• Journal of Sericultural and Entomological Science
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• v.41 no.1
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• pp.41-47
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• 1999

Silk fibroin dissolved in highly concentrated calcium chloride and ethanol mixture aqueous solution turned into gel under suitable conditions. Preparation conditions and properties of gel were investigated as a function of parameters such as pH of solution, fibroin concentration, glycerol concentration and molecular weight. When pH of silk fibroin aqueous solution was near the isoelectronic point(pH 3.9~4.0), gelation occurred rapidly and strength of gel was stonger than that of pH-unadjusted due to electrostatic repulsion decrease between silk fibroin macromolecules. As concentration of silk fibroin and glycerol was higher, gelation occurred more rapid. FT Infra-red spectra of freeze-dried fibroin gel showed that gelation was derived by intermolecular anti-parallel ${\beta}$-sheet structure formation. In addition to, it was found that white-precipitate occurred instead of gelation when aqueous silk fibroin was treated by enzyme(flavouzyme), however, after flavouzyme-treated silk fibroin aqueous solution was centrifugated gelation occurred instantly. The results of differential scanning thermal analysis and infra-red spectroscopy showed that thermal stability and crystallinity of enzyme-hydrolyzed fibroin are superior to those of unhydrolyzed fibroin.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.748-755
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• 2014

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.390-399
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• 2012

Endoglucanase gene egIV was cloned from Trichoderma viride AS 3.3711, an important cellulose-producing fungus, by using an RT-PCR protocol. The egIV cDNA is 1,297 bp in length and contains a 1,035 bp open reading frame encoding a 344 amino acid protein with an estimated molecular mass of 35.5 kDa and isoelectronic point (pI) of 5.29. The expression of gene egIV in T. viride AS 3.3711 could be induced by sucrose, corn straw, carboxymethylcellulose (CMC), or microcrystalline cellulose, but especially by CMC. The transcripts of egIV were regulated under these substrates, but the expression level of the egIV gene could be inhibited by glucose and fructose. Three recombinant vectors, pYES2-xegIV, $pYES2M{\alpha}$-egIV, and $pYES2M{\alpha}$-xegIV, were constructed to express the egIV gene in Saccharomyces cerevisiae H158. The CMCase activity of yeast transformants $IpYES2M{\alpha}$-xegIV was higher than that of transformant IpYES2-xegIV or $IpYES2M{\alpha}$-egIV, with the highest activity of 0.13 U/ml at induction for 48 h, illustrating that the modified egIV gene could enhance CMCase activity and that $MF{\alpha}$ signal peptide from S. cerevisiae could regulate exogenous gene expression more effectively in S. cerevisiae. The recombinant EGIV enzyme was stable at pH 3.5 to 7.5 and temperature of $35^{\circ}C$ to $65^{\circ}C$. The optimal reaction condition for EGIV enzyme activity was at the temperature of $55^{\circ}C$, pH of 5.0, 0.75 mM $Ba^{2+}$, and using CMC as substrate. Under these conditions, the highest activity of EGIV enzyme in transformant $IpYES2M{\alpha}$-xegIV was 0.18 U/ml. These properties would provide technical parameters for utilizing cellulose in industrial bioethanol production.