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1.
Katerina Batsalova Kostadin Kunchev Yana Popova Annie Kozhukharova Nadezhda Kirova 《Applied microbiology and biotechnology》1987,26(3):227-230
Summary Fungal -galactosidase was immobilized in polyvinylalcohol gel formed in pores of contton material. Temperature and pH effects on the activity of free and immobilized enzymes were studied. The optimum temperatures of free and immobilized enzymes were 60° C and 55° C respectively. The pH optimum ranged from 4.5 to 5.0 for both enzymes. The thermal stability of the immobilized -galactosidase was slightly higher. The Km values for soluble and immobilized enzymes were respectively 1.9 mM and 2.5 mM. The optimization of conditions for a highly effective hydrolysis of 4% lactose solution and reusability of the immobilized enzyme resulted in 75% hydrolysis after 5–6 h. The degree of conversion decreased to 50% after 30 repeated runs. The capacity of the immobilized enzyme to hydrolyze lactose in whey was also studied. 相似文献
2.
Song J Abe K Imanaka H Imamura K Minoda M Yamaguchi S Nakanishi K 《Bioscience, biotechnology, and biochemistry》2011,75(2):268-278
The presence of multiple types of β-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four β-galactosidases, β-Gal-A, β-Gal-B, β-Gal-C, and β-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. β-Gal-B, β-Gal-C, and β-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20-30 and 9-12%, while β-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for β-Gal-A, showed biphasic behavior. β-Gal-A was truncated to yield multiple β-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of β-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for β-Gal-D. 相似文献
3.
Zahid Mozaffar Kazuhiro Nakanishi Ryuichi Matsuno 《Applied microbiology and biotechnology》1986,25(3):229-231
Summary The specific activity-dependent stability of the immobilized -galactosidase-2 (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans during the continuous production of galactooligosaccharides from lactose was studied. This was done by measuring the elution pattern of saccharides from the various immobilized Merckogel (controlled pore silica gel) columns and the amount of saccharides remaining in the gel. It was suggested that oligosaccharides produced were trapped inside the three dimensional enzyme aggregate with the immobilized enzyme having a specific activity of 240 units/g of wet gel, causing gradual inactivation, while the immobilized enzyme with 15 units/g of wet gel was stable since the oligosaccharides were not accumulated.Free -galactosidase-2 was stable during continuous reaction in a membrane reactor. 相似文献
4.
5.
R Rech C F Cassini A Secchi M A Z Ayub 《Journal of industrial microbiology & biotechnology》1999,23(2):91-96
We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the
yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and
37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these
conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown
in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS
6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature
of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks.
Received 20 January 1999/ Accepted in revised form 30 April 1999 相似文献
6.
The objective of this work was to compare the properties of free and immobilized β-galactosidase (Aspergillus oryzae), entrapped in alginate–gelatin beads and cross-linked with glutaraldehyde. The free and immobilized forms of the enzyme showed no decrease in enzyme activity when incubated in buffer solutions in pH ranges of 4.5–7.0. The kinetics of lactose hydrolysis by the free and immobilized enzymes were studied at maximum substrate concentrations of 90 g/L and 140 g/L, respectively, a temperature of 35 °C and a pH of 4.5. The Michaelis–Menten model with competitive inhibition by galactose fit the experimental results for both forms. The Km and Vm values of the free enzyme were 52.13 ± 2.8 mM and 2.56 ± 0.3 gglucose/L min mgenzyme, respectively, and were 60.30 ± 3.3 mM and 1032.07 ± 51.6 glactose/min m3catalyst, respectively, for the immobilized form. The maximum enzymatic activity of the soluble form of β-galactosidase was obtained at pH 4.5 and 55 °C. Alternatively, the immobilized form was most active at pH 5.0 at 60 °C. The free and immobilized enzymes presented activation energies of 6.90 ± 0.5 kcal/mol and 7.7 ± 0.7 kcal/mol, respectively, which suggested that the immobilized enzyme possessed a lower resistance to substrate transfer. 相似文献
7.
Recombinant human β-mannosidase (rhMANB) is an important glycosidase enzyme that degrades mannose-linked glycoproteins and mannan polysaccharides. rhMANB was purified and covalently immobilized onto magnetic nanoparticles. The immobilization of the enzyme was confirmed by Fourier-transform infrared spectroscopy (FTIR) and magnetic nanoparticles linked immunosorbent assay (MagLISA). Antibodies against rhMANB were raised, purified and characterized for MagLISA. The binding of rhMANB onto magnetic nanoparticles was found to be 65%. The V( max ) and K( m ) of immobilized rhMANB was observed 3.0-fold higher and 2.024-fold lower, respectively, as compared to unbound rhMANB. The stability and activity of immobilized enzyme was observed at different pH, temperature, and after storage at 4°C. Metal chelators (oxalic acid, citric acid, and ascorbic acid) did not affect the enzyme activity of immobilized enzyme, whereas ethylenediamine tetraacetic acid reduced the activity. The results obtained from thin-layer chromatography indicate that immobilized rhMANB is more efficient than the unbound form to hydrolyze mannobiose, mannotriose, mannotetraose, mannopentose, galactoglucomannan, and locust bean gum. Magnetic nanoparticles suspended gel-permeation chromatography showed that 29% locust bean gum hydrolyzed efficiently during flow in the column. The immobilization of rhMANB will be a good process for gelling and saccharification of mannan polymers at industrial scale. 相似文献
8.
Galacto-oligosaccharides (GalOS) were continuously produced using lactose and immobilized β-galactosidase from Bullera singularis ATCC 24193 in a packed bed reactor. Partially purified β-galactosidase was immobilized in Chitopearl BCW 3510 bead (970 GU/g resin) by simple adsorption. 55% (w/w) oligosaccharides was obtained continuously with a productivity of 4·4 g/(litre-h) from 100 g/litre lactose solution during a 15-day operation. Batch productivity was 6·5 g GalOS/(litre-h) from 300 g/litre lactose. 相似文献
9.
Zahid Mozaffar Kazuhiro Nakanishi Ryuichi Matsuno 《Applied microbiology and biotechnology》1986,25(3):224-228
Summary -Galactosidase-2 (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was purified using hydroxyapatite gel chromatography and immobilized onto Duolite ES-762 (phenolformaldehyde resin) and Merckogel (controlled pore silica gel) for continuous production of galacto-oligosaccharides using lactose as the substrate. The maximum amount of ologosaccharides produced by the immobilized enzyme was 35–40% of the total sugar during hydrolysis of 4.56% lactose. Partially purified -galactosidase from B. circulans was also immobilized onto various supports for the same purpose. The stability of the immobilized -galactosidase-2 or partially purified enzyme during a continuous reaction depended on their supports and specific activity. Of the supports tested, Merckogel was best for operational stability. With this support, the enzyme was quite stable with specific activity up to 15 units/g of wet gel; it was reversibly inactivated with more. 相似文献
10.
A spin-labeled β-galactoside has been synthesized. It is a substrate for β-galactosidase and is accumulated by E. coli ML 308-225 and K12-N2244. The accumulation appears to be active transport via the lactose permease since it is inhibited with lactose and the energy poison, amytal. Induction of the K12 strain with isopropylthiogalactoside results in a 100-fold stimulation of uptake of the spin-labeled β-galactoside. Binding of the spin-labeled sugar to membranes derived from the ML strain is inhibited by lactose. 相似文献
11.
Natalja Genina Heikki Räikkönen Jyrki Heinämäki Peep Veski Jouko Yliruusi 《AAPS PharmSciTech》2010,11(2):959-965
We nano-coated powdered lactose particles with the enzyme β-galactosidase using an ultrasound-assisted technique. Atomization
of the enzyme solution did not change its activity. The amount of surface-attached β-galactosidase was measured through its
enzymatic reaction product D-galactose using a standardized method. A near-linear increase was obtained in the thickness of
the enzyme coat as the treatment proceeded. Interestingly, lactose, which is a substrate for β-galactosidase, did not undergo
enzymatic degradation during processing and remained unchanged for at least 1 month. Stability of protein-coated lactose was
due to the absence of water within the powder, as it was dry after the treatment procedure. In conclusion, we were able to
attach the polypeptide to the core particles and determine precisely the coating efficiency of the surface-treated powder
using a simple approach. 相似文献
12.
Sachio Hayashi Junko Kinoshita Masaharu Nonoguchi Yoshiyuki Takasaki Kiyohisa Imada 《Biotechnology letters》1991,13(6):395-398
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. 相似文献
13.
J. Ortega-Lopez L. H. Morales-Ramos M. C. Montes I. Magaña-Plaza 《Biotechnology Techniques》1993,7(11):775-780
Summary -D-galactosidase from Kluyveromyces lactis immobilized on nylon-6 microbeads was used to hydrolyze lactose in skim milk (containing 28.6% total solids), using a novel spin-basket reactor. More than 75% of lactose was hydrolyzed at 34°C within a short space-time (<7 min) without experiencing any plugging such as typically seen in packed columns. 相似文献
14.
Zahid Mozaffar Kazuhiro Nakanishi Ryuichi Matsuno 《Applied microbiology and biotechnology》1987,25(5):426-429
Summary The oligosaccharide-producing activity of -galactosidase-1, one of the isomers of -galactosidase (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was changed after immobilization onto porous silica gel (Merckogel) by crosslinkage with glutaraldehyde. The reason for this modification was studied by treating the free enzyme with glutaraldehyde. Glutaraldehyde of 0.025% to 3% modified 40% to 90% of the free amino groups with or without intermolecular crosslinking. The maximum yield of oligosaccharides increased from 12% to 40% depending upon degree of modification, while native enzyme gave only 6% trisaccharides during hydrolysis of 127 mM lactose. The K
m
value for the enzyme treated with glutaraldehyde was also increased. 相似文献
15.
High-throughput screens and selections have had profound impact on our ability to engineer proteins possessing new, desired properties. These methods are especially useful when applied to the modification of existing enzymes to create natural and unnatural products. In an advance upon existing methods we developed a high-throughput, genetically regulated screen for the in vivo production of β-lactam antibiotics using a green fluorescent protein (gfp) reporter. This assay proved reliable and sensitive and presents a dynamic range under which a wide array of β-lactam architectural subclasses can be detected. Moreover, the graded response elicited in this assay can be used to rank mutant activity. The utility of this development was demonstrated in vivo and then applied to the first experimental investigation of a putative catalytic residue in carbapenem synthase (CarC). Information gained about the mutability of this residue defines one parameter for enzymatic activity and sets boundaries for future mechanistic and engineering efforts. 相似文献
16.
Summary An activated carbon column was utilized for the synthesis of disaccharides by use of a reversed hydrolysis activity of an immobilized -galactosidase column in order to shift the equilibrium to the direction of condensation. The yields of lactulose and allo-lactulose from galactose and fructose, and N-acetyl lactosamine and N-acetyl allolactosamine from galactose and N-acetyl glucosamine, were 11.3% and 10.0%, respectively. 相似文献
17.
Mita Banerjee Aloka Chakravarty Saroj Kanti Majumdar 《Applied microbiology and biotechnology》1984,20(4):271-274
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. Km 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. 相似文献
18.
《Process Biochemistry》2010,45(4):481-486
A bioprocess with a high conversion rate of limonene to α-terpineol was described. The enzyme hydratase involved in this process was found as being cofactor independent, non-inducible and able to perform the transformation of both R-(+) and S-(−)-limonene. The system used consisted of a biphasic medium in which the aqueous phase contained a concentrated resting cells of Sphingobium sp. and the organic phase was sunflower oil. After 30 h at 30 °C ca. 25 g of R-(+)-α-terpineol per liter of organic phase were obtained from R-(+)-limonene in Erlenmeyer flasks. Performance of the bioconversion in a bioreactor increased the production rate with no changes in yield and maximal R-(+)-α-terpineol concentration, which demonstrated that experiments in flasks were limited by liquid–liquid transport phenomena. A mathematical model able to explain the fact that the reaction always stopped before the precursor became exhausted has also been proposed and validated. Finally, the process reported was the most promising alternative for the biotechnological production of natural R-(+)-α-terpineol published so far and up to ca. 130 g L−1 metabolite could finally be obtained. 相似文献
19.
D. V. Gokhale U. S. Puntambekar A. K. Vyas S. G. Patil Dileep N. Deobagkar 《Biotechnology letters》1984,6(11):719-722
Summary An Aspergillus sp. was isolated which secreted high levels of -glucosidase in growth medium. The maximum activity(10 IU/ml of -glucosidase and 22.6 IU/ml of cellobiase) was obtained in cellulose medium supplemented with wheat bran. The pH and temperature optima for this enzyme were 4.5 and 65°C respectively.NCL Communication No. 3616 相似文献
20.
《Enzyme and microbial technology》1987,9(1):53-56
Bacillus subtillis ATCC 21770 was entrapped in a carrageenan gel, especially formulated for immobilization. Bacterial growth and α-amylase (1,4-α-d-glucan glucanohydrolase EC 3.2.1.1) production were tested. The bead suspensions were submitted to two aeration modes, one consisting of bubbling air into a round flask, the other involving sparging of air into an airlift fermenter. The latter system, which produces microbubbles, gave 40–70% increase in enzyme production over the former and a doubling of bacterial density within the beads was measured. The use of CaCl2instead of KCl as polymerization agent led to a better yield of α-amylase. 相似文献