• BMB Reports
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• v.37 no.4
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• pp.408-415
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• 2004

The expression of the Paenibacillus sp. A11 cyclodextrinase (CDase) gene using the pUC 18 vector in Escherichia coli JM 109 resulted in the formation of an insoluble CDase protein in the cell debris in addition to a soluble CDase protein in the cytoplasm. Unlike the expression in Paenibacillus sp. A11, CDase was primarily observed in cytoplasm. However, by adding 0.5 M sorbitol as an osmolyte, the formation of insoluble CDase was prevented while a three-fold increase in cytoplasmic CDase activity was achieved after a 24 h-induction. The recombinant CDase protein was purified to approximately 14-fold with a 31% recovery to a specific activity of 141 units/mg protein by 40-60% ammonium sulfate precipitation, DEAE-Toyopearl 650 M, and Phenyl Sepharose CL-4B chromatography. It was homogeneous by non-denaturing and SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of 80 kDa, as determined by gel filtration and SDS-PAGE. It showed the highest activity at pH 7.0 and $40^{\circ}C$. The catalytic efficiency ($k_{cat}/K_m$) values for $\alpha$-, $\beta$-, and $\gamma$-CD were $3.0{\times}10^5$, $8.8{\times}10^5$, and $5.5{\times}10^5\;M^{-1}\;min^{-1}$, respectively. The enzyme hydrolyzed CDs and linear maltooligosaccharides to yield maltose and glucose with less amounts of maltotriose and maltotetraose. The rates of hydrolysis for polysaccharides, soluble starch, and pullulan were very low. The cloned CDase was strongly inactivated by N-bromosuccinimide and diethylpyrocarbonate, but activated by dithiothreitol. A comparison of the biochemical properties of the CDases from Paenibacillus sp. A11 and E. coli transformant (pJK 555) indicates that they were almost identical.

• Food Science and Biotechnology
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• v.18 no.3
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• pp.776-781
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• 2009

A putative cyclomaltodextrinase (BHCD) gene was found from the genome of Bacillus halodurans C-125, which encodes 578 amino acids with a predicted molecular mass of 67,279 Da. It shares 42-59% of amino acid sequence identity with common cyclomaltodextrinase (CDase)-family enzymes. The corresponding gene was cloned by polymerase chain reaction (PCR) and the dimeric enzyme with C-terminal 6-histidines was successfully overproduced and purified from recombinant Escherichia coli. BHCD showed the highest activity against ${\beta}-CD$ at pH 7.0 and $50^{\circ}C$. Due to its versatile hydrolysis and transglycosylation activities, BHCD has been confirmed as a member of CDases. However, BHCD can be distinguished from other typical CDases on the basis of its novel multisubstrate specificity. While typical CDases have over 10 times higher activity on ${\beta}-CD$ than starch or pullulan, the CD-hydrolyzing activity of BHCD is only 2.3 times higher than pullulan. In particular, it showed significantly higher activity ratio of maltotriose to acarbose than other common CDase-family enzymes.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.792-799
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• 2007

A gene encoding a putative glycogen-debranching enzyme in Sulfolobus shibatae(abbreviated as SSGDE) was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The recombinant SSGDE was extremely thermostable, with an optimal temperature at $85^{\circ}C$. The enzyme had an optimum pH of 5.5 and was highly stable from pH 4.5 to 6.5. The substrate specificity of SSGDE suggested that it possesses characteristics of both amylo-1,6-glucosidase and $\alpha$-1,4-glucanotransferase. SSGDE clearly hydrolyzed pullulan to maltotriose, and $6-O-\alpha-maltosyl-\beta-cyclodextrin(G2-\beta-CD)$ to maltose and $\beta$-cyclodextrin. At the same time, SSGDE transferred maltooligosyl residues to the maltooligosaccharides employed, and maltosyl residues to $G2-\beta-CD$. The enzyme preferentially hydrolyzed amylopectin, followed in a decreasing order by glycogen, pullulan, and amylose. Therefore, the present results suggest that the glycogen-debranching enzyme from S. shibatae may have industrial application for the efficient debranching and modification of starch to dextrins at a high temperature.

• Journal of Life Science
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• v.7 no.2
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• pp.95-106
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• 1997

The moderately alkalophilic bacterium, identified as Bacillus sp. S-1 , was isolated from soils and effectively secrete extracellular pullulanase. The isolate was moderately alkalophilic since enzyme production occurred at pHs from 6.0 to 10.0. Extracellular crude enzymes of the isolate gave maltotriose as the major product from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes, this isolate secreted extremely high concentration(7.0 units/ml) of pullulanase. The purified pullulanase was moderately alkalophilic and thermoactive; optimal activity was detected at pH 8.0-10.0 and between 50-60$^{\circ}$C. Even at pH 12.0, 10% of S-1 pulluanase activity remained and the strain had broad pH ranges and moderate thermo-stability for their enzyme activities. These results indicate that the new isolate have potential as producer of pullulanase for use in the starch industry.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.765-775
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• 2019

A new ${\alpha}$-amylase-encoding gene (amySL3) of glycoside hydrolase (GH) family 13 was identified in soda lake isolate Alkalibacterium sp. SL3. The deduced AmySL3 shares high identities (82-98%) with putative ${\alpha}$-amylases from the genus Alkalibacterium, but has low identities (<53%) with functionally characterized counterparts. amySL3 was successfully expressed in Escherichia coli, and the recombinant enzyme (rAmySL3) was purified to electrophoretic homogeneity. The optimal temperature and pH of the activity of the purified rAmySL3 were determined to be $45^{\circ}C$ and pH 7.5, respectively. rAmySL3 was found to be extremely halophilic, showing maximal enzyme activity at a nearly saturated concentration of NaCl. Its thermostability was greatly enhanced in the presence of 4 M NaCl, and it was highly stable in 5 M NaCl. Moreover, the enzyme did not require calcium ions for activity, and was strongly resistant to a range of surfactants and hydrophobic organic solvents. The major hydrolysis products of rAmySL3 from soluble starch were maltobiose and maltotriose. The high ratio of acidic amino acids and highly negative electrostatic potential surface might account for the halophilic nature of AmySL3. The extremely halophilic, calcium-independent, and surfactant-resistant properties make AmySL3 a promising candidate enzyme for both basic research and industrial applications.

• Korean Journal of Agricultural Science
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• v.15 no.2
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• pp.153-163
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• 1988

This experiment was carried out to obtain the thermophilic fungus producing amylase and to investigate properties of the amylase. The selected strain, L-11 was obtained from soil in the vicinity of a hot spring and identified as Mocor sp.. And then the conditions for enzyme production in wheat bran cultures and properties of the crude enzyme were investigated. Furthermore, the enzyme was purified and the characteristics of purified enzyme were studied. The results obtained were as follows: 1. On the wheat bran medium added 80-100% water, amylase was effectively produced by the selected strain, L-11 for 48 hrs incubation at $50^{\circ}C$. 2. When the crude enzyme solution of the strain L-11 was passed through DEAE-Sephadex A-50 column chromatography, two peaks having amylase activity were obtained, and one peak was that of the main enzyme (enzyme of B peak). 3. The purified enzyme (enzyme of B peak) was recognized as single protein band on polyacrylamide disc gel electrophoresis. 4. In the hydrolysis reaction of soluble starch by the enzyme of main amylase, oligosaccharides produced at early stage were maltose and maltotriose mainly and procedure of the reaction maltose amount of maltose and glucose was increased. 5. The strain L-11 was recognized as a special strain producing ${\alpha}-amylase$ mainly and scarcely glucoamylase. 6. The optimum pH, optimum temperature, pH stability, and temperature stability of ${\alpha}-amylase$ were pH 4.0, $60-65^{\circ}C$, pH 4.0-9.0, and below$70^{\circ}C$.

• Journal of Oral Medicine and Pain
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• v.34 no.3
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• pp.227-235
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• 2009

Many of the protective functions of saliva can be attributed to the biological, physical, structural, and rheological characteristics of salivary glycoproteins. Therefore, the development of ideal saliva substitutes requires understanding of the rheological as well as biological properties of human saliva. In the present study, we investigated the changes of salivary enzymatic activities by saliva substitutes and compared viscosity of saliva substitutes with human saliva. Five kinds of saliva substitutes such as Moi-Stir, Stoppers4, MouthKote, Saliva Orthana, and SNU were used. Lysozyme activity was determined by the turbidimetric method. Peroxidase activity was determined with an NbsSCN assay. $\alpha$-Amylase activity was determined using a chromogenic substrate, 2-chloro-p-nitrophenol linked with maltotriose. The pH values of saliva substitutes were measured and their viscosity values were measured with a cone-and-plate digital viscometer at six different shear rates. Various types of saliva substitutes affected the activities of salivary enzymes in different ways. Stoppers4 enhanced the enzymatic activities of hen egg-white lysozyme, bovine lactoperoxidase (bLP), and $\alpha$-amylase. Saliva Orthana and SNU inhibited bLP activity and enhanced $\alpha$-amylase activity. MouthKote inhibited $\alpha$-amylase activity. Moi-Stir inhibited the enzymatic activities of bLP and $\alpha$-amylase. The pH values were very different according to the types of saliva substitutes. Stoppers4, MouthKote, and Saliva Orthana showed lower values of viscosity at low shear rates and higher values of viscosity at high shear rates compared with unstimulated and stimulated whole saliva. Moi-Stir and SNU displayed much higher values of viscosity than those of natural whole saliva. Collectively, our results indicate that each saliva substitute has its own biological and rheological characteristics. Each saliva substitute affects the enzymatic activity of salivary enzyme and finally oral health in different ways.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.10
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• pp.1307-1313
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• 2007

In order to evaluate the utilization of immature green wheat kernels as food processing material, these experiments were carried out to analyze general composition, free sugars, free amino acids, mineral content and color of 3 wheat varieties according to their heading dates. Contents such as moisture, crude protein, crude lipid and ash were gradually decreased according to the days after heading of 3 wheat varieties, while crude fiber and starch were gradually increased. Maltose and maltotriose contents in immature green wheat kernels were high but its rapidity decreased by closing to ripeness. Contents of free amino acids showed a tendency to decrease, among which alanine content was the highest around 25 days after heading, and then glutamic acid, GABA, glycine in order. Contents of total free amino acid were decreased by closing to ripeness. Among 3 wheat varieties, its contents of Guru wheat was the highest. As wheat matured, ${\beta}-glucan$ contents were decreased from 1.5% to 0.28% on 43 days after heading. Mineral contents of 3 wheat varieties were generally increased by closing to ripeness after coming into ears, and K, Mg were especially prominent. As wheat matured, lightness was decreased, while redness and yellowness were increased. Considering chemical composition and color, the immature green wheat were produced through blanching the spikes harvested before the yellow ripe stage. The harvested green wheat cereals is able to eat raw wheat or cook it as food processing material.

• KSBB Journal
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• v.19 no.4
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• pp.301-307
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• 2004

The characteristics of the a-amylase of Aspergillus coreanus NR 15-1 isolated from traditional Korean Nuruk have been carried out. The a-amylase of A. coreanus NR 15-1 was purified by ammonium sulfate precipitation followed by column chromatographies on CM-cellulose, DEAE-cellulose, Sephadex G-100 gel filtration and hydroxyapatite. The a-amylase was purified 78-fold with a yield of 8.7%. The molecular weight of the a-amylase was estimated to be 49 kDa by Sephadex G-100 gel filtration and 51 kDa by SDS-polyacrylamide gel eletrophoresis. These experimental results suggested that the purified enzyme might be monomer. The enzyme was stable between pH 4 and 11. The optimum pH was 5.0. The optimum temperature for enzyme was 45$^{\circ}C$ and the enzyme was stable up to 50$^{\circ}C$. The enzyme was significantly inhibited by 1 mM N-bromosuccinimide. These results suggested that tryptophan residue was involved in the active site of a-amylase. The enzyme was identified as a-amylase because the reaction products of soluble starch hydrolyzed by the purified enzyme was oligosaccharide by thin layer chromatography.

• Journal of fish pathology
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• v.20 no.2
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• pp.109-117
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• 2007

This study was performed to identity non hemolytic streptococcus from cultured flounder (Paralichthys olivaceus) with Streptococcosis in the Jeju island. The result of BIOLOGTM test was Streptococcus uberis that simility of 0.5 and 98% identified in MicroLogTM system (Release 4.05). Carbohydrate utility pattern was dextrin, N-acetyl-D-glucosamine, arbutin, maltose, maltotriose, D-cellobiose, D-fructose, D-mannose, α-D-glucose, D-mannitol, β-methyl D-glucoside, salicin, sucrose, D-trehalose, pruvatic acid methyl ester, mono-methyl succinate, glycerol. In addition hemolysis test for S. parauberis and were S. iniae hemolysis in BAP (Blood agar plate). Antibiotic test for S. parauberis were Ampicillin, Amoxicillin and Fluoroquinolone sensitivity. Mutiplex PCR assay were detected S. pauberis (718 bp), S. iniae (870 bp) L. garviae (1,100 bp). Dectected S. parauberis (718 bp) were result of 16S rRNA sequence identified with S. parauberis (Gene bank accession number X89967). All isolated S. parauberis that with bouned by one group. The result were S. pauberis that γ-hemolytic chain form cocci and negative reaction of catalase, Multiplex PCR assay were 718 bp amplicon size.