Showing papers by "Ya-Ping Xue published in 2021"
TL;DR: The Acinetobacter indicus strain ZJB20129 isolated from an urban sewage treatment plant demonstrated the heterotrophic nitrification-aerobic denitrification (HN-AD) ability as discussed by the authors.
26 citations
TL;DR: Results indicated that the inoculant containing only Bacillus was more stable and cost-effective in FW in-situ reduction, and the novel agent greatly improved the stability of in-Situ reduction process.
23 citations
TL;DR: Encouraging results demonstrated that the developed enzyme cascade deracemization process exhibits great potential and economical competitiveness for manufacture of L-PPT from D,L- PPT.
15 citations
TL;DR: The results indicated that DAAO could be applied to the large-scale bioproduction of PPO and provide a promising route for the asymmetric synthesis of L-PPT by bio-enzymatic methods using PPO as the substrate.
Abstract: 2-Oxo-4-(hydroxymethylphosphinyl) butyric acid (PPO) is an important precursor compound for the broad-spectrum herbicide l-glufosinate (L-PPT). In this study, the gene of d-amino acid oxidase (DAAO...
10 citations
TL;DR: A single‐transaminase‐catalyzedBiocatalytic cascade was developed by employing the desired biocatalyst, ATA‐117‐Rd11, that showed high activity toward 2‐oxo‐4‐[(hydroxy)(methyl)phosphinoyl] butyric acid and α‐ketoglutarate, and low activity against pyruvate.
Abstract: A single-transaminase-catalyzed biocatalytic cascade was developed by employing the desired biocatalyst, ATA-117-Rd11, that showed high activity toward 2-oxo-4-[(hydroxy)(methyl)phosphinoyl] butyric acid (PPO) and α-ketoglutarate, and low activity against pyruvate The cascade successfully promotes a highly asymmetric amination reaction for the synthesis of l-phosphinothricin (l-PPT) with high conversion (>95 %) and>99 % ee In a scale-up experiment, using 10 kg pre-frozen E coli cells harboring ATA-117-Rd11 as catalyst, 80 kg PPO was converted to ≈70 kg l-PPT after 24 hours with a high ee value (>99 %)
9 citations
TL;DR: The simultaneous-evolution strategy was applied to a multienzyme-catalyzed reaction for the asymmetric synthesis of l-phosphinothricin, which not only enhanced the catalytic efficiency of Glu DH but also improved the coordination between GluDH and GDH.
Abstract: The traditional strategy to improve the efficiency of an entire coupled enzyme system relies on separate direction of the evolution of enzymes involved in their respective enzymatic reactions. This strategy can lead to enhanced single-enzyme catalytic efficiency but may also lead to loss of coordination among enzymes. This study aimed to overcome such shortcomings by executing a directed evolution strategy on multiple enzymes in one combined group that catalyzes the asymmetric biosynthesis of l-phosphinothricin. The genes of a glutamate dehydrogenase from Pseudomonas moorei (PmGluDH) and a glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), along with other gene parts (promoters, ribosomal binding sites (RBSs), and terminators) were simultaneously evolved. The catalytic efficiency of PmGluDH was boosted by introducing the beneficial mutation A164G (from 1.29 s-1mM-1 to 183.52 s-1mM-1), and the EsGDH expression level was improved by optimizing the linker length between the RBS and the start codon of gdh. The total turnover numbers of the bioreaction increased from 115 (GluDH WTNADPH) to 5846 (A164GNADPH coupled with low expression of EsGDH), and to 33950 (A164GNADPH coupled with high expression of EsGDH). The coupling efficiency was increased from ∼30% (GluDH_WT with low expression of GDH) to 83.3% (GluDH_A164G with high expression of GDH). In the batch production of l-phosphinothricin utilizing whole-cell catalysis, the strongest biocatalytic reaction exhibited a high space-time yield (6410 g·L-1·d-1) with strict stereoselectivity (>99% enantiomeric excess).Importance: The traditional strategy to improve multienzyme-catalyzed reaction efficiency may lead to enhanced single-enzyme catalytic efficiency but may also result in loss of coordination among enzymes. We describe a directed evolution strategy of an entire coupled enzyme system to simultaneously enhance enzyme coordination and catalytic efficiency. The simultaneous evolution strategy was applied to a multienzyme-catalyzed reaction for the asymmetric synthesis of l-phosphinothricin, which not only enhanced the catalytic efficiency of GluDH but also improved the coordination between GluDH and GDH. Since this strategy is enzyme-independent, it may be applicable to other coupled enzyme systems for chiral chemical synthesis.
9 citations
TL;DR: Carboxymethyl cellulose sodium salt and β - d -glucan were the best substrates for CoCel5A among the tested substrates and the kinetic parameters V max, K m, and K cat / K m values against CMC were high, suggesting that CoCel 5A has high affinity and catalytic efficiency.
Abstract: To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene from Colletotrichum orchidophilum was synthesized and a recombinant Pichia pastoris GS115/pPIC9K/cocel5A was constructed for secretory expression of CoCel5A. After purification, the protein CoCel5A was biochemically characterized. The endoglucanase CoCel5A exhibited the optimal activity at 55–75 °C and high thermostability (about 85% residual activity) at the temperature of 55 °C after incubation for 3 h. The highest activity of CoCel5A was detected when 100 mM citric acid buffer (pH 4.0–5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100 mM citric acid buffer (pH 3.0–6.0) at 4 °C for 24 h. Carboxymethyl cellulose sodium salt (n = approx. 500) (CMC) and β-d-glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parameters Vmax, Km, and Kcat/Km values against CMC were 290.70 U/mg, 2.65 mg/mL, and 75.67 mL/mg/s, respectively; and 228.31 U/mg, 2.06 mg/mL, and 76.45 mL/mg/s against β-d-glucan, respectively, suggesting that CoCel5A has high affinity and catalytic efficiency. These properties supported the potential application of CoCel5A in biotechnological and environmental fields.
9 citations
TL;DR: In this article, a high-temperature-resistant microbial consortium was constructed to effectively degrade oily food waste by Fed-in-situ biological reduction treatment (FBRT), and oil degrading bacteria were screened under thermophilic conditions of mineral salt medium with increased oil content.
9 citations
TL;DR: The proposed integrated process avoids substrate inhibition, facilitates the reusability of whole-cell nitrilase-catalyzed asymmetric hydrolysis, and thus shows great potential for the sustainable production of chiral carboxylic acids.
9 citations
TL;DR: An overview of the biological production of NMN with respect to the critical enzymes, reaction conditions, and productivity can be found in this paper, where the authors also present the regulations of enzymes that are not directly involved in the synthesis of NAD.
Abstract: Nicotinamide mononucleotide (NMN) or Nicotinamide-1-ium-1-β-D-ribofuranoside 5'-phosphate is a nucleotide that can be converted into nicotinamide adenine dinucleotide (NAD) in human cells. NMN has recently attracted great attention because of its potential as an anti-aging drug, leading to great efforts for its effective manufacture. The chemical synthesis of NMN is a challenging task since it is an isomeric compound with a complicated structure. The majority of biological synthetic routes for NMN is through the intermediate phosphoribosyl diphosphate (PRPP), which is further converted to NMN by nicotinamide phosphoribosyltransferase (Nampt). There are various routes for the synthesis of PRPP from simple starting materials such as ribose, adenosine, and xylose, but all of these require the expensive phosphate donor adenosine triphosphate (ATP). Thus, an ATP regeneration system can be included, leading to diminished ATP consumption during the catalytic process. The regulations of enzymes that are not directly involved in the synthesis of NMN are also critical for the production of NMN. The aim of this review is to present an overview of the biological production of NMN with respect to the critical enzymes, reaction conditions, and productivity.
7 citations
TL;DR: In this paper, the authors replaced the TEF1 promoter with a weaker promoter (PDog2p300) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression.
Abstract: Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (PTEF1) However, antibiotic-resistant proteins are necessary only for the selection process Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter To address these limitations, we replaced the TEF1 promoter with a weaker promoter (PDog2p300) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression Importantly, the PDog2p300 has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter-based production of recombinant proteins compared with glucose that is usually used for the selection process This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 μg/ml of Zeocin Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteinsKey points• PDog2p300 was identified as a novel glucose-regulated promoter• The expression of antibiotic-resistant gene driven by PDog2p300 was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden• The transformants harboring multiple integrations were cost-effectively selected by using the PDog2p300 for driving antibiotic-resistant genes
TL;DR: In this paper, the factors that inhibit the growth of bacterial cells during a 5-L fed-batch fermentation process were explored, and the fermentation process was optimized by co-expressing catalase (CAT), by balancing the biomass and the enzyme activity, and by adding exogenous D-alanine (D-Ala) to relieve the limitation of DAAO on the cells and optimize fermentation.
Abstract: D-amino acid oxidase (DAAO) is widely used in the industrial preparation of L-amino acids, and cultivating Escherichia coli (E. coli) expressing DAAO for the biosynthesis of L-phosphinothricin (L-PPT) is very attractive. At present, the biomass production of DAAO by fermentation is still limited in large-scale industrial applications because the expression of DAAO during the fermentation process inhibits the growth of host cells, which limits higher cell density. In this study, the factors that inhibit the growth of bacterial cells during a 5-L fed-batch fermentation process were explored, and the fermentation process was optimized by co-expressing catalase (CAT), by balancing the biomass and the enzyme activity, and by adding exogenous D-alanine (D-Ala) to relieve the limitation of DAAO on the cells and optimize fermentation. Under optimal conditions, the DO-STAT feeding mode with DO controlled at 30% ± 5% and the addition of 27.5 g/L lactose mixed with 2 g/L D-Ala during induction at 28 °C resulted in the production of 26.03 g dry cell weight (DCW)/L biomass and 390.0 U/g DCW specific activity of DAAO; an increase of 78% and 84%, respectively, compared with the initial fermentation conditions. The fermentation strategy was successfully scale-up to a 5000-L fermenter.
TL;DR: In this paper, an effective R-PPA synthesis method was established and optimized in order to provide sufficient R2-(4-hydroxyphenoxy)propionic acid (R-HPPA) for the industrial production of R-PNPA, which is a key intermediate for the synthesis of classic herbicides with high selectivity against grassy weed.
TL;DR: A hybrid biomimetic silica particles (HBSPs) with core-shell structure showed a specific activity of 147.2% in the first 10 batches with a productivity of 619.3
TL;DR: The hypothesis that two insertion regions contribute to the unique quaternary structure of the spiral microbial nitrilases, based on cysteine scanning across these two regions, is supported.
TL;DR: In this article, a novel HPLC method coupled with 9-fluorenylmethyl chloroformate derivatization was developed for the quantitative analysis of the two miglitol intermediates, N-2-hydroxyethyl glucamine and 6-(N-hydroxethyl)-amino-6-deoxy-alpha-L-sorbofuranose.
Abstract: In the synthesis of hypoglycemic agent miglitol, the quantitative analysis of two key intermediates, N-2-hydroxyethyl glucamine and 6-(N-hydroxyethyl)-amino-6-deoxy-alpha-L-sorbofuranose, has long been a challenge In this study, derivatization reagents were screened, and then a novel HPLC method coupled with 9-fluorenylmethyl chloroformate derivatization was developed for the quantitative analysis of the two miglitol intermediates The derivatization was performed by reacting Fmoc-Cl and the intermediates at a molar ratio of 16:1 and sodium borate at a final concentration of 02 mol/L at pH 80, 35 °C for 40 min, followed by the addition of 15 times the volume of acetic acid (01%, w/v) The analysis was achieved on Hypersil ODS2 (46 mm × 250 mm) column applying an isocratic mobile phase containing acetonitrile and 01% acetic acid aqueous solution (45:55, v/v) at 08 mL/min flow rate and an injection volume of 10 μL The analyte was detected at 254 nm The method showed good linearity, precision, and accuracy It was capable of detecting both intermediates at low concentrations Interestingly, we found that a small amount (> 5%) of N-2-hydroxyethyl glucamine was not converted to 6-(N-hydroxyethyl)-amino-6-deoxy-alpha-L-sorbofuranose in the biotransformation This method was reliable and efficient for the analysis of miglitol intermediates
28 Oct 2021
TL;DR: In this paper, a new strategy for L-PPT synthesis, based on enzymes from chromosomal integrated expression, which does not depend on antibiotic selection, was demonstrated, and shows a high potential for future industrial application.
Abstract: Phosphinothricin (PPT) is one of the most prevalently using herbicides. The commercial phosphinothricin products are generally in the form of a racemic mixture, of which only the l-phosphinothricin (L-PPT) gives herbicidal function. Synthesis of optically pure L-PPT by deracemization of D/L-PPT is a promising way to cut down the environmental burden and manufacturing cost. To convert D/L-PPT to L-PPT, we expressed the catalytic enzymes by genomic integration in E. coli. The whole production was implemented in two steps in one pot using four catalytic enzymes, namely d-amino acid oxidase, catalase, glutamate dehydrogenase, and glucose dehydrogenase. Finally, after a series of process optimization, the results showed that with our system the overall L-PPT yield reached 86%. Our study demonstrated a new strategy for L-PPT synthesis, based on enzymes from chromosomal integrated expression, which does not depend on antibiotic selection, and shows a high potential for future industrial application.