Bioconversion of butyronitrile to butyramide using whole cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of β-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10°C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10°C for 6 h.     

Bench scale conversion of 3-cyanopyidine to nicotinamide using resting cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

The nitrile hydratase (NHase, EC 3.5.5.1) activity of Rhodococcus rhodochrous PA-34 was explored for the conversion of 3-cyanopyridine to nicotinamide. The NHase activity (∼18 U/mg dry cell weight, dcw) was observed in 0.1 M phosphate buffer, pH 8.0 containing 1M 3-cyanopyridine as substrate, and 0.75 mg of resting cells (dry cell weight) per ml reaction mixture at 40°C. However, 25°C was more suitable for prolonged batch reaction at high substrate (3-cyanopyridine) concentration. In a batch reaction (1 liter), 7M 3-cyanopyridine (729 g) was completely converted to nicotinamide (855 g) in 12h at 25°C using 9.0 g resting cells (dry cell weight) of R. rhodochrous PA-34.     

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.     

Production of acrylamide using immobilized cells of<Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M33

The cellsof Rhodococcus rhodochrous M33, which produce a nitrile hydratase enzyme, were immobilized in acrylamide-based polymer gels. The optimum pH and temperature for the activity of nitrile hydratase in both the free and immobilized cells were 7.4 and 45°C, respectively, yet the optinum temperature for acrylamide production by the immobilized cells was 20°C. The nitrile hydratase of the immobilized cells was more stable with acrylamide than that of the free cells. Under optimal conditions, the final acrylamide concentration reached about 400 g/L with a conversion yield of almost 100% after 8 h of reaction when using 150 g/L of immobilized cells corresponding to a 1.91 g-dry cell weight/L. The enzyme activity of the immobilized cells rapidly decreased with repeated use. However, the quality of the acrylamide produced by the immobilized cells was much better than that produced by the free cells in terms of color, salt content, turbidity, and foam formation. The quality of the aqueous acrylamide solution obtained was found to be of commercial use without further purification.     

The superiority of the third-generation catalyst,Rhodococcus rhodochrous J1 nitrile hydratase,for industrial production of acrylamide

As the third-generation biocatalyst for industrial production of acrylamide, the superiority of Rhodococcus rhodochrous J1 nitrile hydratase was demonstrated in comparison with other acrylamide-producing bacteria. R. rhodochrous J1 enzyme is much more heat stable and more tolerant to a high concentration of acrylonitrile than Pseudomonas chlororaphis B23 and Brevibacterium R312 enzymes. The J1 enzyme is peculiar in its extremely high tolerance to acrylamide. The hydration reaction of acrylonitrile catalysed by J1 cells proceeded even in the presence of 50% (w/v) acrylamide. The tolerance of J1 enzyme to various organic solvents such as n-propanol and isopropanol was prominent. Using R. rhodochrous J1 resting cells, the accumulation reaction was carried out by feeding acrylonitrile to maintain a level of 6%. After 10 h incubation, the accumulation of acrylamide was approximately 65.6% (w/v) at 10°C, 56.7% (w/v) at 15°C, and 56.0 (w/v) at 20°C. The high stability, high catalytic efficiency and other outstanding features of the J1 enzyme are analysed and discussed. Correspondence to: T. Nagasawa     

Construction of a New Shuttle Vector for Zymomonas mobilis

The culture conditions for Rhodococcus sp. N-774 cells showing high nitrile hydratase activity and the reaction conditions for acrylamide production by the resting cells were optimized. Thiamine was essential for the growth of the strain. Yeast extract and Fe^{2 +} or Fe^{3 +} remarkably promoted the formation of nitrile hydratase of the cells. The reaction proceeded optimally at temperatures below 30°C. Incubation for 1 hr at above 40°C resulted in inactivation of the enzyme. Through reaction at a temperature as low as 0°C, the inhibition and inactivation of the enzyme activity by the substrate, acrylonitrile, and the product, acrylamide, were remarkably reduced, and higher accumulation of acrylamide could be attained. Under the optimal conditions, a more than 20% (w/v) acrylamide solution was obtained with a conversion yield of nearly 100%. Thus, the aqueous acrylamide solution obtained showed a high enough quality for use for the commercial preparation of polyacrylamide.     

Bioconversion of benzonitrile to benzoic acid using free and agar entrapped cells of Nocardia globerula NHB-2

The free and agar immobilized cells of Nocardia globerula NHB-2 having nitrilase (EC 3.5.5.1) activity were used to catalyse the transformation of benzonitrile to benzoic acid. The whole cells of N. globerula NHB-2 were immobilized in agar which exhibited maximum conversion of benzonitrile to benzoic acid in 0.1 M potassium phosphate buffer pH 7.5 (free cells) 8.0 (immobilized cells), temperature 40 degrees C, cells 2 mg dcm ml(-1) reaction mixture and benzonitrile (4% v/v) in 4 h (free cells). The effect of temperature on the stability of nitrilase was studied and cells retained 100% activity at 30 degrees C and lost 50% activity at 40 degrees C. In a fed batch mode of reaction 108 and 84 gl(-1) benzoic acid was produced using free and agar entrapped cells (2 g dcm). The agar immobilized cells were recycled up to three times and 80, 62, 20 gl(-1) benzoic acid was again produced respectively in each of three cycles and a total 244 g benzoic acid was produced by recycling the same mass of immobilized biocatalyst.     

Nitrilase-catalysed conversion of acrylonitrile by free and immobilized cells of <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

The biotransformation of acrylonitrile was investigated using thermophilic nitrilase produced from a new isolate Streptomyces sp. MTCC 7546 in both the free and immobilized state. Under optimal conditions, the enzyme converts nitriles to acids without the formation of amides. The whole cells of the isolate were immobilized in agar-agar and the beads so formed were evaluated for 25 cycles at 50°C. The enzyme showed a little loss of activity during reuse. Seventy-one per cent of 0.5 M acrylonitrile was converted to acid at 6 h of incubation at a very low density of immobilized cells, while 100% conversion was observed at 3 h by free cells.     

High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production

The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.     

Methane production from wastewaters by immobilized methanogenic bacteria

A methanogenic population was immobilized onto agar gel, polyacrylamide gel, and collagen membrane. Agar-gel-entrapped methanogenic microorganisms gave the highest activity. The optimum agar concentration was between 1.5 and 3% (w/v), and the optimum microbial content was 20 mg wet cells/g gel. The optimum conditions for methane production by immobilized whole cells were pH 7.0–7.5 and 37–45°C. The rate of methane production was initially 1.8 μmol/g gel/hr. Methane productivity was gradually increased and reached a steady state (4.5μmol/g gel/hr) after 25 days of incubation. The immobilized methanogenic microbial population continuously evolved methane over a 90 day period. No difference in methane productivity was observed after three months of storage at 5°C. Methane was also produced by immobilized whole cells under aerobic conditions. Furthermore, carbohydrates, such as glucose, in wastewater completely decomposed by immobilized whole cells.     

Treatment of simulated wastewater containing toxic amides by immobilized <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> NHB-2 using a highly compact 5-stage plug flow reactor

Biodegradation of toxic amides by immobilized Rhodococcus rhodochrous NHB-2 has been studied to generate data for future development of reactors for the treatment of simulated wastewater containing various toxic amides. The whole resting cells were immobilized in different matrices like agar, polyacrylamide and alginate. Agar gel beads were selected for the treatment of simulated wastewater containing 100mM each acetamide, propionamide, and 10mM of acrylamide and packed in a highly compact five-stage plug flow reactor. The immobilized bacterium worked well in a broad pH range from 5 to 10, with an optimum at 8.7. The apparent K _m-value for the turnover of acetamide for the resting cells was determined to be around 40mM at pH 8.5 and 55°C, whereas the K _m-value of the purified amidase was predicted to be about 20 mM. This organism exhibited greater turnover of aliphatic amides as compared to aromatic amides. Although these cells showed maximal amide-degrading activity at 55°C, simulated wastewater treatment was carried out at 45°C, because of the greater stability of the amidase activity at that temperature. Of note, indices for overall temperature stability, based on the temperature dependence of apparent first order kinetic temperature denaturation constants, were determined to be –7.9±1.1×10^–4, and –13.7±1.3×10^–4, –14.5±0.7×10^–4, and –13.7±0.8×10^–4°Cmin, for free cells and cells immobilized in alginate, agar and polyacrylamide respectively. After 250min the reactor showed maximum degradation of acetamide, propionamide and acrylamide of about 97, 100 and 90%, respectively by using 883 enzyme activity units per reactor stage. The results of this investigation showed that R. rhodochrous NHB-2 expressing thermostable amidase could be used for the efficient treatment of wastewater containing toxic amides. Therefore, we suggest that this microbe has a very high potential for the detoxification of toxic amides from industrial effluents and other wastewaters.     

Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of 42.8%.     

Phytate degradation by immobilized<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phytase in soybean-curd whey

Saccharomyces cerevisiae CY phytase-producing cells were immobilized in calcium alginate beads and used for the degradation of phylate. The maximum activity and immobilization yield of the immobilized phytase reached 280 mU/g-bead and 43%, respectively. The optimal pH of the immobilized cell phytase was not different from that of the free cells. However, the optimum temperature for the immobilized phytase was 50°C, which was 10°C higher than that of the free cells; pH and thermal stability were enhanced as a consequence of immobilization. Using the immobilized phytase, phytate was degraded in a stirred tank bioreactor. Phytate degradation, both in a buffer solution and in soybean-curd whey mixture, showed very similar trends. At an enzyme dosage of 93.9 mU/g-phytate, half of the phytate was degraded after 1 h of hydrolysis. The operational stability of the immobilized beads was examined with repeated batchwise operations. Based on 50% conversion of the phytate and five times of reuse of the immobilized beads, the specific degradation (g phytate/g dry cell weight) for the immobilized phytase increased 170% compared to that of the free phytase.     

A propionitrile-induced nitrilase of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. NDB 1165 and its application in nicotinic acid synthesis

Rhodococcus sp. NDB 1165, a nitrile-transforming organism was isolated from temperate forest soil of Himalayas. The nitrilase (EC 3.5.5.2) activity of this organism had higher substrate specificity toward aromatic nitriles (benzonitrile, 3-cyanopyridine and 4-cyanopyridine) and unsaturated aliphatic nitrile (acrylonitrile) in comparison to saturated aliphatic nitriles (acetonitrile, propionitrile, butyronitrile and isobutyronitrile) nitrile and arylacetonitrile (phenylacetonitrile and indole-3-acetonitrile). The nitrilase of Rhodococcus sp. NDB 1165 was inducible in nature and propionitrile proved to be an efficient inducer. However, the salts of ferrous and cobalt ions had an inhibitory effect. Under optimized reaction conditions (pH 8.0 and temperature 45°C) the nitrilase activity of this organism was 2.39 ± 0.07 U/mg dry cell mass (dcm). The half-life of this enzyme was 150 min and 40 min at 45°C and 50°C respectively. However, it was quite stable at 40°C and around 58 % activity was retained even after 6 h at this temperature. The V _max and K _m value of this nitrilase were 1.67 μmol/ml min and 0.1 M respectively using 3-cyanopyridine as substrate. However, the decrease in V _max and K _m values (0.56 μmol/ml min and 0.02 M, respectively) were ␣observed at >0.05 M 3-cyanopyridine which revealed that this enzyme experienced uncompetitive inhibition at higher substrate concentrations. Under optimized reaction conditions, 1.6 M 3-cyanopyridine was successfully converted in to nicotinic acid using 2.0 mg resting cells (dcm)/ml reaction mixture in 11 h. This is the highest production of nicotinic acid i.e. 8.95 mg/mg resting cells (dcm)/h as compared to nitrilase systems reported hitherto.     

Continuous Production of Dextran from Immobilized Cells of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> KIBGE HA1 Using Acrylamide as a Support

The cells of L. mesenteroides KIBGE HA1 were immobilized for the production of dextran on acrylamide gel and gel concentration was optimized for maximum entrapment. Sucrose at substrate concentration of 10% produced high yield of dextran at 25°C with a percent conversion of 5.82 while at 35°C it was 3.5. However, increasing levels of sucrose diminished dextran yields. The free cells stopped producing dextran after 144 h, while immobilized cells continued to produce dextran even after 480 h. Molecular mass distribution of dextran from free cells indicate that it is identical to that of blue dextran while the molecular mass of dextran from immobilized cells is lower than that of free cells.     

Chemoenzymatic production of 1,5-dimethyl-2-piperidone

A chemoenzymatic process for the preparation of 1,5-dimethyl-2-piperidone (1,5-DMPD) from 2-methylglutaronitrile (MGN) has been demonstrated. MGN was first hydrolyzed to 4-cyanopentanoic acid (4-CPA) ammonium salt using the nitrilase activity of immobilized Acidovorax facilis 72W cells. The hydrolysis reaction produced 4-CPA ammonium salt with greater than 98% regioselectivity at 100% conversion, and at concentrations of 170–210 g 4-CPA/l. Catalyst productivities of at least 1000 g 4-CPA/g dry cell weight (dcw) of immobilized cells were achieved by recycling the immobilized-cell catalyst in consecutive stirred-batch reactions. After recovery of the immobilized cell catalyst for reuse, the 4-CPA ammonium salt in the aqueous product mixture was directly converted to 1,5-DMPD by low-pressure catalytic hydrogenation in the presence of added methylamine.     

Characterization of microbial endo-β-glucanase immobilized in alginate beads

Endo-β-glucanase (endo-β-1,4-glucano-glucanase EC 3.2.1.4), isolated from Trichoderma reesei, was immobilized in calcium alginate beads, retaining 75% of its original activity. The polyanionic moiety surrounding the immobilized enzyme displaced the pH-activity profile to alkaline regions with respect to that of the free enzyme. The enzyme was inhibited by carboxymethylcellulose, but this inhibition appeared to be decreased by immobilizatíon. The enzyme immobilized in alginate beads showed a K_m value (1.02% w/v) lower than that of the enzyme (1.31%). The apparent V_max of immobilized cellulase preparations (238.3 μmol glucose/ml × h) decreased by a factor of 0.59 with respect to that of the soluble enzyme. The optimum temperature (60°C) of the free and entrapped enzymes remained unaltered. In contrast, the half-life of the endoglucanase immobilized in calciumalginate beads was 4.6 h at 55°C and 5.4 h at 60°C, while that of the free enzyme was 3.0 h at 55°C and 1.2 h at 60°C. A technological application of the immobilized enzymes was tested using wheat straw as a source of fermentable sugars. The hydrolytic degradation of straw, by means of a crude extract of free and immobilized cellulases and β-glucosidase, released a large amount of reducing sugars from wheat straw after 48 h (between 250–720 mg glucose/g straw), carrying out more than a 90% saccharification. A mixture of immobilized β-glucosidase and free cellulases maintained 80% of the activity of the soluble counterparts, and the co-immobilization of both types of enzymes reduced by hydrolytic efficiency to half.     

Increased erythromycin production by alginate as a medium ingredient or immobilization support in cultures of <Emphasis Type="BoldItalic">Saccharopolyspora erythraea</Emphasis>

Erythromycin production by Saccharopolyspora erythraea immobilized in 2% (w/v) calcium alginate or grown in medium containing 20 g sodium alginate/l inoculated with free cells was almost twice more than that of the control. S. erythraea did not consume alginate, agar, dextran, silicon antifoaming agent or cyclodextrin as a carbon source, although, all of these increased the production of erythromycin. Highest titer of erythromycin (2.3 times more than that of the control) was achieved in medium containing 1 g agar/l.     

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     

Immobilization of glucose oxidase on Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>/SiO<Subscript>2</Subscript> magnetic nanoparticles

Glucose oxidase (GOD) was covalently immobilized onto Fe₃O₄/SiO₂ magnetic nanoparticles (FSMNs) using glutaraldehyde (GA). Optimal immobilization was at pH 6 with 3-aminopropyltriethoxysilane at 2% (v/v), GA at 3% (v/v) and 0.143 g GOD per g carrier. The activity of immobilized GOD was 4,570 U/g at pH 7 and 50°C. The immobilized GOD retained 80% of its initial activity after 6 h at 45°C while free enzyme retained only 20% activity. The immobilized GOD maintained 60% of its initial activity after 6 cycles of repeated use and retained 75% of its initial activity after 1 month at 4°C whereas free enzymes retained 62% of its activity.