A Transferable IncC-IncX3 Hybrid Plasmid Cocarrying blaNDM-4, tet(X), and tmexCD3-toprJ3 Confers Resistance to Carbapenem and Tigecycline.

Tigecycline is a last-resort antimicrobial against carbapenemase-producing Enterobacterales (CPE). However, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, have emerged in China and have spread possibly worldwide. Tet(X) family proteins function as tigecycline-inactivating enzymes, and TMexCD-TOprJ complexes function as efflux pumps for tigecycline. Here, to the best of our knowledge we report a CPE isolate harboring both emerging tigecycline resistance factors for the first time. A carbapenem- and tigecycline-resistant Klebsiella aerogenes strain, NUITM-VK5, was isolated from an urban drainage in Vietnam in 2021, and a plasmid, pNUITM-VK5_mdr, cocarrying tet(X) and tmexCD3-toprJ3 along with the carbapenemase gene blaNDM-4 was identified in NUITM-VK5. pNUITM-VK5_mdr was transferred to Escherichia coli by conjugation and simultaneously conferred high-level resistance against multiple antimicrobials, including carbapenems and tigecycline. An efflux pump inhibitor reduced TMexCD3-TOprJ3-mediated tigecycline resistance, suggesting that both tigecycline resistance factors independently and additively contribute to the high-level resistance. The plasmid had the IncX3 and IncC replicons and was estimated to be a hybrid of plasmids with different backbones. Unlike IncX3 plasmids, IncC plasmids are stably maintained in an extremely broad range of bacterial hosts in humans, animals, and the environment. Thus, the future global spread of multidrug resistance plasmids such as pNUITM-VK5_mdr poses a public health crisis. IMPORTANCE Tigecycline is important as a last-resort antimicrobial and effective against antimicrobial-resistant bacteria, such as carbapenem-producing Enterobacterales (CPE), whose infections are difficult to treat with antimicrobials. Since 2019, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, and their variants have been reported mainly from China, and it has become important to understand their epidemiological situation and detailed genetic mechanisms. In this study, we identified a bacterial isolate coharboring tet(X) and tmexCD-toprJ on the same plasmid. A Klebsiella aerogenes isolate in Vietnam carried both these tigecycline resistance genes on a transferable plasmid leading to high-level resistance to multiple clinically important antimicrobials, including carbapenem and tigecycline, and could actually transfer the plasmid to other bacteria. The spread of such a multidrug resistance plasmid among bacterial pathogens should be of great concern because there are few antimicrobials to combat bacteria that have acquired the plasmid.

Improving the catalytic efficiency and substrate affinity of a novel esterase from marine Klebsiella aerogenes by random and site-directed mutation.

A novel esterase (EstKa) from marine Klebsiella aerogenes was characterized with hydrolytic activity against p-nitrophenyl caprylate (pNPC, C8) under optimum conditions (50 °C and pH 8.5). After two rounds of mutagenesis, two highly potential mutants (I6E9 and L7B11) were obtained with prominent activity, substrate affinity and thermostability. I6E9 (L90Q/P96T) and L7B11 (A37S/Q100L/S133G/R138C/Q156R) were 1.56- and 1.65-fold higher than EstKa in relative catalytic efficiency. The influence of each amino acid on enzyme activity was explored by site-directed mutation. The mutants Pro96Thr and Gln156Arg showed 1.29- and 1.48-fold increase in catalytic efficiency (Kcat/Km) and 54.4 and 36.2% decrease in substrate affinity (Km), respectively. The compound mutant Pro96Thr/Gln156Arg exhibited 68.9% decrease in Km and 1.41-fold increase in Kcat/Km relative to EstKa. Homology model structure analysis revealed that the replacement of Gln by hydrophilic Arg on the esterase surface improved the microenvironment stability and the activity. The replacement of Pro by Thr enabled the esterase enzyme to retain 90% relative activity after 3 h incubation at 45 °C. Structural analysis confirmed that the formation of a hydrogen bond leads to a notable increase of catalytic efficiency under high temperature conditions.

Rapid Broad Spectrum Detection of Carbapenemases with a Dual Fluorogenic-Colorimetric Probe.

Carbapenems stand as one of the last-resort antibiotics; however, their efficacy is threatened by the rising number and rapid spread of carbapenemases. Effective antimicrobial stewardship thus calls for rapid tests for these enzymes to aid appropriate prescription and infection control. Herein, we report the first effective pan-carbapenemase reporter CARBA-H with a broad scope covering all three Ambler classes. Using a chemical biology approach, we demonstrated that the absence of the 1ß-substituent in the carbapenem core is key to pan-carbapenemase recognition, which led to our rational design and probe development. CARBA-H provides a dual colorimetric-fluorogenic response upon carbapenemase-mediated hydrolysis. A clear visual readout can be obtained within 15 min when tested against a panel of carbapenemase-producing Enterobacteriaceae (CPE) clinical isolates that notably includes OXA-48 and OXA-181-producing strains. Furthermore, CARBA-H can be applied to the detection of carbapemenase activity in CPE-spiked urine samples.

Gene expression of microbial gelatinase activity for porcine gelatine identification.

In order to invent a porcine gelatine detection device using microbial resources, bacterial enzymes with a preference towards porcine gelatine and their candidate genes were evaluated. Five (n = 5) bacterial strains isolated from hot spring water and wet clay, Malaysia were screened for their gelatinase activity. The gelatinase enzyme was extracted and purified using ammonium sulphate precipitation prior to performing gelatinase assay on porcine, bovine and fish gelatine medium substrates. The G2 strain or Enterobacter aerogenes (Strain EA1) was selected for whole genome sequenced after showing a consistent trend of preference towards porcine gelatine. The gelatinase candidate gene gelEA1_9 was cloned and expressed. Based on one-way analysis of variance (ANOVA) with POST-HOC Duncan test (α = 0.05), the final product of gelEA1_9 was identified as a novel gelatinase. This gelatinase presented no significant difference in activity towards porcine gelatine. Hence, the present study demonstrated an enzyme-substrate interaction for porcine gelatine identification.

Production and immobilization of ß-galactosidase isolated from Enterobacter aerogenes KCTC2190 by entrapment method using agar-agar organic matrix.

In the present study, Enterobacter aerogenes KCTC2190 was isolated from soil around a cattle shed area, which was capable of producing intracellular ß-galactosidase. Partially purified ß-galactosidase was immobilized by entrapment method in agar-agar gel matrix. Agar-agar entrapped beads were prepared by dropping the enzyme-agar solution to ice-cooled toluene-chloroform ((3:1 (v/v)). 45.88±0.11% activity of partially purified ß-galactosidase was retained after immobilization (bead shape). Maximum immobilization yield was observed in the presence of 2.5% agar-agar concentration. After immobilization, optimum temperature required for the enzyme-substrate reaction was shifted from 50 to 60 °C and the optimum reaction time was shifted from 15 to 25 min. The optimum pH for both free and immobilized ß-galactosidase was pH 7. Free enzyme showed lower activation energy in comparison with the immobilized one. For free as well as immobilized ß-galactosidase thermal deactivation, rate constant (kd) increased with increasing temperature while the values of decimal reduction time (D-values) and half-lives (t1/2) decreased. Immobilization process increased the t1/2 and D-values of ß-galactosidase while it decreased the kd. Thermostability of immobilized ß-galactosidase was higher as they showed higher enthalpy (ΔΗ0) and Gibb's free energy (ΔG0)value than those of the free ß-galactosidase. The negative entropy (ΔS0) of free and immobilized ß-galactosidase established that both were in a more ordered state within the temperature range (50 to 70 °C) studied. Immobilized ß-galactosidase was able to retain 51.65±1.61% of its initial activity after 7 batches of enzyme-substrate reaction. Immobilized ß-galactosidase showed 78.09±3.69% of its initial activity even after 40 days of storage at 4 °C.

The structure-based reaction mechanism of urease, a nickel dependent enzyme: tale of a long debate.

This review is an attempt to retrace the chronicle that starts from the discovery of the role of nickel as the essential metal ion in urease for the enzymatic catalysis of urea, a key step in the biogeochemical cycle of nitrogen on Earth, to the most recent progress in understanding the chemistry of this historical enzyme. Data and facts are presented through the magnifying lenses of the authors, using their best judgment to filter and elaborate on the many facets of the research carried out on this metalloenzyme over the years. The tale is divided in chapters that discuss and describe the results obtained in the subsequent leaps in the knowledge that led from the discovery of a biological role for Ni to the most recent advancements in the comprehension of the relationship between the structure and function of urease. This review is intended not only to focus on the bioinorganic chemistry of this beautiful metal-based catalysis, but also, and maybe primarily, to evoke inspiration and motivation to further explore the realm of bio-based coordination chemistry.

Expression, homology modeling and enzymatic characterization of a new ß-mannanase belonging to glycoside hydrolase family 1 from Enterobacter aerogenes B19.

BACKGROUND: ß-mannanase can hydrolyze ß-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, ß-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. ß-mannanase is widespread in various organisms, however, microorganisms are the main source of ß-mannanases. Microbial ß-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial ß-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce ß-mannanase, which shows more advantages than traditional microbial fermentation in various aspects. RESULTS: A ß-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5-8.5. The ß-mannanase activity was activated by low concentration of Co2+, Mn2+, Zn2+, Ba2+ and Ca2+. Man1E showed the highest affinity for Locust bean gum (LBG). The Km and Vmax values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 µmol/(mL min), respectively. CONCLUSIONS: A new type of ß-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported ß-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.

Bioremediation of malachite green by cyanobacterium Synechococcus elongatus PCC 7942 engineered with a triphenylmethane reductase gene.

Malachite green is a carcinogenic dye that has been detected in fish tissues and freshwater. Here we evaluated the malachite green decoloring ability of a photoautotrophic cyanobacterium, Synechococcus elongatus PCC 7942 (Synechococcus), that lives in freshwater. Results show that 99.5% of the dye was removed by Synechococcus through bioabsorption and bioaccumulation; however, the dye was not degraded or chemically modified. Next, we established an engineered Synechococcus strain to degrade the dye after uptake. The triphenylmethane reductase gene katmr was heterologously expressed, resulting in high production of a soluble recombinant protein. The engineered strain showed advanced decoloring abilities at a low cell density and in stressful environments. It degraded malachite green into the smaller molecules 4-methylaminobenzoic acid and 4-hydroxyl-aniline. After treatment with the engineered cyanobacterium, the growth of wheat seeds was fully recovered in the presence of malachite green. These results demonstrate the potential application of the engineered Synechococcus as a photosynthetic cell factory for the removal of malachite green from wastewater.

Outbreak of Efficiently Transferred Carbapenem-Resistant blaNDM-Producing Gram-Negative Bacilli Isolated from Neonatal Intensive Care Unit of an Indian Hospital.

The emergence of blaNDM particularly in Gram-negative bacteria is a burden on the health care system in developing countries. Hence, this study was initiated to screen New Delhi Metallo-ß-lactamase (NDM)-producing Gram-negative bacterial strains from neonatal intensive care unit (NICU) of an Indian Hospital. A total of 18 blaNDM-producing isolates were detected in the present study. Out of 18 blaNDM variant isolates, 6 were Klebsiella pneumoniae, 4 Escherichia coli, 2 Enterobacter aerogenes, 1 Acinetobacter lwoffii, 1 Enterobacter cloacae, 3 Acinobacter baumannii, and 1 Cedecea davisae from NICU, showing resistance against all antibiotics, except colistin and polymixin. The transferability of resistance determinants was tested by conjugation. Transfer of blaNDM-producing strains was successful in all 18 strains. In the case of transconjugants, the minimum inhibitory concentration values were found to decrease. The blaNDM-producing isolates contained detectable plasmids of size 66, 38, and 6 kb. Plasmi/d-based replicon typing revealed the incompatibility types Inc (A/C, FIIA, FIC, K, F, W, FIA, P, X, FIB, B/O) in blaNDM-carrying isolates. This study revealed the outbreak of multiple variants of blaNDM (13 NDM-1, 4 NDM-5, and 1 NDM-7). Moreover, other resistance markers, viz. blaOXA-1, blaCMY-1, blaVIM-1, and blaSHV-1 coassociated with blaNDM were also found. In this study, we reported NDM-producing C. davisae as a first report to the best of our knowledge. This study is an attempt to reveal the dissemination of blaNDM isolated from neonates in NICU and their efficient transferability among Gram-negative bacilli through horizontal gene transfer.

Structure and catalytic mechanistic insight into Enterobacter aerogenes acetolactate decarboxylase.

α-Acetolactate decarboxylase (ALDC) catalyses α-acetolactate into acetoin (3-hydroxy-2-butanone, AC) and is considered to be the rate-limiting enzyme in the synthesis of 2,3-butanediol. In this work, the enzymatic activity of ALDC from Enterobacter aerogenes ALDC (E.a.-ALDC) was fully characterized with enzyme kinetics, indicating a Km of 14.83 ± 0.87 mM and a kcat of 0.81 ± 0.09 s-1. However, compared with the activities of ALDCs reported from other bacteria, the activity of E.a.-ALDC was determined to present a relatively lower value of 849.08 ± 35.21 U/mg. The enzyme showed maximum activity at pH 5.5. In addition, the activity of E.a.-ALDC was promoted by Mg2+. The crystal structure of E.a.-ALDC firstly solved by X-ray crystallography at resolution of 2.4 Å revealed a chelated zinc ion with conserved His199, His201, His212, Glu70 and Glu259. In the active center, the conservative Arg150 was particularly proven to deviate from the zinc ion of the active centre, by adopting a flexible conformational change, resulting in a weak interaction network of the enzyme and the substrate. Further in silico docking of E.a.-ALDC with two enantiomers, (R)-acetolactate and (S)-acetolactate, unaltered the interaction network of E.a.-ALDC from the apo structure, which confirmed the weakened role of Arg150 in the catalytic properties of E.a.-ALDC. Our results reveal a unique structure-function relationship of acetolactate decarboxylase and provide a fundamental basis for the enzymatic synthesis of acetoin.

Enhancement of alfalfa yield and quality by plant growth-promoting rhizobacteria under saline-alkali conditions.

BACKGROUND: Bacteria with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity could decrease the ethylene level, confer resistance of plant, and stimulate plant growth under biotic and abiotic stress conditions. RESULTS: Plant growth-promoting rhizobacteria (PGPR) strains Enterobacter aerogenes (LJL-5) and Pseudomonas aeruginosa (LJL-13) were obtained from the rhizosphere of alfalfa grown under saline-alkali conditions. The ACC deaminase activity of E. aerogenes (LJL-5) and Ps. aeruginosa (LJL-13) was approximately 2-5 µmol mg-1  h-1 . indole acetic acid synthesis was increased with the increasing concentration of l-tryptophan. Siderophore production and phosphate solubilization in Ps. aeruginosa (LJL-13) were higher than those in E. aerogenes (LJL-5). Compared to the non-inoculated seedlings (1.31 ng mL-1  h-1 ), inoculated alfalfa seedlings with E. aerogenes (LJL-5) (0.90 ng mL-1  h-1 ) and Ps. aeruginosa (LJL-13) (0.78 ng mL-1  h-1 ) emitted lower levels of ethylene. Under saline-alkali conditions in the greenhouse, inoculation with E. aerogenes (LJL-5) and Ps. aeruginosa (LJL-13) increased the biomass, soil and plant analyzer development (SPAD), and P content of alfalfa plants, and also induced the activity of antioxidant enzymes (superoxide dismutase, peroxidase and catalase), promoted the accumulation of antioxidant substances and removed various harmful substances. Under saline-alkali conditions in the field (2012, 2013, and 2014), inoculation of alfalfa with E. aerogenes (LJL-5) and Ps. aeruginosa (LJL-13) significantly increased the shoot height, fresh and dry weights, yield and crude protein content of alfalfa plants, but decreased the fiber content. CONCLUSION: Two PGPR strains were isolated from the rhizosphere of alfalfa in saline-alkali conditions. Both strains could promote alfalfa growth in saline-alkali soil, and could be used as biofertilizer to promote plant growth under stress and reduce environmental pollution caused by fertilizers simultaneously. © 2018 Society of Chemical Industry.

Emergence of IMP-4-Producing Enterobacter aerogenes Clinical Isolate.

BACKGROUND: IMP-4 class B metallo-ß-lactamase-producing Enterobacteriaceae are resistant to carbapenems. The aim of this study was to characterize of IMP-4 metallo-ß-lactamase (MBL)-producing Enterobacter aerogenes clinical isolate. METHODS: IMP-4 MBL-producing E. aerogenes clinical isolate was collected from a Korean Hospital in 2017. Antimicrobial susceptibility was determined by disk diffusion methods. Further, minimum inhibitory concentrations of ß-lactams were determined by Etest. Detection of bla genes was performed by PCR. The genetic organization of class 1 integron carrying the MBL gene cassette was investigated by PCR mapping and sequencing. RESULTS: E. aerogenes strain YN170501 exhibited resistance to penicillins, cephalosporins, and carbapenems and was susceptible to monobactam, aminoglycosides, fluoroquinolone, tigecycline, and trimethoprim-sulfamethoxazole. The blaIMP-4 gene was located in class 1 integron. CONCLUSIONS: The blaIMP-4 gene has never been reported in Enterobacter aerogenes clinical isolate from Korea.

Detection of virulence and ß-lactamase encoding genes in Enterobacter aerogenes and Enterobacter cloacae clinical isolates from Brazil.

Enterobacter cloacae and E. aerogenes have been increasingly reported as important opportunistic pathogens. In this study, a high prevalence of multi-drug resistant isolates from Brazil, harboring several ß-lactamase encoding genes was found. Several virulence genes were observed in E. aerogenes, contrasting with the E. cloacae isolates which presented none.

A Structural Model of the Urease Activation Complex Derived from Ion Mobility-Mass Spectrometry and Integrative Modeling.

The synthesis of active Klebsiella aerogenes urease via an 18-subunit enzyme apoprotein-accessory protein pre-activation complex has been well studied biochemically, but thus far this complex has remained refractory to direct structural characterization. Using ion mobility-mass spectrometry, we characterized several protein complexes between the core urease apoprotein and its accessory proteins, including the 610-kDa (UreABC)3(UreDFG)3 complex. Using our recently developed computational modeling workflow, we generated ensembles of putative (UreABC)3(UreDFG)3 species consistent with experimental restraints and characterized the structural ambiguity present in these models. By integrating structural information from previous studies, we increased the resolution of the ion mobility-mass spectrometry-derived models substantially, and we observe a discrete population of structures consistent with all of the available data for this complex.

Screening, nutritional optimization and purification for phytase produced by Enterobacter aerogenes and its role in enhancement of hydrocarbons degradation and biofilm inhibition.

In this study, a novel isolate of Enterobacter aerogenes isolated from contaminated soils with hydrocarbons had extracellular phytate-degrading activity. Enterobacter aerogenes isolates were identified by biochemical tests and confirmed by16S rRNA gene products (amplified size 211bp) for genotypic detection. The phytase activity was reached to maximum activity when this isolate was cultivated under the optimal conditions which consisted of using minimal salt medium containing 1%(w/v) rice bran as a sole source for carbon and 2% (w/v) yeast extract at pH 5.5 and temperature of 50°C for 48 h. The phytase had purified to homogeneity by 50% ammonium sulphate precipitation, ion exchange and gel filtration chromatography with 75.7 fold of purification and a yield of 30.35%. The purified phytase is a single peptide with approximate molecular mass of 42 kDa as assessed by SDS-PAGE. The highest degradative ability by Enterobacter aerogenes of black oil, white oil and used engine oil had observed after 72 h of incubation. Rapid degradation of black oil and used engine oil had also observed while slow degradation of white oilat all time of incubation. The purified phytase inhibited biofilm formation ability in a dose-dependent manner for all Gram-negative and Gram-positive biofilm-forming bacteria and a significant difference in cell surface hydrophobicity was observed after exposure of planktonic cells to phytase for hour. The hydrolyzing effect of phytase released by Enterobacter aerogenes for complex salts of phosphorus that are insoluble in the soil led to increase of phosphorus concentrations and enhanced the ability of Enterobacter aerogenes to degrade a specific hydrocarbon in contaminated soil so that the phytase has a promising application in bioremediation of contaminated soils with hydrocarbons.

Detection of virulence and ß-lactamase encoding genes in Enterobacter aerogenes and Enterobacter cloacae clinical isolates from Brazil

ABSTRACT Enterobacter cloacae and E. aerogenes have been increasingly reported as important opportunistic pathogens. In this study, a high prevalence of multi-drug resistant isolates from Brazil, harboring several β-lactamase encoding genes was found. Several virulence genes were observed in E. aerogenes, contrasting with the E. cloacae isolates which presented none.

Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its ß site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the ß site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the ß site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the ß site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.

Crystallographic analyses of isoquinoline complexes reveal a new mode of metallo-ß-lactamase inhibition.

Crystallographic analyses of the VIM-5 metallo-ß-lactamase (MBL) with isoquinoline inhibitors reveal non zinc ion binding modes. Comparison with other MBL-inhibitor structures directed addition of a zinc-binding thiol enabling identification of potent B1 MBL inhibitors. The inhibitors potentiate meropenem activity against clinical isolates harboring MBLs.

GMP and IMP Are Competitive Inhibitors of CMY-10, an Extended-Spectrum Class C ß-Lactamase.

Nucleotides were effective in inhibiting the class C ß-lactamase CMY-10. IMP was the most potent competitive inhibitor, with a Ki value of 16.2 µM. The crystal structure of CMY-10 complexed with GMP or IMP revealed that nucleotides fit into the R2 subsite of the active site with a unique vertical binding mode where the phosphate group at one terminus is deeply bound in the subsite and the base at the other terminus faces the solvent.

Optimization of L-asparaginase production from novel Enterobacter sp., by submerged fermentation using response surface methodology.

The culture conditions and nutritional rations influencing the production of extra cellular antileukemic enzyme by novel Enterobacter aerogenes KCTC2190/MTCC111 were optimized in shake-flask culture. Process variables like pH, temperature, incubation time, carbon and nitrogen sources, inducer concentration, and inoculum size were taken into account. In the present study, finest enzyme activity achieved by traditional one variable at a time method was 7.6 IU/mL which was a 2.6-fold increase compared to the initial value. Further, the L-asparaginase production was optimized using response surface methodology, and validated experimental result at optimized process variables gave 18.35 IU/mL of L-asparaginase activity, which is 2.4-times higher than the traditional optimization approach. The study explored the E. aerogenes MTCC111 as a potent and potential bacterial source for high yield of antileukemic drug.