• Journal of Microbiology and Biotechnology
• /
• v.18 no.3
• /
• pp.457-464
• /
• 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
• /
• v.17 no.9
• /
• pp.1521-1526
• /
• 2007

A maltogenic amylase gene from Lactobacillus gasseri ATCC33323 (LGMA) was expressed in Lactococcus lactis MG1363 using the P170 expression system. The successful production of recombinant LGMA (rLGMA) was confirmed by the catalytic activity of the enzyme in liquid and solid media. The N-terminal amino acid sequencing analysis of the rLGMA showed that it was Met-Gln-Leu-Ala-Ala-Leu-, which was the same as that of genuine protein, meaning the signal peptide was efficiently cleaved during secretion to the extracellular milieu. The optimal reaction temperature and pH of rLGMA ($55^{\circ}C$ and pH 5, respectively) and enzymatic hydrolysis patterns on various substrates (${\beta}$-cyclodextrin, starch, and pullulan) supported that rLGMA was not only efficiently secreted from the Lactococcus lactis MG1363 but was also functionally active. Finally, the branched maltooligosaccharides were effectively produced from liquefied com starch, by using rLGMA secreted from Lactococcus lactis, with a yield of 53.1%.

• Applied Biological Chemistry
• /
• v.13 no.3
• /
• pp.181-186
• /
• 1970

1) Conditions for the hydrolysis of starch by bacterial liquefying amylase (BLA), saccharifying amylase (BSA) and isoamylase were investigated. Out of four syrups prepared by different combinations of these enzymes, those made by BLA followed by BSA and/or isoamylase were comparable to sucrose syrup in canning of orange segments. 2) Two branched maltooligosaccharides were isolated from the hydrolyzate of starch by BLA and BSA, and their structures were tentatively identified as pentaose and hexaose having an ${\alpha}-1$, 6-linkage at the branching point.

• Microbiology and Biotechnology Letters
• /
• v.19 no.5
• /
• pp.444-449
• /
• 1991

Synthesis of maltosyl-$\beta$-cyclodextrin using maltose ($G_2$) and $\beta$-cyclodextrin ($\beta$-CD) as substrates through the reverse reaction of pullulanase was investigated. The optimal conditions for the condensation reaction were as below: mixing ratio of maltose to $\beta$-CD of 12.7, mixed substrate concentration of 70% (w/w, 70 g/100 ml $H_2O$), and amount of pullulanse of 350 units/100 ml. The concentration of synthesized maltosyl-P-CD concentration was reached up to 2.31 g/100 rnl at above reaction conditions, which corresponded the conversion yield of 43% (w/w, g of branched-CD/g of CD). The synthesis of maltosyl-$\alpha >\gamma >\beta$-CD was also attempted, and conversion yield was in the order of a>y>J3-CDs. Condensation reaction between various maltooligosaccharides ($G-1\sim G_6$ showed that maltose was the most effective oligorner for condensation reaction with $\beta$-CD. To increase the conversion yield various alcohols were added into the reaction mixture, amyl alcohol was found to be the most acceptable alcohol for increasement of convesion yield which increased from 43.0 to 83.0% upon addition of same volume of amyl alcohol into the reaction mixture.

• Food Science and Biotechnology
• /
• v.18 no.2
• /
• pp.570-573
• /
• 2009

The debranching enzyme of Nostoc punctiforme (NPDE) is a novel enzyme that catalyzes the hydrolysis of $\alpha$-1,6-glycosidic linkages in starch, followed by the sequential hydrolysis of $\alpha$-1,4-glycosidic linkages. The debranching activity of NPDE is highly specific for branched chains with a degree of polymerization (DP)>8. Moreover, the rate of hydrolysis of $\alpha$-1,4-linkages by NPDE is greatly enhanced for maltooligosaccharides (MOs) with a DP>8. An analysis of reaction mixtures containing various starches revealed the accumulation of maltooctaose (G8) with glucose and maltose. Based on the novel enzymatic properties of NPDE, an MO mixture containing more than 60% G8 with yield of 18 g G8 for 100 g starch was prepared by the reaction of NPDE with soluble starch, followed by ethanol precipitation and gel permeation chromatography (GPC). The yield of the G8-rich mixture was significantly improved by the addition of isoamylase. In summary, a 4-step process for the production of a G8-rich mixture was developed involving the enzymatic hydrolysis of starch by NPDE.