• Microbiology and Biotechnology Letters
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• v.6 no.1
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• pp.33-40
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• 1978

Immobilization of alkaline protease was investigated by absorbing the enzyme on adsorbents. Alkaline protease was adsorbed on silica gel selected as a carrier to immobilize the enzyme. In this study, properties of the immobilized enzyme were compared with those of the soluble enzyme. 1) The optimum pH (10.0) of the enzyme was not changed, but the activity was increased at alkaline pH by immobilization. 2) The optimum temperature of the immobilized enzyme was shifted from 50$^{\circ}C$ to 45$^{\circ}C$, while the temperature-activity Profile became broader than those of the soluble enzyme. 3) The pH stability of the immobilized enzyme was significantely increased at pH 4.0, althouth it did not change in the neutral and alkaline pH region. 4) The heat stability of the enzyme was enhanced in the temperature range of 55$^{\circ}C$∼65$^{\circ}C$ by the immobilization. 5) The immobilized enzyme retained 40％ of its original activity after repetitive use for 6 times. 6) The enzyme stability was greately improved for a prolonged storage at 4$^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.818-825
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• 2012

Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support ($q_m$) and dissociation constant ($K_d$) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at $65^{\circ}C$. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1250-1256
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• 2011

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.

• Journal of Life Science
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• v.20 no.11
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• pp.1656-1661
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• 2010

Alkaline protease for the hydrolysis of egg yolk protein was immobilized on five carriers - Duolite A568, Celite R640, Dowex-1, Dowex 50W and Silica gel R60. Duolite A568 showed a maximum immobilization yield of 24.7%. Optimum pH for the free and immobilized enzyme was pH 8 and 9, respectively. However, no change was observed in optimum temperature ($50^{\circ}C$). Thermal stability was observed in immobilized enzymes compared to free enzymes. The immobilized enzyme retained 86% activity after 10 cycle operations in a repeated batch process. The effect of flow rate on the stability of enzyme activity in continuous packed-bed reactor was investigated. Lowering flow rate increased the stability of the immobilized enzyme. After 96 hr of continuous operation in a packed-bed reactor, the immobilized enzyme retained 83 and 61% activity when casein and egg yolk were used as a raw materials, respectively.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.3
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• pp.466-472
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• 1997

Peptides separated from fish paste washing liquid of an Alaska pollak (Theragria chalcogramma) were purified and characterized. The fish paste washing liquid (supernatant) was separated by centrifugation of fish paste homogenate. The fish paste washing liquid of $0.5\%$ concentration was hydrolyzed for 24 hour at $50^{\circ}C$ by immobilized protease in bioreactor and decomposing liquid of protein having $50\%$ decomposing rate (OPA method) was obtained. The crude peptide fractions were obtained from this liquid by Dowex 50w $(H^+)$ column chromatograpy. Purified peptides (SP-fraction peptides) were fractionated by using SP-Sepadex C-25 $(H^+)$ column chromatography. Molecular weights and amino acid compositions of these peptides were estimated by Sephadex G-50 column chromatography and HPLC, respectively. when the washed peptides was eluated with $0.6\~0.9\%\;and\;1.2\~2.0\%$ of NaCl, peptides composed of weakly basic amino acids and strongly basic amino acid were respectively eluted. Molecular weights of each peptide fractions showed the broad distribution from 1,000 Da to 3,000 Da in the order of SP-4>SP-3>SP-2>SP-1. Peptides contained a large quantity of glycine, arginine, glutamic acid, and alanine in the washed peptide and its SP-tractions, respectively.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.599-602
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• 2005

Truncated form of UBP1, an ubiquitin-specific protease of Saccharomyces cerevisiae, was overexpressed in Escherichia coli. The hexahistidine residue $(His_6)$ was fused to the N-terminus of truncated UBP1 and the corresponding recombinant protein was purified with high yield by immobilized metal affinity chromatography. The truncated form of UBP1 protein was functional to cleave ubiquitinated human growth hormone as substrate. Effects of pH and temperature were investigated in order to optimize deubiquitinating reactions for the truncated UBP1. Optimum temperature and pH for the cleavage reaction were $40^{\circ}C$ and pH 8.0, respectively.

• Korean Journal of Food Science and Technology
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• v.47 no.4
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• pp.438-445
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• 2015

The production of GABA was optimized by co-cultivation of immobilized Lactobacillus plantarum K154 (ILK) with Ceriporia lacerata cultures. The mycelial culture of C. lacerata was performed in a defined medium containing 3% glucose, 3% soybean flour, and 0.15% $MgSO_4$ in a submerged condition for 7 days at $25^{\circ}C$, resulting in the production of 29.7 g/L mycelia, 3.1 g/L exopolysaccharides, 2% (w/w) ${\beta}$-glucan, 68.96 unit/mL protease, and 10.37 unit/mL ${\alpha}$-amylase. ILK in C. lacerata culture showed viable cell counts of $3.13{\time}10^9CFU/mL$ for immobilized cells and $1.48{\time}10^8CFU/mL$ for free cells after 1 day. GABA production in the free and immobilized cells was 9.96 mg/mL and 6.30 mg/mL, respectively, after 7 days. A recycling test of ILK in the co-fermentation was consequently performed five times at $30^{\circ}C$ for 15 days, resulting in the highest production of GABA. GABA could also be efficiently overproduced by co-cultivation with the produced polysaccharides, ${\beta}$-glucan, peptides, and probiotics.

• Korean Journal of Food Science and Technology
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• v.22 no.7
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• pp.805-811
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• 1990

Hydrolysis of soybean proteins was carried out by immobilized trypsin and/or alpha-chymotrypsin. The partially hydrolyzed products of soybean proteins were evaluated for their molecular weights and molecular charges by using Ferguson's plot. The ratio of average molecular weights to average molecular charges($\bar{M}/log\;\bar{Y}_o$) of modified soybean proteins could be used to predict functional properties such as solubility, water holding capacity, oil holding capacity, and emulsifying ability. The low ratio of modified soybean proteins indicated high solubility. while the high ratio showed high water holding capacity. The appropriate ranges of the ratios were necessary for maximun oil holding capacity and emulsifying ability.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.56-60
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• 1990

A bacterial strain of Brevibacterium sp. CHI was isolated from soil and used to produce an enzyme (nitrile hydratase) necessary for carrying out the bioconversion of acrylonitrile to acrylamide. Various immobilization methods and enzyme stability were investigated. The nitrile hydratase showed the maximum stability at pH 7 for the free cells. EDTA and phenyl methyl sulfonyl fluoride were selected as the protease inhibitor and the enzyme stability was evaluated by changing inhibitor concentration. Acrylamide beads were the best carriers among four carriers we tested in terms of stability and physicoehemical strength. The storage stability of the immobilized cells decreased with increasing acrylamide concentration of the gel phase at 4$^{\circ}C$, and was very low at acrylarnide concentration above 25%.

• BMB Reports
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• v.43 no.5
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• pp.319-324
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• 2010

Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value.