Optimization of medium components using response surface methodology for production of thermostable amylopullulanase in submerged fermentation by Clostridium thermosulfurogenes SVM17

Response surface methodology (RSM) based on central composite rotatable design (CCRD) was used to determine the optimal levels of medium components, viz., soluble starch, tapioca flour, peptone, magnesium chloride and ferrous sulphate for enhanced thermostable amylopullulanase production by Clostridium thermosulfurogenes SVM17 in submerged fermentation. The design contains a total of 54 experimental trials with first 32 organized in a fractional factorial design and experimental trials from 33-40 and 51-54 involving the replication of the central points. Within the tested range of concentrations, all medium components were found significant. The optimum levels of nutrients for maximum production of enzyme were (% w/v): potato starch, 5.2; tapioca flour, 6.3; peptone, 2.5; MgCl2·6H2O, 0.015 and FeSO4·7H2O, 6.0 ppm. After optimization of medium components, the strain SVM17 showed 96 and 409 % increased amylase and pullulanase activities, respectively when compared with the non-optimized conditions

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Not AvailableShelf-life studies were carried out on ribbonfish (Trichiurus haumela) cured with red halophilic bacteria (Salinicoccus roseus). Results revealed that the use of salt treatment with sodium metabisulphite, sodium acid phosphate, sodium benzoate, sodium tripolyphosphate, sodium hexametaphosphate and brifisol extended the shelf-life of cured ribbonfish from 3 to 12 months. The red discolouration was monitored up to the point of spoilage. The TVN and PV in crystalline salt cured ribbonfish control samples increased from 25.3 to 416.7 mg N% and 31.5 to 235.0 meq O2/kg, respectively during a storage period of 12 months. The treatment effectiveness for TVN and PV was more in semi-ground salt cured fish than in crystallized salt-cured fish. The bacteriological, physicochemical and sensory quality of the fish cured with treated salt was better than those of the control fish.Not Availabl

Effect of Substrate and Culture Conditions on the Production of Amylase and Pullulanase by Thermophilic Clostridium thermosulforegenes SVM17 in Solid State Fermentation

The endo acting enzyme with dual specificity towards α-1,4- and α-1,6-glycosidic linkages are named as amylopullulanase. The production of extracellular thermostable amylopullulanase by Clostridium thermosulfurogenes SVM17 was investigated in solid state fermentation (SSF). Coarse type wheat bran was found to be the best substrate among ten easily available complex organic substrates evaluated. The production of enzyme reached a peak in 72 h. A high level of enzyme was produced in wheat bran moistened with PYE medium with a moisture content of 73 %. The optimum temperature and pH for amylopullulanase production was 60 °C and 7.5, respectively. An inoculum size of 20 % resulted in maximum production of amylopullulanase. Under the optimum conditions the strain showed a maximum of 17,227 and 21,526 U of amylase and pullulanase activity, respectively per kilogram of bacterial bran (BB). The enzyme production was high in SSF than that in SmF. The use of SSF for the production of thermostable amylopullulanase by C. thermosulfurogenes SVM17 could, therefore led to reduction in the overall cost of enzyme production

Production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of nutrients levels using response surface methodology

Abstract The optimization of nutrient levels for the production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation (SSF) was carried out using response surface methodology based on the central composite rotatable design. The design contains a total of 54 experimental trials with the ®rst 32 organized in a fractional factorial design and experimental trials from 33±40 and 51±54 involving the replication of the central points. The design was employed by selecting potato starch, magnesium chloride, ferrous sulfate, corn steep liquor and pearl millet¯our as model factors. Among the ®ve independent variables studied, except magnesium chloride, all the nutrients were found signi®cant. 16.5% potato starch, 2.5% corn steep, 0.015% ferrous sulfate and 14% pearl millet¯our have been found optimal for the production of thermostable pullulanase. The strain SV2 produced 10% more pullulanase in the nutritionally optimized solid-state fermentation medium containing only four nutrients. Introduction In recent years, pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41), a debranching enzyme, has been gaining importance in starch conversion processes In recent times, the bacterial systems are increasingly investigated for the production of enzymes and metabolites by solid-state fermentation (SSF

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Not AvailableIn the present study, the quality and shelf life of salt cured ribbonfish (Trichiurus lepturus) using heat-treated salt artificially contaminated with known density of Salinicoccus roseus was carried out. To determine whether crystal size of the salt had any effect on the quality of cured fish, semi-ground salt was used along with crystalline (natural) salt. Salinicoccus roseus, a dominant halophilic bacteria among red halophiles isolated from the commercial salt-cured fish showing red discoloration, was used to contaminate the sterile crystalline and semi-ground salts. The salts were further heat treated to test the efficacy of heat treatment of the salt on the bacteria. Shelf life studies were carried till signs of spoilage were evident. During the storage period, total bacterial count, moisture, total volatile nitrogen (TVN), peroxide value (PV) and free fatty acids (FFA) were monitored at three months intervals for one year. During three months of storage, TVN in ribbon fish cured with crystalline salt (control) reached 416.7 mg N% from an initial value of 25.33 mg N%. On the contrary, ribbon fish cured with heat treated crystalline salt (CS) and semi-ground (SS) salt had TVN 204.82 and 168.48 mg N% after 6 and 12 months storage periods respectively. The treatment effect in terms of TVN was 0.62 and 0.65 for CS and SS cured ribbon fish. The peroxide values (PV) increased in crystalline salt cured (control) samples from an initial value of 31.46 to 235 milliequivalents of O sub(2) per Kg of fat at the end of three month storage period. However, these values were 288.46 and 127 milliequivalents of O sub(2) per Kg of fat for heat treated CS and SS cured ribbon fish after 6 and 12 months storage period, respectively. The treatment effect in relation to PV was 0.60 and 0.90 for CS and SS cured ribbon fish. The present study revealed that heat treatment of solar salt used for curing of fish enhanced the shelf life of cured fish from 6 to 12 months when compared to the control. The use of semi-ground salt for curing of fish has the added advantage of superior quality, treatment effectiveness and longer shelf life than that of crystalline salt cured fishes.Not Availabl

Purification and Characterization of Highly Thermostable Amylopullulanase from a Thermophilic, Anaerobic Bacterium Clostridium thermosulfurogenes SVM17

A highly thermostable amylopullulanase was purified to homogeneity from the culture filtrate of the Clostridium thermosulfurogenes SVM17. On SDS-PAGE, the purified fraction having both amylase and pullulanase activities were observed as a single band. The molecular weight of the purified amylopullulanase on SDS-PAGE was 97 kDa. The optimum temperature for both amylase and pullulanase was 70 °C. The enzyme was completely stable at 70 °C for 2 h. The presence of 5% starch increased the thermal stability of the enzyme at 100 °C up to 2 h. Both amylase and pullulanase activities were optimum at pH 5.5 to 6.0 and were stable over a pH range of 4.0 to 6.5. The TLC analysis of the reaction products on starch showed that maltose was the main product along with trace amounts of glucose. The analysis of hydrolysis product of pullulan showed that maltotriose was the main product. At 5 mM concentration, Mn2+ and Ag+ strongly stimulated both amylase and pullulanase activities, where as Mg2+, Ca2+, Cu2+, Fe3+, Zn2+, Hg2+, EDTA, Cd2+ and Li2+ inhibited both amylase and pullulanase activities. When the concentration of metal ions was increased from 5 to10 mM, a further increase in amylase activity was observed in the presence of Ni2+, Mn2+ and Co2+. Where as substantial decrease was observed at 10 mM concentration of Ag+, Pb2+ and Ca2+

L-selective dipeptide synthesis using novel thermophilic enzyme from Clostridium sp.

A novel, inexpensive, thermophilic protease-type enzyme isolated from Clostridium thermohydrosulfuricum was used for dipeptide synthesis. The enzyme showed broad substrate selectivity and enantioselectivity towards L-amino acids in peptide bond formation