• BMB Reports
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• v.31 no.1
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• pp.53-57
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• 1998

The CMCax gene from Acetobacter xylinum ATCC 23769 was cloned and expressed in E. coli. With this gene, three gene products - mature CMCax, CMCax containing signal peptide(pre-CMCax), and a glutathione-S-transferase(GST)-CMCax fusion enzyme - were expressed. CMCax and pre-CMCax are aggregated to multimeric forms which showed high CMC hydrolysis activity, whereas GST-CMCax was less aggregated and showed lower activity, indicating that oligomerization of CMCax controbutes to the cellulose hydrolysis activity to achieve greater efficiency. The enzyme was identified to be an $\beta$-1,4-endoglucanase, which catalyzes the cleavage of internal $\beta$-1,4-glycosidic bonds of cellulose. The reaction products, cellobiose and cellotriose, from cellopentaose as a substrate, were identified by HPLC. Substrate specificity of cellotetraose by this enzyme was poor, and the reaction products consisted of glucose, cellobiose, and cellotriose in a very low yield. Theses results suggested that cellopentaose might be the oligosaccharide substrate consisting of the lowest number of glucose. The optimum pH of CMCax and pre CMCax was about 4.5, whereas that of GST-CMCas was rather broad at pH 4.5-8. The physiological significance of cellulose-hydrolyzing enzyme, CMCax, having such low $\beta$-1,4-endoglucanase activity and low optimum pH in cellulose-producing A. xylinum is not clearly known yet, but it seems to be closely related to the production of cellulose.

• Plant Biotechnology Reports
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• v.4 no.4
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• pp.329-334
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• 2010

Multiple sequence alignments showed that the prolines at the 25th, 129th, 153rd, 242nd, 322nd, and 434th amino acids in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 are strongly conserved in various prokaryotic EPSPS proteins. Single point mutations of the conserved prolines to alanine (P25A, P153A, P242A, P322A, and P434A) were introduced in the CP4 EPSPS in order to investigate the importance of the conserved prolines for the enzyme properties. The point mutations caused decreases in substrate binding affinity and catalytic efficiency as well as the glyphosate resistance, in general. Especially, the 25th and 242nd prolines located in the polypeptide hinges connecting top and bottom domains of CP4 EPSPS as well as the 434th proline at the C-terminus of the enzyme turned out to be crucial for the enzyme activity.

• Journal of Microbiology and Biotechnology
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• v.31 no.3
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• pp.419-428
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• 2021

To efficiently recycle GH78 thermostable rhamnosidase (TpeRha) and easily separate it from the reaction mixture and furtherly improve the enzyme properties, the magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNcZ8) was prepared by modifying Fe3O4-NH2 with tetraethyl silicate (TEOS), microcrystalline cellulose and zinc nitrate hexahydrate. FSNcZ8 displayed better magnetic stability and higher-temperature stability than unmodified Fe3O4-NH2 (FN), and it was used to adsorb and immobilize TpeRha from Thermotoga petrophilea 13995. As for properties, FSNcZ8-TpeRha showed optimal reaction temperature and pH of 90℃ and 5.0, while its highest activity approached 714 U/g. In addition, FSNcZ8-TpeRha had better higher-temperature stability than FN. After incubation at 80℃ for 3 h, the residual enzyme activities of FSNcZ8-TpeRha, FN-TpeRha and free enzyme were 93.5%, 63.32%, and 62.77%, respectively. The organic solvent tolerance and the monosaccharides tolerance of FSNcZ8-TpeRha, compared with free TpeRha, were greatly improved. Using naringin (1 mmol/l) as the substrate, the optimal conversion conditions were as follows: FSNcZ8-TpeRha concentration was 6 U/ml; induction temperature was 80℃; the pH was 5.5; induction time was 30 min, and the yield of products was the same as free enzyme. After repeating the reaction 10 times, the conversion of naringin remained above 80%, showing great improvement of the catalytic efficiency and repeated utilization of the immobilized α-L-rhamnosidase.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.163-170
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• 2021

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

• Journal of the East Asian Society of Dietary Life
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• v.22 no.1
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• pp.88-94
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• 2012

Histamine is a kind of primary biogenic amine arising from the decarboxylation of the amino acid L-histidine. The toxicology of histamine and its occurrence and formation in foods are especially emphasized in fermented foods. In this study, the biosensor for detection of histamine with functionalized multi-walled carbon nanotubes (MWCNT) was developed. We also searched for an appropriate insoluble substrate to immobilize the enzyme. The developed biosensor showed a detection limit of $0.1{\mu}M$ hydrogen peroxide. The enzyme reactor was prepared with diamine oxidase immobilized on insoluble carriers including CNBr-activated sepharose 4B, calcium alginate, and controlled pore size glass beads. The coupling efficiency of CNBr-activated sepharose 4B, calcium alginate, and controlled pore size glass beads were 48.5%, 40.3%, and 51.0%, respectively. In addition, the response currents on histamine with each immobilized enzyme reactor prepared with CNBr-activated sepharose 4B, calcium alginate, and controlled pore size glass beads were 120 nA, 110 nA, and 140 nA at $100{\mu}M$ of histamine concentration, respectively. Therefore, it is suggested that controlled pore size glass beads are the best carriers for immobilizing diamine oxidase to detect histamine in this biosensor.

• Journal of Animal Science and Technology
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• v.61 no.4
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• pp.192-203
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• 2019

A study was conducted to determine the influence of feed form and microbial enzyme supplementation on energy utilization, bone quality, and amino acid and mineral digestibility of broiler chickens. Four hundred and eighty Ross 308, day-old broiler chickens were randomly assigned to eight diets formulated from commonly used ingredients in Tanzania. A 2 (pellet or mash) ${\times}$ 4 (control, Axtra XB, Quantum Blue (QB) and Axtra XB + QB enzyme) factorial array in a completely randomized design having six replicates per treatment (10 birds per replicate) was used. Birds were raised in climate-controlled rooms in a 3-phase; starter (0-10 days), grower (11-24 days) and finisher (25-35 days). Apparent metabolizable energy (AME), metabolizable energy intake, net energy of production, energy retained as protein (REp), and efficiency of metabolizable energy use for energy and protein retention were higher (p < 0.05) in birds fed pelleted diets. The AME and REp was higher (p < 0.05) with enzyme supplementation. Ash content, weight, length, width and breaking strength of tibia bones were highest (p < 0.05) in birds on pelleted diets. Tibia bone traits were improved (p < 0.05) when enzymes were included, particularly in a combination of QB and Axtra XB. However, potassium, magnesium, and zinc contents were highest (p < 0.05) when QB was supplemented. Digestibility of all amino acids was higher (p < 0.05) in birds supplied with pellets and with enzyme supplementation for most amino acids, except for serine. There was a positive interaction (p < 0.05) between feed form and enzymes on lysine and phenylalanine digestibility. Digestibility of Ca, P, K, S, Zn, and Fe was higher (p < 0.05) in birds fed pelleted diets, while those on mashed diets had higher (p < 0.05) digestibility of Cu and B. The digestibility of P, K, and Zn was highest (p < 0.001) when QB was added, while Ca, P, S, and B digestibility was highest when a combination of Axtra XB + QB was applied. Pelleted diets with or without enzymes improved energy utilization, digestibility of amino acids, and minerals, and increased bone strength in broiler chickens.

• Journal of Plant Biology
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• v.26 no.4
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• pp.191-196
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• 1983

The isolatin and culture of protoplasts from hypocotyl originated callus of Phaseolus vulgaris cv. Damyang were carried out. The maximum protoplast yield of 4.6$\times$105 per gram fresh callus, using the 13-day-old callus, was obtained by digeston for 6 hours in the enzyme solution. After 10 day-culture of the isolated callus protoplsts, plating efficiency was 50%. Thereafter, cell cluster medium, and followed by leading to callus formation on an agar medium after 3 weeks of the liquid culture.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.2
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• pp.59-62
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• 2006

Seaweed biotechnology is a multidisciplinary subject to produce food, pharmaceuticals, chemicals, and environmental remediation materials from seaweed resources. It uses various techniques of cell culture, enzyme reaction and genetic manipulation to increase the production efficiency of useful seaweeds or their products. Firstly, an overview of key topics will be introduced in the fields of seaweed tissue culture, strain improvement, genetic analysis briefly as basic techniques. Secondly, some biologically active substances such as anti-inflammatory and antifouling substances that have been screened in my laboratory will be focused.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.2
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• pp.248-256
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• 2003

Two experiments were conducted to determine the effects of expander processing and enzyme supplementation of wheat-based diets on growth performance and nutrient digestibility in finishing pigs. For Exp. 1, 60 finishing pigs (average initial BW of 49.5 kg) were fed meal, standard pellets and expanded pellets in a 70 d growth assay. From 49.5 to 79.0 kg, 79.0 to 111.8 kg, and overall (49.5 to 111.8 kg), ADG and ADFI were not affected by pelleting or standard vs expander conditioning (p>0.22). However, from 49.5 to 79.0 kg, pigs fed pellets have greater gain/feed than pigs fed mash (p<0.04), and pigs fed expanded pellets tended to have greater (p<0.10) gain/feed than pigs fed standard pellets. Overall (i.e. from 49.5 to 111.8 kg), gain/feed (p<0.02) and apparent fecal digestibilities of DM (p<0.001) and N (p<0.02) were improved by pelleting the diets. Also, expander processing further improved gain/feed (p<0.06) and digestibility of DM (p<0.04) compared to standard steam conditioning. Scores for keratinization (p<0.002) and ulceration (p<0.003) of the stomach were increased by pelleting, but the mean scores for the various treatments ranged only from 0.05 to 1.08 (i.e., low to mild keratosis and ulceration). For Exp. 2, 80 pigs (average initial BW of 54.1 kg) were fed mash and pellets (standard or expander) without and with xylanase. The enzyme was added to supply 4,000 units of xylanase activity/kg of diet. Adding xylanase to the mash diet improved gain/feed from 90.7 to 115.9 kg (p<0.04) of the growth assay and digestibility of DM (p<0.05) on d 39. However, in pelleted diets, adding the enzyme did not improve growth performance or digestibility of nutrients. Pelleting tended to increase scores for ulceration (p<0.06), and enzyme supplementation decreased stomach keratinization scores for pigs fed the standard pellets (p<0.01). However, as in Exp. 1, the mean scores for all treatment groups were quiet low (i.e., ranging from normal to mild). In conclusion, pelleting improved efficiency of growth, but additional benefits from expander conditioning were observed only in Exp. 1. Finally, xylanase tended to improve growth performance and nutrient digestibility, only in pigs fed mash diets but not in pigs fed pellets.

• Applied Biological Chemistry
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• v.22 no.4
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• pp.191-197
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• 1979

${\beta}-Tyrosinase$ was purified and crystallized from cells of Escherichia intermedia A-21 grown in a medium supplemented with 0.2% L-tyrosine. Molecular weight of its subunit, Km value and absorption spectra were determined. Crystallization methods were also studied to eliminate any unnecessary procedures. The results obtained were as follows: 1. The purification procedure included ammonium sulfate fractionation, dialysis against potassium phosphate buffer, pH 6.0 and pH 7.0, and DEAE-Sephadex column chromatography. In the column chromatography, 11 mg of protein was applied per ml of DEAE-Sephadex for efficiency. 2. Steps of protamine sulfate treatment and Sephadex G-150 gel filtration could be eliminated for this enzyme from the known procedures. 3. The purified enzyme was dissolved in 0.01M potassium phosphate buffer containing 2-mercaptoethanol, with a concentration of 20mg/ml. Crystalline enzyme, which appears as hexagonal rods, was obtained by adding solid fine powdered ammonium sulfate to the enzyme solution. 4. Absorption maxima of the enzyme appeared at 340 and 430nm when associated with pyridoxal phosphate. 5. Km value of the enzyme for L-tyrosine was $2.31{\times}10^{-4}M$ and the molecular weight of its subunit was determined by SDS-polyacrylamide electrophoresis to be approximately 50,000.