Adsorption of aspartase onto hydrophobic cloth for conversion of ammonium fumarate

Summary A simple aspartase assay was developed. Aspartase fromEscherichia coli Crooks strain was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme did not desorb in 1 M ammonium fumarate. The adsorbed enzyme exhibited the same pH vs. activity curve as free enzyme and had a half life of approx. 40 weeks. A column packed with the adsorbed aspartase showed 100% conversion of 1 M ammonium fumarate at a space velocity of approx. 2.     

Resistance of bacterial film to contamination during use as inoculum in repeated batch fermentation

Summary AnEscherichia coli strain constitutive for -galactosidase was immobilized onto cotton cloth. The resultingE.coli film was used as a resident inoculum in repeated batch fermentations for 30 days in the presence ofBrevibacterium ammoniagenes added as a contaminant. Analysis of -galactosidase production shows that contamination did not decrease the capacity of the film to generateE.coli cells, or decrease theE.coli population on the film.     

Amylolytic enzymes produced by a color variant strain ofAureobasidium pullulans

A color variant strain ofAureobasidium pullulans (NRRL Y-12974) produced amylase and -glucosidase activities when grown at 28°C for 4 days in liquid culture on a wide variety of carbon sources such as starch, pullulan, glucose, maltose, cyclodextrins, sucrose, xylose, and xylan. An -glucosidase was separated by Q-Sepharose adsorption from the cell-free culture broth and partially purified by hydroxylapatite and octyl-Sepharose chromatography. After ammonium sulfate treatment of the culture supernatant (obtained after Q-Sepharose adsorption), the amylase fraction was separated into three active fractions by hydroxylapatite column chromatography, which were identified as -amylase, glucoamylase A, and glucoamylase B. The glucoamylase A was further purified by octyl-Sepharose column chromatography. The pH optima for the action of -amylase, glucoamylase A, glucoamylase B, and -glucosidase were 5.0, 4.5, 4.0–4.5, and 4.5, respectively. The -amylase and glucoamylase B were fully stable at pH 3.0–6.0, glucoamylase A at pH 4.5–5.5, and -glucosidase at pH 3.5–7.0 for 1 h at 50°C. The optimum temperatures for the action of these enzymes were 55°, 50°–60°, 65°, and 65°C, respectively. The -amylase, glucoamylase A, and glucoamylase B were adsorbed onto raw corn starch and degraded it. Glucoamylase B readily cleaved pullulan. The -glucosidase was not adsorbed onto raw starch and did not degrade it at all. It hydrolyzed both -1,4 and -1,6 linkages in oligosaccharides. All four enzymes did not require any metal ion for activity and were inhibited by cyclodextrins (-and -, 10mm).     

Characteristics of yeast β-galactosidase immobilized on calcium alginate gels

Summary Saccharomyces anamensis having -galactosidase activity, has been immobilized in calcium alginate gel matrix that retained 78.6% enzyme activity to that of native cells. Optimum pH(7.0) was negligibly affected by immobilization. K_m values for immobilized and native cells were 119 mM and 102 mM respectively. Protective agents like dithioerythritol, bovine serum albumin, enhance the enzyme activity when added prior to immobilization. Immobilized cells can be stored in refrigeration(4°C) for 42 days without a significant loss of enzyme activity.     

Glucoamylase and α-amylase production by immobilized Aspergillus niger

Summary Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and -amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and -amylase produced was altered by immobilization.     

Thermal Stability of Immobilized Glucoamylase Entrapped in Polyacrylamide Gels and Bound to SP-Sephadex C–50

Glucoamylase[α-1,4: 1,6-glucan-4: 6-glucohydroease, EC 3.2.1.3] from Rhizopus niveus was entrapped in polyacrylamide gels and adsorbed onto SP-Sephadex C–50 to elucidate the thermostability mechanism of immobilized enzymes. The thermal stability of immobilized glucoamylase entrapped in polyacrylamide gels was enhanced slightly compared with glucoamylase in free solution, and was independent of the acrylamide monomer concentration and N, N′-methylene-bis (acrylamide) content. To explain this phenomenon, the cellular structure of polyacrylamide gel was taken into consideration in addition to interactions between glucoamylase and gel, and a decrease in dielectric constant in the gel [S. Moriyama et al., Agric. Biol. Chem., 41, 1985 (1977)¹⁾]. On the other hand, immobilized glucoamylase bound to SP-Sephadex by ionic interaction showed lower stability than free glucoamylase, and much greater stability than glucoamylase in the presence of dextran sulfate, a constituent of SP-Sephadex. Thermal stabilities for the free and immobilized enzymes were also compared at the pH not in the bulk solution, but in the SP-Sephadex.     

Continuous ethanol production by immobilized cells of Zymomonas mobilis

Summary Studies have been carried out with a highly productive strain of Zymomonas mobilis in an immobilized cell reactor using both Ca alginate and -carrageenan as supporting matrices. Productivities above 50 g/l/h have been found at ethanol concentrations in excess of 60 g/l. With immobilized cells of Z. mobilis, there was a decline of approximately 30s% in activity after 800 h operation.     

Extracellular invertase from Aspergillus athecius: Isolation and immobilization

Summary A fungal strain isolated from soil and identified asAspergillus athecius, when grown on moistened wheat bran produced large amounts of extracellular invertase. Most of the invertase from the moldy bran was easily extracted by low ionic strength buffer (0.005 M, pH 5.7). The crude invertase immobilized on DEAE cellulose showed not only increased activity (45%) but also greater thermal and storage stability than the free enzyme. The free and the bound enzymes showed a temperature optimum of 50–55°C and a pH optimum of 5.7 and 4.8 respectively. The K_m app. of the bound enzyme was lower than that of the free enzyme.     

Isolation and immobilization ofSclerotium rolfsii protoplasts

Summary Protoplasts fromSclerotium rolfsii were prepared usingTrichoderma harzianum lytic enzymes and immobilized in Ca alginate gels. The immobilized protoplasts when incubated with 1% carboxymethylcellulose in osmotically stabilized induction medium, could secrete endoglucanase and -glucosidase. On repeated use the immobilized preparation retained 36% endoglucanase and 26% -glucosidase activity after 5 cycles.NCL Communication No. 3798     

Immobilization of β-Galactosidase by physical adsorption on Chromosorb-W

Summary -Galactosidase was immobilized by physical adsorption on Chromosorb-W. 94.8 % of the initial protein was adsorbed at 4^ºC, pH=5.5 and ionic strength=0.31. The specific activity of the immobilized derivative was 105.3 U/mg and K_M for o-nitrophenyl- -D-galactopyranoside was 0.12 mM.     

Isolation of aClostridium thermocellum gene encoding a thermostable β-1, 3-glucanase (laminarinase)

Summary AClostridium thermocellum gene directing the synthesis of a thermostable -glucanase was localized on a 1.9-kb DNA fragment by subcloning intoEscherichia coli plasmid vectors. The enzyme was highly efficient in degrading glucans with alternating -1, 3- and -1,4-linkages such as lichenan and barley glucan. It was also active towards the -1, 3-glucan laminarin, but lacked activity on cellulosic substrates and -glucans. The enzyme was therefore classified as -1, 3-glucanase (laminarinase) and the corresponding gene was designatedlicA. With barley -glucan as substrate the enzyme had a pH optimum around pH 6.5 and a temperature optimum at 65°C. It was stable for several hours at 60°C in the absence of substrate.     

Stability of plasmid PBR322 in Escherichia coli immobilized on cotton cloth during use as resident inoculum

Summary E. coli cells harbouring plasmid pBR322 which confers ampicillin resistance were immobilized on cotton cloth. The resulting film was used as an inoculum in daily repeated batch culture in ampicillin-free medium. During two months, the film was able to produce cultures which, at the late log phase, showed little sensitivity to 10 mg/ml ampicillin. Thus such a bacterial film can effectively be used as an inoculum for the production of recombinant DNA products by means of pBR322 or its derivatives in the absence of ampicillin.     

Spectrophotometric titrations of ferricytochrome C with ferrohexacyanide in the pH range 5 to 7

Spectrophotometric titrations were conducted on the system horse heart ferricytochromec plus ferrohexacyanide in the pH range 5 to 7 and at temperatures 8, 18, 22 and 28°C. A difference extinction coefficient for reducedvs. oxidized cytochromec at 550 nm of 21 mmol^–1cm^–1 was used in part of the evaluations. On the assumption that only one electron-transferlinked proton dissociation is effective for both ferro- and ferricytochromec in this pH range, various possible models are developed with only three conforming with the experimental pH dependence of the spectrophotometric equilibrium constant. The data conform best to a model with protonic dissociation constants between pH 5 and 7 such that the reduced cytochromec species is at least a factor of 3 more acidic than the one for oxidized cytochromec (with pK_H 6). This interpretation holds least for the data at 22°C, which points to a structural rearrangement at about this temperature (Czerlinski and Bracokova, 1973; Zabinski and Czerlinski, 1974; Zabinski, et al., 1974). While the extinction coefficient of ferrocytochromec shows no significant change with pH and temperature, the one for ferricytochromec does: it is about 5% larger at pH 5 than at pH 7 (550 nm). Graphs for the absorption change of ferricytochromec (pH 7 as reference) document the details over the wavelength range 500 to 750 nm.     

Solvent production from starch: effect of pH on α-amylase and glucoamylase localization and synthesis in synthetic medium

Summary The fermentation of starch by Clostridium acetobutylicum ATCC 824 has been reviewed in an optimised synthetic medium. A progressive increase of pH from 4.4 to 5.2 led to a higher production of extracellular -amylase whereas glucoamylase was poorly affected. A portion of these enzymes was cell-associated and on increasing the pH from 4.4 to 5.8 a decrease was noted in cell-bound enzymes. The association was higher for the glucoamylase than for the -amylase. The highest rate of starch consumption was at pH 5.2 whereas due to the earlier shift to solvent production at low pH, the highest solvent production was at pH 4.4. This study suggested that the level of -amylase and then the rate of starch hydrolysis was the limiting step of sugar catabolism in C. acetobutylicum ATCC 824. Correspondence to: P. Soucaille     

Continous production of 1-kestose by β-fructofuranosidase immobilized onshirasu porous glass

Summary 1-Kestose was produced continously and selectively from 40% (w/v) sucrose solution at fast flow rate by a column packed with an immobilized -fructofuranosidase onshirasu porous glass.     

Bioconversion of gitoxigenin by immobilized plant cells in a column bioreactor

Summary Daucus carota cells immobilized in a Ca-alginate performed the bioconversion of gitoxigenin to 5-hydroxygitoxigenin in a column bioreactor. The smooth spherical shape of the alginate beads was preserved for more than three weeks. The bioreactor was functional for more than thirty days as detected by the bioconversion activity. The rate of bioconversion was influenced by means of aeration.Issued as National Research Council of Canada Publication Number 19556     

Diacetyl production by co-immobilized citrate-positiveLactococcus lactis subsp.lactis 3022 and homogenized bovin liver in alginate fibers with double gel layers

Summary Diacetyl production by (Citr⁺)Lactococcus lactis subsp.lactis 3022 cells immobilized in Ca-alginate fine fibers with single layer in the presence of catalase was three times higher than that in the absence of catalase. A co-immobilized culture system of the lactic acid bacterial cells (outer) and the homogenized bovine liver (inner layer) in Ca-alginate fibers with double gel layers was developed. The culture system gave high diacetyl productivity (30 mg/l) for ten repeated batch cultures.     

Preparation of hydrophobic cotton cloth

Summary Hydrophobic cotton cloths were prepared by heating cotton flannel in a mixture of alcohols or phenols, epichlorohydrin and 4 M NaOH. These cloths adsorbed as much bovine serum albumin as did a commercial preparation of phenyl agarose. -Galactosidase and -glucosidase adsorbed on the cloths were about 50% as active as free enzymes. Glucoamylase immobilized on naphthyl cloth in a packed bed column efficiently hydrolyzed soluble starch to glucose. These inexpensive media would be useful for commercial-scale hydrophobic chromatography and enzyme immobilization.     

Repeated batch antibiotic fermentation by streptomycetes immobilized on cotton cloth

Summary Streptomyces cattleya and Streptomyces clavuligerus were found to form immobilized populations on cotton cloth. The resulting bacterial films were used as resident inocula in batch fermentations of thienamycin and cephalosporin C. During 20 cycles of 3-day batch fermentation, these antibiotics were produced at the constant yields.     

Glucose production using immobilized mycelial-associated β-glucosidase of Trichoderma E58

Summary The immobilization of the mycelial-associated -glucosidase of Trichoderma E-58 has been carried out by encapsulating, in calcium alginate beads, the fungal mycelium obtained durinq liquid culture. The activity of this immobilized -glucosidase was found to vary with culture age and to be more thermally stable than the extracellular -glucosidase produced by this organism. The activity of the immobilized enzyme was successfully demonstrated in both static and shake-flask batch reaction mixtures at 50°C using both cellobiose and salicin as substrates.