Production Of Isoamylase Research Articles

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.

Production of Extracellular Isoamylase and Endoglucanase by Entrapped Spores of Rhizopus oryzae PR7

The encapsulation of spores of Rhizopus oryzae PR7 by ionotropic gelation and production of isoamylase and endoglucanase was observed in the present study. The immobilized spores showed maximum production of both the enzymes in beads made up of 9 % of alginate concentration. Maximum production of isoamylase and endoglucanase was found when immobilization of spores involved BaCl2 and CaCl2 respectively. Linear negative correlation was found between isoamylase production and number of spores used for the inoculum, while in case of endoglucanase, production value was more or less independent of the inoculum size. The immobilized spores were reutilized effectively for isoamylase production by recycling of the entrapped spores for four consecutive cycles.

Induction and carbon catabolite repression of isoamylase production in Rhizopus oryzae PR7

The growth and the extracellular isoamylase production by Rhizopus oryzae PR7 MTCC 9642 were studied in a stationary culture at 28°C, with maximum isoamylase production obtained after 72 hours. Glycogen was found to be the best inducer for isoamylase synthesis, followed by maltose and dextrin. The enzyme was found to be repressed by glucose and this repression was not overcome by the addition of cGMP. The abrupt reduction in enzyme synthesis after the addition of exogenous glucose in a glycogen-induced culture medium confirmed the repressive action of glucose. An almost similar rate of repression was found to be exerted by α- and β-cyclodextrins. The inhibition of enzyme production after the addition of cycloheximide, a translation blocker, indicated the existence of de novo synthesis of the enzyme.

Optimum conditions for isoamylase production by Pseudomonas sp.

Different nutritional and cultural factors were studied to determine the optimum conditions for isoamylase production by Pseudomonas sp. in a batch culture of the production medium. These factors include carbon, nitrogen and phosphate sources and their concentrations, temperature and pH. Results showed that the optimum conditions for isoamylase production by Pseudomonas sp. were achieved when the production medium was supplemented with maltose 1%, peptone 0.4%, and K2HPO4 0.4% as a carbon, nitrogen, and phosphate sources respectively, at initial medium pH 6, and incubation at 28°C for 24 hours. Under these conditions isoamylase productivity reaches the maximum, at which enzyme specific activity was 0.85 U/mg proteins.

Extra-cellular isoamylase production by Rhizopus oryzae in solid-state fermentation of agro wastes

Extra-cellular isoamylase was produced by Rhizopus oryzae PR7 in solid-state fermentations of various agro wastes, among which millet, oat, tapioca, and arum (Colocasia esculenta) showed promising results. The highest amount of enzyme production was obtained after 72 h of growth at 28°C. The optimum pH for enzyme production was - 8.0. Among the various additives tested, enzyme production increased with ions such as Ca2+, Mg2+ and also with cysteine, GSH, and DTT. The enzyme synthesis was reduced in the presence of thiol inhibitors like Cu2+ and pCMB. The surfactants like Tween-40, Tween-80 and Triton X-100 helped in enhancing the enzyme activity. The production could be further increased by using the combinations of substrates. The ability to produce high amount of isoamylase within a relatively very short period and the capability of degrading wastes could make the strain suitable for commercial production of the enzyme.

Optimization of Acidic Isoamylase Production by Isolated Bacillus coagulans

Optimization of Acidic Isoamylase Production by Isolated Bacillus coagulans

Production enhancement of Pseudomonas amyloderamosa isoamylase

Isoamylase production in batchwise and fed-batch cultures of Pseudomonas amyloderomosa (strain WU7211-2) was investigated. By feeding maltose in a mode motivated by its structure gene (iam), the final isoamylase activity of 5100 U/ml was achieved, as compared to 4100 U/ml in batch cultivation and 3800 U/ml in shaken flasks. The enhancement may be due to the fact that the production of isoamylase can be induced effectively by the maltose only if the glucose concentration is maintained below the inhibitory level. Also, cultivations based on 500-ml shaken flasks, performed with different amount of proteimax HE90, showed that higher amounts of proteimax HE90 resulted in an increased value of pH that had an adverse effect on isoamylase yield.

Effect of pH on isoamylase production by Pseudomonas amyloderamosa WU 5315

The isoamylase activity of Pseudomonas amyloderamosa WU 5315 was stable over the pH range from 5.5 to 6.25 while only about 30% of the activity remained at pH 6.5. Low isoamylase activity (418 U ml‐1) was produced by the cells grown at high pH. Activity reached almost 3000 U ml‐1 when pH was kept below 6.0 during the fermentation. With 1% glucose plus 2% maltose instead of 3% maltose as carbon source, however, no pH control was required and the isoamylase activity of Ps. amyloderamosa WU 5315 increased to 3400 U ml‐1.

Optimization of cultivation medium composition for isoamylase production

The medium composition for production of isoamylase by Pseudomonas amyloderamosa JD210 was optimized using response surface methodology. The factors chosen for optimization were maltose, soybean protein hydrolysate (SPH), isoleucine, proline, KH2PO4 and MgSO4. Fractional factorial designs (FFD) and the path of steepest ascent were effective in searching for the major factors and optimum medium composition. By a 26–1 FFD, supplementary isoleucine was shown to have a negative effect on enzyme production. The effects of the other five factors were further investigated by a central composite design and the optimum composition was found to be 1.10% maltose, 0.13% SPH, 0.15% proline, 0.38% KH2PO4, and 0.05% MgSO4. When the strain was cultivated in the optimum medium, enzyme production increased 60% compared with ordinary medium. Proline was verified as being a significant factor in promoting enzyme production.

Production of isoamylase by Escherichia intermedia.

A culture of a coliform bacteria isolated from soil produced isoamylase when grown in peptone-maltose-salts broth. The enzyme preparation was partially purified. Its similarity with isoamylase produced by Aerobacter aerogenes was established by studying its action upon various polysaccharides. A study of the organism producing isoamylase showed that it differed from A. aerogenes in some of its physiological properties. This organism was identified as Escherichia intermedia from morphological and cultural characteristics.

Production of Isoamylase by Escherichia intermedia

A culture of a coliform bacteria isolated from soil produced isoamylase when grown in peptone-maltose-salts broth. The enzyme preparation was partially purified. Its similarity with isoamylase produced by Aerobacter aerogenes was established by studying its action upon various polysaccharides. A study of the organism producing isoamylase showed that it differed from A. aerogenes in some of its physiological properties. This organism was identified as Escherichia intermedia from morphological and cultural characteristics.