• Journal of Microbiology and Biotechnology
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• v.18 no.9
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• pp.1544-1549
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• 2008

MhMTS and MhMTH are trehalose ($\alpha$-D-glucopyranosyl-[1,1]-$\alpha$-D-glucopyranose) biosynthesis genes of the thermophilic microorganism Metallosphaera hakonensis, and encode a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively. In this study, the two genes were fused in-frame in a recombinant DNA, and expressed in Escherichia coli to produce a bifunctional fusion enzyme, MhMTSH. Similar to the two-step reactions with MhMTS and MhMTH, the fusion enzyme catalyzed the sequential reactions on maltopentaose, maltotriosyltrehalose formation, and following hydrolysis, producing trehalose and maltotriose. Optimum conditions for the fusion enzyme-catalyzed trehalose synthesis were around $70^{\circ}C$ and pH 5.0-6.0. The MhMTSH fusion enzyme exhibited a high degree of thermostability, retaining 80% of the activity when pre-incubated at $70^{\circ}C$ for 48 h. The stability was gradually abolished by incubating the fusion enzyme at above $80^{\circ}C$. The MhMTSH fusion enzyme was active on various sizes of maltooligosaccharides, extending its substrate specificity to soluble starch, the most abundant natural source of trehalose production.

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.1-8
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• 1999

Most carbohydrates exist in nature in an insoluble state, which reduces their susceptibility towards various carbohydrases. Accordingly, they require intensive pretreatment for structural modification to enhance an enzyme reaction. The direct conversion of insoluble carbohydrates has distinct advantages for special types of reaction, especially exo-type carbohydrase; however, its application is limited due to structural constraints. This paper introduces two novel heterogeneous enzyme reaction systems for direct conversion of insoluble carbohydrates; one is an attrition coupled enzyme reaction system containing attrition-milling media for enhancing the enzyme reaction, and the other is a heterogeneous enzyme reaction system using extruded starch as an insoluble substrate. The direct conversion of typically insoluble carbohydrates, including cellulose, starch, and chitin with their corresponding carbohydrases, including cellulase, amylase, chitinase, and cyclodextrin glucanotransferase, was carried out using two proposed enzyme reaction systems. The conceptual features of the systems, their reaction characteristics and mechanism, and the industrial applications of the various carbohydrates are analyzed in this review.

• Food Science and Biotechnology
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• v.14 no.5
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• pp.681-684
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• 2005

A maltopentaose-producing amylase (G5-amylase) from Bacillus megaterium KSM B-404 was applied to retard bread retrogradation. Retrogradation rates were determined by differential scanning calorimetry. Gel permeation chromatography determined changes in maltooligosaccharide composition and the molecular weight profiles of carbohydrate tractions. The baking process produced maltopentaose and maltotriose by the hydrolysis of starch molecules into small units. Amylose and amylopectin degradation as well as maltooligosaccharides produced by the enzyme were likely responsible for retarding starch retrogradation. Overall, addition of G5-amylase reduced the starch retrogradation rate, and was as effective as Novamyl(R), a commercial enzyme.

• BMB Reports
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• v.35 no.6
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• pp.568-575
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• 2002

An $\alpha$-amylase (EC 3.2.1.1) was purified that catalyses the production of a high level of maltose from starch without the attendant production of glucose. The enzyme was produced extracellularly by thermophilic Streptomyces sp. that was isolated from Thailand's soil. Purification was achieved by alcohol precipiation, DEAE-Cellulose, and Gel filtration chromatographies. The purified enzyme exhibited maximum activity at pH 6-7 and $60^{\circ}C$. It had a relative molecular mass of 45 kDa, as determined by SDS-PAGE. The hydrolysis products from starch had $\alpha$-anomeric forms, as determined by $^1H$-NMR. This maltose-forming $\alpha$-amylase completely hydrolyzed the soluble starch to produce a high level of maltose, representing up to 90%. It hydrolyzed maltotetrose and maltotriose to primarily produce maltose (82% and 62%, repectively) without the attendant production of glucose. The high maltose level as a final end-product from starch and maltooligosaccharides, and the unique action pattern of this enzyme, indicate an unusual maltose-forming system. After the addition of the enzyme in the bread-baking process, the bread's volume increased and kept its softness longer than when the bread had no enzyme.

• The Korean Journal of Food And Nutrition
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• v.11 no.2
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• pp.150-158
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• 1998

Limit dextrin of Korean beer(3 brands) and Japanese beer(21 brands) were separated by ethanol fractionation. Limit dextrin of Korean and Japanese beer was estimated to be 1.1%. 1H-NMR analysis revealed that the limit dextrin showed both signal of $\alpha$-1, 4- and $\alpha$-1, 6- glucosidic linkage with its estimation ratio of average 5.5:1. Limit dextrin was hydrolyzed to glucose with the yield of 57.22% by Aspergillus awamori $\alpha$-glucosidase(24.7 unit) plus human salivay $\alpha$-amylase(2.4 unit) in 100${mu}ell$ of 0.043M acetate buffer at 37$^{\circ}C$ for 5 hour. Among them, limit dextrin of Korean beer showed the highest hydrolysis rate of 76%. Small size sugars (64.8%) removed by ethanol fractionation and limit dextrin(21.4%) hydrolyzed by amylases that is digestable sugar. Non hydrolyzed limit dextrin(13.8%) by the amylases which can be a growth factor of Bifidobacterium in human intestine.

• 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.