1,3,6-Trigalloylglucose: A Novel Potent Anti-Helicobacter pylori Adhesion Agent Derived from Aqueous Extracts of Terminalia chebula Retz.

1,3,6-Trigalloylglucose is a natural compound that can be extracted from the aqueous extracts of ripe fruit of Terminalia chebula Retz, commonly known as "Haritaki". The potential anti-Helicobacter pylori (HP) activity of this compound has not been extensively studied or confirmed in scientific research. This compound was isolated using a semi-preparative liquid chromatography (LC) system and identified through Ultra-high-performance liquid chromatography-MS/MS (UPLC-MS/MS) and Nuclear Magnetic Resonance (NMR). Its role was evaluated using Minimum inhibitory concentration (MIC) assay and minimum bactericidal concentration (MBC) assay, scanning electron microscope (SEM), inhibiting kinetics curves, urea fast test, Cell Counting Kit-8 (CCK-8) assay, Western blot, and Griess Reagent System. Results showed that this compound effectively inhibits the growth of HP strain ATCC 700392, damages the HP structure, and suppresses the Cytotoxin-associated gene A (Cag A) protein, a crucial factor in HP infection. Importantly, it exhibits selective antimicrobial activity without impacting normal epithelial cells GES-1. In vitro studies have revealed that 1,3,6-Trigalloylglucose acts as an anti-adhesive agent, disrupting the adhesion of HP to host cells, a critical step in HP infection. These findings underscore the potential of 1,3,6-Trigalloylglucose as a targeted therapeutic agent against HP infections.

A chemoenzymatic process for preparation of highly purified dehydroepiandrosterone in high space-time yield.

Dehydroepiandrosterone (DHEA) is an important neurosteroid hormone to keep human hormonal balance and reproductive health. However, DHEA was always produced with impurities either by chemical or biological method and required high-cost purification before the medical use. To address this issue, a novel chemoenzymatic process was proposed and implemented to produce DHEA. An acetoxylated derivate of 4-androstene-3,17-dione (4-AD) was generated by chemical reaction and converted into DHEA by an enzyme cascade reaction combining a hydrolysis reaction with a reduction reaction. The hydrolysis reaction was catalyzed by a commercial esterase Z03 while the reduction reaction was catalyzed by E. coli cells co-expressing a 3ß-hydroxysteroid dehydrogenase SfSDR and a glucose dehydrogenase BtGDH. After the condition optimization, DHEA was synthesized at a 100 mL scale under 100 mM of substrate loading and purified as white powder with the highest space-time yield (4.80 g/L/h) and purity (99 %) in the biosynthesis of DHEA. The successful attempt in this study provides a new approach for green synthesis of highly purified DHEA in the pharmaceutical industry.

"Nonpolarity paving" in substrate tunnel of a Limnobacter sp. Phenylacetone monooxygenase for efficient single whole-cell synthesis of esomeprazole.

Baeyer-Villiger monooxygenase (BVMO) mediated sulfoxidation is a sustainable approach for the synthesis of esomeprazole. In this work, a novel phenylacetone monooxygenase from Limnobacter sp. (LnPAMO) was found to have trace activity for synthesis of enantiopure esomeprazole. Through engineering in the substrate tunnel using a mutagenesis strategy called "nonpolarity paving" and some modifications in cofactor binding domains, a mutant harboring 15 mutations (LnPAMO Mu15) was obtained with 6.6 × 103-fold higher activity to convert omeprazole sulfide into esomeprazole. The activities of the mutant for synthesis of (S)-methyl phenyl sulfoxide and (S)-pantoprazole also increased much, indicating the versatility of the mutant for sulfoxide synthesis. Importantly, no over-oxidation byproduct omeprazole sulfone was detected in the sulfoxidation products by both mass spectrometry and HPLC analysis. Then NADP-dependent Burkholderia stabili formate dehydrogenase was ligated behind Mu15 along with a ribosome binding site sequence in pET-28a for co-expression. By single whole-cell of recombinant Escherichia coli BL21 coexpressing Mu15 and formate dehydrogenase, omeprazole sulfide was efficiently converted into esomeprazole without production of sulfone (16 g/L substrate, enantiomeric excess > 99.9% (S) and > 99% conversion) and the space-time-yield reached 1.67 g product/L/h.

Improving the Cß Stereoselectivity of l-Threonine Aldolase for the Synthesis of l-threo-4-Methylsulfonylphenylserine by Modulating the Substrate-Binding Pocket To Control the Orientation of the Substrate Entrance.

l-Threonine aldolase from Actinocorallia herbida (AhLTA) is an ideal catalyst for producing l-threo-4-methylsulfonylphenylserine [(2S,3R)-1 b], a key chiral precursor for florfenicol and thiamphenicol. The moderate Cß stereoselectivity is the main obstacle to the industrial application of AhLTA. To address this issue, a combinatorial active-site saturation test (CAST) together with sequence conservatism analysis was applied to engineer the AhLTA toward improved Cß stereoselectivity. The optical mutant Y314R could asymmetrically synthesize l-threo-4-methylsulfonylphenylserine with 81 % diastereomeric excess (de), which is 23 % higher than wild-type AhLTA. Molecular dynamic (MD) simulations revealed that the mechanism for the improvement in Cß stereoselectivity of Y314R is due to the acylamino group of residues Arg314 controlling the orientation of substrate 4-methylsulfonyl benzaldehyde (1 a) in the active pocket by directed interaction with the methylsulfonyl group; this leads to asymmetric synthesis of l-threo-4-methylsulfonylphenylserine. The success in this study demonstrates that direct control of substrates in an active pocket is an attract strategy to address the Cß stereoselectivity problem of LTA and contribute to the industrial application of LTA.

Efficient biosynthesis of (2S, 3R)-4-methylsulfonylphenylserine by artificial self-assembly of enzyme complex combined with an intensified acetaldehyde elimination system.

(2S, 3R)-4-methylsulfonylphenylserine [(2S, 3R)-MPS], a key chiral precursor for antibiotics florfenicol and thiamphenicol, could be asymmetrically synthesized by l-threonine transaldolase (LTTA) coupled with an acetaldehyde elimination system. The low efficiency of acetaldehyde elimination system blocked further accumulation of (2S, 3R)-MPS. To address this issue, strengthening acetaldehyde elimination system and enzyme self-assembly strategy were combined to accelerate biosynthesis of (2S, 3R)-MPS. The new multi-enzyme cascade with intensified acetaldehyde elimination system BL21 (PsLTTAD2/ScADH/BtGDH) could produce (2S, 3R)-MPS with a titer of 157.6 mM, 1.7-folds than that produced by the original system BL21 (PsLTTAD2/ApADH/CbFDH). Moreover, self-assembly of PsLTTAD2 and ScADH by respective fusion of SpyTag and SpyCatcher were carried out to develop a self-assembled multi-enzyme cascade BL21 (ST-PsLTTAD2/SC-ScADH/BtGDH). As a result, the yield of (2S, 3R)-MPS was up to 248.1 mM with 95% de. As far as we knew, that represented the highest yield of (2S, 3R)-MPS by enzymatic synthesis, and therefore was a promising and green route for industrial production of this valuable compound.

Rational design of the carbonyl reductase EbSDR8 for efficient biosynthesis of enantiopure (R)-3-chloro-1-phenyl-1-propanol.

(R)-3-Chloro-1-phenyl-1-propanol ((R)-CPPO) is an important chiral intermediate for antidepressants. For its efficient biosynthesis, the carbonyl reductase EbSDR8 was engineered to asymmetrically reduce the unnatural substrate 3-chloro-1-phenyl-1-propanone (3-CPP) at high concentrations. Molecular docking and molecular dynamics simulations of the resulting mutants suggested enlarged substrate binding pocket and more reasonable interactions between the enzyme and the substrate or cofactor as the reasons for the enhanced catalytic activity and thus the remarkably improved conversion of high-concentration 3-CPP. Using the best mutant EbSDR8G94A/L153I/Y188A/Y202M as the whole-cell biocatalyst, reduction of 3-CPP (1.0 M) was conducted using 100% isopropanol as both the solvent and co-substrate for NADH regeneration, delivering (R)-CPPO with ˃ 99% eep and 95.5% conversion. This result suggests EbSDR8G94A/L153I/Y188A/Y202M as a potential biocatalyst for green production of (R)-CPPO at the industrial scale. KEY POINTS: • Rational design of EbSDR8 by modulating steric hindrance and molecular interactions; • Non-aqueous biocatalysis using isopropanol as both the solvent and co-substrate; • Whole-cell catalyzed production of 161 g/L enantiopure (R)-CPPO from 1.0 M of 3-CPP. Graphical Abstract.

Improved enantioselectivity of E. coli BioH in kinetic resolution of methyl (S)-3-cyclohexene-1-carboxylate by combinatorial modulation of steric and aromatic interactions.

As a chiral precursor for the important anticoagulant Edoxaban, enantioselective synthesis of (S)-3-cyclohexene-1-carboxylic acid is of great significance. The complicated procedures and generation of massive solid waste discourage its chemical synthesis, and the alternative biocatalysis route calls for an enzyme capable of asymmetric hydrolysis of racemic methyl-3-cyclohexene-1-carboxylate. To this end, we engineered the E. coli esterase BioH for improved S-enantioselectivity via rational design. By combinatorial modulation of steric and aromatic interactions, a positive mutant Mu3 (L24A/W81A/L209A) with relatively high S-selectivity in hydrolyzing racemic methyl-3-cyclohexene-1-carboxylate was obtained, improving the enantiomeric excess from 32.3% (the wild type) to 70.9%. Molecular dynamics simulation was conducted for both (R)- or (S)- complexes of the wild type and Mu3 to provide hints for the mechanism behind the increased S-selectivity. Moreover, the reaction conditions of Mu3 in methyl-3-cyclohexene-1-carboxylate hydrolysis was optimized to improve the conversion rate to 2 folds.

A study on the role that quorum sensing play in antibiotic-resistant plasmid conjugative transfer in Escherichia coli.

Horizontal genes transfer (HGT) plays an important role in the dissemination of antibiotic resistance genes (ARGs) in the environment. However, the mechanisms of HGT of ARGs under the influence of antibiotics in sub-MIC remain rarely explored. Moreover, given its collective nature, HGT was considered to be relative to quorum sensing (QS) system. To investigate whether QS has any impact on horizontal gene transfer of ARGs, experiments were conducted to determine the conjugative efficiency of plasmid RP4 on Escherichia coli (E.coli) under the influences of tetracyclines (TCs), quorum sensing autoinducers (AIs) and quorum sensing inhibitors (QSIs). The results indicated that the sub-MIC TCs could facilitate the conjugative transfer of RP4, a process which could be enhanced by AIs but inhibited by QSIs. This study demonstrated the roles that QS played in the dissemination of ARGs, and provided theoretical insights into the mechanism of HGT of ARGs in the environment.

Efficiently manufacturing ectoine via metabolic engineering and protein engineering of L-2,4-diaminobutyrate transaminase.

Ectoine, so-called tetrahydropyrimidine, is an important osmotic adjustment solute and widely applied in cosmetics and protein protectant. Some attempts have been made to improve the ectoine productivity. However, the strains with both high ectoine production capacity and high glucose conversion were still absent so far. Aim to construct a strain for efficiently producing ectoine, ectoine synthetic gene cluster ectABC from Pseudomonas stutzeri was overexpressed in E. coli BL21 (DE3). The ection production was improved by 382 % (ectoine titer increased from 1.73 g/L to 8.33 g/L) after the rational design of rate-limiting enzyme L-2,4-diaminobutyrate transaminase EctBps (protein engineering) combined with the metabolic engineering that focused on the enrichment and conversion of precursors. The final strain YW20 was applied to overproduce ectoine in fed-batch fermentation and yield 68.9 g/L of ectoine with 0.88 g/L/h of space-time yield and the highest glucose conversion reported [34 % (g/g)]. From the fermentation broth, ectoine was purified with 99.7 % purity and 79.8 % yield. This study successfully provided an engineered strain as well as an efficient method for the industrial bio-synthesis and preparation of ectoine.

Development of whole cell biocatalytic system for asymmetric synthesis of esomeprazole with enhancing coenzyme biosynthesis pathway.

Esomeprazole is the most popular proton pump inhibitor for treating gastroesophageal reflux disease. Previously, a phenylacetone monooxygenase mutant LnPAMOmu15 (LM15) was obtained by protein engineering for asymmetric synthesis of esomeprazole using pyrmetazole as substrate. To scale up the whole cell asymmetric synthesis of esomeprazole and reduce the cost, in this work, an Escherichia coli whole-cell catalyst harboring LM15 and formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) were constructed through optimized gene assembly patterns. CRISPR/Cas9 mediated insertion of Ptrc promoter in genome was done to enhance the expression of key genes to increase the cellular NADP supply in the whole cell catalyst, by which the amount of externally added NADP+ for the asymmetric synthesis of esomeprazole decreased to 0.05 mM from 0.3 mM for reducing the cost. After the optimization of reaction conditions in the reactor, the scalable synthesis of esomeprazole was performed using the efficient LM15-BstFDH whole-cell as catalyst, which showed the highest reported space-time yield of 3.28 g/L/h with 50 mM of pyrmetazole loading. Isolation procedure was conducted to obtain esomeprazole sodium of 99.55 % purity and > 99.9 % ee with 90.1 % isolation yield. This work provides the basis for production of enantio-pure esomeprazole via cost-effective whole cell biocatalysis.

Single-cell enzymatic cascade synthesis of testolactone enabled by engineering of polycyclic ketone monooxygenase and multi-gene expression fine-tuning.

The synthesis of steroids is challenging through multistep steroidal core modifications with high site-selectivity and productivity. In this work, a novel enzymatic cascade system was constructed for synthesis of testolactone by specific C17 lactonization/Δ1-dehydrogenation from inexpensive androstenedione using an engineered polycyclic ketone monooxygenase (PockeMO) and an appropriate 3-ketosteroid-Δ1-dehydrogenase (ReKstD). The focused saturation mutagenesis in the substrate binding pocket was implemented for evolution of PockeMO to eliminate the bottleneck effect. A best mutant MU3 (I225L/L226V/L532Y) was obtained with 20-fold higher specific activity compared to PockeMO. The catalytic efficiency (kcat/Km) of MU3 was 171-fold higher and the substrate scope shifted to polycyclic ketones. Molecular dynamic simulations suggested that the activity was improved by stabilization of the pre-lactonization state and generation of productive orientation of 4-AD mediated by distal L532Y mutation. Based on that, the three genes, MU3, ReKstD and a ketoreductase for NADPH regeneration, were rationally integrated in one cell via expression fine-tuning to form the efficient single cell catalyst E. coli S9. The single whole-cell biocatalytic process was scaled up and could generate 9.0 g/L testolactone with the high space time yield of 1 g/L/h without steroidal by-product, indicating the potential for site-specific and one-pot synthesis of steroid.

An artificial biocatalytic cascade for efficient synthesis of norepinephrine by combination of engineered L-threonine transaldolase with multi-enzyme expression fine-tuning.

Norepinephrine, a kind of ß-adrenergic receptor agonist, is commonly used for treating shocks and hypotension caused by a variety of symptoms. The development of a straightforward, efficient and environmentally friendly biocatalytic route for manufacturing norepinephrine remains a challenge. Here, we designed and realized an artificial biocatalytic cascade to access norepinephrine starting from 3, 4-dihydroxybenzaldehyde and L-threonine mediated by a tailored-made L-threonine transaldolase PsLTTA-Mu1 and a newly screened tyrosine decarboxylase ErTDC. To overcome the imbalance of multi-enzymes in a single cell, engineering of PsLTTA for improved activity and fine-tuning expression mode of multi-enzymes in single E.coli cells were combined, leading to a robust whole cell biocatalyst ES07 that could produce 100 mM norepinephrine with 99% conversion, delivering a highest time-space yield (3.38 g/L/h) ever reported. To summarized, the current study proposed an effective biocatalytic approach for the synthesis of norepinephrine from low-cost substrates, paving the way for industrial applications of enzymatic norepinephrine production.

Multienzymatic Cascade for Synthesis of Hydroxytyrosol via Two-Stage Biocatalysis.

Hydroxytyrosol, a naturally occurring compound with antioxidant and antiviral activity, is widely applied in the cosmetic, food, and nutraceutical industries. The development of a biocatalytic approach for producing hydroxytyrosol from simple and readily accessible substrates remains a challenge. Here, we designed and implemented an effective biocatalytic cascade to obtain hydroxytyrosol from 3,4-dihydroxybenzaldehyde and l-threonine via a four-step enzymatic cascade composed of seven enzymes. To prevent cross-reactions and protein expression burden caused by multiple enzymes expressed in a single cell, the designed enzymatic cascade was divided into two modules and catalyzed in a stepwise manner. The first module (FM) assisted the assembly of 3,4-dihydroxybenzaldehyde and l-threonine into (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid, and the second module (SM) entailed converting (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid into hydroxytyrosol. Each module was cloned into Escherichia coli BL21 (DE3) and engineered in parallel by fine-tuning enzyme expression, resulting in two engineered whole-cell catalyst modules, BL21(FM01) and BL21(SM13), capable of converting 30 mM 3,4-dihydroxybenzaldehyde to 28.7 mM hydroxytyrosol with a high space-time yield (0.88 g/L/h). To summarize, the current study proposes a simple and effective approach for biosynthesizing hydroxytyrosol from low-cost substrates and thus has great potential for industrial applications.

Studies on the mechanisms of Helicobacter pylori inhibition by Syzygium aromaticum aqueous extract.

BACKGROUND: The aqueous extract of the dried buds of Syzygium aromaticum (SAAE) have the potential to alleviate Helicobacter pylori infection, but the specific molecular mechanism has not been fully elucidated. PURPOSE: This study aimed to investigate the underlying mechanisms of SAAE on H. pylori pathogenicity. METHODS: The inhibitory kinetics and anti-H. pylori adhesive capacity assays were conducted to examine the effects of SAAE on the growth and adhesive capability of H. pylori. The H. pylori outer membrane vesicles (OMVs) were purified from the culture supernatant through high-speed centrifugation, filtration, and two rounds of ultracentrifugation. Their characteristics and protein composition were then identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and qualitative proteomics study. Subsequently, the effect of SAAE on the pathogenicity of H. pylori OMVs was investigated using the Griess reagent assay, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics study, TEM, and western blotting assay. RESULTS: SAAE exhibited inhibitory effects on H. pylori growth and adhesion. The isolated H. pylori OMVs showed particle size of 27-242 nm and Zeta potential of -9.67 ± 0.53 mV. A total of 599 proteins were identified in the OMVs. Proteomics study indicated that the differential expressed proteins induced by OMVs with or without SAAE commonly enriched in P53 and autophagy pathways. Besides, SAAE counteracted the increased production of pro-inflammatory cytokines and attenuated the induction of cell autophagy caused by H. pylori OMVs. Furthermore, SAAE normalized the abnormal regulation of downstream targets (AIFM2 and IGFBP3) in the P53 signaling pathway caused by H. pylori OMVs. CONCLUSION: SAAE can inhibit the growth and adhesion of H. pylori, reduce the inflammation and autophagy induced by H. pylori OMVs, and combated the abnormal regulation of P53 signaling pathway caused by H. pylori OMVs. These findings may help elucidate the mechanisms through which SAAE reduces the pathogenicity of H. pylori.

Phellodendron chinense C.K.Schneid: An in vitro study on its anti-Helicobacter pylori effect.

ETHNOPHARMACOLOGICAL RELEVANCE: Phellodendron chinense C.K.Schneid(P. chinense Schneid) is known in TCM as Huang Bo, is traditionally used to support gastrointestinal function and alleviate stomach-related ailments, including gastric ulcer bleeding and symptoms of gastroesophageal reflux disease. Helicobacter pylori (H. pylori) is classified by the WHO as a Group 1 carcinogen. However, the specific activity and mechanism of action of P. chinense Schneid against H. pylori infection remain unclear. It has been noted that Huangjiu processing may alter the bitter and cold properties of P. chinense Schneid, but its effect on antimicrobial activity requires further investigation. Additionally, it remains uncertain whether berberine is the sole antimicrobial active component of P. chinense Schneid. AIM OF STUDY: This study aims to elucidate the anti-H. pylori infection activity of P. chinense Schneid, along with its mechanism of action and key antimicrobial active components. MATERIALS AND METHODS: Phytochemical analysis was carried out by UPLC-MS/MS. HPLC was employed to quantify the berberine content of the extracts. Antimicrobial activity was assessed using the micro broth dilution method. Morphology was observed using SEM. The impact on urease activity was analyzed through in vitro urease enzyme kinetics. RT-qPCR was employed to detect the expression of virulence genes, including adhesin, flagellum, urease, and cytotoxin-related genes. The adhesion effect was evaluated by immunofluorescence staining and agar culture. RESULTS: P. chinense Schneid exhibited strong antimicrobial activity against both antibiotic-sensitive and resistant H. pylori strains, with MIC ranging from 40 to 160 µg/mL. Combination with amoxicillin, metronidazole, levofloxacin, and clarithromycin did not result in antagonistic effects. P. chinense Schneid induced alterations in bacterial morphology and structure, downregulated the expression of various virulence genes, and inhibited urease enzyme activity. In co-infection systems, P. chinense Schneid significantly attenuated H. pylori adhesion and urease relative content, thereby mitigating cellular damage caused by infection. Huangjiu processing enhanced the anti-H. pylori activity of P. chinense Schneid. Besides berberine, P. chinense Schneid contained seven other components with anti-H. pylori activity, with palmatine exhibiting the strongest activity, followed by jatrorrhizine. CONCLUSIONS: This study sheds light on the potential therapeutic mechanisms of P. chinense Schneid against H. pylori infection, demonstrating its capacity to disrupt bacterial structure, inhibit urease activity, suppress virulence gene transcription, inhibit adhesion, and protect host cells. The anti-H. pylori activity of P. chinense Schneid was potentiated by Huangjiu processing, and additional components beyond berberine were identified as possessing strong anti-H. pylori activity. Notably, jatrorrhizine, a core component of P. chinense Schneid, exhibited significant anti-H. pylori activity, marking a groundbreaking discovery.

Improved Antimicrobial Activity of Bovine Lactoferrin Peptide (LFcinB) Based on Rational Design.

Bovine lactoferrin peptide (LFcinB), as an antimicrobial peptide, is expected to be an alternative of antibiotics owing to its broad-spectrum antimicrobial activity and specific mechanism. However, the weak antimicrobial activity, high hemolysis, and poor stability of LFcinB limited its applications in the field of biomedicine, food and agriculture. In order to improve the antimicrobial activity of LFcinB, five mutants were designed rationally, of which mutant LF4 (M10W/P16R/A24L) showed highest antimicrobial activity. The bioinformatics analysis indicated that the improved antimicrobial activity of LF4 was related to its increased cations, higher amphiphilicity and the extension of the ß-sheet in the structure. These studies will highlight the important role of bioinformatic tools in designing ideal biopeptides and lay a foundation for further development of antimicrobial peptides.

Construction of efficient enzyme systems for preparing chiral ethyl 3-hydroxy-3-phenylpropionate.

Ethyl 3-hydroxy-3-phenylpropionate (EHPP), (R)-EHPP or (S)-EHPP, is an important chiral intermediate for pharmaceuticals. Its synthesis from ethyl benzoyl acetate (EBA) by alcohol dehydrogenase is regarded as a green method. However, scarcely any alcohol dehydrogenase has been reported competent in asymmetric synthesis of chiral EHPP at high EBA loading. Present study developed two robust and efficient bio-catalysts Mu-S2 and Mu-R4 for preparation of (S)-EHPP and (R)-EHPP respectively by rational design of alcohol dehydrogenase PcSDR from Pedobacter chitinilyticus based on molecular dynamics (MD) simulation analysis. BtGDH, a glucose dehydrogenase from Bacillus toyonensis catalyzing the oxidation of glucose for cofactor regeneration, was co-expressed with the screened mutants to form enzyme systems Mu-S2-BtGDH and Mu-R4-BtGDH. After reaction condition optimization, Mu-S2-BtGDH and Mu-R4-BtGDH were efficient in the synthesis of (S)-EHPP (94% conv. and 99% e.e.) and (R)-EHPP (99% conv. and 98% e.e.) respectively in 100 mL scale under 500 mM of EBA loading in 10 h following a substrate continuous feeding mode. After purifying, the isolated yield for each EHPP enantiomer is > 93%. This work not only provides potential biocatalysts for the industrial production of (R)-EHPP and (S)-EHPP, but also enriches the constructure-function relationship of alcohol dehydrogenases.

Design of a PL18 alginate lyase with flexible loops and broader entrance to enhance the activity and thermostability.

Alginate oligosaccharides are enzymolysis products of alginate with versatile bioactivities and their industrial preparation was limited by the insufficient activity and unsatisfying thermostability of alginate lyases. The structure-function information about PL18 alginate lyases was seldom reported since which few positive mutants of PL18 alginate lyases were generated. In present study, a mutant of PL18 alginate lyase E226K was expressed intracellularly and taken as parent for further modification. Site I211 at the lid loop 1 and sites E276, Y292 and R294 at the predicted entrance were chosen as engineering targets based on the E226K-PM4 binding mode in prereaction-state MD simulation and 29 mutants were constructed, from those, the variant E226K/I211T/R294V was screened out as the best mutant (showing 4.78-fold increased catalytic efficiency and the half-time t1/245℃ increased up to 557 min from 89 min). MD simulations indicated that the affinity of E226K/I211T/R294V towards alginate was improved due to the optimized energy distribution of active center, more flexible loops around catalytic cleft and larger substrate entrance. The more efficient proton transmitting endowed E226K/I211T/R294V higher activity and the more complicated intraprotein interactions together with stronger backbone rigidity were responsible for the improved thermostability of E226K/I211T/R294V than E226K. The success in this study enriches the structure-function information of PL18 alginate lyases and provides hints for their further design.

Multi-enzyme cascade for improving ß-hydroxy-α-amino acids production by engineering L-threonine transaldolase and combining acetaldehyde elimination system.

L-threonine transaldolase(PsLTTA) could asymmetric synthesize ß-hydroxy-α-amino acids (HAAs) with excellentstereoselectivity, while the poor yield limited its further application. Here we provided a combinatorial strategy to improve HAAs production, by directed evolution of PsLTTA towards enhanced activity and introducing an acetaldehyde elimination system to avoid acetaldehyde over-accumulation. A novel high throughput screening (HTS) method for evaluating PsLTTA activity was developed andapplied for directed evolution of PsLTTA. Subsequently, we co-expressedalcohol dehydrogenase andformate dehydrogenase to construct an acetaldehyde elimination system toremove acetaldehyde inhibition.Moreover, the above positive strategies were integrated. As a result,the (2S,3R)-p-methylsulfonyl phenylserine yield reached 154.0 mM andwith 94.6% devalue, the highest productivity and stereoselectivity of (2S,3R)-HAAs reported by enzymatic synthesis so far. Taken together, our studies provided an efficient and green route for chiral synthesis of (2S,3R)-HAAs, which might contribute to the industrialization production of these useful building blocks.

Mutagenesis on the surface of a ß-agarase from Vibrio sp. ZC-1 increased its thermo-stability.

The recombinant rAgaZC-1 was a family GH50 ß-agarase from Vibrio sp. ZC-1 (CICC 24670). In this paper, the mutant D622G (i.e., mutate the aspartic acid at position 622 to glycine) had better thermo-stability than rAgaZC-1, showing 1.5℃ higher T5010 (the temperature at which the half-time is 10 min) and 4-folds of half-time at 41℃, while they had almost same optimum temperature (38.5℃), optimum pH (pH6.0) and catalytic efficiency. Thermal deactivation kinetical analysis showed that D622G had higher activation energy for deactivation, enthalpy and Gibbs free energy than rAgaZC-1, indicating that more energy is required by D622G for deactivation. Substrate can protect agarase against thermal inactivation, especially D622G. Hence the yield of agarose hydrolysis catalyzed by D622G was higher than that by rAgaZC-1. The models of D622G and rAgaZC-1 predicted by homology modeling were compared to find that it is the improved distribution of surface electrostatic potential, great symmetric positive potential and more hydrophobic interactions of D622G that enhance the thermo-stability.