize a large amount of the methyl donor S-adenosylmethionine, which promoted methyl anthranilate yield. This provides a theoretical basis for in-depth research on the applications of the flavor formation mechanisms of fermented foods.

Sustainable asymmetric synthesis of diltiazem precursor enabled by recombinant Escherichia coli whole cells co-expressing an engineered ketoreductase and glucose dehydrogenase.

As a key synthetic intermediate of the cardiovascular drug diltiazem, methyl (2R,3S)-3-(4-methoxyphenyl) glycidate ((2R,3S)-MPGM) (1) is accessible via the ring closure of chlorohydrin (3S)-methyl 2-chloro-3-hydroxy-3-(4-methoxyphenyl)propanoate ((3S)-2). We report the efficient reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate (3) to (3S)-2 using an engineered enzyme SSCRM2 possessing 4.5-fold improved specific activity, which was obtained through the structure-guided site-saturation mutagenesis of the ketoreductase SSCR by reliving steric hindrance and undesired interactions. With the combined use of the co-expression fine-tuning strategy, a recombinant E. coli (pET28a-RBS-SSCRM2 /pACYCDuet-GDH), co-expressing SSCRM2 and glucose dehydrogenase, was constructed and optimized for protein expression. After optimizing the reaction conditions, whole-cell-catalyzed complete reduction of industrially relevant 300 g L-1 of 3 was realized, affording (3S)-2 with 99% ee and a space-time yield of 519.1 g∙L-1 ∙d-1 , representing the highest record for the biocatalytic synthesis of (3S)-2 reported to date. The E-factor of this biocatalytic synthesis was 24.5 (including water). Chiral alcohol (3S)-2 generated in this atom-economic synthesis was transformed to (2R,3S)-MPGM in 95% yield with 99% ee.

Asymmetric Synthesis of Sterically Hindered 1-Substituted Tetrahydro-ß-carbolines Enabled by Imine Reductase: Enzyme Discovery, Protein Engineering, and Reaction Development.

We report the discovery of a new imine reductase (IRED), named AtIRED, by genome mining. Site-saturation mutagenesis on AtIRED generated two single mutants M118'L and P120'G and the double mutant M118'L/P120'G with improved specific activity toward sterically hindered 1-substituted dihydro-ß-carbolines. The synthetic potential of these engineered IREDs was showcased by the preparative-scale synthesis of nine chiral 1-substituted tetrahydro-ß-carbolines (THßCs), including (S)-1-t-butyl-THßC and (S)-1-t-pentyl-THßC, in 30-87% isolated yields with excellent optical purities (98-99% ee).

Biocatalytic dynamic reductive kinetic resolution of aryl α-chloro ß-keto esters: divergent, stereocontrolled synthesis of diltiazem, clentiazem, and siratiazem.

The first systematic study of ketoreductase (KRED)-catalyzed dynamic reductive kinetic resolution (DYRKR) on aryl α-chloro ß-keto esters was performed, and 15 structurally diverse chiral anti-aryl α-chloro ß-hydroxy esters were synthesized in 74-98% isolated yields, along with moderate-to-excellent diastereoselectivity (up to >99 : 1 dr) and good-to-excellent enantioselectivity (mostly >99% ee). LfSDR1-catalyzed complete reduction of 100 g L-1 of substrate 6b at a ten-gram scale was achieved with a continuous fed-batch strategy, affording anti-(2S,3S)-1b, the key intermediate of diltiazem, in a record-breaking space-time yield of 96 g L-1 d-1. An eight-step synthesis of diltiazem, clentiazem, and siratiazem was accomplished in 32-45% overall yields, featuring this versatile biocatalytic reduction reaction as well as an efficient, green chlorination reaction in flow.

Simple, Skin-Attachable, and Multifunctional Colorimetric Sweat Sensor.

In situ analysis of sweat provides a simple, convenient, cost-effective, and noninvasive approach for the early diagnosis of physical illness in humans and is particularly useful in family care. In this study, a flexible and skin-attachable colorimetric sweat sensor for multiplexed analysis is developed using a simple, cost-effective, and convenient method. The obtained sweat sensor can be used to simultaneously detect glucose, lactate, urea, and pH value in sweat, as well as sweat loss and skin temperature. Only 2.5 µL of sweat is enough for the whole test, and the sweat loss and chemical-sensing results can be read out conveniently by naked eyes or a smartphone. In addition, body temperature can also be detected with an additional electrical circuit. Our sweat sensor provides a new, cost-effective, and convenient approach for in vitro diagnosis of multiple components in sweat, and the easy fabrication and cost-effectiveness make our sensor commercializable in the near future.

Synthesis of Thin Bi9 O7.5 S6 Nanosheets for Improved Photodetection in a Wide Wavelength Range.

Bismuth-based compounds possess layered structures with a variety of stacking modes, endowing the compounds with diverse properties. As one type of bismuth oxysulfides, Bi9 O7.5 S6 nanocrystals has great applications in photodetection; however, the responsivity of bulky Bi9 O7.5 S6 is limited due to the poor charge separation. Herein, single-crystalline Bi9 O7.5 S6 thin nanosheets are successfully synthesized by using a solvothermal method. The thickness of the obtained Bi9 O7.5 S6 nanosheets is down to 15 nm and can be easily tuned by varying the reaction period. Moreover, the Bi9 O7.5 S6 nanosheets show strong light absorption in the visible and near infrared range, making it a promising candidate in optoelectronics. As a demonstration, the thin Bi9 O7.5 S6 nanosheets are used as active layer in an optoelectronic device, which exhibits sensitive photoelectric response to light in a wide range of 400-800 nm. The responsivity of the device reaches up to 1140 µA W-1 , and the performance of the device is stable after long-period illumination. This work demonstrates a great potential of the thin Bi9 O7.5 S6 nanosheets in optoelectronic devices, and these nanosheets may also be extended to various optoelectronic applications.

Preparation and applications of freestanding Janus nanosheets.

In the family of Janus nanomaterials, Janus nanosheets possess not only the advantages of Janus nanomaterials, but also the advantages of two-dimensional nanosheets, endowing them with many extraordinary properties. Therefore, Janus nanosheets have great potential in the fields of interfacial engineering, catalysis, and molecular recognition. This review summarizes and discusses the recent advances in both the preparation and applications of freestanding Janus nanosheets. After a short introduction to different types of Janus nanosheets, a variety of methods for preparing freestanding Janus nanosheets are introduced, including the surface reaction, interface reaction, emulsion reaction, self-assembly, and stripping of non-Janus nanosheets, as well as selective grafting of existing Janus nanosheets. Then, the wide applications of Janus nanosheets in the fields of emulsification, catalysis, polymer reinforcement, nanomotors, and molecular recognition are summarized in detail. Finally, a discussion on the remaining challenges and future perspectives in this field is included. This review will not only deepen the understanding of Janus nanosheets, but also benefit the designs and fabrications of extraordinary and multi-functional Janus nanosheets.

Embedding Silver Nanowires into a Hydroxypropyl Methyl Cellulose Film for Flexible Electrochromic Devices with High Electromechanical Stability.

Transparent conductive films (TCFs) based on silver nanowires (AgNWs) are becoming one of the best candidates in realizing flexible optoelectronic devices. The AgNW-based TCF is usually prepared by coating AgNWs on a transparent polymer film; however, the coated AgNWs easily detach from the polymer underneath because of the weak adhesion between them. Herein, a network of AgNWs is embedded in the transparent hydroxypropyl methyl cellulose film, which has a strong adhesion with the AgNWs. The obtained TCF shows high optical transmittance (>85%), low roughness (rms = 4.8 ± 0.5 nm), and low haze (<0.2%). More importantly, owing to the embedding structure and strong adhesion, this TCF also shows excellent electromechanical stability, which is superior to the reported ones. Employing this TCF in a flexible electrochromic device, the obtained device exhibits excellent cyclic electromechanical stability and high coloring efficiency. Our work demonstrates a promising TCF with superior electromechanical stability for future applications in flexible optoelectronics.

[Heterologous expression and characterization of Aspergillus oryzae acidic protease in Pichia pastoris].

Acid protease, an important aspartic protease, has been widely used in food, pharmaceutical and tanning industries. To promote the research and application of acid protease, an acid protease gene (pepA) from Aspergillus oryzae was obtained from fermented soy based on metagenome sequencing, and then cloned and transformed into Pichia pastoris GS115 for heterologous expression. The characteristic of recombinant PepA was also investigated. The activity of acid protease in the culture supernatant of P. pastoris was 50.62 U/mL. The molecular mass of PepA was about 50 kDa, and almost no other proteins in the supernatant were observed, as shown by SDS-PAGE. The optimum pH and temperature of PepA were determined as pH 4.5 and 50 ℃. Mn²âº and Cu²âº enhanced the activity of PepA, whereas Fe³âº, Fe²âº and Ca² had inhibitory effects on its activity. The above findings can provide guidance for heterologous expression and industrial application of acid protease from Aspergillus oryzae.

[Inhibition of hTERT antisense oligodeoxynucleotide on proliferation and telomerase activity in HL-60 cells].

This study was purposed to investigate the inhibition of hTERT antisense oligodeoxynucleotide (ASODN) on the proliferation and telomerase activity in HL-60 cells and to explore the relativity between the telomerase activity and the expression of hTERT gene in HL-60 cells. After treated by hTERT ASODN the expression of hTERT was detected by RT-PCR, the morphological changes of HL-60 cells was observed with inverted microscopy, the cell proliferation was measured by MTT method, and the telomerase activity was determined with TRAP-ELISA and TRAP-PAGE. The results showed that after sealing hTERT gene with ASODN for 72 hours, the expression of hTERT gene was significantly inhibited, the cell growth was repressed and the ability of proliferation decreased, and the effect was specific in sequence and dependent in dose and time. OD(450-690) values were 2.648 +/- 0.42, 1.504 +/- 0.47, 1.223 +/- 0.39, 0.944 +/- 0.16 respectively, as the cells were treated with 0, 10, 20, 30 micromol/L ASODN for 72 hours. The difference was significant as compared 10, 20, 30 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 20 micromol/L SODN group (2.376 +/- 0.65) with untreated group (2.648 +/- 0.42) (P > 0.05). TRAP-PAGE detection revealed that comparing ASODN groups with SODN groups the telomerase image bands were decreased and least was found in groups of 30 +/- mol/L. It is concluded that the hTERT ASODN may inhibit the proliferation and down-regulate the telomerase activity in HL-60 cells by sealing the expression of hTERT gene.