Discovery and directed evolution of C-C bond formation enzymes for the biosynthesis of ß-hydroxy-α-amino acids and derivatives.

ß-Hydroxy-α-amino acids (ß-HAAs) have extensive applications in the pharmaceutical, chemical synthesis, and food industries. The development of synthetic methodologies aimed at producing optically pure ß-HAAs has been driven by practical applications. Among the various synthetic methods, biocatalytic asymmetric synthesis is considered a sustainable approach due to its capacity to generate two stereogenic centers from simple prochiral precursors in a single step. Therefore, extensive efforts have been made in recent years to search for effective enzymes which enable such biotransformation. This review provides an overview on the discovery and engineering of C-C bond formation enzymes for the biocatalytic synthesis of ß-HAAs. We highlight examples where the use of threonine aldolases, threonine transaldolases, serine hydroxymethyltransferases, α-methylserine aldolases, α-methylserine hydroxymethyltransferases, and engineered alanine racemases facilitated the synthesis of ß-HAAs. Additionally, we discuss the potential future advancements and persistent obstacles in the enzymatic synthesis of ß-HAAs.

Preparation of pectin-coated and chitosan-coated phenylethanoside liposomes: Studies on characterization, stability, digestion and release behavior.

In this paper, the effects of extrusion, ultrasound on physicochemical properties of liposomes were studied, and the liposomes were prepared by ethanol injection combined with extrusion-ultrasound. In addition, the quality of PhGs lips, pectin-coated PhGs lips (P-lips) and chitosan-coated PhGs lips (C-lips) was evaluated by the average particle size, encapsulation efficiency (EE) and other indicators, which indicated that the nanoparticles had been successfully prepared. Compared with extrusion or ultrasonic operation alone, the EEs of ethanol injection combined with extrusion-ultrasonic increased by 8 % and 18 % respectively. Subsequently, transmission electron microscopy, Fourier transform infrared spectroscopy and DSC thermal analysis showed that PhGs in PhGs lips may produce hydrogen bonding forces with phospholipids, and pectin and chitosan in P-lips and C-lips were not only coated on the surface of PhGs lips, but also might have some interaction between them. Cell experiments showed that PhGs lips, P-lips and C-lips can effectively improve the bioavailability of PhGs. In addition, the storage stability of P-lips and C-lips was not significantly improved compared to PhGs lips, but their digestive stability was significantly improved, and the final retention rate in simulated intestinal fluid was about 25 % higher than that of PhGs lips.

NMGMDA: a computational model for predicting potential microbe-drug associations based on minimize matrix nuclear norm and graph attention network.

The prediction of potential microbe-drug associations is of great value for drug research and development, especially, methods, based on deep learning, have been achieved significant improvement in bio-medicine. In this manuscript, we proposed a novel computational model named NMGMDA based on the nuclear norm minimization and graph attention network to infer latent microbe-drug associations. Firstly, we created a heterogeneous microbe-drug network in NMGMDA by fusing the drug and microbe similarities with the established drug-microbe associations. After this, by using GAT and NNM to calculate the predict scores. Lastly, we created a fivefold cross validation framework to assess the new model NMGMDA's progressiveness. According to the simulation results, NMGMDA outperforms some of the most advanced methods, with a reliable AUC of 0.9946 on both MDAD and aBioflm databases. Furthermore, case studies on Ciprofloxacin, Moxifoxacin, HIV-1 and Mycobacterium tuberculosis were carried out in order to assess the effectiveness of NMGMDA even more. The experimental results demonstrated that, following the removal of known correlations from the database, 16 and 14 medications as well as 19 and 17 microbes in the top 20 predictions were validated by pertinent literature. This demonstrates the potential of our new model, NMGMDA, to reach acceptable prediction performance.

Synergistic mediating effect of edible fungal polysaccharides (Auricularia and Tremellan) and Crataegus flavonoids in hyperlipidemic rats.

Both edible fungal polysaccharides (Auricularia and Tremellan) and Crataegus flavonoids promote the balance of dyslipidemia, which have a positive biological regulating effect on intestinal flora. In this study, the extraction of water-soluble polysaccharides from Auricularia and Tremellan was investigated and optimized firstly. Polysaccharides and flavonoids were then combined to study the effects on the mediating role of abnormal blood lipid concentration and intestinal flora in vivo. The rats were divided into 10 groups, the NC (normal control), HM (model), PCI (Simvastatin control), PCII (Fenofibrate control), AAP (Auricularia auricular Polysaccharide), TFP (Tremella fuciformis Polysaccharide), HF (Crataegus Flavonoid), LDC (Low-dose combination), MDC (Medium dose combination), and HDC (High-dose combination), used to explore the impact of polysaccharides and flavonoids complex on state of blood lipid, liver, and intestinal flora of dyslipidemia rats. The results showed that the combination of polysaccharides and flavonoids could significantly decrease the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), and increase the level of high-density lipoprotein cholesterol (HDL-C). It also significantly decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and improved liver morphology. What is more, the HDC favorably alters the intestinal microflora balance, promotes intestinal integrity and mobility, and inhibits the growth of harmful bacteria such as Escherichia coli/Shigella and Clostridium compared with HM group. In brief, the combination of polysaccharides and flavonoids had a synergistic effect on the remission of dyslipidemia, and promoted health by improving lipid metabolism, protecting liver tissue, and regulating the intestinal flora in hyperlipidemia rats.

[Teaching reform and practice of "food enzymology and enzyme engineering" based on cutting-edge researches].

Food enzymology and enzyme engineering is an important professional course of food science. The course includes the basic theory of enzymology, enzyme engineering technology and the application of enzymes in food industry. Considering the knowledge gap between the teaching contents and the cutting-edge researches, the team constantly adjusted and optimized the course contents to enable students to keep up with state-of-the-art progress by carefully mining the cutting-edge researches. Taking cutting-edge researches as the breakthrough point, we explored the problem-based learning (PBL) teaching model under the guidance of outcome-based education (OBE) concept, and highlighted the importance of the teacher-student and student-student interactions to improve students' enthusiasm and participation. A diversified assessment system was established to evaluate the performance of students in the learning process. The teaching reform consolidated the basic knowledge and expanded the academic frontiers, and fostered students' ability in analyzing problems, designing solutions and achieving team communication. The course may give new insights into the teaching reform of food enzymology and enzyme engineering and other related courses.

[Catalytic mechanism, molecular engineering and applications of threonine aldolases].

Threonine aldolases catalyze the aldol condensation of aldehydes with glycine to furnish ß-hydroxy-α-amino acid with two stereogenic centers in a single reaction. This is one of the most promising green methods for the synthesis of optically pure ß-hydroxy-α-amino acid with high atomic economy and less negative environmental impact. Several threonine aldolases from different origins have been identified and characterized. The insufficient -carbon stereoselectivity and the challenges of balancing kinetic versus thermodynamic control to achieve the optimal optical purity and yield hampered the application of threonine aldolases. This review summarizes the recent advances in discovery, catalytic mechanism, high-throughput screening, molecular engineering and applications of threonine aldolases, with the aim to provide some insights for further research in this field.

Engineering of l-threonine aldolase for the preparation of 4-(methylsulfonyl)phenylserine, an important intermediate for the synthesis of florfenicol and thiamphenicol.

l-Threonine aldolases (l-TAs) catalyze the aldol condensation of aldehyde and glycine, offering direct enzymatic synthesis of ß-hydroxy-α-amino acids under mild conditions. However, this method suffers from moderate yield and low stereoselectivity at the ß-carbon. Given the importance of 4-(methylsulfonyl)phenylserine for the synthesis of florfenicol and thiamphenicol, mutations of a l-threonine aldolase from Pseudomonas sp. (l-PsTA) were performed in this study by error-prone PCR and combinatorial mutation. Some beneficial mutants were obtained by screening the mutant library using a stepwise visual method. 4-(Methylsulfonyl)phenylserine was synthesized in up to 71 % diastereomeric excess (de), which are much higher than the previously reported 2 % de value, by using the newly identified mutants. The mutants V200I and C187S/V200I were found to improve the product yield and stereoselectivity for the aldol condensation of various benzaldehydes with glycine. These results show that the amino acid residues outside of the substrate-binding cavity of l-PsTA play an important role in determining its Cß-stereoseletivity.

Molecular Characterization of a Novel N-Acetylneuraminate Lyase from a Deep-Sea Symbiotic Mycoplasma.

N-acetylneuraminic acid (Neu5Ac) based novel pharmaceutical agents and diagnostic reagents are highly required in medical fields. However, N-acetylneuraminate lyase（NAL）for Neu5Ac synthesis is not applicable for industry due to its low catalytic efficiency. In this study, we biochemically characterized a deep-sea NAL enzyme (abbreviated form: MyNal) from a symbiotic Mycoplasma inhabiting the stomach of a deep-sea isopod, Bathynomus jamesi. Enzyme kinetic studies of MyNal showed that it exhibited a very low Km for both cleavage and synthesis activities compared to previously described NALs. Though it favors the cleavage process, MyNal out-competes the known NALs with respect to the efficiency of Neu5Ac synthesis and exhibits the highest kcat/Km values. High expression levels of recombinant MyNal could be achieved (9.56 mol L-1 culture) with a stable activity in a wide pH (5.0-9.0) and temperature (40-60 °C) range. All these features indicated that the deep-sea NAL has potential in the industrial production of Neu5Ac. Furthermore, we found that the amino acid 189 of MyNal (equivalent to Phe190 in Escherichia coli NAL), located in the sugar-binding domain, GX189DE, was also involved in conferring its enzymatic features. Therefore, the results of this study improved our understanding of the NALs from different environments and provided a model for protein engineering of NAL for biosynthesis of Neu5Ac.

On Priming Action: Conclusions from a Meta-Analysis of the Behavioral Effects of Incidentally-Presented Words.

This paper presents a summary of the conclusions drawn from a meta-analysis of the behavioral impact of presenting words connected to an action or a goal representation (Weingarten et al., 2016). The average and distribution of 352 effect sizes from 133 studies (84 reports) revealed a small behavioral priming effect (dFE = 0.332, dRE = 0.352), which was robust across methodological procedures and only minimally biased by the publication of positive (vs. negative) results. More valued behavior or goal concepts (e.g., associated with important outcomes or values) were associated with stronger priming effects than were less valued behaviors. In addition, opportunities for goal satisfaction appeared to decrease priming effects.

A sialic acid aldolase from Peptoclostridium difficile NAP08 with 4-hydroxy-2-oxo-pentanoate aldolase activity.

Sialic acid aldolases (E.C.4.1.3.3) catalyze the reversible aldol cleavage of N-acetyl-d-neuraminic acid (Neu5Ac) to from N-acetyl-d-mannosamine (ManNAc) and pyruvate. In this study, a sialic acid aldolase (PdNAL) from Peptoclostridium difficile NAP08 was expressed in Escherichia coli BL21 (DE3). This homotetrameric enzyme was purified with a specific activity of 18.34U/mg for the cleavage of Neu5Ac. The optimal pH and temperature for aldol addition reaction were 7.4 and 65°C, respectively. PdNAL was quite stable at neutral and alkaline pH (6.0-10.0) and maintained about 89% of the activity after incubation at pH 10.0 for 24h. After incubation at 70°C for 15min, almost no activity loss was observed. The high thermostability simplified the purification of this enzyme. Interestingly, substrate profiling showed that PdNAL not only accepted ManNAc but also short chain aliphatic aldehydes such as acetaldehyde, propionaldehyde and n-butyraldehyde as the substrates. This is the first example that a sialic acid aldolase is active toward aliphatic aldehyde acceptors with two or more carbons. The amino acid sequence analysis indicates that PdNAL belongs to the NAL subfamily rather than 4-hydroxy-2-oxopentanoate (HOPA) aldolase, but it is interesting that the enzyme possesses the activity of HOPA aldolase.

[Expression and characterization of a novel ω-transaminase from Burkholderia phytofirmans PsJN].

Production of chiral amines and unnatural amino-acid using ω-transaminase can be achieved by kinetic resolution and asymmetric synthesis, thus ω-transaminase is of great importance in the synthesis of pharmaceutical intermediates. By genomic data mining, a putative ω-transaminase gene hbp was found in Burkholderia phytofirmans PsJN. The gene was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme (HBP) was purified by Ni-NTA column and its catalytic properties and substrate profile were studied. HBP showed high relative activity (33.80 U/mg) and enantioselectivity toward ß-phenylalanine (ß-Phe). The optimal reaction temperature and pH were 40 ℃ and 8.0-8.5, respectively. We also established a simpler and more effective method to detect the deamination reaction of ß-Phe by UV absorption method using microplate reader, and demonstrated the thermodynamic property of this reaction. The substrate profiling showed that HBP was specific to ß-Phe and its derivatives as the amino donor. HBP catalyzed the resolution of rac-ß-Phe and its derivatives, the products (R)-amino acids were obtained with about 50% conversions and 99% ee.

Action Tweets Linked to Reduced County-Level HIV Prevalence in the United States: Online Messages and Structural Determinants.

HIV is uncommon in most US counties but travels quickly through vulnerable communities when it strikes. Tracking behavior through social media may provide an unobtrusive, naturalistic means of predicting HIV outbreaks and understanding the behavioral and psychological factors that increase communities' risk. General action goals, or the motivation to engage in cognitive and motor activity, may support protective health behavior (e.g., using condoms) or encourage activity indiscriminately (e.g., risky sex), resulting in mixed health effects. We explored these opposing hypotheses by regressing county-level HIV prevalence on action language (e.g., work, plan) in over 150 million tweets mapped to US counties. Controlling for demographic and structural predictors of HIV, more active language was associated with lower HIV rates. By leveraging language used on social media to improve existing predictive models of geographic variation in HIV, future targeted HIV-prevention interventions may have a better chance of reaching high-risk communities before outbreaks occur.

From primed concepts to action: A meta-analysis of the behavioral effects of incidentally presented words.

A meta-analysis assessed the behavioral impact of and psychological processes associated with presenting words connected to an action or a goal representation. The average and distribution of 352 effect sizes (analyzed using fixed-effects and random-effects models) was obtained from 133 studies (84 reports) in which word primes were incidentally presented to participants, with a nonopposite control group, before measuring a behavioral dependent variable. Findings revealed a small behavioral priming effect (dFE = 0.332, dRE = 0.352), which was robust across methodological procedures and only minimally biased by the publication of positive (vs. negative) results. Theory testing analyses indicated that more valued behavior or goal concepts (e.g., associated with important outcomes or values) were associated with stronger priming effects than were less valued behaviors. Furthermore, there was some evidence of persistence of goal effects over time. These results support the notion that goal activation contributes over and above perception-behavior in explaining priming effects. In summary, theorizing about the role of value and satisfaction in goal activation pointed to stronger effects of a behavior or goal concept on overt action. There was no evidence that expectancy (ease of achieving the goal) moderated priming effects. (PsycINFO Database Record

Future-oriented tweets predict lower county-level HIV prevalence in the United States.

OBJECTIVE: Future orientation promotes health and well-being at the individual level. Computerized text analysis of a dataset encompassing billions of words used across the United States on Twitter tested whether community-level rates of future-oriented messages correlated with lower human immunodeficiency virus (HIV) rates and moderated the association between behavioral risk indicators and HIV. METHOD: Over 150 million tweets mapped to U.S. counties were analyzed using 2 methods of text analysis. First, county-level HIV rates (cases per 100,000) were regressed on aggregate usage of future-oriented language (e.g., will, gonna). A second data-driven method regressed HIV rates on individual words and phrases. RESULTS: Results showed that counties with higher rates of future tense on Twitter had fewer HIV cases, independent of strong structural predictors of HIV such as population density. Future-oriented messages also appeared to buffer health risk: Sexually transmitted infection rates and references to risky behavior on Twitter were associated with higher HIV prevalence in all counties except those with high rates of future orientation. Data-driven analyses likewise showed that words and phrases referencing the future (e.g., tomorrow, would be) correlated with lower HIV prevalence. CONCLUSION: Integrating big data approaches to text analysis and epidemiology with psychological theory may provide an inexpensive, real-time method of anticipating outbreaks of HIV and etiologically similar diseases.

Highly Efficient Synthesis of Optically Pure (S)-1-phenyl-1,2-ethanediol by a Self-Sufficient Whole Cell Biocatalyst.

Terminal vicinal diols are important chiral building blocks and intermediates in organic synthesis. Reduction of α-hydroxy ketones provides a straightforward approach to access these important compounds. In this study, it has been found that asymmetric reduction of a series of α-hydroxy aromatic ketones and 1-hydroxy-2-pentanone, catalyzed by Candida magnolia carbonyl reductase (CMCR) with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, afforded 1-aryl-1,2-ethanediols and pentane-1,2-diol, respectively, in up to 99 % ee. In order to evaluate the efficiency of the bioreduction, lyophilized recombinant Escherichia coli whole cells coexpressing CMCR and GDH genes were used as the biocatalyst and α-hydroxy acetophenone as the model substrate, and the reaction conditions, such as pH, cosolvent, the amount of biocatalyst and the presences of a cofactor (i.e., NADP(+)), were optimized. Under the optimized conditions (pH 6, 16 h), the bioreduction proceeded smoothly at 1.0 m substrate concentration without the external addition of cofactor, and the product (S)-1-phenyl-1,2-ethanediol was isolated with 90 % yield and 99 % ee. This offers a practical biocatalytic method for the preparation of these important vicinal diols.