Hydrolysis of animall fats by lipase at temperatures below their melting points

Summary We have studied the hydrolysis of high melting animal fats by the lipase fromCandida rugosa at temperatures between 20°C and 37°C without the addition of surfactants or organic solvents. To establish the practical applications of this process we investigated the optimal conditions of the reaction at high substrate concentrations (50% fat w/v) to achieve 95% hydrolysis (or better) in 24 hours. Experiments were conducted in solid emulsions without constant stirring (500 ml total reaction volume). Under all conditions tested, edible pork lard was a better substrate than inedible beef tallow yielding up to 96% hydrolysis with as low as 0.3 g lipase/Kg fat or 98% hydrolysis with 0.5 g lipase/Kg fat. The optimum temperature for the hydrolysis of edible pork lard was around 30°C. Inedible beef tallow and pork lard did not exhibit a clear optimum temperature. Inedible lard gave results intermediate between those of edible lard and inedible beef tallow.     

Selection of yeast able to produce ethanol from glucose at 40° C

Summary A total of 55 yeast strains selected from 7 genera known to ferment carbohydrates to ethanol were screened for their ability to ferment glucose to ethanol in shaken flask culture at 37°, 40° and 45°C. Yields of more than 50% of the theoretical maximum were obtained with 28 strains at 37°C, but only 12 at 40°C. Only 6 could grow at 45°C, but they produced poor yields. In general Kluyveromyces strains were more thermotolerant than Saccharomyces and Candida strains, but Saccharomyces strains produced higher ethanol yields. The 8 strains with the highest yields at 40°C were evaluated in batch fermentations. Three of these, two Saccharomyces and one Candida, were able to meet minimum commercial targets set at 8% (v/v) ethanol from 14% (w/v) glucose at 40°C.     

Characterization of a superior thermotolerant mutant ofZymomonas mobilis ZM4 for ethanol production in glucose medium

Summary Nitrosoguanidine-induced, stable theromotolerant mutant (ZMI₂) ofZymomonas mobilis ZM4 was found to possess almost normal cell morphology, and a better ethanol tolerance at 42°C than the parent strain (ZM4). Its kinetic parameters, in converting different concentrations of glucose to ethanol, were comparable to ZM4 at 30°C, and significantly superior at 42°C. In a 200 g/L glucose medium in a pH-stat (5.0) at 42°C, the mutant yielded more ethanol (71.0 g/L) (improved to 73.7 g/L at pH 5.5) and alcohol dehydrogenase (ADH) than the parent strain. The ADH levels in both the strains were repressed, depending upon the increased level of sugar and degree of temperature.     

Effect of temperature on plasmid stability and expression of cloned cellulase gene in a recombinantBacillus megaterium

Summary The expression of carboxymethyl cellulase gene inBacillus megaterium (pCK108) was investigated with respect to temperature in batch culture. The suboptimal temperature supporting maximal cell growth rate was determined to be 30 °C at which stability of the plasmid pCK108 could be maintained stable. However, cellular plasmid contents, production rate of cellulase of the cell, and efficiency of the gene expression increased significantly with increase of the temperature from 30 °C to 44 °C, even though the plasmid stability decreased up to 60% level at the end of the culture.     

The isomaltulose synthesising enzyme ofSerratia plymuthica

Summary An intracellular enzyme was located inSerratia plymuthica which produced isomaltulose from sucrose. The enzyme was purified giving a preparation with a specific activity of 1,285. It has pH and temperature optima of 6.0 and 30°C, respectively. The enzyme was stable retaining 100% activity after 2 weeks at 30°C. It had an isoelectric point at pH 9.0, a Mr of 79,500 and the Km for sucrose was 65.3mM. The enzyme converted 40% (w/v) sucrose to isomaltulose with an efficiency of 87%.     

The effect of temperature,pH, and initial glucose concentration on the kinetics of ethanol production by Zymomonas mobilis in batch fermentation

Summary Zymomonas mobilis, strain ATCC 10988, was used to evaluate the effects of pH (5.0 to 8.0), temperature (30°C to 40°C), and initial glucose concentration (75 g/l to 150 g/l) on the kinetics of ethanol production from glucose using batch fermentation. Specific ethanol production rate was maximum and nearly constant over a pH range of 6.0 to 7.5. End-of-batch ethanol yield and specific growth rate were insensitive to pH in the range of 5.0 to 7.5. End-of-batch ethanol yield was maximum and nearly constant between 30°C and 37°C but decreased by 24% between 37°C and 40°C. All other kinetic parameters are greatest at 34°C. End-of-batch ethanol yield is maximum at an initial glucose concentration of 100 g/l. Specific growth rate reaches a maximum at 75 g/l, but specific ethanol production rate decreases throughout the range. The optimum initial glucose concentration of 100 g/l gives the highest ethanol yield at a specific ethanol production rate less than 10% below the maximum observed.     

Production of active human interferon-β inE. coli I. Preferential production by lower culture temperature

Summary Unusually low culture temperature, such as 20°C, was shown to be preferable for the synthesis of active human interferon- (IFN-) inE. coli harboring a recombinant plasmid. TheE. coli cells cultured at 20°C gave 8.6-fold higher IFN- activity than those cultured at 37°C. However, almost the equal amounts of IFN- protein were accumulated in both cells cultured at 20°C and at temperature higher than 20°C, suggesting that IFN- might exist as an active form in the cells cultured at 20°C, while as a rather denatured form in the cells cultured at higher temperature.     

Kinetic behavior of Candida rugosa in the batch fermentation of sugar beet stillage; Temperature dependence of growth and flocculation characteristics

Summary The aerobic fermentation of sugar beet stillages to yeast biomass was carried out byC.rugosa at 20, 30, and 40°C. At 40°C,C.rugosa grew faster with corresponding COD-reduction, and showed better flocculation characteristics.     

Improvement of phytase thermostability by using sorghum liquor wastes supplemented with starch

When a phytase solution, soluble starch, and sorghum liquor wastes were mixed at the ratio of 1:1:10 (v/w/w), the residual phytase activities after 30 min of treatment at 70 and 80 °C were respectively, about 90% and 18% of that at 37 °C. After 10 min treatment, the residual activity was 67% at 80 °C and 10% at 90 °C.     

Production of dextranase byLipomyces starkeyi

Summary The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.     

Continuous production of β-cyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan

Cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on glutaraldehyde-activated chitosan spheres and used in a packed bed reactor to investigate the continuous production of β-cyclodextrin (β-CD). The optimum temperatures were 75 °C and 85 °C at pH 6.0, respectively for free and immobilized CGTase, and the optimum pH (5.0) was the same for both at 60 °C. In the reactor, the effects of flow rate and substrate concentration in the β-CD production were evaluated. The optimum substrate concentration was 4% (w/v), maximizing the β-CD production (1.32 g/L) in a flow rate of 3 mL/min. In addition, the biocatalyst had good operational stability at 60 °C, maintaining 61% of its initial activity after 100 cycles of batch and 100% after 100 h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.     

Plantlet production through high frequency somatic embryogenesis in long term cultures ofEucalyptus citriodora

A highly embryogenic culture ofEucalyptus citriodora was obtained by repetitive embryogenesis from somatic embryos cultured in the dark on a medium containing 500 mg/l each of glutamine and casein hydrolysate, 30 g/l of sucrose and 5 mg/l of 1-napthaleneacetic acid. Cultures retained morphogenetic ability for upto 36 months when maintained at 27°C by subculture at intervals of 4–5 weeks. The subculture period could be extended beyond 9 months if cultures were incubated at 10°C. On a hormone free medium incubated in light 50% of the embryos germinated to plantlets of which 70% survived when transferred to a sand and soil mixture.Abbreviations NAA 1-naphthal eneacetic acid NCL Communication No: 4480     

Hydrogenation with entrapped clostridium spec. LA 1 and observation on its stability

Summary Clostridium spec. La 1 can be entrapped in ENT-110 with reasonable activity for the hydrogenation of ²-enoates or aldehydes with hydrogen gas. This catalyst can be stored for several months. In operation at 35° C it has a half-life of about 10 days. The catalyst can be reisolated and reused. The presence of an organic solvent such as decalin does not reduce its stability.     

Solid state fermentation of straw withNeurospora crassa for CMCase and β-glucosidase production

Summary CMCase and -glucosidase were produced by the mutantNeurospora crassa 40b cultivated on untreated wheat straw in a solid state fermentation. Best enzyme activities were observed when the growth medium was composed of wheat straw mixed with certain mineral solutions at a ratio 1:2 (w/v). A partially purified enzyme preparation showed optimum enzyme activities of CMCase and -glucosidase at pH 4.0 and 5.0 and temperature 50 and 60°C respectively. The apparent Km values for the same enzymes were 16.8 g/l and 1.03x10^–4 M respectively. At optimum growth and enzyme assay conditions yields as high as 586.2 U CMCase and 58.4 U -glucosidase per gram of straw were obtained.     

Partial purification and properties of proteases from fig (Ficus carica) callus cultures

Summary Cell culture of fig (Ficus carica) was evaluated as a source of thermostable cysteine proteases. Extracts of 28 day old callus contained a benzoyl-arginine-p-nitroanilide (BAPNA) hydrolase activity of 2200 U/g dry wt (49.5 U/mg protein) at 25°C. Thermal stability and thermal denaturation (at 70°C) properties showed some similarities to those of commercial ficin (Sigma). High performance gel filtration revealed the presence of a peak with BAPNA hydrolase and caseinolytic activity which matched commercial ficin. Covalent chromatography using Thiopropyl Sepharose 6B (Pharmacia) demonstrated that approx 50% of the total BAPNA hydrolase activity could be attributed to thiol-proteins.Abbreviations BAPNA benzoyl-arginine-p-nitroanilide - BAPNAse BAPNA hydrolase Contribution No. 147.     

Production and stabilization of amylase preparations from rice bran solid medium

A low-cost amylase preparation of dried fermented bran was developed from rice bran solid cultures of Aspergillus oryzae supplemented with soya bean flour (SBF) and cassava starch (3:1) and dried at 50 °C for 4 h. Storage stability of preparations at 4 °C or 30 °C was significantly enhanced (P 0.05) by adding SBF or partially hydrolyzed starch (PHS). While amylase preparations without stabilizer retained 59 and 48% of their activity after 12 weeks storage at 4 and 30 °C respectively, the same preparations fortified with SBF (5% w/v) retained 95 and 94% stability respectively, during the same period. PHS at 5% (w/v) also gave a maximum stability of 94 and 91.8% at 4 and 30 °C, respectively. The unstabilized preparation retained only 42% of its activity compared to the stabilized forms, which retained 82–90% activity after 15 min incubation at 100 °C.     

Influence of temperature on solvents production from whey

Summary The influence of temperature on solvent production from whey was investigated by using strains ofClostridium acetobutylicum andbutylicum. Higher yields of solvents were observed at 37°C or at 30°C depending on the strain used.     

Effect of fermentation variables on cellulase production by Trichoderma Sp.

Summary Batch cultivation ofTrichodermma reesei QM9414 was carried out in Mandels medium containing(w/v) 1% beech wood cellulose and 0.05% yeast extract at 29°C. Use of 36 hours old inoculum(10% v/v),3.2 1/min aeration rate at 400 rpm(K_La 220/h) and pH cycling strategy produced 4 g/1 cell mass and 21.5 IU/1/h FPA cellulase.     

Response surface optimization ofLactobacillus plantarum batch growth

Summary Optimal conditions for batch growth ofLactobacillus plantarum, ATCC 8014, are a pH of 6.0, a temperature of 33°C, and an initial glucose concentration of 24 g/l. A maximum biomass concentration of 6.0 g/l was achieved. Growth parameters were also determined.     

The combined relationship of temperature and molybdenum concentration to nitrogen fixation byAnabaena cylindrica

The joint effects of growth temperature, incubation temperature, and molybdenum concentration on the nitrogen fixation rate ofAnabaena cylindrica were determined using the acetylene-reduction technique. The nitrogen-fixation response to increased molybdenum concentration varied among three growth temperatures (15°, 23°, and 30° C). The pattern of rate change was similar within a growth temperature but increased overall in magnitude with the three incubation temperatures (also 15°, 23°, and 30° C). The maximum rate of nitrogen fixation occurred at 30°C regardless of previous growth temperature. The minimum molybdenum concentration necessary to yield substantial acetylene reduction varied with growth temperature: at 15°C, 15g 1^–1 was effective; at 23°C, less than 5g 1^–1 was effective; and at 30°C, 50g 1^–1 was effective. At all three growth temperatures, increases in molybdenum concentration above the minimum effective concentration produced increases in acetylene reduction. However, at higher molybdenum concentrations inhibition of nitrogen fixation occurred.