Characterization and Application of BiLA, a Psychrophilic α-Amylase from <i>Bifidobacterium longum</i>

Lee, Hye Won; Jeon, Hye-Yeon; Choi, Ho‐Young; Kim, Na-Ri; Choung, Woo-Jae; Koo, Ye-Seul; Ko, Dam-Seul; You, SangGuan; Shim, Jae‐Hoon

doi:10.1021/acs.jafc.5b05904

Cited by 18 publications

(8 citation statements)

References 54 publications

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“…In general, the measured kinetic parameters were consistent with prior values observed for bacterial enzymes that process these substrates (glycogen 38,39,40 , amylose 41,42 , pullulan 43,41 ). Comparing the specificity constants ( k cat /K m ) for each substrate revealed that glycogen is the preferred substrate for the G. vaginalis and L. iners PulA enzymes.…”

Section: Resultssupporting

confidence: 87%

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Identification and characterization of bacterial glycogen-degrading enzymes in the vaginal microbiome

Woolston

Jenkins

Hood-Pishchany

et al. 2021

Preprint

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The healthy human vaginal microbiota is generally dominated by Lactobacilli, and the transition to a more diverse community of anaerobic microbes is associated with a number of health risks. While the mechanisms underlying the stability of Lactobacillus-dominated vaginal communities are not fully understood, competition for nutrients is a likely contributing factor. Glycogen secreted by epithelial cells is widely believed to support the growth of vaginal microbes. However, the mechanism by which bacteria access sugars from this complex polymer is unclear, with evidence to support a role for both microbial and human enzymes. To shed light on the potential contribution from microbial enzymes, here we biochemically characterize six glycogen-degrading enzymes predicted to be secreted by vaginal bacteria and confirm their ability to support the growth of an amylase-deficient strain of L. crispatus on glycogen. We reveal significant differences in the pH tolerance between enzymes from different organisms, suggesting the adaptation of Lactobacilli to an acidic vaginal environment. Using a simple assay specific for the microbial enzymes, we confirm their presence in cervicovaginal lavage samples. Finally, we demonstrate the selective inhibition of glycogen-degrading enzymes from two vaginal microbes associated with dysbiosis. This work provides biochemical evidence to support the role of vaginal bacterial amylase enzymes in the breakdown of glycogen, providing insight into factors that shape the vaginal microbiota and highlighting the possibility of manipulating community structure via non-antibiotic small molecules.

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Section: Resultssupporting

confidence: 87%

Section: Kinetic Characterization Reveals Different Glycosidic Linkag...supporting

confidence: 86%

Identification and characterization of bacterial glycogen-degrading enzymes in the vaginal microbiome

Woolston

Jenkins

Hood-Pishchany

et al. 2021

Preprint

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“…6 The purified enzyme was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich Co.; destaining solution was composed of 20% methanol, 10% (v/v) acetic acid, and water). 18 Enzyme Assay. The hydrolysis enzyme reaction was performed at 45 °C in sodium acetate buffer (50 mM, pH 6.0) containing 0.5% (w/ v) soluble starch, and the hydrolytic activity of CGTase was measured with the 3,5-dinitrosalicylic acid (DNS) method using 1% (w/v) β-CD as a substrate.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The supernatant was purified using a β-CD affinity column with washing (50 mM sodium acetate buffer, pH 6.0) and elution (50 mM sodium acetate buffer (pH 6.0), and 1% (w/v) β-CD) buffers. , The purified enzymes were concentrated using a Centricon filter (Millipore, Bedford, MA, USA) with 50 mM sodium acetate buffer (pH 6.0) . The purified enzyme was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich Co.; destaining solution was composed of 20% methanol, 10% (v/v) acetic acid, and water) …”

Section: Methodsmentioning

confidence: 99%

Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes β- and γ-CD Selectively

Koo

Jeong

et al. 2017

J. Agric. Food Chem.

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Cyclodextrins (CDs) are produced from starch by cyclodextrin glucanotransferase (CGTase), which has cyclization activity. Specifically, α-CD is an important biomolecule, as it is a molecular carrier and soluble dietary fiber used in the food industry. Upon inspection of the conserved regions of the glycoside hydrolase (GH) 13 family amylases, the amino acids K232 and H233 of CGTase were identified as playing an important role in enzyme reaction specificity. A novel CD hydrolyzing enzyme, cyclodextrin glycosyl transferase (CGTase)-alpha, was developed using site-directed mutagenesis at these positions. Action pattern analysis using various substrates revealed that CGTase-alpha was able to hydrolyze β- and γ-CD, but not α-CD. This selective CD hydrolyzing property was employed to purify α-CD from a CD mixture solution. The α-CD that remained after treatment with CGTase-alpha and exotype glucoamylase was purified using hydrophobic interaction chromatography with 99% purity.

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“…The side chain length distribution of starch samples was determined using a method that we described previously [31]. Before the analysis, the sample was pretreated to facilitate the structural analysis of the starch.…”

Section: Side Chain Length Distribution Analysismentioning

confidence: 99%

Development of Freeze-Thaw Stable Starch through Enzymatic Modification

Woo

Kim

Jeong

et al. 2021

Foods

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The use of unmodified starch in frozen foods can cause extremely undesirable textural changes after the freeze-thaw process. In this study, using cyclodextrin glucanotransferase (CGTase) and branching enzymes, an amylopectin cluster with high freeze-thaw stability was produced, and was named CBAC. It was found to have a water solubility seven times higher, and a molecular weight 77 times lower, than corn starch. According to the results of a differential scanning calorimetry (DSC) analysis, dough containing 5% CBAC lost 19% less water than a control dough after three freeze-thaw cycles. During storage for 7 days at 4 °C, bread produced using CBAC-treated dough exhibited a 14% smaller retrogradation peak and 37% less hardness than a control dough, suggesting that CBAC could be a potential candidate for clean label starch, providing high-level food stability under repeated freeze-thaw conditions.

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Characterization and Application of BiLA, a Psychrophilic α-Amylase from Bifidobacterium longum

Cited by 18 publications

References 54 publications

Identification and characterization of bacterial glycogen-degrading enzymes in the vaginal microbiome

Identification and characterization of bacterial glycogen-degrading enzymes in the vaginal microbiome

Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes β- and γ-CD Selectively

Development of Freeze-Thaw Stable Starch through Enzymatic Modification

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