Bioconversion of acrylonitrile to acrylamide using polyacrylamide entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

The nitrile hydratase (NHase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The resting cells (having NHase activity) (8 %; 1 mL corresponds to 22 mg dry cell mass, DCM) were immobilized in polyacrylamide gel containing 12.5 % acrylamide, 0.6 % bisacrylamide, 0.2 % diammonium persulfate and 0.4 % TEMED. The polyacrylamide entrapped cells (1.12 mg DCM/mL) completely converted acrylonitrile in 3 h at 10 °C, using 0.1 mol/L potassium phosphate buffer. In a partitioned fed batch reactor, 432 g/L acrylamide was accumulated after 1 d. The polyacrylamide discs were recycled up to 3×; 405, 210 and 170 g/L acrylamide was produced in 1st, 2nd and 3rd recycling reactions. In four cycles, a total of 1217 g acrylamide was produced by recycling the same mass of entrapped cells.     

Methods for increasing nitrile biotransformation into amides using Mesorhizobium sp.

Nitriles are potential soil pollutants from industrial wastewater. There has been increased demand for an efficient process for the nitrile degradation process. Nitrile hydratase (NHase) has been extensively used in the production of acrylamide and treatment of organocyanide-contaminated industrial effluents. The NHase of Mesorhizobium sp., isolated from polyacrylonitrile (PAN) activated sludge from fiber manufacturing wastewater treatment systems was studied in the whole bacterial cells. Different chemicals were added to observe the variation in the percentage of acrylonitrile converted into acrylamide. The result indicated that cobalt ions were the NHase cofactor and could increase the NHase activity. The addition of propionaldehyde, or butyraldehyde, could enhance the acrylonitrile conversion rate. Therefore, acrylamide could be accumulated effectively and the percentage of acrylonitrile converted into acrylamide increased. Propionaldehyde was the most effective NHase activator. The percentage of acrylonitrile converted into acrylamide was nearly 100% at 3.8 h when propionaldehyde was added at about 207.4 mg/l. The addition of benzaldehyde was unable to increase the percentage of acrylonitrile converted into acrylamide. EDTA and acrylamide showed no effect on NHase activity. However, 0.1 mg/l of Ag₂SO₄ would slightly inhibit NHase activity, producing an acrylonitrile conversion rate of 492.9 mg/l with 54.9% converted at 29.1 h. The ability of the acrylonitrile biotransformation was completely inhibited if the Ag₂SO₄ concentration was above 0.5 mg/l. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 3, pp. 304–307. The text was submitted in English.     

Bioprocess engineering to produce 9-(nonanoyloxy) nonanoic acid by a recombinant <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>-based biocatalyst

Here, Corynebacterium glutamicum ATCC13032 expressing Baeyer–Villiger monooxygenase from Pseudomonas putida KT2440 was designed to produce 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid. Diverse parameters including cultivation and reaction temperatures, type of detergent, and pH were found to improve biotransformation efficiency. The optimal temperature of cultivation for the production of 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid using whole cells of recombinant C. glutamicum was 15 °C, but the reaction temperature was optimal at 30 °C. Enhanced conversion efficiency was obtained by supplying 0.05 g/L of Tween 80 at pH 7.5. Under these optimal conditions, recombinant C. glutamicum produced 0.28 mM of 9-(nonanoyloxy) nonanoic acid with a 75.6% (mol/mol) conversion yield in 2 h. This is the first report on the biotransformation of 10-ketostearic acid to 9-(nonanoyloxy) nonanoic acid with a recombinant whole-cell C. glutamicum-based biocatalyst and the results demonstrate the feasibility of using C. glutamicum as a whole-cell biocatalyst.     

Methods for increasing nitrile biotransformation into amides using Mesorhizobium sp

Nitriles are potential soil pollutants from industrial wastewater. There has been increased demand for efficient process for nitrile degradation process. Nitrile hydratase (NHase) has been extensively used in the production of acrylamide and treatment of organocyanide contaminated industrial effluents. The NHase of Mesorhizobium sp., isolated from polyacrylonitrile activated sludge from fiber manufacturing wastewater treatment systems was studied in the whole bacterial cells. Different chemicals were added to observe the variation in the percentage of acrylonitrile converted into acrylamide. The result indicated that cobalt ions were the NHase cofactor and could increase the NHase activity. The addition of propionaldehyde, or butyraldehyde could enhance the acrylonitrile conversion rate. Therefore, acrylamide could be accumulated effectively and the percentage of acrylonitrile converted into acrylamide increased. Propionaldehyde was the most effective NHase activator. The percentage of acrylonitrile converted into acrylamide was nearly 100% at 3.8 h when propionaldehyde was added at about 207.4 mg/l. The addition of benzaldehyde was unable to increase the percentage of acrylonitrile converted into acrylamide. EDTA and acrylamide showed no effect on NHase activity. However, 0.1 mg/l of Ag2SO4 would slightly inhibit NHase activity, producing an acrylonitrile conversion rate of 492.9 mg/l with 54.9% converted at 29.1 h. The ability of the acrylonitrile biotransformation was completely inhibited if the Ag2SO4 concentration was above 0.5 mg/l.     

Nitrile bioconversion by Microbacterium imperiale CBS 498-74 resting cells in batch and ultrafiltration membrane bioreactors

The biohydration of acrylonitrile, propionitrile and benzonitrile catalysed by the NHase activity contained in resting cells of Microbacterium imperiale CBS 498-74 was operated at 5, 10 and 20°C in laboratory-scale batch and membrane bioreactors. The bioreactions were conducted in buffered medium (50 mM Na₂HPO₄/NaH₂PO₄, pH 7.0) in the presence of distilled water or tap-water, to simulate a possible end-pipe biotreatment process. The integral bioreactor performances were studied with a cell loading (dry cell weight; DCW) varying from 0.1 mg_DCW per reactor to 16 mg_DCW per reactor, in order to realize near 100% bioconversion of acrylonitrile, propionitrile and benzonitrile without consistent loss of NHase activity.     

Characterization of a newly synthesized carbonyl reductase and construction of a biocatalytic process for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate with high space-time yield

A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg^?1. The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent K _m and V _max values of purified enzyme for COBE are 6.4 mM and 63.3 μmol min^?1 mg^?1, respectively. The biocatalytic process for the synthesis of (S)-CHBE was constructed by this SCR2 in an aqueous–organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, (S)-CHBE with yield of 95.3 % and e.e. of 99 % was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP⁺ to (S)-CHBE were 26.5 mmol L^?1 h^?1 g^?1 DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works.     

Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization

Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11?±?0.004 h^?1 and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.     

Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli.     

Kinetics of manganese transport and gene expressions of manganese transport carriers in Caco-2 cell monolayers

Two experiments were conducted to investigate the kinetics of manganese (Mn) transport in Caco-2 cell monolayers and the gene expressions of Mn transport carriers in apical (AP) and basolateral (BL) membranes. In experiment 1, the cells were treated with the medium containing 146 μmol/L of Mn (MnSO₄·H₂O). Both the uptake and transport of Mn from AP–BL or from BL–AP at different time-points were assessed to determine the optimal time for kinetics of Mn transport. The transport of Mn increased linearly with higher efficiency values in AP–BL than in BL–AP direction, however, the uptake of Mn revealed an asymptotic pattern within 120 min. In experiment 2, the kinetics of Mn transport in AP–BL was determined with media containing Mn concentrations from 0 to 2,500 μmol/L at 40 and 120 min, respectively, and mRNA levels of divalent metal transporter 1 (DMT1) and ferroportin (FPN1) were determined in Caco-2 cells treated with the medium containing 0 or 800 μmol/L of Mn for 120 min. The kinetics of Mn transport showed a carrier-mediated process when Mn concentrations were lower than 1,000 μmol/L and a linear increment when Mn concentrations exceeded 1,000 μmol/L at either 40 or 120 min. Mn treatment decreased (P < 0.01) DMT1 mRNA level and increased (P < 0.01) FPN1 mRNA level. The results from the present study suggested that Mn transport in AP–BL fit both carrier-mediated saturable and non-saturable diffusion processes, and Mn transport carriers DMT1 and FPN1 mediate the apical uptake and basolateral exit of Mn in Caco-2 cells.     

Next-generation sequencing-based genome-wide mutation analysis of l-lysine-producing Corynebacterium glutamicum ATCC 21300 strain

In order to identify single nucleotide polymorphism and insertion/deletion mutations, we performed whole-genome re-sequencing of the enhanced l-lysine-producing Corynebacterium glutamicum ATCC 21300 strain. In total, 142 single nucleotide polymorphisms and 477 insertion/deletion mutations were identified in the ATCC 21300 strain when compared to 3,434 predicted genes of the wild-type C. glutamicum ATCC 13032 strain. Among them, 110 transitions and 29 transversions of single nucleotide polymorphisms were found from genes of the ATCC 21300 strain. In addition, 11 genes, involved in the L-lysine biosynthetic pathway and central carbohydrate metabolism, contained mutations including single nucleotide polymorphisms and insertions/deletions. Interestingly, RT-PCR analysis of these 11 genes indicated that they were normally expressed in the ATCC 21300 strain. This information of genome-wide gene-associated variations will be useful for genome breeding of C. glutamicum in order to develop an industrial amino acid-producing strain with minimal mutation.     

Cloning of the nitrile hydratase gene from Nocardia sp. in Escherichia coli and Pichia pastoris and its functional expression using site-directed mutagenesis

To obtain a recombinant Rhodococcus or Nocardia with not only higher enzymatic activity but also better operational stability and product-tolerance ability for bioconversion of acrylamide from acrylonitrile, an active and stable expression system of nitrile hydratase (NHase) was tried to construct as the technical platform of genetic manipulations. Two NHase genes, NHBA and NHBAX, from Nocardia YS-2002 were successfully cloned, based on bioinformatics design of PCR primers, and inserted into plasmid pUC18 and pET32a, respectively. Then, two recombinant Escherichia coli strains, JM105 (pUC18-NHBA) and BL21 (DE3) (pET32a-NHBAX) were constructed and their expressions of NHase were focused. The induction results showed that there was either no NHase activity in JM105 (pUC18-NHBA), or as low as 0.04 U (1 U=1 μmol acrylamide min⁻¹ mg⁻¹ dry cell) in BL21 (DE3) (pET32a-NHBAX). SDS-PAGE results showed that the -subunit of NHBA and NHBAX could not be efficiently expressed in both recombinant E. coli strains. The novel Pichia pastoris system was also applied to express NHase, but the expression level remained quite low (0.5–0.6 U) and the protein was unstable. For solving this problem, a possible genetic strategy, site-directed mutagenesis of the -subunit of the NHase was carried out. After the successful mutagenesis of the original rare start codon gtg into atg, a new recombinant strain, E. coli XL1-Blue (pUC18-NHBA^M), was screened and the NHase activity stably reached as high as 51 U under the same induction conditions.     

Overproduction, purification, and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli

Lipoxygenase (LOX; EC 1.13.11.12,) is an enzyme that is widely used in food industry to improve aroma, rheological, or baking properties of foods. In this study, we described the expression and characterization of Pseudomonas aeruginosa LOX in Escherichia coli. The recombinant LOX was successfully expressed and secreted by E. coli using its endogenous signal peptide. When induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (final concentration) at 20 °C for 47 h, the titer of the recombinant enzyme reached 3.89 U/mL. In order to characterize the catalytic properties, the recombinant LOX was purified to homogeneity on Q High Performance and Mono Q5/50GL sequentially. The molecular weight of the LOX was estimated as 70 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The K _m and V _max of the recombinant enzyme were 48.9 μM and 0.226 μmol/min, respectively. The purified enzyme exhibited a maximum activity at 25 °C and pH 7.5. High-performance liquid chromatography analysis of the linoleic acid hydroperoxides produced by recombinant LOX revealed that the LOX from P. aeruginosa falls into linoleic acid 13(S)-LOX. To the best of our knowledge, this is the first report on the overexpression of extracellular LOX in microorganisms, and the achieved LOX yield is the highest ever reported.     

Stability study on the nitrile hydratase of Nocardia sp. 108: From resting cells to crude enzyme preparation

In recent years nitrile hydratases (NHases) have drawn increasing attention due to their critical roles in organic synthesis. In the present paper an extensive investigation on the stability and activity of NHase from Nocardia sp. 108, which has succeeded in industrial application in China, was conducted by bioconversion of acrylonitrile to acrylamide in a batch manner. A study of cultivation demonstrated that biosynthesis of NHase changed significantly with the time of the culture, and the optimal NHase biosynthesis phase was 45 h after inoculation with NHase activity of a biomass of 1209.8 U/g. A stability study indicated that both crude enzyme preparations exhibited a good stability when exposed to a pH 7.2 tris-HCl buffer at 4°C for 4 h. The text was submitted by the authors in English.     

Rhodococcus pyridinovorans MW3, a bacterium producing a nitrile hydratase

Rhodococcus pyridinovorans MW3 was isolated from an arable land of manioc from the Congo for its ability to transform acrylonitrile to acrylamide. This strain contains a cobalt nitrile hydratase (NHase) showing high sequence homology with NHases so far described. The specific NHase activity was 97 U mg(-1) dry wt. NHase production by R. pyridinovorans MW3 was urea and Co-dependent. The NHase was active for acrylamide up to 60% (w/v) indicating its potential for acrylamide production.     

Recombinant expression and characterization of a novel endoglucanase from Bacillus subtilis in Escherichia coli

The goal of this work was to produce high levels of endoglucanase in Escherichia coli for its potential usage in different industrial applications. Endoglucanase gene was amplified from genomic DNA of Bacillus subtilis JS2004 by PCR. The isolated putative endoglucanase gene consisted of an open reading frame of 1,701 nucleotides and encoded a protein of 567 amino acids with a molecular mass of 63-kDa. The gene was cloned into pET-28a(+) and expressed in E. coli BL21 (DE3). Optimum temperature and pH of the recombinant endoglucanase were 50 °C and 9, respectively which makes it very attractive for using in bio-bleaching and pulp industry. It had a K _M of 1.76 μmol and V _max 0.20 μmol/min with carboxymethylcellulose as substrate. The activity of recombinant endoglucanse was enhanced by Mg²⁺, Ca²⁺, isopropanol and Tween 20 and inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ni²⁺ and SDS. The activity of this recombinant endoglucanase was significantly higher than wild type. Therefore, this recombinant enzyme has potential for many industrial applications involving biomass conversions, due to characteristic of broad pH and higher temperature stability.     

Redirecting carbon flux through <Emphasis Type="Italic">pgi</Emphasis>-deficient and heterologous transhydrogenase toward efficient succinate production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum is particularly known for its potentiality in succinate production. We engineered C. glutamicum for the production of succinate. To enhance C₃–C₄ carboxylation efficiency, chromosomal integration of the pyruvate carboxylase gene pyc resulted in strain NC-4. To increase intracellular NADH pools, the pntAB gene from Escherichia coli, encoding for transhydrogenase, was chromosomally integrated into NC-4, leading to strain NC-5. Furthermore, we deleted pgi gene in strain NC-5 to redirect carbon flux to the pentose phosphate pathway (PPP). To solve the drastic reduction of PTS-mediated glucose uptake, the ptsG gene from C. glutamicum, encoding for the glucose-specific transporter, was chromosomally integrated into pgi-deficient strain resulted in strain NC-6. In anaerobic batch fermentation, the production of succinate in pntAB-overexpressing strain NC-5 increased by 14% and a product yield of 1.22 mol/mol was obtained. In anaerobic fed-batch process, succinic acid concentration reached 856 mM by NC-6. The yields of succinate from glucose were 1.37 mol/mol accompanied by a very low level of by-products. Activating PPP and transhydrogenase in combination led to a succinate yield of 1.37 mol/mol, suggesting that they exhibited a synergistic effect for improving succinate yield.     

微生物法生产丙烯酰胺的研究（Ⅱ）—腈水合酶催化反应动力学与失活动力学

在以丙烯腈为原料 ,微生物转化生产丙烯酰胺的过程中 ,酶催化反应是过程的关键。为了了解酶催化的动力学 ,本研究以自由细胞的酶为催化剂 ,进行了腈水合酶的反应动力学和失活动力学的研究。首先研究了菌体浓度、温度、pH值、丙烯腈浓度、丙烯酰胺浓度等对腈水合酶催化反应速度的影响。结果表明 ,在这些因素中 ,温度和丙烯酰胺浓度是最主要的影响因素。 2 8℃时酶活为 5 6 5 9u mL(菌液 ) ,在 5℃时的反应速率仅为 2 8℃时的11 72 % ,相应的表观酶活为 6 6 3u mL(菌液 )。而在丙烯酰胺 45 %浓度条件下的酶活大约只有丙烯酰胺 5 %浓度下的酶活的 1 2。经过对不同温度下的反应速度的研究 ,得到腈水合酶水合反应的活化能为 6 5 5 7kJ·mol- 1 。本文进一步研究了自由细胞状态下 ,菌体浓度、pH值、温度、丙烯腈浓度、丙烯酰胺浓度对腈水合酶失活的影响 ,得到了失活动力学。结果表明 ,在这些因素中 ,对酶失活影响的最主要因素还是温度和丙烯酰胺浓度。尤其当丙烯酰胺浓度到达 35 %时 ,酶活下降得很快 ,在 5 5h后 ,酶活几乎为零。而在丙烯酰胺浓度为 10 %的情况下 ,5 5h的酶活仍然还存在约 5 0 %。试验结果还表明 ,丙烯腈对酶的稳定性的影响很小。经过数据处理 ,得到的 2 8℃的酶失活速率常数为 5℃下的 2 1 7     

Selenium and Zinc against Aβ<Subscript>25–35</Subscript>-Induced Cytotoxicity and Tau Phosphorylation in PC12 Cells and Inhibits γ-cleavage of APP

Amyloid beta (Aβ) is the main component of the amyloid plaques that accumulate in the brains of Alzheimer patients. The present study was conducted to investigate whether the combined treatment with selenium (Se) and zinc (Zn) offers more beneficial effects than that provided by either of them alone in reversing Aβ_25–35-induced neurotoxicity in PC12 cells. Cells were pretreated with 0.1 μmol/L of Se and Zn for 4 h, after treated with 10 mmol/L Aβ_25–35 for 24 h. Cells were divided into control and five treated groups, and received either 10 mmol/L Aβ_25–35,10 mmol/L Aβ_25–35 + 0.1 μmol/L Se, 10 mmol/L Aβ_25–35 + 0.1 μmol/L Zn, 10 mmol/LAβ_25–35 + 0.1 μmol/L Se + 0.1 μmol/L Zn, or 0.1 μmol/L Se + 0.1 μmol/L Zn. The result showed that cell viability was decreased in MTT metabolic rate; LDH release and MDA, H₂O₂, and NO levels were increased and the GSK-3β and phosphorylated tau protein level were increased in Aβ_25–35-treated group (P < 0.05 or P < 0.01), which whole changes were attenuated by Se and Zn and Se combined Zn. In order to evaluate whether the Se and Zn have an effect on processing pathway of amyloid precursor protein (APP), we examined the activity of γ-secretase in primary cultured cortical neuron cells. ELISA analysis showed that Se and Zn could inhibit the activity of γ-secretase. Then we also investigated the effect of Se and Zn on the Aβ_1–40 concentration and APP-N-terminal fragment expression from APP695 stably transfected Chinese hamster ovary (CHO) cells. APP695 stably transfected CHO cells were treated with 0.1 μmol/L Se and Zn; cells were divided into control and four treated groups, which received either 0.5 M DAPT, 0.1 μmol/L Se, 0.1 μmol/L Zn, or 0.1 μmol/L Se + 0.1 μmol/L Zn. Se and Zn could decrease Aβ_1–40 production and increase the APP-N-terminal fragment protein expression. These experiments indicate that Se and Zn have a protective effect on AD pathology that a possible mechanism is inhibiting the activity of γ-secretase to decreasing Aβ_1–40 production further influencing the APP processing. Altogether, our findings may provide a novel therapeutic target to treat AD sufferers.