Raw starch degrading α-amylases: an unsolved riddle

Amylase ◽  
2017 ◽  
Vol 1 (1) ◽  
Author(s):  
Nataša Božić ◽  
Nikola Lončar ◽  
Marinela Šokarda Slavić ◽  
Zoran Vujčić
Keyword(s):  
Glycoside Hydrolase Family ◽  
Food Ingredient ◽  
Raw Starch ◽  
Functional Studies ◽  
Structures And Properties ◽  
Structural Peculiarities ◽  
To Come ◽  
Starch Conversion ◽  
Maltose Syrup ◽  
Hydrolysis Of

AbstractStarch is an important food ingredient and a substrate for the production of many industrial products. Biological and industrial processes involve hydrolysis of raw starch, such as digestion by humans and animals, starch metabolism in plants, and industrial starch conversion for obtaining glucose, fructose and maltose syrup or bioethanol. Raw starch degrading α-amylases (RSDA) can directly degrade raw starch below the gelatinization temperature of starch. Knowledge of the structures and properties of starch and RSDA has increased significantly in recent years. Understanding the relationships between structural peculiarities and properties of RSDA is a prerequisite for efficient application in different aspects of human benefit from health to the industry. This review summarizes recent advances on RSDA research with emphasizes on representatives of glycoside hydrolase family GH13. Definite understanding of raw starch digesting ability is yet to come with accumulating structural and functional studies of RSDA.

scholarly journals The thermostable 4,6-α-glucanotransferase of Bacillus coagulans DSM 1 synthesizes isomaltooligosaccharides

Amylase ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 13-22
Author(s):  
Gang Xiang ◽  
Piet L. Buwalda ◽  
Marc J.E.C van der Maarel ◽  
Hans Leemhuis
Keyword(s):  
Bacillus Coagulans ◽  
Glycoside Hydrolase Family ◽  
Rat Intestine ◽  
Glycaemic Response ◽  
Food Ingredient ◽  
Starch Conversion ◽  
Identification And Characterization ◽  
Hydrolase Family

Abstract The 4,6-α-glucanotransferases of the glycoside hydrolase family 70 can convert starch into isomaltooligosaccharides (IMOs). However, no thermostable 4,6-α-glucanotransferases have been reported to date, limiting their applicability in the starch conversion industry. Here we report the identification and characterization of a thermostable 4,6-α-glucanotransferase from Bacillus coagulans DSM 1. The gene was cloned and the recombinant protein, called BcGtfC, was produced in Escherichia coli. BcGtfC is stable up to 66 °C in the presence of substrate. It converts debranched starch into an IMO product with a high percentage of α-1,6-glycosidic linkages and a relatively high molecular weight compared to commercially available IMOs. Importantly, the product is only partly and very slowly digested by rat intestine powder, suggesting that the IMO will provide a low glycaemic response in vivo when applied as food ingredient. Thus, BcGtfC is a thermostable 4,6-α-glucanotransferase suitable for the industrial production of slowly digestible IMOs from starch.

scholarly journals Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

2010 ◽  
Vol 9 (11) ◽  
pp. 1650-1660 ◽  
Author(s):  
Encarnación Dueñas-Santero ◽  
Ana Belén Martín-Cuadrado ◽  
Thierry Fontaine ◽  
Jean-Paul Latgé ◽  
Francisco del Rey ◽  
...  
Keyword(s):  
Cell Wall ◽  
Schizosaccharomyces Pombe ◽  
Protein Characterization ◽  
Wall Material ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Hydrolysis Of ◽  
Controlled Hydrolysis ◽  
Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

scholarly journals Study of the mode of action of a polygalacturonase from the phytopathogen Burkholderia cepacia

2007 ◽  
Vol 407 (2) ◽  
pp. 207-217 ◽  
Author(s):  
Claudia Massa ◽  
Mads H. Clausen ◽  
Jure Stojan ◽  
Doriano Lamba ◽  
Cristiana Campa
Keyword(s):  
Mode Of Action ◽  
Burkholderia Cepacia ◽  
Time Course ◽  
Glycoside Hydrolase Family ◽  
Enzymatic Mechanism ◽  
Processive Enzyme ◽  
Chain Lengths ◽  
Methylation Patterns ◽  
Hydrolysis Of ◽  
Hydrolase Family

We have recently isolated and heterologously expressed BcPeh28A, an endopolygalacturonase from the phytopathogenic Gram-negative bacterium Burkholderia cepacia. Endopolygalacturonases belong to glycoside hydrolase family 28 and are responsible for the hydrolysis of the non-esterified regions of pectins. The mode of action of BcPeh28A on different substrates has been investigated and its enzymatic mechanism elucidated. The hydrolysis of polygalacturonate indicates that BcPeh28A is a non-processive enzyme that releases oligomers with chain lengths ranging from two to eight. By inspection of product progression curves, a kinetic model has been generated and extensively tested. It has been used to derive the kinetic parameters that describe the time course of the formation of six predominant products. Moreover, an investigation of the enzymatic activity on shorter substrates that differ in their overall length and methylation patterns sheds light on the architecture of the BcPeh28A active site. Specifically the tolerance of individual sites towards methylated saccharide units was rationalized on the basis of the hydrolysis of hexagalacturonides with different methylation patterns.

scholarly journals Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

2004 ◽  
Vol 186 (15) ◽  
pp. 4885-4893 ◽  
Author(s):  
Takane Katayama ◽  
Akiko Sakuma ◽  
Takatoshi Kimura ◽  
Yutaka Makimura ◽  
Jun Hiratake ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Sugar Chain ◽  
Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

scholarly journals Camel Gelatin Composition, Properties, Production, and Applications

2020 ◽  
pp. 306-327
Author(s):  
Irwandi Jaswir ◽  
Hassan Abdullah Al-Kahtani ◽  
Fitri Octavianti ◽  
Widya Lestari ◽  
Nurlina Yusof
Keyword(s):  
Body Temperature ◽  
High Water ◽  
Partial Hydrolysis ◽  
High Water Content ◽  
Food Ingredient ◽  
Normal Body Temperature ◽  
Food Industries ◽  
Reversible Gel ◽  
Composition Properties ◽  
Hydrolysis Of

Gelatin is an important protein produced through partial hydrolysis of collagen from animal parts and byproducts such as cartilage, bones, tendons, and hides. The ability of gelatin to form a thermo-reversible gel at normal body temperature and high water content make it an exceptional food ingredient. A good quality gelatin is translucent, brittle, colorless (sometimes slightly yellow), bland in taste, and odorless. Gelatin has been found useful as stabilizer and filler in dairy products and other food industries. Recently, the global gelatin production net over 300,000 metric tons: 46% were from pigskin, 29.4% from bovine hides, 23.1% from bones, and 1.5% from other parts. Although camels have been recognized as source of meat and milk, utilization of camel bones and skins for gelatin production has not been fully explored. This chapter will discuss the processing of camel gelatin extraction.

scholarly journals Identification of a Key Enzyme for the Hydrolysis of β-(1→3)-Xylosyl Linkage in Red Alga Dulse Xylooligosaccharide from Bifidobacterium Adolescentis

Marine Drugs ◽  
2020 ◽  
Vol 18 (3) ◽  
pp. 174 ◽  
Author(s):  
Manami Kobayashi ◽  
Yuya Kumagai ◽  
Yohei Yamamoto ◽  
Hajime Yasui ◽  
Hideki Kishimura
Keyword(s):  
Main Product ◽  
Specific Activity ◽  
Red Alga ◽  
Glycoside Hydrolase Family ◽  
Bifidobacterium Adolescentis ◽  
Glycoside Hydrolase Family 43 ◽  
Key Enzyme ◽  
Hydrolysis Of ◽  
Low Activity ◽  
Hydrolase Family

Red alga dulse possesses a unique xylan, which is composed of a linear β-(1→3)/β-(1→4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (β-(1→3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides sp. and Bifidobacterium adolescentis, Bacteroides Ksp. grew slowly as compared with β-(1→4)-xylotriose (X3) but B. adolescentis grew similar to X3. Therefore, we aimed to find the key DX3 hydrolysis enzymes in B. adolescentis. From bioinformatics analysis, two enzymes from the glycoside hydrolase family 43 (BAD0423: subfamily 12 and BAD0428: subfamily 11) were selected and expressed in Escherichia coli. BAD0423 hydrolyzed β-(1→3)-xylosyl linkage in DX3 with the specific activity of 2988 mU/mg producing xylose (X1) and xylobiose (X2), and showed low activity on X2 and X3. BAD0428 showed high activity on X2 and X3 producing X1, and the activity of BAD0428 on DX3 was 1298 mU/mg producing X1. Cooperative hydrolysis of DX3 was found in the combination of BAD0423 and BAD0428 producing X1 as the main product. From enzymatic character, hydrolysis of X3 was completed by one enzyme BAD0428, whereas hydrolysis of DX3 needed more than two enzymes.

Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori

2002 ◽  
Vol 362 (1) ◽  
pp. 131-135 ◽  
Author(s):  
Michael ARAND ◽  
Alexander M. GOLUBEV ◽  
J. R. Brandao NETO ◽  
Igor POLIKARPOV ◽  
R. WATTIEZ ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Solid Phase ◽  
Aspergillus Awamori ◽  
Glycoside Hydrolase Family ◽  
Orthorhombic Space Group ◽  
Ph Optimum ◽  
X Ray ◽  
Cell Parameters ◽  
Hydrolysis Of

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69±1kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the β-(2 → 1)-fructan (inulin) and β-(2 → 6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants Km and Vmax in the hydrolysis of inulin were 0.003±0.0001mM and 175±5μmol·min−1·mg−1. The same parameters in the hydrolysis of levan were 2.08±0.04mg/ml and 1.2±0.02μmol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P212121 with cell parameters a = 64.726 Å (1Å = 0.1 nm), b = 82.041 Å and c = 136.075 Å. Crystals diffracted beyond 1.54 Å, and useful X-ray data were collected to a resolution of 1.73 Å.

scholarly journals Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases

2013 ◽  
Vol 6 (1) ◽  
pp. 167 ◽  
Author(s):  
Marko J Viktor ◽  
Shaunita H Rose ◽  
Willem H van Zyl ◽  
Marinda Viljoen-Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Aspergillus Tubingensis ◽  
Raw Starch ◽  
Starch Conversion

scholarly journals β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

Catalysts ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 365 ◽  
Author(s):  
Jan Muschiol ◽  
Marlene Vuillemin ◽  
Anne S. Meyer ◽  
Birgitte Zeuner
Keyword(s):  
Cell Wall ◽  
Biomimetic Synthesis ◽  
Glycoside Hydrolase Family ◽  
Reaction Conditions ◽  
Substrate Activation ◽  
Donor And Acceptor ◽  
Hydrolysis Of ◽  
Synthesis Reactions ◽  
Hydrolase Family ◽  
Novel Protein

β-N-acetylhexosaminidases (EC 3.2.1.52) are retaining hydrolases of glycoside hydrolase family 20 (GH20). These enzymes catalyze hydrolysis of terminal, non-reducing N-acetylhexosamine residues, notably N-acetylglucosamine or N-acetylgalactosamine, in N-acetyl-β-D-hexosaminides. In nature, bacterial β-N-acetylhexosaminidases are mainly involved in cell wall peptidoglycan synthesis, analogously, fungal β-N-acetylhexosaminidases act on cell wall chitin. The enzymes work via a distinct substrate-assisted mechanism that utilizes the 2-acetamido group as nucleophile. Curiously, the β-N-acetylhexosaminidases possess an inherent trans-glycosylation ability which is potentially useful for biocatalytic synthesis of functional carbohydrates, including biomimetic synthesis of human milk oligosaccharides and other glycan-functionalized compounds. In this review, we summarize the reaction engineering approaches (donor substrate activation, additives, and reaction conditions) that have proven useful for enhancing trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. We provide comprehensive overviews of reported synthesis reactions with GH20 enzymes, including tables that list the specific enzyme used, donor and acceptor substrates, reaction conditions, and details of the products and yields obtained. We also describe the active site traits and mutations that appear to favor trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. Finally, we discuss novel protein engineering strategies and suggest potential “hotspots” for mutations to promote trans-glycosylation activity in GH20 for efficient synthesis of specific functional carbohydrates and other glyco-engineered products.

scholarly journals Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yuya Kuritani ◽  
Kohei Sato ◽  
Hideo Dohra ◽  
Seiichiro Umemura ◽  
Motomitsu Kitaoka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Metabolic Pathway ◽  
Glycoside Hydrolase ◽  
Gene Locus ◽  
Glycoside Hydrolase Family ◽  
Sequential Reaction ◽  
Layer Chromatography ◽  
Hydrolysis Of ◽  
Hydrolase Family

AbstractLevoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-d-glucose by LgdB2; next, LgdC reduces 3-keto-d-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

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