Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

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.     

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.     

Biotransformation of acrylonitrile to acrylamide using immobilized whole cells of Brevibacterium CH1 in a recycle fed-batch reactor

Acrylamide was produced from acrylonitrile using immobilized Brevibacterium CH1 cells that were isolated from soil and found to possess nitrile hydratase activity. The reaction conditions and stability of the enzyme activity were studied. The conversion yield was nearly 100%, including a trace amount of acrylic acid. This strain showed strong activity of nitrile hydratase toward acrylonitrile and extremely low activity of amidase toward acrylamide. A packed bed reactor was operated in a fed-batch manner for acrylamide production of high concentration. The acrylonitrile concentration was maintained below 3% and the operating temperature at 4 degrees C to minimize enzyme deactivation.     

A study of the catalytic properties of the nitrile hydratase immobilized on aluminum oxides and carbon-containing adsorbents

The nitrile hydratase isolated from Rhodococcus ruber strain gt1, displaying a high nitrile hydratase activity, was immobilized on unmodified aluminum oxides and carbon-containing adsorbents, including the carbon support Sibunit. The activity and operational stability of the immobilized nitrile hydratase were studied in the reaction of acrylonitrile transformation into acrylamide. It was demonstrated that an increase in the carbon content in the support led to an increase in the amount of adsorbed enzyme and, concurrently, to a decrease in its activity. The nitrile hydratase immobilized on Sibunit and carbon-containing aluminum α-oxide having a “crust” structure displayed the highest operational stability in acrylonitrile hydration. It was shown that the thermostability of adsorbed nitrile hydratase increased by one order of magnitude.     

Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.     

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.     

Nitrile-metabolizing potential of Amycolatopsis sp. IITR215

Strain Amycolatopsis sp. IITR215 was isolated from a sewage sample using polyacrylonitrile powder as the sole nitrogen source. Identification was performed by 16S rDNA analysis. The isolated strain harbored multiple nitrile-metabolizing enzymes having a wide range of substrate specificities. It metabolized nitrile and amide compounds with constitutive enzymes. Studies using an amidase inhibitor showed that hydrolysis of acrylonitrile and acrylamide occurred due to nitrile hydratase and amidase, respectively, while hydrolysis of hexanenitrile was due to the action of either nitrilase or a second nitrile hydratase/amidase system. The inhibitory effects of N-bromosuccinimide and N-ethylmaleimide on enzymes of this culture were also studied and this further indicated the involvement of either a nitrilase or a second nitrile hydratase/amidase system for hydrolysis of hexanenitrile. Interestingly, hexanenitrile hydrolysis exhibited an optimum temperature of 55 °C, whereas acrylonitrile and acrylamide hydrolysis showed an optimum temperature of 45 °C. The optimum pH was 5.8 for hexanenitrile hydrolysis and 7.0 for acrylonitrile and acrylamide hydrolysis. Hexanenitrile hydrolysis by enzymes of this strain showed better organic solvent tolerance in the presence of alcohols. The maximum enzyme activity of nitrile-metabolizing enzymes was found using media containing isobutyramide as the nitrogen source. This is the first report on constitutive multiple enzymes from the Amycolatopsis genus.     

Immobilization of <Emphasis Type="Italic">Rhodococcus ruber</Emphasis> strain gt1, possessing nitrile hydratase activity,on carbon supports

Rhodococcus ruber strain gt1, possessing nitrile hydratase activity, was immobilized by adsorption on carbon supports differing in structure and porosity. The adsorption capacity of the supports towards cells, the substrate of the nitrile hydratase reaction (acrylonitrile), and the product (acrylamide) was studied. Also, the effect of immobilization on nitrile hydratase activity of bacteria was investigated, and the operational stability of the immobilized biocatalyst was determined. It was shown that crushed and granulated active coals were more appropriate for immobilization than fibrous carbon adsorbents.     

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     

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.     

Solvent Effects on Circular Dichroism of Colominic Acid

To produce acrylamide from acrylonitrile by use of a new enzyme, nitrile hydratase, a number of nitrile-utilizing microorganisms were screened for the enzyme activity by an intact cell system. An isobutyronitrile-utilizing bacterium, strain B23, showed the best productivity among 186 strains tested. The strain was identified taxonomically as Pseudomonas chlor or aphis. The culture and reaction conditions for the production were studied for the strain. Under the optimum conditions, 400 grams/liter of acrylamide was produced in 7.5 hr. The yield was nearly 100% with a trace amount of acrylic acid. The cell-free extract of the strain showed strong activity of nitrile hydratase toward acrylonitrile and extremely low activity of amidase toward acrylamide.     

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.     

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.     

Catalytic properties of a nitrile hydratase immobilized on activated chitosan

The catalytic properties of a nitrile hydratase, isolated from a strain of Rhodococcus ruber gt1 and immobilized by covalent cross-linking with chitosan activated with 0.1% benzoquinone solution, have been investigated. The kinetic parameters of acrylonitrile hydration catalyzed by immobilized nitrile hydratase and the enzyme in a solution have been determined. It is found that the immobilization does not lead to a decrease in the maximum reaction rate (V _max), whereas the Michaelis constant (K _M) is reduced by a factor of 2.4. The possibility of reusing an immobilized enzyme for 50 consecutive cycles of acrylonitrile transformation was shown, and the nitrile hydratase activity in the 50th cycle exceeded that in the first cycle by 3.5 times. It is shown that the effect of temperature on activity depended on the concentration of the enzyme, which confirms the dissociative nature of nitrile hydratase inactivation. It was found that immobilized nitrile hydratases remain active at pH 3.0–4.0, whereas the enzyme is inactivated in a solution under these conditions. The resulting biocatalyst can be effectively used to receive acrylamide from acrylonitrile.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

Immobilization of Xylan-Degrading Enzymes from Scytalidium thermophilum on Eudragit L-100

Summary Xylanase from Scytalidium thermophilum was immobilized on Eudragit L-100, a pH sensitive copolymer of methacrylic acid and methyl methacrylate. The enzyme was non-covalently immobilized and the system expressed 70% xylanase activity. The immobilized preparation had broader optimum temperature of activity between 55 and 65 °C as compared to 65 °C in case of free enzyme and broader optimum pH between 6.0 and 7.0 as compared to 6.5 in case of free enzyme. Immobilization increased the t_1/2 of enzyme at 60 °C from 15 to 30 min with a stabilization factor of 2. The K_m and V_max values for the immobilized and free xylanase were 0.5% xylan and 0.89 μmol/ml/min and 0.35% xylan and 1.01 μmol/ml/min respectively. An Arrhenius plot showed an increased value of activation energy for immobilized xylanase (227 kcal/mol) as compared to free xylanase (210 kcal/mol) confirming the higher temperature stability of the free enzyme. Enzymatic saccharification of xylan was also improved by xylanase immobilization.     

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.     

Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties

Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E _A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E _A = 1.59 and 1.99 Kcal mol⁻¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K _m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.     

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.