Leuconostoc mesenteroides and Liquorilactobacillus mali strains, isolated from Algerian food products, are producers of the postbiotic compounds dextran, oligosaccharides and mannitol.

Six lactic acid bacteria (LAB) isolated from Algerian sheep's milk, traditional butter, date palm sap and barley, which produce dextran, mannitol, oligosaccharides and vitamin B2 have been characterized. They were identified as Leuconostoc mesenteroides (A4X, Z36P, B12 and O9) and Liquorilactobacillus mali (BR201 and FR123). Their exopolysaccharides synthesized from sucrose by dextransucrase (Dsr) were characterized as dextrans with (1,6)-D-glucopyranose units in the main backbone and branched at positions O-4, O-2 and/or O-3, with D-glucopyranose units in the side chain. A4X was the best dextran producer (4.5 g/L), while the other strains synthesized 2.1-2.7 g/L. Zymograms revealed that L. mali strains have a single Dsr with a molecular weight (Mw) of ~ 145 kDa, while the Lc. mesenteroides possess one or two enzymes with 170-211 kDa Mw. As far as we know, this is the first detection of L. mali Dsr. Analysis of metabolic fluxes from sucrose revealed that the six LAB produced mannitol (~ 12 g/L). The co-addition of maltose-sucrose resulted in the production of panose (up to 37.53 mM), an oligosaccharide known for its prebiotic effect. A4X, Z36P and B12 showed dextranase hydrolytic enzymatic activity and were able to produce another trisaccharide, maltotriose, which is the first instance of a dextranase activity encoded by Lc. mesenteroides strains. Furthermore, B12 and O9 grew in the absence of riboflavin (vitamin B2) and synthesized this vitamin, in a defined medium at the level of ~ 220 µg/L. Therefore, these LAB, especially Lc. mesenteroides B12, are good candidates for the development of new fermented food biofortified with functional compounds.

Molecular docking and dynamics of a dextranase derived from Penicillium cyclopium CICC-4022.

This study aimed to investigate the recognition mechanism of dextranase (PC-Edex) produced by Penicillium cyclopium CICC-4022 on dextran. Whole genome information of P. cyclopium CICC-4022 was obtained through genome sequencing technology. The coding information of PC-Edex was determined based on the annotation of the protein-coding genes using protein databases. The three-dimensional structure of PC-Edex was obtained via homology modelling. The active site and binding free energy between PC-Edex and dextran were calculated by molecular docking and molecular dynamics techniques. The results showed that the total sequence length and GC content of P. cyclopium CICC-4022 were 29,710,801 bp and 47.02 %, respectively. The annotation of protein-encoding genes showed that P. cyclopium CICC-4022 is highly active and has many carbohydrate transport and metabolic functions, and most of its proteases are glycolytic anhydrases. Furthermore, the gene encoding PC-Edex was successfully annotated. Molecular dynamics simulations indicated that van der Waals interaction was the main driving force of interaction. Residues Ile114, Asp115, Tyr332, Lys344, and Gln403 significantly promoted the binding between dextran and PC-Edex. In summary, this study explored the active site catalyzed by PC-Edex based on the binding pattern of PC-Edex and dextran. Therefore, this study provides genomic information on dextranase and data supporting the rational modification and enhancement of PC-Edex.

Review on recent advances in the properties, production and applications of microbial dextranases.

Dextranase is a type of hydrolase that is responsible for catalyzing the breakdown of high-molecular-weight dextran into low-molecular-weight polysaccharides. This process is called dextranolysis. A select group of bacteria and fungi, including yeasts and likely certain complex eukaryotes, produce dextranase enzymes as extracellular enzymes that are released into the environment. These enzymes join dextran's α-1,6 glycosidic bonds to make glucose, exodextranases, or isomalto-oligosaccharides (endodextranases). Dextranase is an enzyme that has a wide variety of applications, some of which include the sugar business, the production of human plasma replacements, the treatment of dental plaque and its protection, and the creation of human plasma replacements. Because of this, the quantity of studies carried out on worldwide has steadily increased over the course of the past couple of decades. The major focus of this study is on the most current advancements in the production, administration, and properties of microbial dextranases. This will be done throughout the entirety of the review.

Molecular Docking and Site-Directed Mutagenesis of GH49 Family Dextranase for the Preparation of High-Degree Polymerization Isomaltooligosaccharide.

The high-degree polymerization of isomaltooligosaccharide (IMO) not only effectively promotes the growth and reproduction of Bifidobacterium in the human body but also renders it resistant to rapid degradation by gastric acid and can stimulate insulin secretion. In this study, we chose the engineered strain expressed dextranase (PsDex1711) as the research model and used the AutoDock vina molecular docking technique to dock IMO4, IMO5, and IMO6 with it to obtain mutation sites, and then studied the potential effect of key amino acids in this enzyme on its hydrolysate composition and enzymatic properties by site-directed mutagenesis method. It was found that the yield of IMO4 increased significantly to 62.32% by the mutant enzyme H373A. Saturation mutation depicted that the yield of IMO4 increased to 69.81% by the mutant enzyme H373R, and its neighboring site S374R IMO4 yield was augmented to 64.31%. Analysis of the enzymatic properties of the mutant enzyme revealed that the optimum temperature of H373R decreased from 30 °C to 20 °C, and more than 70% of the enzyme activity was maintained under alkaline conditions. The double-site saturation mutation results showed that the mutant enzyme H373R/N445Y IMO4 yield increased to 68.57%. The results suggest that the 373 sites with basic non-polar amino acids, such as arginine and histidine, affect the catalytic properties of the enzyme. The findings provide an important theoretical basis for the future marketable production of IMO4 and analysis of the structure of dextranase.

Purification and characterization of dextranase from Penicillium cyclopium CICC-4022 and its degradation of dextran.

A dextranase was purified from Penicillium cyclopium CICC-4022 by ammonium sulfate fractionation and secondary tangential flow filtration, and the enzymatic properties were studied. The purified dextranase was used to regulated the molecular mass and homogeneity of dextran. Weight-average molecular mass (Mw) and polydispersity index (Mw/Mn) of dextran were measured by gel permeation chromatography (GPC) coupled with a triple-detector array (GPC-TDA), which is composed of a multiple-angle light scattering, a viscometer, and a refractive-index detector. The dextranase was purified by 2.24-fold, the recovery rate was 45.84%, the specific activity was 1442.05 U/mg, and the Mw was 77 KDa. Dextranase showed maximum activity at pH of 5.0 and 55 °C. Na+, K+ and NH4+ can effectively improve the dextranase activity, Cu2+ and Pb2+ can strongly inhibit the dextranase activity. Dextranase specifically degraded the α-1,6 glycosidic bonds of dextran. By controlling the dextranase activity, substrate concentration, and time, the specific Mw dextran with good homogeneity was obtained. The structure of dextran was not altered before or after dextranase hydrolysis, but its conformation changed from a spherical chain to a compliant chain. When the Mw of the dextran product was about 5 KDa, it was a compact spherical chain conformation in solution.

Alkalic dextranase produced by marine bacterium Cellulosimicrobium sp. PX02 and its application.

The enzyme dextranase is widely used in the sugar and food industries, as well as in the medical field. Most land-derived dextranases are produced by fungi and have the disadvantages of long production cycles, low tolerance to environmental conditions, and low safety. The use of marine bacteria to produce dextranases may overcome these problems. In this study, a dextranase-producing bacterium was isolated from the Rizhao seacoast of Shandong, China. The bacterium, denoted as PX02, was identified as Cellulosimicrobium sp. and its growing conditions and the production and properties of its dextranase were investigated. The dextranase had a molecular weight of approximately 40 kDa, maximum activity at 40°C and pH 7.5, with a stability range of up to 45°C and pH 7.0-9.0. High-performance liquid chromatography showed that the dextranase hydrolyzed dextranT20 to isomaltotriose, maltopentaose, and isomaltooligosaccharides. Hydrolysis by dextranase produced excellent antioxidant effects, suggesting its potential use in the health food industry. Investigation of the action of the dextranase on Streptococcus mutans biofilm and scanning electron microscopy showed that it to be effective both for removing and inhibiting the formation of biofilms, suggesting its potential application in the dental industry.

Effects of hulless barley and exogenous beta-glucanase levels on ileal digesta soluble beta-glucan molecular weight, digestive tract characteristics, and performance of broiler chickens.

The reduced use of antibiotics in poultry feed has led to the investigation of alternatives to antibiotics, and one such substitution is fermentable carbohydrates. Exogenous ß-glucanase (BGase) is commonly used in poultry fed barley-based diets to reduce digesta viscosity. The effects of hulless barley (HB) and BGase levels on ileal digesta soluble ß-glucan molecular weight, digestive tract characteristics, and performance of broiler chickens were determined. A total of 360 day-old broilers were housed in battery cages (4 birds per cage) and fed graded levels of high ß-glucan HB (CDC Fibar; 0, 30, and 60% replacing wheat) and BGase (Econase GT 200 P; 0, 0.01, and 0.1%) in a 3 × 3 factorial arrangement. Beta-glucan peak molecular weight in the ileal digesta was lower with 30 and 60 than 0% HB, whereas the peak decreased with increasing BGase. The weight average molecular weight was lower at 0.1 than 0% BGase in wheat diets, whereas in HB diets, it was lower at 0.01 and 0.1 than 0% BGase. The maximum molecular weight was lower with 0.01 and 0.1 than 0% BGase regardless of the HB level. The maximum molecular weight was lower with HB than wheat at 0 or 0.01% BGase. Overall, empty weights and lengths of digestive tract sections increased with increasing HB, but there was no BGase effect. Hulless barley decreased the duodenum and jejunum contents, whereas increasing the gizzard (diets with BGase), ileum, and colon contents. The jejunum and small intestine contents decreased with increasing BGase. Ileal and colon pH increased with increasing HB, but there was no BGase effect. Treatment effects were minor on short-chain fatty acids levels and performance. In conclusion, exogenous BGase depolymerized the ileal digesta soluble ß-glucan in broiler chickens in a dose-dependent manner. Overall, feed efficiency was impaired by increasing HB levels. However, HB and BGase did not affect carbohydrate fermentation in the ileum and ceca, although BGase decreased ileal viscosity and improved feed efficiency at the 0.1% dietary level.

Accumulation and cross-linkage of ß-1,3/1,6-glucan lead to loss of basal stipe cell wall extensibility in mushroom Coprinopsis cinerea.

The mature basal stipe of mushroom Coprinopsis cinerea loses wall extensibility. We found that an endo-ß-1,3-glucanase ENG from C. cinerea could restore mature basal stipe wall extensibility via pretreatment such that the ENG-pretreated basal stipe walls could be induced to extend by chitinase ChiIII. ENG pretreatment released glucose, laminaribiose, and 3-O-D-gentiobiose-D-glucose from the basal stipe walls, consistent with ENG-digested products of ß-1,6-branched ß-1,3-glucan. Different effects of endo-ß-1,3-glucanase ENG and exo-ß-1,3-glucanase EXG pretreatment on the structure, amount and ratio (ß-1,3-glucoside bonds to ß-1,6-glucoside bonds) of products from the basal stipe and the apical stipe cell walls, respectively, and on the cell wall extensibility and the cell wall ultra-architecture of the basal stipes were analyzed. All results demonstrate that the more accumulation and cross-linkage of ß-1,6-branched ß-1,3-glucan with wall maturation lead to loss of wall extensibility of the basal stipe regions compared to the apical stipe cell walls.

A novel dextranase gene from the marine bacterium Bacillus aquimaris S5 and its expression and characteristics.

Dextranase specifically hydrolyzes dextran and is used to produce functional isomalto-saccharide prebiotics. Moreover, dextranase is used as an additive in mouthwash to remove dental plaque. We cloned and expressed the dextranase gene of the marine bacterium Bacillus aquimaris S5. The length of the BaDex gene was 1788 bp, encoding 573 amino acids. Using bioinformatics to predict and analyze the amino acid sequence of BaDex, we found the isoelectric point and instability coefficient to be 4.55 and 29.22, respectively. The average hydrophilicity (GRAVY) was -0.662. The secondary structure of BaDex consisted of 145 alpha helices, accounting for 25.31% of the protein; 126 extended strands, accounting for 21.99%; and 282 random coils, accounting for 49.21%. The 3D structure of the BaDex protein was predicted and simulated using SWISS-MODEL, and BaDex was classified as a Glycoside Hydrolase Family 66 protein. The optimal temperature and pH for BaDex activity were 40°C and 6.0, respectively. The hydrolysates had excellent antioxidant activity, and 8 U/mL of BaDex could remove 80% of dental plaque in MBRC experiment. This recombinant protein thus has great promise for applications in the food and pharmaceutical industries.

Enzymatic-fingerprinting workflow of polysaccharides in Hericium erinaceus fruiting bodies: From HILIC-ESI--MS screening to targeted MIM profiling.

In this study, an uncommon enzymatic-fingerprinting workflow, was proposed for characterization and discrimination of mushroom polysaccharides (MPs) by hydrophilic interaction liquid chromatography-negative electrospray mass spectrometry (HILIC-ESI--MS). Firstly, the HILIC-ESI--MS was used to screen and identify the enzymatic digestion products of MPs using HILIC-Orbitrap based on full scan and MS/MS modes. Secondly, a targeted structural-fingerprinting of polysaccharides (SFP) was built in a multiple-ion monitoring (MIM) mode using the same HILIC separation with a triple quadrupole MS. Thirdly, a case study of polysaccharides in Hericium erinaceus fruiting bodies (HEP) was performed to obtain the expected SFP based on dextranase digestion that allows for visual discrimination of polysaccharides from other five edible mushrooms attributed to Agrocybe cylindracea, Arimillaria mellea, Flammulina velutipes, Pleurotus eryngii, and Lentinula edodes. Furthermore, a major structural backbone of HEP was unveiled by occurrence of → 6(Hex)1 → along with multiple possible substitutions including of terminal GalA, Fuc, acetyl, → 4Hex1 →, and → 3Hex1 →. Finally, the similarity analysis, hierarchical cluster analysis (HCA), and partial least squares discriminant analysis (PLS-DA) were performed to visualize various MPs. As a result, the enzymatic-fingerprinting workflow presents an effective example for quality evaluation of fungi polysaccharides using a SFP strategy.

Food-Grade Expression and Characterization of a Dextranase from Chaetomium gracile Suitable for Sugarcane Juice Clarification.

The microbial production of dextranase using cheap carbon sources is beneficial to solve the economic loss caused by the accumulation of dextran in syrup. A food-grade microbial cell factory was constructed by introducing the dextranase encoding gene DEX from Chaetomium gracile to the chromosome of Bacillus subtilis, and the antibiotic resistance marker gene was subsequently deleted via the Cre/loxP strategy. The dual-promoter system with a sequentially arranged constitutive P43 promoter resulted in an 85 % increase in DEX expression. Under the optimal fermentation conditions of 10 g/L maltose, 15 g/L casein, 1 g/L Na2 HPO4 , 1 g/L FeSO4 and 8 g/L NaCl, DEX activity was increased from 2.625 to 64.34 U/mL. Recombinant DEX was purified 5.98-fold with a recovery ratio of 26.67 % and specific activity of 3935.02 U/mg. Enzyme activity was optimal at 55 °C and pH 5.0 and remained 80.34 % and 71.36 % of the initial activity at 55 °C and pH 4.0 after 60 min, respectively. The enzyme possessed high activity in the presence of Co2+ , while Ag+ showed the strongest inhibition ability. The optimal substrate was 20 g/L dextran T-2000. The findings could facilitate the low-cost, large-scale production of food-grade DEX for use in the sugar industry.

The role of dextran production in the metabolic context of Leuconostoc and Weissella Tunisian strains.

High molecular weight dextrans improve the rheological properties of fermented products and have immunomodulatory and antiviral activity. We report on 5.84 × 107-2.61 × 108 Da dextrans produced by Leuconostoc lactis AV1n, Weissella cibaria AV2ou and Weissella confusa V30 and FS54 strains. Dextransucrases catalyze dextran synthesis by sucrose hydrolysis concomitant with fructose generation. The four bacteria have dextransucrases with molecular weight of about 160 kDa detected by zymograms. Each bacterium showed different interplay of dextran production and metabolic fluxes. All bacteria produced lactate, and AV2ou apart, synthesized mannitol from fructose. FS54 hydrolyzed dextran blue and the concentration of dextran produced by this bacterium decreased during the stationary phase. The AV1n binding to Caco-2 cells and polystyrene plates was higher under conditions for dextran synthesis. Thus, this is the first instance of a Weissella dextranase, associated with a dextransucrase ability, and of a positive influence of dextran on adhesion and aggregation properties of a bacterium.

Near-Infrared-Induced Contractile Proteinosome Microreactor with a Fast Control on Enzymatic Reactions.

Inspired by the compartmentalized structure of cells, self-regulating responsive hollow microcapsules are highly desirable for the modulation of enzymatic reactions. Here, we report a strategy to fabricate gold nanorod embedded proteinosomes by covalently grafting gold nanorods onto the surface of proteinosomes. The excellent photothermal conversion efficiency of the embedded gold nanorod and the thermal phase transition of the grafted PNIPAAm allow the constructed hybrid proteinosomes to show reversible contraction behaviors triggered by near-infrared light with the molecular weight cutoff of the membrane decreased to ca. 50 kDa, and importantly, the contraction frequency of the constructed proteinosomes could be as fast as 1 min and last for at least 15 cycles. Subsequently, the effective encapsulation of three cascade enzymes into the proteinosomes realizes the construction of a near-infrared responsive microreactor that allows control of the cascade reaction by near-infrared illumination, thereby enabling reversible on and off of the enzymatic reaction. Such microcapsule-based reactors demonstrate the potential to alter the membrane molecular weight cutoff, and it is believed that the development of such responsive microcapsules will have great potential for studying cellular responses and provide a platform for future applications in biosensing and drug delivery.

Enzymatic synthesis of non-digestible oligosaccharide catalyzed by dextransucrase and dextranase from maltose acceptor reaction.

Non-digestible oligosaccharides have wide food industrial applications as dietary fibers and prebiotics. The aim of this study is to realize the effective biosynthesis of isomalto-oligosaccharides (IMOs) and reduce the production of by-product dextran. In the presence of acceptors improved the dextransucrase reaction shifting to oligosaccharides formation but a number of by-products dextran appeared. Maltose acceptor performed stronger inhibition behaviors in dextran synthesis than lactose and glucose acceptor due to its higher efficiencies. Acceptors had no influence on the structure of by-product dextran which mainly composed of α-(1,6)-glycosidic linkages and low α-(1,3)-glycosidic branch. In addition, the Mw and contents of IMOs and oligodextrans synthesized by dual-enzyme were hard to control. Addition of maltose acceptor in the dual-enzyme reaction, the adequate dextranase preferentially degraded dextran than the acceptor products to yield the IMOs. Results indicated that the combined use of the dual-enzyme and the maltose acceptor is a simple and effective method to promote the high-quality of functional IMOs.

Structural insights into polysaccharide recognition by Flavobacterium johnsoniae dextranase, a member of glycoside hydrolase family 31.

Glycoside hydrolase family (GH) 31 contains a large variety of enzymes, but the major members are enzymes that act on relatively small oligosaccharides such as α-glucosidase. Here, we determined the crystal structure of Flavobacterium johnsoniae dextranase (FjDex31A), an enzyme from F. johnsoniae that hydrolyzes a polysaccharide, dextran. FjDex31A is composed of four domains: an N-terminal domain, a catalytic domain, a proximal C-terminal domain, and a distal C-terminal domain, as observed in typical GH31 enzymes. However, the architecture of active site residues in FjDex31A, other than subsite -1, is markedly different from that of other GH31 enzymes. The FjDex31A structure in complex with isomaltotriose shows that Gly273 and Tyr524, both of which interact with an α-glucose residue at subsite -2, as well as Trp376 and Leu308-cisGln309, are especially unique to FjDex31A. Site-directed mutagenesis of Gly273 and Tyr524 resulted in a decrease in the hydrolysis of polysaccharides dextran and pullulan, as well as that of the disaccharide isomaltose. These results suggest that, regardless of the length of sugar chains of the substrates, binding of FjDex31A to the substrates at subsite -2 is likely to be important for its activity. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6JR6, 6JR7, and 6JR8.

Synthesis and characterization of stevioside having low degree polymerized glucosides using dextransucrase and dextranase.

Transglycosylation is one of enzymatic methods to improve the physical and biochemical properties of various functional compounds. In this study, stevioside glucosides were synthesized using sucrose as a substrate, stevioside (Ste) as an acceptor, and dextransucrase from Leuconostoc mesenteroides B-512 F/KM. The highest Ste conversion yield of 98% was obtained with 50 mg/mL Ste, 800 mM sucrose, and dextransucrase 4 U/mL at 28 °C for 6 h. The concentration of Ste was unchanged while of Ste-G1 was increased from 7.7 mM to 9.1 mM as the Ste acceptor reaction digest was treated with dextranase from Lipomyces starkeyi. Ste-G1 (13-O-ß-sophorosyl-19-O-ß-isomaltosyl-steviol), Ste-G2 (13-O-(ß-(1→6) glucosyl)-ß-glucosylsophorosyl-19-O-ß-isomaltosyl-steviol), and Ste-G2' (13-O-ß-sophorosyl-19-O-ß-isomaltotriosyl-steviol) were determined by NMR. These glucosylated Ste showed increased stabilities at pH 2, 60 °C for 48 h as compared to Ste. Ste-G1, Ste-G2, and Ste-G2' inhibited the insoluble glucan synthesis from sucrose by mutansucrase from Streptococcus muntans by the transfer of the glucosyl group of sucrose to Ste-G1, Ste-G2, and Ste-G2'. The relative water solubility of curcumin, pterostilbene or idebenone was increased by Ste or Ste glucosides treatment. Ste and Ste-G1 restored cell viability in RAW264.7 cells at concentrations up to 8 mg/mL and inhibited nitric oxide production in LPS-induced RAW264.7 cells with IC50 of 3.29 and 1.87 mg/mL.

Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis cinerea.

This study reports that a high concentration of the endo-ß-1,3-glucanase ENG (200 µg ml-1) induced heat-inactivated stipe wall extension of Coprinopsis cinerea, whereas a high concentration of the extracellular ß-glucosidase BGL2 (1,000 µg ml-1) did not; however, in combination, low concentrations of ENG (25 µg ml-1) and BGL2 (260 µg ml-1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, ß-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60 min; the stipe cell wall extension induced by a high concentration of ß-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the ß-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of ß-1,6 branched ß-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the ß-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases.IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis cinerea In this study, we explored that ß-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of ß-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, ß-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of ß-1,3-glucanases is necessary for the cell wall remodeling and the addition of new ß-glucans during stipe elongation growth.

A novel intracellular dextranase derived from Paenibacillus sp. 598K with an ability to degrade cycloisomaltooligosaccharides.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more α-(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 α-(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.

Crystal Structure of GH49 Dextranase from Arthrobacter oxidans KQ11: Identification of Catalytic Base and Improvement of Thermostability Using Semirational Design Based on B-Factors.

The crystal structure of Dextranase from the marine bacterium Arthrobacter oxidans KQ11 (Aodex) was determined at a resolution of 1.4 Å. The crystal structure of the conserved Aodex fragment (Ala52-Thr638) consisted of an N-terminal domain N and a C-terminal domain C. The N-terminal domain N was identified as a ß-sandwich, connected to a right-handed parallel ß-helix at the C-terminus. Sequence comparisons, cavity regions, and key residues of the catalytic domain analysis all suggested that the Aodex was an inverting enzyme, and the catalytic acid and base were Asp439 and Asp420, respectively. Asp440 was not a general base in the Aodex catalytic domain, and Asp396 in Dex49A may not be a general base in the catalytic domain. The thermostability of the S357F mutant using semirational design based on B-factors was clearly better than that of wild-type Aodex. This process may promote the aromatic-aromatic interactions that increase the thermostability of mutant Phe357.

Purification, Characterization and Degradation Performance of a Novel Dextranase from Penicillium cyclopium CICC-4022.

A novel dextranase was purified from Penicillium cyclopium CICC-4022 by ammonium sulfate fractional precipitation and gel filtration chromatography. The effects of temperature, pH and some metal ions and chemicals on dextranase activity were investigated. Subsequently, the dextranase was used to produce dextran with specific molecular mass. Weight-average molecular mass (Mw) and the ratio of weight-average molecular mass/number-average molecular mass, or polydispersity index (Mw/Mn), of dextran were measured by multiple-angle laser light scattering (MALS) combined with gel permeation chromatography (GPC). The dextranase was purified to 16.09-fold concentration; the recovery rate was 29.17%; and the specific activity reached 350.29 U/mg. Mw of the dextranase was 66 kDa, which is similar to dextranase obtained from other Penicillium species reported previously. The highest activity was observed at 55 °C and a pH of 5.0. This dextranase was identified as an endodextranase, which specifically degraded the α-1,6 glucosidic bonds of dextran. According to metal ion dependency tests, Li⁺, Na⁺ and Fe2+ were observed to effectively improve the enzymatic activity. In particular, Li⁺ could improve the activity to 116.28%. Furthermore, the dextranase was efficient at degrading dextran and the degradation rate can be well controlled by the dextranase activity, substrate concentration and reaction time. Thus, our results demonstrate the high potential of this dextranase from Penicillium cyclopium CICC-4022 as an efficient enzyme to produce specific clinical dextrans.