Isomaltotetraose

Immobilization of Dextranase Obtained from the Marine Cellulosimicrobium sp. Y1 on Nanoparticles: Nano-TiO(2) Improving Hydrolysate Properties and Enhancing Reuse.[Pubmed:36985959]

Nanomaterials (Basel). 2023 Mar 15;13(6):1065.

Dextranase is widely used in sugar production, drug synthesis, material preparation, and biotechnology, among other fields. The immobilization of dextranase using nanomaterials in order to make it reusable, is a hot research topic. In this study, the immobilization of purified dextranase was performed using different nanomaterials. The best results were obtained when dextranase was immobilized on titanium dioxide (TiO(2)), and a particle size of 30 nm was achieved. The optimum immobilization conditions were pH 7.0, temperature 25 degrees C, time 1 h, and immobilization agent TiO(2). The immobilized materials were characterized using Fourier-transform infrared spectroscopy, X-ray diffractometry, and field emission gun scanning electron microscopy. The optimum temperature and pH of the immobilized dextranase were 30 degrees C and 7.5, respectively. The activity of the immobilized dextranase was >50% even after 7 times of reuse, and 58% of the enzyme was active even after 7 days of storage at 25 degrees C, indicating the reproducibility of the immobilized enzyme. The adsorption of dextranase by TiO(2) nanoparticles exhibited secondary reaction kinetics. Compared with free dextranase, the hydrolysates of the immobilized dextranase were significantly different, and consisted mainly of isomaltotriose and Isomaltotetraose. The highly polymerized Isomaltotetraose levels could reach >78.69% of the product after 30 min of enzymatic digestion.

Purification and characterization of cold-adapted and salt-tolerant dextranase from Cellulosimicrobium sp. THN1 and its potential application for treatment of dental plaque.[Pubmed:36439846]

Front Microbiol. 2022 Nov 11;13:1012957.

The cold-adapted and/or salt-tolerant enzymes from marine microorganisms were confirmed to be meritorious tools to enhance the efficiency of biocatalysis in industrial biotechnology. We purified and characterized a dextranase CeDex from the marine bacterium Cellulosimicrobium sp. THN1. CeDex acted in alkaline pHs (7.5-8.5) and a broad temperature range (10-50 degrees C) with sufficient pH stability and thermostability. Remarkably, CeDex retained approximately 40% of its maximal activities at 4 degrees C and increased its activity to 150% in 4 M NaCl, displaying prominently cold adaptation and salt tolerance. Moreover, CeDex was greatly stimulated by Mg(2+), Na(+), Ba(2+), Ca(2+) and Sr(2+), and sugarcane juice always contains K(+), Ca(2+), Mg(2+) and Na(+), so CeDex will be suitable for removing dextran in the sugar industry. The main hydrolysate of CeDex was isomaltotriose, accompanied by Isomaltotetraose, long-chain IOMs, and a small amount of isomaltose. The amino acid sequence of CeDex was identified from the THN1 genomic sequence by Nano LC-MS/MS and classified into the GH49 family. Notably, CeDex could prevent the formation of Streptococcus mutans biofilm and disassemble existing biofilms at 10 U/ml concentration and would have great potential to defeat biofilm-related dental caries.

Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability.[Pubmed:36032722]

Front Bioeng Biotechnol. 2022 Aug 10;10:961776.

Obtaining high-degree polymerized isomaltose is more difficult while achieving better prebiotic effects. We investigated the mutation specificity and enzymatic properties of SP5-Badex, a dextranase from the GH66 family of Bacillus aquimaris SP5, and determined its mutation sites through molecular docking to obtain five mutants, namely E454K, E454G, Y539F, N369F, and Y153N. Among them, Y539F and Y153N exhibited no enzymatic activity, but their hydrolysates included Isomaltotetraose (IMO4). The enzymatic activity of E454G was 1.96 U/ml, which was 3.08 times higher than that before mutation. Moreover, 70% of the enzymatic activity could be retained after holding at 45 degrees C for 180 min, which was 40% higher than that of SP5-Badex. Furthermore, its IMO4 content was 5.62% higher than that of SP5-Badex after hydrolysis at 30 degrees C for 180 min. To investigate the effect of different amino acids on the same mutation site, saturation mutation was induced at site Y153, and the results showed that the enzyme activity of Y153W could be increased by 2 times, and some of the enzyme activity could still be retained at 50 degrees C. Moreover, the enzyme activity increased by 50% compared with that of SP5-Badex after holding at 45 degrees C for 180 min, and the IMO4 content of Y153W was approximately 64.97% after hydrolysis at 30 degrees C for 180 min, which increased by approximately 12.47% compared with that of SP5-Badex. This site is hypothesized to rigidly bind to nonpolar (hydrophobic) amino acids to improve the stability of the protein structure, which in turn improves the thermal stability and simultaneously increases the IMO4 yield.

NMR analysis and molecular dynamics conformation of alpha-1,6-linear and alpha-1,3-branched isomaltose oligomers as mimetics of alpha-1,6-linked dextran.[Pubmed:33813322]

Carbohydr Res. 2021 May;503:108296.

The conformational preferences of several alpha-1,6-linear and alpha-1,3-branched isomalto-oligosaccharides were investigated by NMR and MD-simulations. Right-handed helical structure contributed to the solution geometry in isomaltotriose and Isomaltotetraose with one nearly complete helix turn and stabilizing intramolecular hydrogen bonds in the latter by MD-simulation. Decreased helix contribution was observed in alpha-1,3-glucopyranosyl- and alpha-1,3-isomaltosyl-branched saccharide chains. Especially the latter modification was predicted to cause a more compact structure consistent with literature rheology measurements as well as with published dextranase-resistant alpha-1,3-branched oligosaccharides. The findings presented here are significant because they shed further light on the conformational preference of isomalto-oligosaccharides and provide possible help for the design of dextran-based drug delivery systems or for the targeted degradation of capsular polysaccharides by dextranases in multi-drug resistant bacteria.

Purification and characterization of cycloisomaltotetraose-forming glucanotransferases from Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006.[Pubmed:33624786]

Biosci Biotechnol Biochem. 2021 Feb 24;85(3):600-610.

Glucanotransferases that can synthesize cyclo--->6)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1--> (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 degrees C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 degrees C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 degrees C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 degrees C. Both enzymes catalyzed 4 reactions, namely, intramolecular alpha-1,6-transglycosylation (cyclization), intermolecular alpha-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from alpha-1,6-linked glucan synthesized from starch by 6-alpha-glucosyltransferase. These findings will enable the production of CI4 from starch.

Products Released from Structurally Different Dextrans by Bacterial and Fungal Dextranases.[Pubmed:33530339]

Foods. 2021 Jan 26;10(2):244.

Dextran hydrolysis by dextranases is applied in the sugar industry and the medical sector, but it also has a high potential for use in structural analysis of dextrans. However, dextranases are produced by several organisms and thus differ in their properties. The aim of this study was to comparatively investigate the product patterns obtained from the incubation of linear as well as O3- and O4-branched dextrans with different dextranases. For this purpose, genes encoding for dextranases from Bacteroides thetaiotaomicron and Streptococcus salivarius were cloned and heterologously expressed in Escherichia coli. The two recombinant enzymes as well as two commercial dextranases from Chaetomium sp. and Penicillium sp. were subsequently used to hydrolyze structurally different dextrans. The hydrolysis products were investigated in detail by HPAEC-PAD. For dextranases from Chaetomium sp., Penicillium sp., and Bacteroides thetaiotaomicron, isomaltose was the end product of the hydrolysis from linear dextrans, whereas Penicillium sp. dextranase led to isomaltose and Isomaltotetraose. In addition, the latter enzyme also catalyzed a disproportionation reaction when incubated with isomaltotriose. For O3- and O4-branched dextrans, the fungal dextranases yielded significantly different oligosaccharide patterns than the bacterial enzymes. Overall, the product patterns can be adjusted by choosing the correct enzyme as well as a defined enzyme activity.

A cyclic tetrasaccharide, cycloisomaltotetraose, was enzymatically produced from dextran and its crystal structure was determined.[Pubmed:32795710]

Carbohydr Res. 2020 Oct;496:108104.

Two bacterial strains isolated from soil, namely Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006, were found to produce a novel oligosaccharide. The oligosaccharide was enzymatically produced from dextran using the culture supernatant of Agreia sp. D1110 or M. trichothecenolyticum D2006. LC-MS and NMR analysis identified the novel oligosaccharide as cyclo--->6)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->, which was named cycloIsomaltotetraose, and abbreviated as CI4. CI4 was subsequently crystalized and its X-ray crystallographic structure was determined. CI4 crystals were shown to be pentahydrate, with the CI4 molecules in the crystal structure displaying a unique 3D structure, in which two glucosyl residues in the molecule were facing each other. This unique 3D structure was quite different from the 3D structure of known cyclic tetrasaccharides. This is the first report of CI4 molecules and their unique crystal structure.

Characterization of an Alkaline GH49 Dextranase from Marine Bacterium Arthrobacter oxydans KQ11 and Its Application in the Preparation of Isomalto-Oligosaccharide.[Pubmed:31430863]

Mar Drugs. 2019 Aug 19;17(8):479.

A GH49 dextranase gene DexKQ was cloned from marine bacteria Arthrobacter oxydans KQ11. It was recombinantly expressed using an Escherichia coli system. Recombinant DexKQ dextranase of 66 kDa exhibited the highest catalytic activity at pH 9.0 and 55 degrees C. kcat/Km of recombinant DexKQ at the optimum condition reached 3.03 s(-1) muM(-1), which was six times that of commercial dextranase (0.5 s(-1) muM(-1)). DexKQ possessed a Km value of 67.99 microM against dextran T70 substrate with 70 kDa molecular weight. Thin-layer chromatography (TLC) analysis showed that main hydrolysis end products were isomalto-oligosaccharide (IMO) including Isomaltotetraose, isomaltopantose, and isomaltohexaose. When compared with glucose, IMO could significantly improve growth of Bifidobacterium longum and Lactobacillus rhamnosus and inhibit growth of Escherichia coli and Staphylococcus aureus. This is the first report of dextranase from marine bacteria concerning recombinant expression and application in isomalto-oligosaccharide preparation.

A novel intracellular dextranase derived from Paenibacillus sp. 598K with an ability to degrade cycloisomaltooligosaccharides.[Pubmed:31273396]

Appl Microbiol Biotechnol. 2019 Aug;103(16):6581-6592.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more alpha-(1 --> 6)-linked-D-glucose residues. The extracellular enzyme CI glucanotransferase (PsCITase), which is the member of glycoside hydrolase family 66, catalyzes the final stage of CI production and produces mainly cycloisomaltoheptaose. We have discovered a novel intracellular CI-degrading dextranase (PsDEX598) from Paenibacillus sp. 598K. The 69.7-kDa recombinant PsDEX598 does not digest Isomaltotetraose or shorter isomaltooligosaccharides, but digests longer ones of at least up to isomaltoheptaose. It also digests oligoCIs of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononaose better than it does with megaloCIs of cycloisomaltodecaose, cycloisomaltoundecaose, and cycloisomaltododecaose, as well as an alpha-(1 --> 6)-D-glucan of dextran 40. PsDEX598 is produced intracellularly when culture medium is supplemented with cycloisomaltoheptaose or dextran, but not with isomaltooligosaccharides (a mixture of isomaltose, isomaltotriose, and panose), starch, or glucose. The whole genomic DNA sequence of the strain 598K implies that it harbors two genes for enzymes belonging to glycoside hydrolase family 66 (PsCITase and PsDEX598), and PsDEX598 is the only dextranase in the strain. PsDEX598 does not have any carbohydrate-binding modules (CBMs) and has a low similarity (< 30%) with other family 66 dextranases, and the catalytic amino acids of this enzyme are predicted to be Asp191, Asp303, and Glu368. The strain Paenibacillus sp. 598K appears to take up CI-7, so these findings indicate that this bacterium can degrade CIs using a dextranase within the cells and so utilize them as a carbon source for growth.

Futile Encounter Engineering of the DSR-M Dextransucrase Modifies the Resulting Polymer Length.[Pubmed:31140266]

Biochemistry. 2019 Jun 25;58(25):2853-2859.

The factors that define the resulting polymer length of distributive polymerases are poorly understood. Here, starting from the crystal structure of the dextransucrase DSR-M in complex with an Isomaltotetraose, we define different anchoring points for the incoming acceptor. Mutation of one of these, Trp624, decreases the catalytic rate of the enzyme but equally skews the size distribution of the resulting dextran chains toward shorter chains. Nuclear magnetic resonance analysis shows that this mutation influences both the dynamics of the active site and the water accessibility. Monte Carlo simulation of the elongation process allows interpretation of these results in terms of enhanced futile encounters, whereby the less effective binding increases the pool of effective seeds for the dextran chains and thereby directly determines the length distribution of the final polymers.

Identification of Catalytic Amino Acid Residues by Chemical Modification in Dextranase.[Pubmed:26907761]

J Microbiol Biotechnol. 2016 May 28;26(5):837-45.

A novel endodextranase isolated from Paenibacillus sp. was found to produce Isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7-14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3- (dimethylamino)-propyl]-carbodiimide (EDC) and alpha-epoxyalkyl alpha-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity.

Crystal structures of starch binding domain from Rhizopus oryzae glucoamylase in complex with isomaltooligosaccharide: insights into polysaccharide binding mechanism of CBM21 family.[Pubmed:24108499]

Proteins. 2014 Jun;82(6):1079-85.

Glucoamylases are responsible for hydrolysis of starch and polysaccharides to yield beta-D-glucose. Rhizopus oryzae glucoamylase (RoGA) is composed of an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain connected by an O-glycosylated linker. Two carbohydrate binding sites in RoSBD have been identified, site I is created by three highly conserved aromatic residues, Trp47, Tyr83, and Tyr94, and site II is built up by Tyr32 and Phe58. Here, the two crystal structures of RoSBD in complex with only alpha-(1,6)-linked isomaltotriose (RoSBD-isoG3) and Isomaltotetraose (RoSBD-isoG4) have been determined at 1.2 and 1.3 A, respectively. Interestingly, site II binding is observed in both complexes, while site I binding is only found in the RoSBD-isoG4 complex. Hence, site II acts as the recognition binding site for carbohydrate and site I accommodates site II to bind isoG4. Site I participates in sugar binding only when the number of glucosyl units of oligosaccharides is more than three. Taken together, two carbohydrate binding sites in RoSBD cooperate to reinforce binding mode of glucoamylase with polysaccharides as well as the starch.

Bacteroides thetaiotaomicron VPI-5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions.[Pubmed:22776355]

FEBS J. 2012 Sep;279(17):3185-91.

Bacteroides thetaiotaomicron VPI-5482 harbors a gene encoding a putative cycloisomaltooligosaccharide glucanotransferase (BT3087) belonging to glycoside hydrolase family 66. The goal of the present study was to characterize the catalytic properties of this enzyme. Therefore, we expressed BT3087 (recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482) in Escherichia coli and determined that recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482 preferentially synthesized Isomaltotetraose and isomaltooligosaccharides (degree of polymerization > 4) from dextran. The enzyme also generated large cyclic isomaltooligosaccharides early in the reaction. We conclude that members of the glycoside hydrolase 66 family may be classified into three types: (a) endo-dextranases, (b) dextranases possessing weak cycloisomaltooligosaccharide glucanotransferase activity, and (c) cycloisomaltooligosaccharide glucanotransferases.

Production of isomaltooligosaccharides from banana flour.[Pubmed:22689251]

J Sci Food Agric. 2013 Jan 15;93(1):180-6.

BACKGROUND: Banana is one of the important crops native to tropical Southeast Asia. Since overproduction frequently leads to excessive waste of produce, alternative uses are continuously sought in order to utilise fruits at all stages of maturity. The aim of this study was to investigate the production of isomaltooligosaccharides (IMOs) from banana flour. RESULTS: Banana slurries liquefied by Termamyl SC and saccharified by either Fungamyl 800 L or barley beta-amylase were used for IMO synthesis by Transglucosidase L. After 12 h of transglucosylation, maximum IMO yields of 76.67 +/- 2.71 and 70.74 +/- 4.09 g L(-1) respectively were achieved. Although the yields were comparable, the IMO profiles obtained through the use of the two saccharification enzymes were different. Glucose and maltose were removed by 10 g L(-1) bakers' yeast fermentation for 12 h. Regarding total sugars, the final IMO mixture was composed of 53% isomaltotriose, 21% Isomaltotetraose and 26% maltooligoheptaose and larger oligomers. CONCLUSION: Banana flour could be used as a potential raw material for IMO synthesis.

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K.[Pubmed:22542750]

Biochim Biophys Acta. 2012 Jul;1824(7):919-24.

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.