Characterization of a recombinant sucrose isomerase and its application to enzymatic production of isomaltulose.

OBJECTIVE: To characterize a recombinant isomerase that can catalyze the isomerization of sucrose into isomaltulose and investigate its application for the enzymatic production of isomaltulose. RESULTS: A sucrose isomerase gene from Erwinia sp. Ejp617 was synthesized and expressed in Escherichia coli BL21(DE3). The enzymatic characterization revealed that the optimal pH and temperature of the purified sucrose isomerase were 6.0 and 40 °C, respectively. The enzyme activity was slightly activated by Mn2+and Mg2+, but partially inhibited by Ca2+, Ba2+, Cu2+, Zn2+ and EDTA. The kinetic parameters of Km and Vmax for sucrose were 69.28 mM and 118.87 U/mg, respectively. The time course showed that 240.9 g/L of isomaltulose was produced from 300 g/L of sucrose, and the yield reached 80.3% after bioreaction for 180 min. CONCLUSIONS: This recombinant enzyme showed excellent capability for biotransforming sucrose to isomaltulose at the substrate concentration of 300 g/L. Further investigations should be carried out focusing on selection of suitable heterologous expression system with the aim to improve its expression level.

A Randomized Phase I Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of Recombinant Erwinia Asparaginase (JZP-458) in Healthy Adult Volunteers.

L-asparaginase has been an important component of acute lymphoblastic leukemia (ALL) therapy for over 40 years, and is standard therapy during ALL induction and consolidation treatment. L-asparaginases are immunogenic and can induce hypersensitivity reactions; inability to receive asparaginase has been associated with poor patient outcomes. There are L-asparaginases of varied bacterial origins, with the most commonly used being Escherichia coli (E. coli); therefore, to ensure that patients who develop hypersensitivity to E. coli-derived asparaginases receive an adequate therapeutic course, alternative preparations are warranted. JZP-458 is a recombinant Erwinia asparaginase produced using a novel Pseudomonas fluorescens expression platform that yields an enzyme with no immunologic cross-reactivity to E. coli-derived asparaginases. To evaluate the safety, tolerability, and pharmacokinetics (PK) of a single dose of JZP-458, a randomized, single-center, open-label, phase I study was conducted with JZP-458 given via i.m. injection or i.v. infusion to healthy adult volunteers. At the highest doses tested for each route of administration (i.e., 25 mg/m2 i.m. and 37.5 mg/m2 i.v.), JZP-458 achieved serum asparaginase activity (SAA) levels ≥ 0.1 IU/mL at 72 hours postdose for 100% of volunteers. Bioavailability for i.m. JZP-458 was estimated at 36.8% based on SAA data. All dose levels were well-tolerated, with no unanticipated adverse events (AEs), no serious AEs, and no grade 3 or higher AEs. Based on PK and safety data, the recommended JZP-458 starting dose for the pivotal phase II/III study in adult and pediatric patients is 25 mg/m2 i.m. and 37.5 mg/m2 i.v. on a Monday/Wednesday/Friday dosing schedule.

Higher plasma asparaginase activity after intramuscular than intravenous Erwinia asparaginase.

It is unclear if dosing intervals for Erwinase can be extended with intramuscular (i.m.) versus intravenous (i.v.) dosing. Children with acute lymphoblastic leukemia received Erwinase at 30 000-42 000 IU/m2 i.v. or i.m. I.m. Erwinase (n = 22) achieved activity above 0.1 IU/mL for longer than i.v. Erwinase (n = 33) (3.4 vs 2.9 days, P = 0.0007). With 30 000 IU/m2 Monday, Wednesday, Friday, more patients achieved adequate concentrations over the weekend with i.m. vs i.v. dosing (P = 5 × 10-36 ). A schedule with i.v. doses on Monday and Wednesday and i.m. doses on Friday of 30 000 IU/m2 maintained activity > 0.1 IU/mL over the weekend in 80% of patients.

Impact of Asparaginase Discontinuation on Outcome in Childhood Acute Lymphoblastic Leukemia: A Report From the Children's Oncology Group.

PURPOSE: Asparaginase (ASNase) is an important component of acute lymphoblastic leukemia (ALL) treatment, but is often discontinued because of toxicity. Erwinia chrysanthemi ASNase (Erwinia) substitution was approved in 2011 for allergic reactions. Erwinia has, however, been intermittently unavailable because of drug supply issues. The impact of Erwinia substitution or complete ASNase discontinuation is unknown. METHODS: Patients aged 1-30.99 years in frontline Children's Oncology Group trials for B-cell acute lymphoblastic leukemia between 2004 and 2011 (National Cancer Institute [NCI] standard risk [SR]: AALL0331; NCI high risk: AALL0232) were included. The number of prescribed pegaspargase (PEG-ASNase) doses varied by trial and strata. Maintenance therapy did not contain ASNase. Landmark analyses at maintenance compared disease-free survival (DFS) among those receiving all prescribed PEG-ASNase doses versus switching to Erwinia but receiving all doses versus not receiving all ASNase doses. RESULTS: We included 5,195 AALL0331 and 3,001 AALL0232 patients. The cumulative incidence of PEG-ASNase discontinuation was 12.2% ± 4.6% in AALL0331 and 25.4% ± 0.8% in AALL0232. In multivariable analyses, NCI high-risk patients not receiving all prescribed ASNase doses had inferior DFS (hazard ratio [HR], 1.5; 95% CI, 1.2 to 1.9; P = .002) compared with those receiving all prescribed PEG-ASNase doses. Patients with Erwinia substitution who completed subsequent courses were not at increased risk (HR, 1.1; 95% CI, 0.7 to 1.6; P = .69). NCI SR patients who discontinued ASNase were not at elevated risk (HR, 1.2; 95% CI, 0.9 to 1.6; P = .23), except when restricted to those with slow early response, who were prescribed more ASNase because of therapy intensification (HR, 1.7; 95% CI, 1.1 to 2.7; P = .03). CONCLUSION: Discontinuation of ASNase doses is associated with inferior DFS in higher-risk patients. Our results illustrate the severe consequences of Erwinia shortages.

A host target of a bacterial cysteine protease virulence effector plays a key role in convergent evolution of plant innate immune system receptors.

Some virulence effectors secreted from pathogens target host proteins and induce biochemical modifications that are monitored by nucleotide-binding and leucine-rich repeat (NLR) immune receptors. Arabidopsis RIN4 protein (AtRIN4: RPM1-interacting protein 4) homologs are present in diverse plant species and targeted by several bacterial type III effector proteins including the cysteine protease AvrRpt2. RIN4 is 'guarded' by several independently evolved NLRs from various plant species, including Arabidopsis RPS2. Recently, it was shown that the MR5 NLR from a wild apple relative can recognize the AvrRpt2 effector from Erwinia amylovora, but the details of this recognition remained unclear. The present contribution reports the mechanism of AvrRpt2 recognition by independently evolved NLRs, MR5 from apple and RPS2, both of which require proteolytically processed RIN4 for activation. It shows that the C-terminal cleaved product of apple RIN4 (MdRIN4) but not AtRIN4 is necessary and sufficient for MR5 activation. Additionally, two polymorphic residues in AtRIN4 and MdRIN4 are identified that are crucial in the regulation of and physical association with NLRs. It is proposed that polymorphisms in RIN4 from distantly related plant species allow it to remain an effector target while maintaining compatibility with multiple NLRs.

Economical production of isomaltulose from agricultural residues in a system with sucrose isomerase displayed on Bacillus subtilis spores.

A safe, efficient, environmentally friendly process for producing isomaltulose is needed. Here, the biocatalyst, sucrose isomerase (SIase) from Erwinia rhapontici NX-5, displayed on the surface of Bacillus subtilis 168 spores (food-grade strain) was applied for isomaltulose production. The anchored SIase showed relatively high bioactivity, suggesting that the surface display system using CotX as the anchoring protein was successful. The stability of the anchored SIase was also significantly better. Thermal stability analysis showed that 80% of relative activity was retained after incubation at 40 °C and 45 °C for 60 min. To develop an economical industrial fermentation medium, untreated beet molasses (30 g/L) and cold-pressed soybean powder (50 g/L) were utilised as the main broth components for SIase pilot-scale production. Under the optimal conditions, the productive spores converted 92% of sucrose after 6 h and the conversion rate was 45% after six cycles. Isomaltulose production with this system using the agricultural residues, untreated beet molasses and soybean powder, as substrates is cost-effective and environmentally friendly and can help to overcome issues due to the genetic background.

Production of L-tyrosine using tyrosine phenol-lyase by whole cell biotransformation approach.

L-tyrosine is an amino acid that has been widely used in the food, agriculture and pharmaceutical industries. In order to screen a tyrosine phenol-lyase (TPL) with excellent catalytic performance for L-tyrosine production, TPL genes from Citrobacter freundii (CfTPL), Erwinia herbicola (EhTPL) and Rhodobacter capsulatus (TutA) were codon-optimized and overexpressed in Escherichia coli. The results showed that EhTPL had the highest whole cell catalysis activity and tyrosine yield (3-fold that of CfTPL). The results of RT-qPCR and a stability analysis also revealed that EhTPL had a higher transcriptional level in whole cell catalysis, while CfTPL possessed greater stability. Conditions for the production by whole cell transformation were optimized in terms of reaction conditions and fed-batch strategy. Finally, the maximum production was obtained with a titer of 48.5 g·L-1 by intermittent feeding with a conversion ratio of 75%.

Delineation of the functional and structural properties of the glutathione transferase family from the plant pathogen Erwinia carotovora.

Erwinia carotovora, a widespread plant pathogen that causes soft rot disease in many plants, is considered a major threat in agriculture. Bacterial glutathione transferases (GSTs) play important roles in a variety of metabolic pathways and processes, such as the biodegradation of xenobiotics, protection against abiotic stress, and resistance against antimicrobial drugs. The GST family of canonical soluble enzymes from Erwinia carotovora subsp. atroseptica strain SCRI1043 (EcaGSTs) was investigated. Genome analysis showed the presence of six putative canonical cytoplasmic EcaGSTs, which were revealed by phylogenetic analysis to belong to the well-characterized GST classes beta, nu, phi, and zeta. The analysis also revealed the presence of two isoenzymes that were phylogenetically close to the omega class of GSTs, but formed a distinct class. The EcaGSTs were cloned and expressed in Escherichia coli, and their catalytic activity toward different electrophilic substrates was elucidated. The EcaGSTs catalyzed different types of reactions, although all enzymes were particularly active in reactions involving electrophile substitution. Gene and protein expression profiling conducted under normal culture conditions as well as in the presence of the herbicide alachlor and the xenobiotic 1-chloro-2,4-dinitrobenzene (CDNB) showed that the isoenzyme EcaGST1, belonging to the omega-like class, was specifically induced at both the protein and mRNA levels. EcaGST1 presumably participates in counteracting the xenobiotic toxicity and/or abiotic stress conditions, and may therefore represent a novel molecular target in the development of new chemical treatments to control soft rot diseases.

A cost-effectiveness analysis of Erwinia asparaginase therapy in children with acute lymphoblastic leukemia.

OBJECTIVES: Erwinia asparaginase is used as a second-line formulation after a neutralizing hypersensitivity reaction to the first-line formulation of asparaginase. Here, we have performed a cost-effectiveness analysis of Erwinia asparaginase treatment. METHODS: Children with acute lymphoblastic leukemia treated according to the Dutch Childhood Oncology ALL-10 or ALL-11 protocol were included and initially treated with PEGasparaginase in the intensification phase. The total treatment costs of this treatment phase, quality of life (QoL), and life years saved (LYS) were studied for two scenarios: (a) patients were switched to Erwinia asparaginase treatment after a hypersensitivity reaction, or (b) asparaginase would have been permanently stopped. RESULTS: Sixty-eight patients were included. There was no difference in QoL between patients with and without a hypersensitivity reaction. The mean costs of the intensification phase per patient were $40,925 if PEGasparaginase could be continued, $175,632 if patients had to switch to Erwinia asparaginase, and $21,190 if asparaginase would have been permanently stopped. An extrapolation of the literature suggests that the 5-year event-free survival would be 10.3% lower without intensive asparaginase treatment if asparaginase is stopped after a reaction. Thus, the costs per LYS were $1892 for scenario 1 and $872 for scenario 2. CONCLUSIONS: Switching to Erwinia asparaginase increases the costs per LYS by $1020, which is modest in view of the total costs. Moreover, when asparaginase treatment can be completed by switching to Erwinia asparaginase, relapses-and consequential costs-will be avoided. Therefore, from a cost perspective, we recommend a switch to Erwinia asparaginase to complete asparaginase treatment.

Probing the specificity of CYP112 in bacterial gibberellin biosynthesis.

Biosynthesis of the gibberellin A (GA) plant hormones evolved independently in plant-associated fungi and bacteria. While the relevant enzymes have distinct evolutionary origins, the pathways proceed via highly similar reactions. One particularly complex transformation involves combined demethylation and γ-lactone ring formation, catalyzed in bacteria by the cytochrome P450 CYP112 in three individual steps, which involves large structural changes in the transition from substrate to product, with further divergence in the recently demonstrated use of two separate mechanistic routes. Here, the substrate specificity of the isozyme from Erwinia tracheiphila, EtCYP112, was probed via UV-Vis spectral binding studies and activity assays with alternate substrates from the GA biosynthetic pathway. EtCYP112 tightly binds its native substrate GA12 and reaction intermediates GA15 and GA24, as well as the methylated derivatives of GA12 and GA15 It, however, only poorly binds methylated GA24, its GA9 final product and the C-20 carboxylate side product GA25 These distinct affinities are consistent with the known reactivity of EtCYP112. However, while it binds to the immediately preceding pathway metabolite GA12-aldehyde and even earlier oxygenated ent-kaurene precursors, EtCYP112 only reacts with GA12-aldehyde and not the earlier ent-kaurene-derived metabolites. Even with GA12-aldehyde conversion is limited to the first two steps, and the full combined demethylation and γ-lactone ring-forming transformation is not catalyzed. Thus, CYP112 has evolved specificity at the catalytic rather than substrate-binding level to enable its role in GA biosynthesis.

Purification, characterization, and gene cloning of a new cold-adapted ß-galactosidase from Erwinia sp. E602 isolated in northeast China.

ß-Galactosidases are widely used in industry for elimination of lactose from milk products. A new ß-galactosidase was obtained from bacterial strain Erwinia sp. E602, newly isolated in northeast China. The enzyme was purified with the methods of ammonium sulfate fractionation, ion exchange, and gel filtration chromatography for further study of the enzymatic characteristics. The purified enzyme had a molecular weight of near 110 kDa. The optimum reaction temperature and pH of this enzyme was determined to be 40°C and 7.0, respectively, indicating that this enzyme was a mesophilic neutral ß-galactosidase. Furthermore, the enzyme retained near 10% of the activity at 0°C, which also suggested its cold-adapted property. Kinetics of the ß-galactosidase was studied, and the Km (Michaelis constant) and Vmax (maximum enzymatic reaction rate) of this enzyme were 0.21 mmol/L and 263.16 µmol/mg per minute, respectively. The effects of metal ions on the enzymatic activity and the lactose hydrolysis efficiency in milk, as well as its trans-glycosylation activity, were studied in this work. The ß-galactosidase coding gene was cloned to be a 3-kb length fragment, which shared at most 81% of identity with the published sequences in NCBI Blast database (https://blast.ncbi.nlm.nih.gov). Results in this work suggested it is a new ß-galactosidase and it has potential to be used in dairy and food processing.

Outcome of pediatric patients with acute lymphoblastic leukemia/lymphoblastic lymphoma with hypersensitivity to pegaspargase treated with PEGylated Erwinia asparaginase, pegcrisantaspase: A report from the Children's Oncology Group.

BACKGROUND: Erwinia asparaginase is a Food and Drug Administration approved agent for the treatment of acute lymphoblastic leukemia (ALL) for patients who develop hypersensitivity to Escherichia coli derived asparaginases. Erwinia asparaginase is efficacious, but has a short half-life, requiring six doses to replace one dose of the most commonly used first-line asparaginase, pegaspargase, a polyethylene glycol (PEG) conjugated E. coli asparaginase. Pegcristantaspase, a recombinant PEGylated Erwinia asparaginase with improved pharmacokinetics, was developed for patients with hypersensitivity to pegaspargase. Here, we report a series of patients treated on a pediatric phase 2 trial of pegcrisantaspase. PROCEDURE: Pediatric patients with ALL or lymphoblastic lymphoma and hypersensitivity to pegaspargase enrolled on Children's Oncology Group trial AALL1421 (Jazz 13-011) and received intravenous pegcrisantaspase. Serum asparaginase activity (SAA) was monitored before and after dosing; immunogenicity assays were performed for antiasparaginase and anti-PEG antibodies and complement activation was evaluated. RESULTS: Three of the four treated patients experienced hypersensitivity to pegcrisantaspase manifested as clinical hypersensitivity reactions or rapid clearance of SAA. Immunogenicity assays demonstrated the presence of anti-PEG immunoglobulin G antibodies in all three hypersensitive patients, indicating a PEG-mediated immune response. CONCLUSIONS: This small series of patients, nonetheless, provides data, suggesting preexisting immunogenicity against the PEG moiety of pegaspargase and poses the question as to whether PEGylation may be an effective strategy to optimize Erwinia asparaginase administration. Further study of larger cohorts is needed to determine the incidence of preexisting antibodies against PEG-mediated hypersensitivity to pegaspargase.

Plasma asparaginase activity, asparagine concentration, and toxicity after administration of Erwinia asparaginase in children and young adults with acute lymphoblastic leukemia: Phase I/II clinical trial in Japan.

BACKGROUND: A phase I/II study of Erwinia asparaginase in Japanese children and young adults with acute lymphoblastic leukemia (ALL) was performed to investigate its activity and toxicity. PROCEDURE: Eligible patients were in remission and had developed allergy to Escherichia coli asparaginase. Erwina asparaginase was intramuscularly administrated on days 2, 5, 7, 9, 11, and 13. To measure the plasma l-asparagine concentration (PAC), amino acids were derivatized with Nα -(5-fluoro-2,4-dinitrophenyl)-l-leucinamide. RESULTS: Six consecutive patients completed the phase I study with 25,000 IU/m2 per dose without dose-limiting toxicity and 18 patients completed the phase II study with 25,000 IU/m2 per dose. Median age of 24 patients was 7.5 (range 2-16) years. The half-life of plasma asparaginase activity (PAA) was 16.9 ± 7.5 hr and the maximum PAA was 3.10 ± 1.47 IU/ml (n = 23, noncompartment model). PAA of 0.1 IU/ml or more was achieved in all 23 patients (100%) 48 hr and in 18 of 23 patients (78.3%) 72 hr after the first administration. During the 2-week study, 94.2% (65 of 69) of the 48-hr samples and 80.4% (37 of 46) of the 72-hr samples had PAA of 0.1 IU/ml or more. PAC less than 1.0 µM was achieved in 95.7% patients 48 and 72 hr after administration. PAC values in all the samples were greater than the limit of quantitation (0.0625 µM). Karnofsky performance status of all patients was good during the 2-week study. CONCLUSIONS: Erwinia asparaginase 25,000 IU/m2 per dose × six intramuscular administrations in 2 weeks was well tolerated, pharmacologically efficacious, and safe in Japanese patients with ALL/lymphoblastic lymphoma.

Robust quantitation of basic-protein higher-order aggregates using size-exclusion chromatography.

Detection of higher-order aggregates (HOA) using size-exclusion chromatography (SEC) was found to be variable for a basic protein, using exposed-silanol or diol-silica-based SEC columns. Preparations of the tetrameric biopharmaceutical enzyme Erwinia chrysanthemil-asparaginase (ErA), which has an isoelectric point of 8.6, were analysed using a diol-silica SEC column. Although the proportions of ErA main peak and octamer species were unaffected, HOA recovery and detection were extremely variable and had poor agreement with an orthogonal measurement technique, analytical ultracentrifugation (AUC). The observation that only HOA was selectively affected by non-specific silanol interactions was unexpected, so alternatives were sought. Coated-silica SEC columns improved the resolution and reproducibility of HOA detection for this alkaline-pI protein, and improved the agreement of HOA with the AUC method. Basic proteins, such as ErA, should be thoroughly evaluated in SEC method development, to ensure that resolution of larger aggregate species is not compromised.

The role of autophagy in asparaginase-induced immune suppression of macrophages.

Erwinia asparaginase, a bacteria-derived enzyme drug, has been used in the treatment of various cancers, especially acute lymphoblastic leukemia (ALL). One of the most significant side effects associated with asparaginase administration is immune suppression, which limits its application in clinic. Macrophages are phagocytic immune cells and have a central role in inflammation and host defense. We reported here that asparaginase disturbed the function of macrophages including phagocytosis, proliferation, ROS and nitric oxide secretion, interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) secretion, and major histocompatibility complex II (MHC-II) molecule expression, thus induced immune suppression in interferon-γ and lipopolysaccharide-stimulated macrophages. We also observed that asparaginase inhibited autophagy in macrophages via activating Akt/mTOR and suppressing Erk1/2 signaling pathway as evidenced by less formation of autophagosomes, downregulation of autophagy-related protein LC3-II, and decreased number of autophagy-like vacuoles. Further study discovered that treatment with autophagy inhibitor 3-MA in place of asparaginase on activated macrophages could also downregulate phagocytosis, cytokine secretion, and MHC-II expression. Moreover, incubation with autophagy inducer trehalose restored the capacity of phagocytosis, IL-6 and TNF-α secretion, and MHC-II expression in macrophages. These results prove the important role of autophagy in the function of macrophages, and activation of autophagy can overcome asparaginase-induced immune suppression in macrophages.

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass.

Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a ß-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5-6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

The use of Erwinia asparaginase for adult patients with acute lymphoblastic leukemia after pegaspargase intolerance.

Asparaginase administration has become a crucial component of front-line pediatric and pediatric-insipired multi-agent regimens for the treatment of acute lymphoblastic leukemia (ALL). The aim of this retrospective study was to assess the safety and feasibility of switching to Erwinia asparaginase after pegaspargase intolerance in adult ALL patients treated at Memorial Sloan Kettering Cancer Center. Our analysis included 10 patients, with a median age of 39 years (range 20-72), male predominance (90%), and a typical B-cell to T-cell ratio (70:30%) for ALL. Nine patients were switched to Erwinia asparaginase after pegaspargase hypersensitivity and one patient after grade 4 hyperbilirubinemia secondary to pegaspargase. With Erwinia asparaginase, no hypersensitivity reactions occurred and no patient developed other known clinical asparaginase-related toxicities. Laboratory adverse effects consisted of mostly mild elevation in liver enzymes. No morphologic relapses have occurred in any patient switched to Erwinia asparaginase in first remission at a follow up of 0.4-34.6 months. These findings are unique in that all of our patients received Erwinia asparaginase after hypersensitivity or intolerance to pegaspargase and 50% of them were older than 40 years of age, a population with very limited Erwinia asparaginase data. Our observations provide preliminary information that treatment with Erwinia asparaginase can proceed as scheduled in adult patients, despite pegaspargase hypersensitivity and possibly liver intolerance.

Overview on the biotechnological production of L-DOPA.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) has been widely used as a drug for Parkinson's disease caused by deficiency of the neurotransmitter dopamine. Since Monsanto developed the commercial process for L-DOPA synthesis for the first time, most of currently supplied L-DOPA has been produced by the asymmetric method, especially asymmetric hydrogenation. However, the asymmetric synthesis shows critical limitations such as a poor conversion rate and a low enantioselectivity. Accordingly, alternative biotechnological approaches have been researched for overcoming the shortcomings: microbial fermentation using microorganisms with tyrosinase, tyrosine phenol-lyase, or p-hydroxyphenylacetate 3-hydroxylase activity and enzymatic conversion by immobilized tyrosinase. Actually, Ajinomoto Co. Ltd commercialized Erwinia herbicola fermentation to produce L-DOPA from catechol. In addition, the electroenzymatic conversion system was recently introduced as a newly emerging scheme. In this review, we aim to not only overview the biotechnological L-DOPA production methods, but also to briefly compare and analyze their advantages and drawbacks. Furthermore, we suggest the future potential of biotechnological L-DOPA production as an industrial process.