• KSBB Journal
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• 제16권3호
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• pp.246-252
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• 2001

We isolated a bacterium that produces an extracellular maltopentaose(G5)-forming amylase from amylose and soluble starch. The bacterium was identified and assigned as a Bacillus sp. AIR-5. The amylase did not hydrolyze maltose, maltotriose, maltotetraose or maltopentaose. Optimum medium composition for maltopentaose production in flask culture was 2%(w/v) soluble starch, 0.4%(w/v) tryptone, 0.5%(w/v) NaCl, 0.5%(w/v) K$_2$HPO$_4$, and 3 mM CaCl$_2$at pH 8.0, 28$^{\circ}C$. The highest yield for maltopentaose production in this condition was 6.45 g/L and was 32.55% of theoretical yield.

• 한국식품영양과학회지
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• 제27권4호
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• pp.639-647
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• 1998

This study was carried out to produce the maltooligosaccharides directly from the culture medium containing high concentration of soluble starch as carbohydrate source by Bacillus cereus IAM 1072. Optimum conditions for the production of maltopentaose and maltooligosaccharides were predicted as 10.62 and 10.92 in C/N ration, 115.74 and 116.51 rpm in agitation speed, 30.19 and 30.9$0^{\circ}C$, respectively. And at these conditions, products of maltopentaose and maltooligosaccharides were 23.23 and 50.33g/L, respectively. From the results of continuous culture for maltopentaose, the productivity increased up to 6.9 times, showing 6.6g/L/hr compared with 0.96g/L/hr batch culture. Maltopentaose showed lower sweetness at 3% concentration representing 1/5 of that sugar. Also, swelling power of maltooligosaccharides was reached to the same point with sugar after fermentation.

• Journal of Microbiology and Biotechnology
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• 제11권4호
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• pp.636-643
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• 2001

Bacillus sp. AIR-5, a strain from soil, produced an extracellular maltopentaose-forming amylase from amylose and soluble starch. This bacterium produced 8.9 g/l of maltopentaose from 40 g/l of soluble starch in a batch fermentation and the maltopentaose made up 90 % of the maltooligosaccharides produced (from maltose to maltoheptaose). The culture supernatant was concentrated using a 30 K molecular weight cut-off membrane and purified by DEAE-Cellulose and Sephadex G-150 column chromatographies. The purified protein showed one band on a native-PAGE and its molecular mass was estimated as 250 kDa. The 250-kDa protein was composed of tetramers of a 63-kDa protein. the isoelectric point of the purified protein was pH 6.9, and the optimum temperature for the enzyme activity was $45^{\circ}C$. The enzyme was quickly inactivated above $55^{\circ}C$, and showed a maximum activity at pH 8.5 and over 90% stability between a pH of 6 to 10. The putative N-terminal amino acid sequence of AIR-5 amylase, ATINNGTLMQYFEWYVPNDG, showed a 96% sequence similarity with that of BLA, a general liquefying amylase.

• Journal of Applied Biological Chemistry
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• 제43권4호
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• pp.240-245
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• 2000

Two genes encoding maltooligosyltrehalose synthase (SaMTS) and maltooligosyltrehalose trehalohydrolase (SaMTH) were isolated from a hyperthermophilic microorganism, Sulfolobus acidocaldarius (ATCC 49462). ORFs of the SaMTS and SaMTH genes are 2,163 and 1,671 bp long and encode 720 and 556 amino acid residues, respectively. A bifunctional fusion enzyme (SaMTSH) was constructed through the gene fusion of SaMTS and SaMTH. Recombinant SaMTS, SaMTH, and SaMTSH fusion enzyme were overexpressed in E. coli BL21. SaMTS and SaMTH produced trehalose and maltotriose from maltopentaose in a sequential reaction. SaMTSH fusion enzyme catalyzed the sequential reaction in which the formation of maltotriosyltrehalose was followed by hydrolysis leading to the synthesis of trehalose and maltotriose. The SaMTSH fusion enzyme showed the highest activity at pH 5.0-5.5 and $70-75^{\circ}C$. SaMTS, SaMTH, and SaMTSH fusion enzyme were active in soluble starch, which resulted in the production of trehalose.

• Journal of Microbiology and Biotechnology
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• 제18권9호
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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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• 제18권3호
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• pp.457-464
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• 2008

An extracellular enzyme (RMEBE) possessing ${\alpha}-(1{\rightarrow}4)-(1{\rightarrow}6)$-transferring activity was purified to homogeneity from Rhodothermus marin us by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex-200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at $80^{\circ}C$. Its half-life was determined to be 73.7 and 16.7 min at 80 and $85^{\circ}C$, respectively. The enzyme was also halophilic and highly halotolerant up to about 2M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial ${\alpha}-(1{\rightarrow}4)$-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited ${\alpha}-(1{\rightarrow}4)-(1{\rightarrow}6)$-transferase activity for small maltooligosaccharides, producing disproportionated ${\alpha}-(1{\rightarrow}6)$-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.

• Journal of Microbiology and Biotechnology
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• 제29권12호
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• pp.1938-1946
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• 2019

Isomaltooligosaccharides (IMOs) have good prebiotic effects, and long IMOs (LIMOs) with a degree of polymerization (DP) of 7 or above show improved effects. However, they are not yet commercially available, and require costly enzymes and processes for production. The N-terminal region of the thermostable Thermoanaerobacter thermocopriae cycloisomaltooligosaccharide glucanotransferase (TtCITase) shows cyclic isomaltooligosaccharide (CI)-producing activity owing to a catalytic domain of glycoside hydrolase (GH) family 66 and carbohydrate-binding module (CBM) 35. In the present study, we elucidated the activity of the C-terminal region of TtCITase (TtCITase-C; Met740-Phe1,559), including a CBM35-like region and the GH family 15 domain. The domain was successfully cloned, expressed, and purified as a single protein with a molecular mass of 115 kDa. TtCITase-C exhibited optimal activity at 40℃ and pH 5.5, and retained 100% activity at pH 5.5 after 18-h incubation. TtCITase-C synthesized α-1,6 glucosyl products with over seven degrees of polymerization (DP) by an α-1,6 glucosyl transfer reaction from maltopentaose, isomaltopentaose, or commercialized maltodextrins as substrates. These results indicate that TtCITase-C could be used for the production of α-1,6 glucosyl oligosaccharides with over DP7 (LIMOs) in a more cost-effective manner, without requiring cyclodextran.

• Asian-Australasian Journal of Animal Sciences
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• 제22권4호
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• pp.591-597
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• 2009

Egg white protein (EWP) was glycated with maltopentaose (MP) through the Maillard reaction and subsequently phosphorylated by $85^{\circ}C$ dry-heating at pH 4.0 for 1 d in the presence of pyrophosphate. The functional properties of glycated, phosphorylated EWP were compared with those of native EWP and with EWP which was phosphorylated by dry-heating in the presence of pyrophosphate under the same conditions. The phosphorus content of EWP was increased to ~0.60% by phosphorylation, and to ~0.74% by glycation with MP and subsequent phosphorylation. The electrophoretic mobility of EWP increased through phosphorylation. The stability of EWP against heat-induced insolubility at pH 7.0 was considerably improved by phosphorylation alone and further by phosphorylation after glycation. The anti-ovalbumin antibody response was reduced significantly by glycation and phosphorylation, and further reduced by phosphorylation after glycation. The anti-ovomucoid antibody response was reduced significantly by glycation, phosphorylation and phosphorylation after glycation. The calcium phosphate-solubilizing ability of EWP was enhanced by both phosphorylation methods.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.123-129
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• 2007

The trehalose $({\alpha}-D-glucopyranosyl-[1,1]-{\alpha}-D-glucopyranose)$ biosynthesis genes MhMTS and MhMTH, encoding a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively, have been cloned from the hyperthermophilic archaebacterium Metallosphaera hakonesis. The ORF of MhMTS is 2,142 bp long, and encodes 713 amino acid residues constituting a 83.8 kDa protein. MhMTH is 1,677 bp long, and encodes 558 amino acid residues constituting a 63.7 kDa protein. The deduced amino acid sequences of MhMTS and MhMTH contain four regions highly conserved for MTSs and three for MTHs that are known to constitute substrate-binding sites of starch-hydrolyzing enzymes. Recombinant proteins obtained by expressing the MhMTS and MhMTH genes in E. coli catalyzed a sequential reaction converting maltooligosaccharides to produce trehalose. Optimum pH of the MhMTS/MhMTH enzyme reaction was around 5.0 and optimum temperature was around 70 C. Trehalose-producing activity of the MhMTS/ MhMTH was notably stable, retaining 80% of the activity after preincubation of the enzyme mixture at $70^{\circ}C$ for 48 h, but was gradually abolished by incubating at above $85^{\circ}C$. Addition of thermostable $4-{\alpha}-glucanotransferase$ increased the yield of trehalose production from maltopentaose by 10%. The substrate specificity of the MhMTS/MhMTH-catalyzed reaction was extended to soluble starch, the most abundant maltodextrin in nature.