L-lysine Α-oxidase Research Articles

Evaluation of high-value bioproducts production by marine endophytic fungus Arthrinium sp. FAKSA 10 under solid state fermentation using agro-industrial wastes

BackgroundTo address public health challenges, there is increasing interest in bioactive metabolites from unconventional sources for targeted cancer and viral treatments with minimal side effects. Endophytic fungi are promising for these therapies. This study focused on maximizing bioactive agent yields from these fungi using a low-cost medium and optimized fermentation system. ResultsForty-three endophytic fungi isolated from Sinularia polydactyla were cultivated on wheat bran medium and evaluated for bioactive metabolites under solid-state fermentation. Strain FAKSA 10, identified as Arthrinium sp. FAKSA 10, exhibited the highest levels of pharmaceutical metabolites, including L-glutaminase, L-methioninase, L-arginase, L-asparaginase, L-tyrosinase, L-lysine α-oxidase, and ribonuclease, with a 79.12% hepatitis C virus knockdown rate. This strain produced 46 metabolites with anticancer, antioxidant, antiviral, and cytotoxic properties, including major compounds like hexadecanoic acid methyl ester; hexadecanoic acid ethyl ester; 9, 12-octadecadienoic acid (Z, Z), methyl ester; 9-octadecenoic acid (Z)-, methyl ester, and 11, 14-eicosadienoic acid, methyl ester. A cost effective strategy using 11 agro-industrial residues was applied, following by optimization of the solid-state fermentation system. This optimization increased enzyme yields and enhanced antiviral and antioxidant activities. The optimal conditions for solid-state fermentation were a 10 d incubation, 1 mm particle size, 60% initial moisture, 28°C temperature, and 2 × 107 CFU/mL inoculum size. ConclusionsThis study exploited Arthrinium sp. FAKSA 10 metabolites as natural pharmaceuticals against cancer and viral diseases, highlighting their significant antioxidant properties. Among various residues, oil cakes emerged as an effective and cost-efficient medium, capable of significantly enhancing the production of valuable bioactive metabolites.

Novel Bioproduction of 1,6-Hexamethylenediamine from l-Lysine Based on an Artificial One-Carbon Elongation Cycle.

1,6-hexamethylenediamine (HMD) is an important precursor for nylon-66 material synthesis, while research on the bioproduction of HMD has been relatively scarce in scientific literature. As concerns about climate change, environmental pollution, and the depletion of fossil fuel reserves continue to grow, the significance of producing fundamental chemicals from renewable sources is becoming increasingly prominent. In recent investigations, the bioproduction of HMD from adipic acid has been reported but with lingering challenges concerning costly raw materials and low yields. Here, we have undertaken the reconstruction of the HMD synthetic pathway within Escherichia coli, which was constituted with l-lysine α-oxidase (Raip), LeuABCD, α-ketoacid decarboxylase (KivD), and transaminases (Vfl), leveraging a carbon chain extension module and a metabolic pathway of transaminase-decarboxylase cascade catalysis within the strain WD20, which successfully produce 46.7 ± 2.0 mg/L HMD. To increase the cascade activity and create a higher tolerance to external environmental disturbance for l-lysine to convert into HMD, another two enzymes d-alanine aminotransferase (Dat) and alpha-ketoacid decarboxylase (KdcA) were introduced into WD21 to provide flux flexibility for α-ketoacid metabolization, which was named "Smart-net metabolic engineering" in our research, and high-efficiency synthesis of HMD utilizing l-lysine as the substrate has been successfully achieved. Finally, we established a + 1C bioconversion multienzyme cascade catalyzing up to 65% conversion of l-lysine to HMD. Notably, our fermentation process yielded an impressive 213.5 ± 8.7 mg/L, representing the highest reported yield to date for the bioproduction of HMD from l-lysine.

Metabolic engineering of Escherichia coli for de novo production of 5-hydroxyvalerate via L-lysine α-oxidase pathway

5-hydroxyvalerate (5-HV) is a crucial C5 platform chemical with versatile applications, yet its efficient production remains a challenge. The Raip, gabT, and yahK genes were integrated into the E. coli LE genome, deleted gabD, and enhanced gabP expression, resulting in the QluMG strain. Additionally, the impact of ethanol and H2O2 on 5-HV production was investigated. Further enhancement was achieved by incorporating an NADPH supplementation system, resulting in the QluMG strain. In the 5 L fermenter, the QluMGD strain produced 21.7 g/L of 5-HV from 50 g/L glucose, with a conversion rate of 43.4 %. The successful integration of the RaiP pathway into the E. coli genome significantly enhanced 5-HV production. The QluMG strain achieved the highest reported yield from glucose in engineered E. coli to date. This study provides a new strategy for the efficient production of 5-HV and other chemicals using 5-HV as a precursor, demonstrating potential for industrial application.

The recombinant l-lysine α-oxidase from the fungus Trichoderma harzianum promotes apoptosis and necrosis of leukemia CD34 + hematopoietic cells

BackgroundIn hematologic cancers, including leukemia, cells depend on amino acids for rapid growth. Anti-metabolites that prevent their synthesis or promote their degradation are considered potential cancer treatment agents. Amino acid deprivation triggers proliferation inhibition, autophagy, and programmed cell death. l-lysine, an essential amino acid, is required for tumor growth and has been investigated for its potential as a target for cancer treatment. l-lysine α-oxidase, a flavoenzyme that degrades l-lysine, has been studied for its ability to induce apoptosis and prevent cancer cell proliferation. In this study, we describe the use of l-lysine α-oxidase (LO) from the filamentous fungus Trichoderma harzianum for cancer treatment.ResultsThe study identified and characterized a novel LO from T. harzianum and demonstrated that the recombinant protein (rLO) has potent and selective cytotoxic effects on leukemic cells by triggering the apoptotic cascade through mitochondrial dysfunction.ConclusionsThe results support future translational studies using the recombinant LO as a potential drug for the treatment of leukemia.

A new biosynthesis route for production of 5-aminovalanoic acid, a biobased plastic monomer

5-aminovalanoic acid (5AVA) can be used as the precursor of new plastics nylon 5 and nylon 56, and is a promising platform compound for the synthesis of polyimides. At present, the biosynthesis of 5-aminovalanoic acid generally is of low yield, complex synthesis process and high cost, which hampers large-scale industrial production. In order to achieve efficient biosynthesis of 5AVA, we developed a new pathway mediated by 2-keto-6-aminohexanoate. By combinatory expression of L-lysine α-oxidase from Scomber japonicus, α-ketoacid decarcarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli, the synthesis of 5AVA from L-lysine in Escherichia coli was achieved. Under the initial conditions of glucose concentration of 55 g/L and lysine hydrochloride of 40 g/L, the final consumption of 158 g/L glucose and 144 g/L lysine hydrochloride, feeding batch fermentation to produce 57.52 g/L of 5AVA, and the molar yield is 0.62 mol/mol. The new 5AVA biosynthetic pathway does not require ethanol and H2O2, and achieved a higher production efficiency as compared to the previously reported Bio-Chem hybrid pathway mediated by 2-keto-6-aminohexanoate.

Nanosized magnetite modified with poly(ethylene glycol) for efficient sorption of L-lysine-α-oxidase from the culture fluid

Fe3O4@PEG have been proposed for sorption of L-lysine-α-oxidase (LO) from the culture fluid of Trichoderma harzianum Rifai F-180 for the first time. To synthesize the Fe3O4@PEG nanoparticles, an original method based on aqueous biphasic systems has been developed. The PEG-modified magnetite provide a high sorption ability towards LO in contrast to the non-modified Fe3O4 synthesized by the traditional precipitation method. The morphology and structure of the prepared nanoparticles were characterized by TEM, FTIR and XRD. The data on magnetic properties and stability in physiological media are presented. The synthesized nanoparticles ensure quantitative sorption and desorption of LO during at least 3 cycles.

Monoclonal Antibody Functionalized, and L-lysine α-Oxidase Loaded PEGylated-Chitosan Nanoparticle for HER2/Neu Targeted Breast Cancer Therapy.

Simple SummaryBreast cancer is one of the dominant cancers that threaten human beings worldwide. Moreover, the treatment of HER2+ breast cancer is challenging due to heterogeneity. The L-lysine α-oxidase (LO) enzyme is a well-known antitumor enzyme, but its clinical utility has been limited due to side effects, decreased stability, and inability to target tumor cells. To overcome the clinical challenges in delivery of LO enzymes and improve HER2+ breast cancer therapeutics, the present study developed the dual stimuli responsive nanocarrier system (CS-LO-PEG-HER NPs) for pH sensitive and HER2/neu targeted breast cancer therapy.Herein, we designed a nanocarrier to deliver the LO specifically to HER2+ breast cancer (BC) cells, where functionalization of mAb (anti-HER2+) with PEGylated chitosan enabled it to target the HER2+ BC cells. Taking advantage of overexpression of HER2+ in cancer cells, our nanocarrier (CS-LO-PEG-HER NPs) exhibited promising potency and selectivity against HER2+ BC cells (BT474). The CS-LO-PEG-HER NPs demonstrated the cytotoxicity in BT474 cells by promoting reactive oxygen species, mitochondrial membrane potential loss, and nucleus damage. The biocompatibility of CS-LO-PEG-HER NPs was evidenced by the hemolysis assay and H & E staining of major organs. The CS-LO-PEG-HER NPs showed anticancer potency against the BT474-xenograft tumor-bearing mice, as evident by the reduction of tumor size and cell density. These results indicate that CS-LO-PEG-HER NPs are biocompatible with mice while inhibiting tumor growth through alter the oxidative stress. Overall, this work provides a promising approach for the delivery of LO for good therapeutic effect in combination with mAb.

L-Lysine α-Oxidase: Enzyme with Anticancer Properties.

L-lysine α-oxidase (LO), one of L-amino acid oxidases, deaminates L-lysine with the yield of H2O2, ammonia, and α-keto-ε-aminocaproate. Multiple in vitro and in vivo studies have reported cytotoxic, antitumor, antimetastatic, and antitumor activity of LO. Unlike asparaginase, LO has a dual mechanism of action: depletion of L-lysine and formation of H2O2, both targeting tumor growth. Prominent results were obtained on murine and human tumor models, including human colon cancer xenografts HCT 116, LS174T, and T47D with maximum T/C 12, 37, and 36%, respectively. The data obtained from human cancer xenografts in immunodeficient mice confirm the potential of LO as an agent for colon cancer treatment. In this review, we discuss recently discovered molecular mechanisms of biological action and the potential of LO as anticancer enzyme.

Coproduction of 5-Aminovalerate and δ-Valerolactam for the Synthesis of Nylon 5 From L-Lysine in Escherichia coli.

The compounds 5-aminovalerate and δ-valerolactam are important building blocks that can be used to synthesize bioplastics. The production of 5-aminovalerate and δ-valerolactam in microorganisms provides an ideal source that reduces the cost. To achieve efficient biobased coproduction of 5-aminovalerate and δ-valerolactam in Escherichia coli, a single biotransformation step from L-lysine was constructed. First, an equilibrium mixture was formed by L-lysine α-oxidase RaiP from Scomber japonicus. In addition, by adjusting the pH and H2O2 concentration, the titers of 5-aminovalerate and δ-valerolactam reached 10.24 and 1.82 g/L from 40 g/L L-lysine HCl at pH 5.0 and 10 mM H2O2, respectively. With the optimized pH value, the δ-valerolactam titer was improved to 6.88 g/L at pH 9.0 with a molar yield of 0.35 mol/mol lysine. The ratio of 5AVA and δ-valerolactam was obviously affected by pH value. The ratio of 5AVA and δ-valerolactam could be obtained in the range of 5.63:1–0.58:1 at pH 5.0–9.0 from the equilibrium mixture. As a result, the simultaneous synthesis of 5-aminovalerate and δ-valerolactam from L-lysine in Escherichia coli is highly promising. To our knowledge, this result constitutes the highest δ-valerolactam titer reported by biological methods. In summary, a commercially implied bioprocess developed for the coproduction of 5-aminovalerate and δ-valerolactam using engineered Escherichia coli.

High-efficiency production of 5-aminovalerate in engineered Escherichia coli controlled by an anaerobically-induced nirB promoter

Biobased production of 5-aminovalerate (5AVA) from biomass can support a sustainable and economic biorefinery process to produce bio-based nylon 5 for food packaging materials. Cost-competitive production of 5AVA from biomass is a key factor in the successful commercialization of nylon 5. Bioproduction of 5AVA is a promising candidate for the industrial process to the current petrochemical route. In this study, we developed an artificial 2-keto-6-aminocaproate-mediated pathway for cost-competitive and high efficiency production of 5AVA in engineered Escherichia coli. Firstly, the combination of native l-lysine α-oxidase (RaiP) from Scomber japonicas, α-ketoacid decarboxylase (KivD) from Lactococcus lactis and aldehyde dehydrogenase (PadA) from Escherichia coli could efficiently convert l-lysine into 5AVA. Moreover, the engineered strains ML03-PnirB-RKP, ML03-PPL-PR-RKP, ML03-PM1-93-RKP induced by anaerobic condition, temperature-induced, constitutive expression instead of expensive isopropyl β-D-thiogalactoside were constructed, respectively. The use of nirB promoter induced by anaerobic condition not only could attain a higher titer of 5AVA than PL-PR and M1-93 promoters, but omit cost of expensive exogenous inducers. After the replacement of industrial materials, 5AVA titer successfully reached 33.68 g/L in engineered strain ML03-PnirB-RKP via biotransformation. This biotransformation process conduces to the cosmically industrial 5AVA bioproduction.

A High-Efficiency Artificial Synthetic Pathway for 5-Aminovalerate Production From Biobased L-Lysine in Escherichia coli.

Bioproduction of 5-aminovalerate (5AVA) from renewable feedstock can support a sustainable biorefinery process to produce bioplastics, such as nylon 5 and nylon 56. In order to achieve the biobased production of 5AVA, a 2-keto-6-aminocaproate-mediated synthetic pathway was established. Combination of L-Lysine α-oxidase from Scomber japonicus, α-ketoacid decarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli could achieve the biosynthesis of 5AVA from biobased L-Lysine in E. coli. The H2O2 produced by L-Lysine α-oxidase was decomposed by the expression of catalase KatE. Finally, 52.24 g/L of 5AVA were obtained through fed-batch biotransformation. Moreover, homology modeling, molecular docking and molecular dynamic simulation analyses were used to identify mutation sites and propose a possible trait-improvement strategy: the expanded catalytic channel of mutant and more hydrogen bonds formed might be beneficial for the substrates stretch. In summary, we have developed a promising artificial pathway for efficient 5AVA synthesis.

Plasma pharmacokinetics and tissue distribution of L-lysine α-oxidase from Trichoderma cf. aureoviride RIFAI VKM F- 4268D in mice.

L-lysine α-oxidase (LO) is an L-amino acid oxidase with antitumor, antimicrobial and antiviral properties. Pharmacokinetic (PK) studies were carried out by measuring LO concentration in plasma and tissue samples by enzyme immunoassay. L-lysine concentration in samples was measured spectrophotometrically using LO. After single i.v. injection of 1.0, 1.5, 3.0mg/kg the circulating T1/2 of enzyme in mice varied from 51 to 74min and the AUC0-inf values were 6.54 ± 0.46, 8.66 ± 0.59, 9.47 ± 1.45μg/ml × h, respectively. LO was distributed in tissues and determined within 48h after administration with maximal accumulation in liver and heart tissues. Mean time to reach the maximum concentration was highest for the liver-9h, kidney-1h and 15min for the tissues of heart, spleen and brain. T1/2 of LO in tissues ranged from 7.75 ± 0.73 to 26.10 ± 2.60h. In mice, plasma L-lysine decreased by 79% 15min after LO administration in dose 1.6mg/kg. The serum L-lysine levels remained very low from 1 to 9h (< 25μM, 17%), indicating an acute lack of L-lysine in animals for at least 9h. Concentration of L-lysine in serum restored only 24h after LO administration. The results of LO PK study show that it might be considered as a promising enzyme for further investigation as a potential anticancer agent.

Structural basis of enzyme activity regulation by the propeptide of l-lysine α-oxidase precursor from Trichoderma viride

Harmuful proteins are usually synthesized as inactive precursors and are activated by proteolytic processing. l-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid to produce a 2-oxo acid with ammonia and highly toxic hydrogen peroxide and, therefore, is expressed as a precursor. The LAAO precursor shows significant variation in size and the cleavage pattern for activation. However, the molecular mechanism of how the propeptide suppresses the enzyme activity remains unclear except for deaminating/decarboxylating Pseudomonasl-phenylalanine oxidase (PAO), which has a short N-terminal propeptide composed of 14 residues. Here we show the inactivation mechanism of the l-lysine oxidase (LysOX) precursor (prLysOX), which has a long N-terminal propeptide composed of 77 residues, based on the crystal structure at 1.97 Å resolution. The propeptide of prLysOX indirectly changes the active site structure to inhibit the enzyme activity. prLysOX retains weak enzymatic activity with strict specificity for l-lysine and shows raised activity in acidic conditions. The structures of prLysOX crystals that soaked in a solution with various concentrations of l-lysine have revealed that prLysOX can adopt two conformations; one is the inhibitory form, and the other is very similar to mature LysOX. The propeptide region of the latter form is disordered, and l-lysine is bound to the latter form. These results indicate that prLysOX uses a different strategy from PAO to suppress the enzyme activity and suggest that prLysOX can be activated quickly in response to the environmental change without proteolytic processing.

Fungal Enzyme l-Lysine α-Oxidase Affects the Amino Acid Metabolism in the Brain and Decreases the Polyamine Level.

The fungal glycoprotein l-lysine α-oxidase (LO) catalyzes the oxidative deamination of l-lysine (l-lys). LO may be internalized in the intestine and shows antitumor, antibacterial, and antiviral effects in vivo. The main mechanisms of its effects have been shown to be depletion of the essential amino acid l-lys and action of reactive oxidative species produced by the reaction. Here, we report that LO penetrates into the brain and is retained there for up to 48 h after intravenous injection, which might be explained by specific pharmacokinetics. LO actively intervenes in amino acid metabolism in the brain. The most significant impact of LO was towards amino acids, which are directly exposed to its action (l-lys, l-orn, l-arg). In addition, the enzyme significantly affected the redistribution of amino acids directly associated with the tricarboxylic acid (TCA) cycle (l-asp and l-glu). We discovered that the depletion of l-orn, the precursor of polyamines (PA), led to a significant and long-term decrease in the concentration of polyamines, which are responsible for regulation of many processes including cell proliferation. Thus, LO may be used to reduce levels of l-lys and PA in the brain.

Allosteric Enzyme-Based Biosensors-Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties.

The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme–substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.

Structural basis of strict substrate recognition of l-lysine α-oxidase from Trichoderma viride.

l-Lysine oxidase (LysOX) is a FAD-dependent homodimeric enzyme that catalyzes the oxidative deamination of l-lysine to produce α-keto-ε-aminocaproate with ammonia and hydrogen peroxide. LysOX shows strict substrate specificity for l-lysine, whereas most l-amino acid oxidases (LAAOs) exhibit broad substrate specificity for l-amino acids. Previous studies of LysOX showed that overall structural similarity to the well-studied snake venom LAAOs. However, the molecular mechanism of strict specificity for l-lysine was still unclear. We here determined the structure of LysOX in complex with l-lysine at 1.7 Å resolution. The structure revealed that the hydrogen bonding network formed by D212, D315, and A440 with two water molecules is responsible for the recognition of the side chain amino group. In addition, a narrow hole formed by five hydrophobic residues in the active site contributes to strict substrate specificity. Mutation studies demonstrated that D212 and D315 are essential for l-lysine recognition, and the D212A/D315A double mutant LysOX showed different substrate specificity from LysOX. Moreover, the structural basis of the substrate specificity change has also been revealed by the structural analysis of the mutant variant and its substrate complexes. These results clearly explain the molecular mechanism of the strict specificity of LysOX and suggest that LysOX is a potential candidate for a template to design LAAOs specific to other l-amino acids.

Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle

Bioplastics produced from microbial source are promising green alternatives to traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids, especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential monomers for the synthesis of polymeric bioplastics as their primary amine and carboxylic acid are ideal functional groups for polymerization. Previous pathways for 5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from L-lysine catabolism and the citric acid cycle, respectively. Here, we show the construction of an artificial iterative carbon-chain-extension cycle in Escherichia coli for simultaneous production of a series of nonnatural amino acids with varying chain length. Overexpression of L-lysine α-oxidase in E. coli yields 2-keto-6-aminocaproate (2K6AC) as a non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-extended α-ketoacid products are decarboxylated and oxidized by an α-ketoacid decarboxylase and an aldehyde dehydrogenase, respectively, to yield their corresponding nonnatural straight-chain amino acids. The engineered system demonstrated simultaneous in vitro production of 99.16 mg/L of 5-aminovalerate, 46.96 mg/L of 6-aminocaproate and 4.78 mg/L of 7-aminoheptanoate after 8 h of enzyme catalysis starting from 2K6AC as the substrate. Furthermore, simultaneous production of 2.15 g/L of 5-aminovalerate, 24.12 mg/L of 6-aminocaproate and 4.74 mg/L of 7-aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising metabolic-engineering strategy to access other medium-chain organic acids with –NH2, –SCH3, –SOCH3, –SH, –COOH, –COH, or –OH functional groups through carbon-chain-elongation chemistry.

Amino Acid Degrading Enzymes and their Application in Cancer Therapy.

Amino acids are essential components in various biochemical pathways. The deprivation of certain amino acids is an antimetabolite strategy for the treatment of amino acid-dependent cancers which exploits the compromised metabolism of malignant cells. Several studies have focused on the development and preclinical and clinical evaluation of amino acid degrading enzymes, namely L-asparaginase, L-methionine γ-lyase, L-arginine deiminase, L-lysine α-oxidase. Further research into cancer cell metabolism may therefore define possible targets for controlling tumor growth. The purpose of this review was to summarize recent progress in the relationship between amino acids metabolism and cancer therapy, with a particular focus on Lasparagine, L-methionine, L-arginine and L-lysine degrading enzymes and their formulations, which have been successfully used in the treatment of several types of cancer. We carried out a structured search among literature regarding to amino acid degrading enzymes. The main aspects of search were in vitro and in vivo studies, clinical trials concerning application of these enzymes in oncology. Most published research are on the subject of L-asparaginase properties and it's use for cancer treatment. L-arginine deiminase has shown promising results in a phase II trial in advanced melanoma and hepatocellular carcinoma. Other enzymes, in particular Lmethionine γ-lyase and L-lysine α-oxidase, were effective in vitro and in vivo. The findings of this review revealed that therapy based on amino acid depletion may have the potential application for cancer treatment but further clinical investigations are required to provide the efficacy and safety of these agents.

A novel approach for the selective analysis of l-lysine in untreated human serum by a co-crosslinked l-lysine–α-oxidase/overoxidized polypyrrole bilayer based amperometric biosensor

An amperometric biosensor based on an l-lysine-α-oxidase (LO) layer immobilized by co-crosslinking onto the surface of an overoxidized polypyrrole modified Pt electrode (Pt/oPPy) and able to analyse l-lysine (Lys) in untreated human serum is described. The sensing electrode has been characterised and a proper enzyme kinetics optimisation permits to use a low specific enzyme as LO from Trichoderma viride for the selective biorecognition of Lys in the presence of other interferent amino acids; a kinetics study of LO evidenced also the allosteric behaviour of this enzyme, a kinetic feature which was never reported before for this enzyme. The biosensor showed a sensitivity of 0.11 μA/mM mm2, linear responses up to 4 mM and a limit of detection of 2 μM; the within-a-day coefficients of variation for replicate (n = 5) were 0.92% and 1.35% at 4 mM and 0.2 mM Lys levels, respectively. The permselective behaviour of Pt/oPPy modified electrode assured an interference- and fouling-free determination of Lys even in untreated serum samples. The determination of Lys in human serum from healthy donors gave Lys levels in good agreement with the expected values so that the use of the proposed biosensor appears promising in the relevant clinical fields.

An economically and environmentally acceptable synthesis of chiral drug intermediate L-pipecolic acid from biomass-derived lysine via artificially engineered microbes.

Deficiency in petroleum resources and increasing environmental concerns have pushed a bio-based economy to be built, employing a highly reproducible, metal contaminant free, sustainable and green biomanufacturing method. Here, a chiral drug intermediate L-pipecolic acid has been synthesized from biomass-derived lysine. This artificial bioconversion system involves the coexpression of four functional genes, which encode L-lysine α-oxidase from Scomber japonicus, glucose dehydrogenase from Bacillus subtilis, Δ1-piperideine-2-carboxylase reductase from Pseudomonas putida, and lysine permease from Escherichia coli. Besides, a lysine degradation enzyme has been knocked out to strengthen the process in this microbe. The overexpression of LysP improved the L-pipecolic acid titer about 1.6-folds compared to the control. This engineered microbial factory showed the highest L-pipecolic acid production of 46.7g/L reported to date and a higher productivity of 2.41g/Lh and a yield of 0.89g/g. This biotechnological L-pipecolic acid production is a simple, economic, and green technology to replace the presently used chemical synthesis.