Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9.

L-asparaginase (L-ASNase) is a versatile anticancer and acrylamide reduction enzyme predominantly used in medical and food industries. However, the high specificity of L-asparaginase formulations for glutamine, low thermostability, and blood clearance are the major disadvantages. Present study describes production, characterization, and applications of glutaminase free extracellular L-asparaginase from indigenous Bacillus halotolerans ASN9 isolated from soil sample. L-asparaginase production was optimized in M9 medium (containing 0.2% sucrose and 1% L-asparagine) that yielded maximum L-ASNase with a specific activity of 256 U mg-1 at pH 6 and 37°C. L-asparaginase was purified through acetone precipitation and Sephadex G-100 column, yielding 48.9 and 24% recovery, respectively. Enzyme kinetics revealed a Vmax of 466 mM min-1 and Km of 0.097 mM. Purified L-ASNase showed no activity against glutamine. The purified glutaminase free L-ASNase has a molecular mass of 60 kDa and an optimum specific activity of 3083 U mg-1 at pH 7 and 37°C. The enzyme retains its activity and stability over a wide range of pH and temperature, in the presence of selected protein inhibitors (SDS, ß-mercaptoethanol), CoCl2, KCl, and NaCl. The enzyme also exhibited antioxidant activity against DPPH radical (IC50 value 70.7 µg mL-1) and anticancer activity against U87 human malignant glioma (IC50 55 µg mL-1) and Huh7 human hepatocellular carcinoma (IC50 37 µg mL-1) cell lines. Normal human embryonic kidney cells (HEK293) had greater than 80% cell viability with purified L-ASNase indicating its least cytotoxicity against normal cells. The present work identified potent glutaminase free L-ASNase from B. halotolerans ASN9 that performs well in a wide range of environmental conditions indicating its suitability for various commercial applications.

Self-Nanoemulsifying Drug Delivery System (SNEDDS) for Improved Oral Bioavailability of Chlorpromazine: In Vitro and In Vivo Evaluation.

Background and Objectives: Lipid-based self-nanoemulsifying drug delivery systems (SNEDDS) have resurged the eminence of nanoemulsions by modest adjustments and offer many valuable opportunities in drug delivery. Chlorpromazine, an antipsychotic agent with poor aqueous solubility-with extensive first-pass metabolism-can be a suitable candidate for the development of SNEDDS. The current study was designed to develop triglyceride-based SNEDDS of chlorpromazine to achieve improved solubility, stability, and oral bioavailability. Materials and Methods: Fifteen SNEDDS formulations of each short, medium, and long chain, triglycerides were synthesized and characterized to achieve optimized formulation. The optimized formulation was characterized for several in vitro and in vivo parameters. Results: Particle size, zeta potential, and drug loading of the optimized SNEDDS (LCT14) were found to be 178 ± 16, -21.4, and 85.5%, respectively. Long chain triglyceride (LCT14) showed a 1.5-fold increased elimination half-life (p < 0.01), up to 6-fold increased oral bioavailability, and 1.7-fold decreased plasma clearance rate (p < 0.01) compared to a drug suspension. Conclusion: The findings suggest that SNEDDS based on long-chain triglycerides (LCT14) formulations seem to be a promising alternative for improving the oral bioavailability of chlorpromazine.

ZntR positively regulates T6SS4 expression in Yersinia pseudotuberculosis.

The type VI secretion system (T6SS) is a widespread and versatile protein secretion system found in most Gram-negative bacteria. Studies of T6SS have mainly focused on its role in virulence toward host cells and inter-bacterial interactions, but studies have also shown that T6SS4 in Yersinia pseudotuberculosis participates in the acquisition of zinc ions to alleviate the accumulation of hydroxyl radicals induced by multiple stressors. Here, by comparing the gene expression patterns of wild-type and zntR mutant Y. pseudotuberculosis cells using RNA-seq analysis, T6SS4 and 17 other biological processes were found to be regulated by ZntR. T6SS4 was positively regulated by ZntR in Y. pseudotuberculosis, and further investigation demonstrated that ZntR regulates T6SS4 by directly binding to its promoter region. T6SS4 expression is regulated by zinc via ZntR, which maintains intracellular zinc homeostasis and controls the concentration of reactive oxygen species to prevent bacterial death under oxidative stress. This study provides new insights into the regulation of T6SS4 by a zinc-dependent transcriptional regulator, and it provides a foundation for further investigation of the mechanism of zinc transport by T6SS.

Molecular characterization of a eukaryotic-like phenol hydroxylase from Corynebacterium glutamicum.

This study focuses on the genetic and biochemical characterization of phenol hydroxylase (Phe, NCgl2588) from Corynebacterium glutamicum that shares 31% identity in amino acids with phenol hydroxylase from yeast Trichosporon cutaneum but less similarity with that from bacteria. The phe deletion mutant significantly reduced its ability to grow with phenol as the sole carbon and energy source. Expression of the phe gene was strongly induced with phenol and also subject to the control of carbon catabolite repression (CCR). The molecular weight of purified Phe protein determined by gel filtration chromatography was 70 kDa, indicating that Phe exists as a monomer in the purification condition. However, Phe protein pre-incubated with phenol showed a molecular weight of 140 kDa, suggesting that Phe is likely active as a dimer. In addition to phenol, the Phe protein could utilize various other phenolic compounds as substrates. Site-directed mutagenesis revealed that D75, P261, R262, R269, C349 and C476 are key amino acid residues closely related to the enzyme activity of Phe.

Ohr Protects Corynebacterium glutamicum against Organic Hydroperoxide Induced Oxidative Stress.

Ohr, a bacterial protein encoded by the Organic Hydroperoxide Resistance (ohr) gene, plays a critical role in resistance to organic hydroperoxides. In the present study, we show that the Cys-based thiol-dependent Ohr of Corynebacterium glutamicum decomposes organic hydroperoxides more efficiently than hydrogen peroxide. Replacement of either of the two Cys residues of Ohr by a Ser residue resulted in drastic loss of activity. The electron donors supporting regeneration of the peroxidase activity of the oxidized Ohr of C. glutamicum were principally lipoylated proteins (LpdA and Lpd/SucB). A Δohr mutant exhibited significantly decreased resistance to organic hydroperoxides and marked accumulation of reactive oxygen species (ROS) in vivo; protein carbonylation was also enhanced notably. The resistance to hydrogen peroxide also decreased, but protein carbonylation did not rise to any great extent. Together, the results unequivocally show that Ohr is essential for mediation of organic hydroperoxide resistance by C. glutamicum.

Corynebacterium glutamicum methionine sulfoxide reductase A uses both mycoredoxin and thioredoxin for regeneration and oxidative stress resistance.

Oxidation of methionine leads to the formation of the S and R diastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown in Actinomycetes. Here, we report that a Corynebacterium glutamicum methionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of the msrA gene in C. glutamicum resulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of an S-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under stress conditions in vivo, the Trx/TrxR pathway alone is sufficient to reduce CgMsrA under normal conditions. Based on these results, a catalytic model for the reduction of CgMsrA by Mrx1 and Trx is proposed.

Functional characterization of Corynebacterium glutamicum mycothiol S-conjugate amidase.

The present study focuses on the genetic and biochemical characterization of mycothiol S-conjugate amidase (Mca) of Corynebacterium glutamicum. Recombinant C. glutamicum Mca was heterologously expressed in Escherichia coli and purified to apparent homogeneity. The molecular weight of native Mca protein determined by gel filtration chromatography was 35 kDa, indicating that Mca exists as monomers in the purification condition. Mca showed amidase activity with mycothiol S-conjugate of monobromobimane (MSmB) in vivo while mca mutant lost the ability to cleave MSmB. In addition, Mca showed limited deacetylase activity with N-acetyl-D-glucosamine (GlcNAc) as substrate. Optimum pH for amidase activity was between 7.5 and 8.5, while the highest activity in the presence of Zn2+ confirmed Mca as a zinc metalloprotein. Amino acid residues conserved among Mca family members were located in C. glutamicum Mca and site-directed mutagenesis of these residues indicated that Asp14, Tyr137, His139 and Asp141 were important for activity. The mca deletion mutant showed decreased resistance to antibiotics, alkylating agents, oxidants and heavy metals, and these sensitive phenotypes were recovered in the complementary strain to a great extent. The physiological roles of Mca in resistance to various toxins were further supported by the induced expression of Mca in C. glutamicum under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH.

Enhancing Corynebacterium glutamicum robustness by over-expressing a gene, mshA, for mycothiol glycosyltransferase.

Over-expression of the gene, mshA, coding for mycothiol glycosyl transferase improved the robustness of Corynebacterium glutamicum to various stresses. Intracellular mycothiol (MSH) content was increased by 114 % in WT(pXMJ19-mshA) compared to WT(pXMJ19). Survival rates increased by 44, 39, 90, 77, 131, 87, 52, 47, 57, 85 and 33 % as compared to WT(pXMJ19) under stress by H2O2 (40 mM), methylglyoxal (5.8 mM), erythromycin (0.08 mg ml(-1)), streptomycin (0.005 mg ml(-1)), Cd(2+) (0.01 mM), Mn(2+) (2 mM), formic acid (0.05 %), acetic acid (0.15 %), levulinic acid (0.25 %), furfural (7.2 mM), and ethanol (10 % v/v), respectively. Increased MSH content also decreased the concentration of reactive oxygen species in the presence of the above stresses. Our results may open a new avenue for enhancing robustness of industrial bacteria for production of commodity chemicals.

Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum.

Corynebacterium glutamicum uses 4-cresol as sole carbon source for growth. Protocatechuate 3,4-dioxygenase activity had been detected when C. glutamicum was grown with 4-cresol. In this work, we found that 4-cresol was catabolized via 4-hydroxybenzoate and protocatechuate as intermediate metabolites, and a genetic cluster called cre (designated for 4-cresol, creABCDEFGHIR, tagged as ncgl0521-ncgl0531 in NCBI) was identified. The cre gene cluster comprises of 11 genes, and six of them were experimentally confirmed to be involving in 4-cresol catabolism. The genes creD, creE, and creJ were involved in oxidation of 4-cresol into 4-hydroxybenzyl alcohol. The creD encoded a protein showing Mg(2+)-dependent phosphohydrolase activity. The genes creE, creF, creJ encoded a putative P450 system. The creG encoded a NAD(+)-dependent dehydrogenase and catalyzed 4-hydroxybenzyl alcohol to 4-hydroxybenzaldehyde. Two other genes creH and creI were involved in conversion of 4-hydroxybenzyl alcohol to 4-hydroxybenzoate, but their catalytic function is still unknown. Similar genetic clusters with high DNA sequence identity were identified in Arthrobacter and additional Corynebacterium species, suggesting that this genetic organization for 4-cresol catabolism might be more widely distributed in Gram-positive bacteria.

Biosorption characteristics of Aspergillus fumigatus for the decolorization of triphenylmethane dye acid violet 49.

This study focuses on the possible use of Aspergillus fumigatus to remove acid violet 49 dye (AV49) from aqueous solution. In batch biosorption experiments, the highest biosorption efficiency was achieved at pH 3.0, with biosorbent dosage of 3.0 gL(-1) within about 30 min at 40 °C. The Langmuir and Freundlich models were able to describe the biosorption equilibrium of AV49 onto fungal biomass with maximum dye uptake capacity 136.98 mg g(-1). Biosorption followed a pseudo-second-order kinetic model with high correlation coefficients (R (2) > 0.99), and the biosorption rate constants increased with increasing temperature. Thermodynamic parameters indicated that the biosorption process was favorable, spontaneous, and endothermic in nature, with insignificant entropy changes. Fourier transform infrared spectroscopy strongly supported the presence of several functional groups responsible for dye-biosorbent interaction. Fungal biomass was regenerated with 0.1 M sodium hydroxide and could be reused a number of times without significant loss of biosorption activity. The effective decolorization of AV49 in simulated conditions indicated the potential use of biomass for the removal of color contaminants from wastewater.

Environmental biotechnology in China.

Environmental biotechnology has emerged as an important measure to tackle the environmental pollution as China experiences great economic success. Over the past decade, much emphasis has been paid to the following fields in environmental biotechnology: microbial degradation of toxic and organic chemicals, bio-treatment of wastewater, waste recycling. The Chinese researchers have done a lot of work to understand the natural degradation processes for organic and toxic compounds and finally to clean these compounds from polluted environments. For the treatment of wastewater, many new processes were proposed and optimized to meet the more strict effluent standards in China. Finally, more and more attention has been paid to the reuse of discharged wastes. In this chapter we review the development in the above fields.

Responses to arsenate stress by Comamonas sp. strain CNB-1 at genetic and proteomic levels.

Comamonas sp. strain CNB-1, a chloronitrobenzene-degrading bacterium, was demonstrated to possess higher arsenate tolerance as compared with the mutant strain CNB-2. pCNB1, a plasmid harboured by CNB-1 but not CNB-2, contained the genetic cluster ars(RPBC)Com, which putatively encodes arsenate-resistance regulator, family II arsenate reductase, arsenite efflux pump and family I arsenate reductase, respectively, in Comamonas strain CNB-1. The arsC-negative Escherichia coli could gain arsenate resistance by transformation with arsPCom or arsCCom, indicating that these two genes might express functional forms of arsenate reductases. Intriguingly, when CNB-1 cells were exposed to arsenate, the transcription of arsPCom and arsCCom was measurable by RT-PCR, but only ArsPCom was detectable at protein level. To explore the proteins responding to arsenate stress, CNB-1 cells were cultured with and without arsenate and differential proteomics was carried out by two-dimensional PAGE (2-DE) and MALDI-TOF MS. A total of 31 differential 2-DE spots were defined upon image analysis and 23 proteins were identified to be responsive specifically to arsenate. Of these spots, 18 were unique proteins. These proteins were identified to be phosphate transporters, heat-shock proteins involved in protein refolding, and enzymes participating in carbon and energy metabolism.

Comparative proteomes of Corynebacterium glutamicum grown on aromatic compounds revealed novel proteins involved in aromatic degradation and a clear link between aromatic catabolism and gluconeogenesis via fructose-1,6-bisphosphatase.

The current study examined the aromatic degradation and central metabolism in Corynebacterium glutamicum by proteomic and molecular methods. Comparative analysis of proteomes from cells grown on gentisate and on glucose revealed that 30% of the proteins of which their abundance changed were involved in aromatic degradation and central carbon metabolism. Similar results were obtained from cells grown on benzoate, 4-cresol, phenol, and resorcinol. Results from these experiments revealed that (i) enzymes involved in degradation of benzoate, 4-cresol, gentisate, phenol, and resorcinol were specifically synthesized and (ii) that the abundance of enzymes involved in central carbon metabolism of glycolysis/gluconeogenesis, pentose phosphate pathway, and TCA cycles were significantly changed on various aromatic compounds. Significantly, three novel proteins, NCgl0524, NCgl0525, and NCgl0527, were identified on 4-cresol. The genes encoding NCgl0525 and NCgl0527 were confirmed to be necessary for assimilation of 4-cresol with C. glutamicum. The abundance of fructose-1,6-bisphosphatase (Fbp) was universally increased on all the tested aromatic compounds. This Fbp gene was disrupted and the mutant WT(Deltafbp) lost the ability to grow on aromatic compounds. Genetic complementation by the Fbp gene restored this ability. We concluded that gluconeogenesis is a necessary process for C. glutamicum growing on various aromatic compounds.

Genome-wide investigation of aromatic acid transporters in Corynebacterium glutamicum.

Genome-wide data mining indicated that six genes (ncgl1031, ncgl2302, ncgl2325, ncgl2326, ncgl2922 and ncgl2953) encoding putative transport proteins are involved in uptake of various aromatic compounds that are further degraded through the beta-ketoadipate, gentisate and resorcinol pathways in Corynebacterium glutamicum. The gentisate (GenK/NCgl2922) and vanillate (VanK/NCgl2302) transporters have been identified previously. In this study, physiological functions of the remaining four putative transporters as well as the vanillate transporter (VanK/NCgl2302) were examined by genetic disruption/complementation and uptake assays. Results indicated that ncgl1031 encodes PcaK for 4-hydroxybenzoate and protocatechuate transport, and ncgl2302 encodes VanK for vanillate transport. Genetic studies and uptake assays indicated that both ncgl2325/benK and ncgl2326/benE are involved in benzoate transport in C. glutamicum. When growth rates were compared for two benzoate transporter mutants, benK and benE, a high growth rate was observed for the benE mutant. Sequence alignments revealed that PcaK, VanK, BenK and GenK belong to the major facilitator superfamily (MFS). Modelling of secondary structures based on previously characterized MFS members revealed that NCgl1031, NCgl2302, NCgl2325 and NCgl2922 are typical 12 helix transmembrane proteins but NCgl2326 contains only 11 alpha-helices. Thus the functionally identified NCgl2326 belongs to a novel type of benzoate transporters. Attempts to identify the phenotype of a hydK/ncgl2953 mutant failed, so the function of ncgl2953 remains unclear.

Genetic characterization of the resorcinol catabolic pathway in Corynebacterium glutamicum.

Corynebacterium glutamicum grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110-NCgl1113 and NCgl2950-NCgl2953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCgl2950-NCgl2953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110-NCgl1113, NCgl2951, and NCgl2952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCgl2951 in Escherichia coli revealed that NCgl1113 and NCgl2951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in E. coli revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor. E. coli cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCgl2950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCgl2953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by C. glutamicum. The function of NCgl2953 remains unclear.