Specific Degree Of Polymerization Research Articles

Secretory production and characterization of a highly effective chitosanase from Streptomyces coelicolor A3(2) M145 in Pichia pastoris.

In this study, a glycoside hydrolase family 46 chitosanase from Streptomyces coelicolor A3(2) M145 was firstly cloned and expressed in Pichia pastoris GS115 (P. pastoris GS115). The recombinant enzyme (CsnA) showed maximal activity at pH 6.0 and 65°C. Both thermal stability and pH stability of CsnA expressed in P. pastoris GS115 were significantly increased compared with homologous expression in Streptomyces coelicolor A3(2). A stable chitosanase activity of 725.7±9.58 U mL-1 was obtained in fed-batch fermentation. It's the highest level of CsnA from Streptomyces coelicolor expressed in P. pastoris so far. The hydrolytic process of CsnA showed a time-dependent manner. Chitosan oligosaccharides (COSs) generated by CsnA showed antifungal activity against Fusarium oxysporum sp. cucumerinum (F. oxysporum sp. cucumerinum). The secreted expression and hydrolytic performance make the enzyme a desirable biocatalyst for industrial controllable production of chitooligosaccharides with specific degree of polymerization, which have potential to control fungi that cause important crop diseases.

How alginate lyase produces quasi-monodisperse oligosaccharides: A normal-mode-based docking and molecular dynamics simulation study

Oligosaccharide degradation products of alginate (AOS) hold significant potential in diverse fields, including pharmaceuticals, health foods, textiles, and agricultural production. Enzymatic alginate degradation is appealing due to its mild conditions, predictable activity, high yields, and controllability. However, the alginate degradation often results in a complex mixture of oligosaccharides, necessitating costly purification to isolate highly active oligosaccharides with a specific degree of polymerization (DP). Addressing this, our study centers on the alginate lyase AlyB from Vibrio Splendidus OU02, which uniquely breaks down alginate into mono-distributed trisaccharides. This enzyme features a polysaccharide lyase family 7 domain (PL-7) and a CBM32 carbohydrate-binding module connected by a helical structure. Through normal-mode-based docking and all-atom molecular simulations, we demonstrate that AlyB's substrate and product specificities are influenced by the spatial conformation of the catalytic pocket and the flexibility of its structure. The helically attached CBM is pivotal in releasing trisaccharides, which is crucial for avoiding further degradation. This study sheds light on AlyB's specificity and efficiency and contributes to the evolving field of enzyme design for producing targeted oligosaccharides, with significant implications for various bioindustries.

Fructooligosaccharides (FOS) Production by Microorganisms with Fructosyltransferase Activity

Fructans are fructose-based polymers, defined as fructooligosaccharides (FOS), when they possess a short chain. These molecules are highly appreciated in the food and pharmaceutical international market and have an increasing demand worldwide, mainly for their prebiotic activity and, therefore, for all their health benefits to those who consume them constantly. Thus, new natural or alternative FOS production systems of industrial scale are needed. In this regard, microorganisms (prokaryotes and eukaryotes) have the potential to produce them through a wide and diverse number of enzymes with fructosyltransferase activity, which add a fructosyl group to sucrose or FOS molecules to elongate their chain. Microbial fructosyltransferases are preferred in the industry because of their high FOS production yields. Some of these enzymes include levansucrases, inulosucrases, and β-fructofuranosidases obtained and used through biotechnological tools to enhance their fructosyltransferase activity. In addition, characterizing new microorganisms with fructosyltransferase activity and modifying them could help to increase the production of FOS with a specific degree of polymerization and reduce the FOS production time, thus easing FOS obtention. Therefore, the aim of this review is to compile, discuss, and propose new perspectives about the microbial potential for FOS production through enzymes with fructosyltransferase activity and describe the modulation of FOS production yields by exogenous stimuli and endogenous modifications.

Adsorption mechanisms of alginate oligosaccharides with specific degree of polymerizations on a novel ion-exchange resin: Experiments and simulations

Alginate oligosaccharides (AOS) with a specific degree of polymerization (DP) have unique physiological activity, but industrialization and high-value applications of AOS are restricted due to the paucity of study on its precise separation mechanism. In the process design of adsorption separation chromatography, the adsorption behaviors of the adsorbent are crucial. Herein, the adsorption properties of AOS with DPs 1–4 (AOS1-4) on novel SM-7 resin were clarified by combining adsorption experiments and theoretical calculations. The effect of various parameters on the adsorption process of AOS1-4 was investigated using a series of static adsorption tests. Molecular dynamics modeling was implemented to determine the interaction energy and diffusion between AOS1-4 and SM-7 resin. The results of the adsorption equilibrium demonstrated that the adsorption of AOS1-4 complied with the Langmuir model and exhibited ion dependency and pH stability. The saturation adsorption capacities of AOS1-4 on SM-7 resin were 1.144 mmol/g, 0.810 mmol/g, 0.730 mmol/g, and 0.582 mmol/g, respectively. The external diffusion was the primary rate-limiting phase in the adsorption kinetics of AOS1-4, which followed the pseudo-second-order kinetic model. As the increasing of DPs of AOS, the hydroxyl, carboxyl, and molecular volume rose, which decreased the adsorption capacity and rate. Simulation results showed that interaction energies between SM-7 resin and AOS enhanced while the diffusion coefficient declined with rising DPs of AOS. Hydrogen bond interaction and electrostatic attraction promoted the adsorption of AOS on the novel resin. The fundamental theoretical data in this study can provide guidance for the large-scale preparation of AOS with specific DPs.

Strategies for efficient extracellular secretion of recombinant cyclomaltodextrinase by Escherichia coli

Cyclomaltodextrinase (CDase, EC 3.2.1.54) is used in the preparation of maltooligosaccharides with a specific degree of polymerization by hydrolyzing cyclodextrins. Extracellular secretion of CDase is beneficial in promoting its application potential. However, a major barrier is that proteins are difficult to cross the inner and outer membranes of cells. Herein, we made an effort to increase the permeability of cell membrane by the deletion of lipopolysaccharide synthesis-related gene (lpxM). In addition, glycine and Ca2+ assisted in the extracellular secretion of recombinant CDase from Bacillus sphaericus E−244. The extracellular fermentation activity of CDase was 2.88 U/mL when ΔycjM-ΔmalS-ΔlpxM was cultured with 1 mM glycine and 5 mM Ca2+ for 20 h, accounting for 78.67% of the total fermentation activity, which was 6.26-fold and 6.45-fold higher than that of the parent strain, respectively. Glycine increased cell lysis and disrupted cell morphology, and Ca2+ repaired glycine-induced cell growth inhibition. Significantly, deletion of lpxM replaced the use of glycine to a considerable extent, and half the CDase extracellular secretion time.

Production of xylo-oligosaccharides (XOS) of tailored degree of polymerization from acetylated xylans through modelling of enzymatic hydrolysis

Xylo-oligosaccharides (XOS) are emerging prebiotics that have recently been gained a great interest in the market of functional foods. Since their beneficial activity strictly depends on their chemical structure and on their degree of polymerization (DP), in this work an enzymatic method was developed to produce XOS with variable and modellable DPs, involving a combination of a commercial endo-β-1,4-xylanase M3 from Trichoderma longibrachiatum and a deacetylase, using a commercial acetylated standard xylan as substrate. A Design of Experiment (DoE) was developed and through the variation of some hydrolysis conditions, some experiments allowed to obtain significant amounts of XOS with DP 7–10, up to 11%, despite XOS with DP 2–4 were always the most abundant (60–96% of total XOS). The most impacting parameter on the XOS distribution was the order of addition of the xylanase and deacetylating enzyme, while pH showed to have a great influence on the total yield. The method was also tested on an acetylated xylan extracted from grape stalks, structurally similar to the commercial standard xylan. The model was found to work in a very similar way also on the non-purified xylan sample, allowing the manipulation of enzymatic hydrolysis on a low-cost by-product, with the potential to obtain on a large scale XOS with high added value and with a specific DP, depending on the final application.

Effect of chitooligosaccharides with a specific degree of polymerization on multiple targets in T2DM mice

Chitooligosaccharides (COS) are found naturally in the ocean and present a variety of physiological activities, of which hypoglycemic action has attracted considerable research attention. This study aimed to assess the therapeutic effect of COS on mice suffering from type 2 diabetes mellitus (T2DM). COS effectively reduced blood glucose and blood lipid levels and improved glucose tolerance. Furthermore, COS revealed strong inhibitory activity against α-glucosidase, reducing postprandial blood glucose levels. Molecular docking data showed that COS might interact with surrounding amino acids to form a complex and decrease α-glucosidase activity. Additionally, COS enhanced insulin signal transduction and glycogen synthesis while restricting gluconeogenesis in the liver and muscles, reducing insulin resistance (IR) as a result. Moreover, COS effectively protected and restored islet cell function to increase insulin secretion. These results indicated that COS exhibited a significant hypoglycemic effect with multi-target participation. Therefore, COS may serve as a new preventive or therapeutic drug for diabetes to alleviate metabolic syndrome.Graphical

Adsorption differences and mechanism of chitooligosaccharides with specific degree of polymerization on macroporous resins with different functional groups

Ion exchange resins are promising materials for separating chitooligosaccharides (COSs) with different degrees of polymerization (DP). The adsorbent functional groups significantly determine the interactions with COSs and affect their adsorption and separation efficiency. This study investigated the adsorption behaviors of COSs on macroporous cation exchange resins with different functional groups to clarify the functional group effect on the adsorption of COSs with different DP, which can also help to provide a theoretical basis for the selection of separation medium and optimization of the separation technics of COSs. The results showed that the adsorption of COSs on sulfonic-acid type resin DX-10 and carboxylic-acid type resin DC-10 were favorable, and the adsorption isotherms were in accord with the Sips model. Adsorption thermodynamics showed that temperature had opposite effects on the two resins' equilibrium adsorption capacity of COSs. The diffusion coefficient of COSs on DX-10 resin was greater, and the affinity gap between COSs on DX-10 resin was larger than that of DC-10 resin, which is beneficial for improving the separation resolution of COSs. In contrast, the interaction energy between COSs with high DP and DC-10 resin was weaker compared with DX-10, which made the elution of COSs with high DP easier.

Characterization of a GH50 β-Agarase: A Biotechnological Tool for Preparing Oligosaccharides from Agarose and Porphyran.

Agarase is of vital significance for functional agaro-oligosaccharides production from algal dived agarose. Especially, the exolytic agarases have the advantage of obtaining agaro-oligosaccharides with a specific degree of polymerization. Herein, we cloned and expressed a novel glycoside hydrolase (GH) 50 family β-agarase OUC-PgJC50 from Photobacterium gaetbulicola. The degradation pattern analysis indicated that OUC-PgJC50 not only showed an exolytic activity with main products of neoagarotetraose from hydrolyzing agarose but also show a hydrolytic activity to transform neoagarotetraose into neoagarobiose. This is the first time that the discovery of a neoagarotetraose-producing exolytic GH50 β-agarase possesses the activity to transform neoagarotetraose into neoagarobiose, which provided new insight into the recognition of the degradation mode of agarases. Molecular docking and sequence alignment analysis further revealed the His654 residue in OUC-PgJC50 may play a vital role in forming a strong force with l-AHG residue at -4 subsite that helps to produce neoagarotetraose from catalyzing agarose. Moreover, the catalytic ability of OUC-PgJC50 toward another agar polysaccharide porphyran was also described that could hydrolyze porphyran into sulfated oligosaccharides, in which the LA6S-d-Gal was the main products. This study is of vital significance for developing the application range of GH50 β-agarases.

Chromatographic preparation of food-grade prebiotic oligosaccharides with defined degree of polymerization

Prebiotic oligosaccharides are of widespread interest in the food industry due to their potential health benefits. This has triggered a need for research into their sensory properties. Such research is currently limited due to the lack of available food-grade oligosaccharide preparations with specific degree of polymerization (DP). The aim of this study was to develop economical approaches for the preparation and characterization of prebiotic oligosaccharides differing with respect to composition and DP. Such preparations were prepared by chromatographic fractionation of commercially available prebiotic mixtures using microcrystalline cellulose stationary phases and aqueous ethanol mobile phases. This approach is shown to work for the preparation of food-grade fructooligosaccharides of DP 3 and 4, galactooligosaccharides of DP 3 and 4, and xylooligosaccharides of DP 2–4. Methods for the characterization of the different classes of oligosaccharides are also presented including those addressing purity, identity, total carbohydrate content, moles per unit mass, and DP.

Adsorption behaviors and kinetics studies of chitooligosaccharides with specific degree of polymerization on a novel ion-exchange resin

Physiological activities of chitooligosaccharides (COSs) are related to their intrinsic structure and purities of products. However, the efficient preparation method of COSs with a specific degree of polymerization (DP) is still lacking, which limits the in-depth research and application of COSs. This work focused on the adsorption characters of COSs on the ion-exchange resin. Static adsorption experiments were conducted to study the effects of initial solution pH and ionic strength on the adsorption behaviors of chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose (COS 2–6). The adsorption equilibrium, thermodynamics, and kinetics of adsorption were investigated, and the molecular dynamics method was used to calculate the interaction energy of COS 2–6 with the resin and the diffusion coefficients of COS 2–6 in solution. The results show that the adsorption equilibrium data of COS 2–6 are satisfied with the Langmuir model, while the adsorption equilibrium capacities decrease with the increase of COSs DP. The COS 2–6 adsorption kinetic data are well fitted to the pseudo-second-order kinetic model. Liquid-film diffusion and intra-practical diffusion are both the adsorption rate-limiting steps. Thermodynamic parameters show that the adsorptions of COS 2–6 on HX-7 resin are spontaneous and feasible. With the increase of DP, the diffusion coefficients of COS 2–6 in solution decrease while the interaction force with resin become stronger, thus leading to more favorable adsorption.

In vitro fermentation of chitooligosaccharides and their effects on human fecal microbial community structure and metabolites

Chitooligosaccharides (COS), with a specific degree of polymerization, were evaluated for their effect on human fecal microbiota, as well as their metabolite, using an in vitro anaerobic batch fermentation model. COS increased the richness and diversity of the gut microbial community. More importantly, the microbial community structure of the COS groups was significantly different from the control and fructooligosaccharides (FOS) groups. COS specifically enriched the butyric and propionic acid-producing microbiota and targeted Faecalibacterium, Clostridium_sensu_stricto_1, C._sensu_stricto_13, and Fusicatenibacter. Furthermore, COS increased the acetic, propionic, and butyric acid contents. The production of propionic acid was greater than that obtained using FOS. Metabolome analysis suggested that COS could upregulate allocholic acid, 1,3-butanediol, 3,4-dihydroxybutanotic acid, and 2-hydroxybutyric acid production and downregulate putrescine, ethanamine, hexanoic acid, and branched chain amino acid (l-valine and l-leucine) production in the fermentation broth. These differential metabolites were closely related to lipid, glucose, and amino acid metabolism. These results should inform further development of COS as potential raw materials for functional foods and health products.

Structure–function analysis of Gynuella sunshinyii chitosanase uncovers the mechanism of substrate binding in GH family 46 members

Chitooligosaccharides (COS) is a kind of functional carbohydrates with great application potential as its various biological functions in food, cosmetics, and pharmaceutical fields. Exploring the relationship between structure and function of chitosanase is essential for the controllable preparation of chitooligosaccharides with the specific degree of polymerization (DP). GsCsn46A is a cold-adapted glycosyl hydrolase (GH) family 46 chitosanase with application potential for the controllable preparation of chitooligosaccharides. Here, we present two complex structures with substrate chitopentaose and chitotetraose of GsCsn46A, respectively. The overall structure of GsCsn46A contains nine α-helices and two β-strands that folds into two globular domains with the substrate between them. The unique binding positions of both chitopentaose and chitotetraose revealed two novel sugar residues in the negatively-numbered subsites of GH family 46 chitosanases. The structure–function analysis of GsCsn46A uncovers the substrate binding and catalysis mechanism of GH family 46 chitosanases. Structural basis mutagenesis in GsCsn46A indicated that altering interactions near +3 subsite would help produce hydrolysis products with higher DP. Specifically, the mutant N21W of GsCsn46A nearly eliminated the ability of hydrolyzing chitotetraose after long-time degradation.

Cyclodextrinase from Thermococcus sp expressed in Bacillus subtilis and its application in the preparation of maltoheptaose

BackgroundMaltoheptaose as malto-oligosaccharides with specific degree of polymerization, has wide applications in food, medicine and cosmetics industries. Currently, cyclodextrinase have been applied as prepared enzyme to prepare maltoheptaose. However, the yield and proportion of maltoheptaose was lower, which is due to limited substrate and product specificity of cyclodextrinase (CDase). To achieve higher maltoheptaose yield, cyclodextrinase with high substrate and product specificity should be obtained.ResultsIn this study, cyclodextrinase derived from Thermococcus sp B1001 (TsCDase) was successfully expressed and characterized in Bacillus subtilis for the first time. The specific activity of TsCDase was 637.95 U/mg under optimal conditions of 90 °C and pH 5.5, which exhibited high substrate specificity for cyclodextrins (CDs). When the concentration of β-CD was 8%, the yield of maltoheptaose achieved by TsCDase was 82.33% across all reaction products, which exceeded the yields of maltoheptaose in other recent reports. Among malto-oligosaccharides generated as reaction products, maltoheptaose was present in the highest proportion, about 94.55%.ConclusionsThis study provides high substrate and product specificity of TsCDase. TsCDase is able to prepare higher yield of maltoheptaose through conversion of β-CD in the food industry.

Chemoenzymatic Production and Engineering of Chitooligosaccharides and N-acetyl Glucosamine for Refining Biological Activities.

Chitooligosaccharides (COS) and N-acetyl glucosamine (GlcNAc) are currently of enormous relevance to pharmaceutical, nutraceutical, cosmetics, food, and agriculture industries due to their wide range of biological activities, which include antimicrobial, antitumor, antioxidant, anticoagulant, wound healing, immunoregulatory, and hypocholesterolemic effects. A range of methods have been developed for the synthesis of COS with a specific degree of polymerization along with high production titres. In this respect, chemical, enzymatic, and microbial means, along with modern genetic manipulation techniques, have been extensively explored; however no method has been able to competently produce defined COS and GlcNAc in a mono-system approach. Henceforth, the chitin research has turned toward increased exploration of chemoenzymatic processes for COS and GlcNAc generation. Recent developments in the area of green chemicals, mainly ionic liquids, proved vital for the specified COS and GlcNAc synthesis with better yield and purity. Moreover, engineering of COS and GlcNAc to generate novel derivatives viz. carboxylated, sulfated, phenolic acid conjugated, amino derived COS, etc., further improved their biological activities. Consequently, chemoenzymatic synthesis and engineering of COS and GlcNAc emerged as a useful approach to lead the biologically-active compound-based biomedical research to an advanced prospect in the forthcoming era.

Compositional analysis and structural characterization of raffinose family oligosaccharides from Eupatorium

Raffinose family oligosaccharides (RFOs) are commonly applied as functional food ingredients. RFOs mixture derived from Eupatorium were analyzed by hydrophilic interaction liquid chromatography-evaporative light scattering detector (HILIC-ELSD) method with a Click Xion column, and then separated by graphitized carbon black (GCB) chromatography. RFOs with a specific degree of polymerization (DP) from 3 to 6 were obtained. The specific ions from glycosidic bond cleavage and cross-ring cleavage of RFOs were produced in Electrospray-collision-induced dissociation-mass spectroscopy/mass spectroscopy (ESI-CID-MS/MS), which are in accordance with the linkage of (1→6) between galactans. The anomeric configuration and glycosidic linkage of RFOs were confirmed to be [α-Gal-(1→6)]n-α-Glc-(1→2)- β-Fru (n = 1–4) by 2D-NMR. In this study, we developed a HILIC-ELSD method for the compositional analysis of RFOs and obtained RFOs with a specific DP and high purity, and their structures were completely elucidated. Our work provides a basis for further research on the structure–activity relationship of RFOs.

Production pectin oligosaccharides using Humicola insolens Y1-derived unusual pectate lyase

The economical production of pectin oligosaccharides with a specific degree of polymerization and structure from agro-food waste is an industrially important process. This study identified a novel pectate lyase gene (plhy1) from the thermophilic cellulolytic fungus H.insolens Y1 and tested its ability to produce pectin oligosaccharides. The recombinant PLHY1 produced in Pichia pastoris was superior to other similar enzymes due to its high thermal and pH stability. PLHY1 demonstrated optimal enzymatic activity at 55°C and pH 10.0 in the presence of 0.4mM Ca2+, and preferred methyl esterified substrates for digestion. High performance anion exchange chromatography-pulsed amperometric detector and ultra high performance liquid chromatography in combination with electrospray ionization tandem mass spectrometry analysis showed that galacturonic acid-oligosaccharides with a small degree of polymerization (4-6) were the major hydrolysates produced by the degradation of apple peel pectin by PLHY1. The properties of PLHY1 make it valuable for application in the agro-food industry for the production of pectin oligosaccharides.

Preparation of malto-oligosaccharides with specific degree of polymerization by a novel cyclodextrinase from Palaeococcus pacificus

An open reading frame from Palaeococcus pacificus was discovered by genomic analysis. The recombinant PpCD was proved as novel extremely thermal-stable cyclodextrinase with potential on production of malto-oligosaccharides with specific degree of polymerization. This enzyme has extremely high substrate specificity toward cyclodextrins, while the rates of hydrolysis toward macromolecular or linear substrates such as starch, pullulan and amylose were pretty slow. The optimal pH and temperature of PpCD were determined as 6.0 and 95 °C respectively. It was confirmed that PpCD could produce malto-oligosaccharides with a certain degree of polymerization in consistent with that of cyclodextrin substrates. In this study, the preparation of high purity maltoheptaose was achieved and the suitable β-cyclodextrin concentration was about 8–10%. Therefore, PpCD may have a potential application in the production of specific degree of polymerization malto-oligosaccharides.

Biochemical properties of a novel chitosanase from Bacillus amyloliquefaciens and its use in membrane reactor

Chitooligosaccharides are widely used in functional food, medicaments and agriculture. Controllable conversion of chitosan to chitooligosaccharides with desired degree of polymerization (DP) is of great value. Herein, a novel glycoside hydrolase family 46 chitosanase (BaCsn46A) was cloned and expressed from Bacillus amyloliquefaciens. BaCsn46A exhibited good enzymatic properties with high specific activity (1031.2 U/mg) under optimal hydrolysis conditions of pH 6.0 and 50 °C. BaCsn46A is an endo-type chitosanase, which could be utilized to yield abundant chitooligosaccharides. Furthermore, the preparation of high degree of polymerization chitooligosaccharides (DP 5–10) was investigated by BaCsn46A in an enzymatic reactor with a subsequent series of membranes. The maximum concentration of total chitooligosaccharides (DP 2–10) was 28.11 g/L (93.70% yield). The proportion of high degree of polymerization chitooligosaccharides (DP 5–10) occupied 56.17% (w/w) of total chitooligosaccharides. This study provided a cleaner production process for the controllable preparation of chitooligosaccharides with specific degree of polymerization.

Expression and characterization of a novel cold-adapted chitosanase suitable for chitooligosaccharides controllable preparation

Chitooligosaccharide is widely used as a functional food additive and a valuable pharmacological agent. The transformation of chitinous biomass into valuable bioactive chitooligosaccharides is one of the most exciting applications of chitosanase. A novel glycoside hydrolase (GH) family 46 chitosanase (GsCsn46A) from rhizobacterium Gynuella sunshinyii was cloned and heterologously expressed in Escherichia coli. GsCsn46A showed maximal activity at pH 5.5 and 30 °C. GsCsn46A featured remarkable cold-adapted property, which controllably hydrolyzed chitosan to three types of chitooligosaccharides at the mild reaction condition (reaction condition: pH 5.5 at 30 °C; method for stopping the reaction: 50 °C for 30 min). The yields of three types of chitooligosaccharides products (degree of polymerization (DP): 2–7, 2–5 and 2–3) were 70.9%, 87.1% and 94.6% respectively. This novel cold-adapted chitosanase provides a cleaner production process for the controllable preparation of chitooligosaccharides with the specific DP.