Recombinant expression of a chitosanase and its application in chitosan oligosaccharide production

Recently, considerable attention has been focused on chitosan oligosaccharides (COSs) due to their various biological activities. COSs can be prepared by enzymatic degradation of chitosan, which is the deacetylation product of chitin, one of the most abundant biopolymers in nature. In the current study, we recombinantly expressed a chitosanase and used it for COS preparation. A bacillus-derived GH8 family chitosanase with a 6×His tag fused at its N-terminal was expressed in the Escherichia coli strain BL21(DE3) as a soluble and active form. Its expression level could be as high as 500 mg/L. Enzymatic activity could reach approximately 140,000 U/L under our assay conditions. The recombinant chitosanase could be purified essentially to homogeneity by immobilized metal-ion affinity chromatography. The enzyme could efficiently convert chitosan into monomer-free COS: 1 g of enzyme could hydrolyze about 100 kg of chitosan. Our present work has provided a cheap chitosanase for large-scale COS production in industry.     

Combinatorial pathway engineering of Bacillus subtilis for production of structurally defined and homogeneous chitooligosaccharides

Chitooligosaccharides (COSs) have a widespread range of biological functions and an incredible potential for various pharmaceutical and agricultural applications. Although several physical, chemical, and biological techniques have been reported for COSs production, it is still a challenge to obtain structurally defined COSs with defined polymerization (DP) and acetylation patterns, which hampers the specific characterization and application of COSs. Herein, we achieved the de novo production of structurally defined COSs using combinatorial pathway engineering in Bacillus subtilis. Specifically, the COSs synthase NodC from Azorhizobium caulinodans was overexpressed in B. subtilis, leading to 30 ± 0.86 mg/L of chitin oligosaccharides (CTOSs), the homo-oligomers of N-acetylglucosamine (GlcNAc) with a well-defined DP lower than 6. Then introduction of a GlcNAc synthesis module to promote the supply of the sugar acceptor GlcNAc, reduced CTOSs production, which suggested that the activity of COSs synthase NodC and the supply of sugar donor UDP-GlcNAc may be the limiting steps for CTOSs synthesis. Therefore, 6 exogenous COSs synthase candidates were examined, and the nodCM from Mesorhizobium loti yielded the highest CTOSs titer of 560 ± 16 mg/L. Finally, both the de novo pathway and the salvage pathway of UDP-GlcNAc were engineered to further promote the biosynthesis of CTOSs. The titer of CTOSs in 3-L fed-batch bioreactor reached 4.82 ± 0.11 g/L (85.6% CTOS5, 7.5% CTOS4, 5.3% CTOS3 and 1.6% CTOS2), which was the highest ever reported. This is the first report proving the feasibility of the de novo production of structurally defined CTOSs by synthetic biology, and provides a good starting point for further engineering to achieve the commercial production.     

Enzymatic production and biological activities of chitosan oligosaccharides (COS): A review

Many researchers have focused chitosan as a source of potential bioactive material during past few decades. However, chitosan has several drawbacks to be utilized in biological applications, including poor solubility under physiological conditions. Therefore, a new interest has recently been emerged on partially hydrolyzed chitosan, chitosan oligosaccharides (COS). During the resent past, several technological approaches have been taken to prepare COS and, enzymatic preparation methods captured a great interest due to safe and non-toxic concerns. With time, new improvements were introduced to enzymatic production and presently it has been developed to a continuous production process. Many of the biological activities reported for COS, such as antimicrobial, anticancer, antioxidant, and immunostimulant effects are depend on their physico-chemical properties. In this review, we have summarized different enzymatic preparation methods of COS and some of their reported biological activities.     

膜分离技术在寡糖制备与分离中的应用*

寡糖是多糖经过降解后得到的小分子活性物质,具有抗氧化、抗肿瘤、抗病毒和免疫调节等多种生物活性,是功能食品开发领域研究的热点。目前,寡糖的分离和制备主要采用离子交换色谱、凝胶渗透色谱以及两者联用的方法,分离时间长、制备成本高,难以实现寡糖的规模化分离和制备。膜分离技术(membrane separation technology,MST)是一种利用膜的选择性渗透作用,实现两组分或者多组分分离的技术,具有操作简单、分离效果好、高效节能等优点,特别是能够直接放大应用于规模化的分离工程,因此在寡糖等小分子的分离和制备等方面具有巨大的应用潜力。系统总结了膜分离技术在寡糖分离与制备领域的最新进展,综述了用于分离和制备寡糖的膜分离技术分类、分离工艺及其应用现状,并对目前膜分离技术用于大规模分离和制备寡糖过程中面临的挑战进行了讨论。     

Tuning chitosan’s chemical structure for enhanced biological functions

Chitosan, an amino polysaccharide mostly derived from crustaceans, has been recently highlighted for its biological activities that depend on its molecular weight (MW), degree of deacetylation (DD), and acetylation pattern (AP). More importantly, for some advanced biomaterials, the homogeneity of the chitosan structure is an important factor in determining its biological activity. Here we review emerging enzymes and cell factories, respectively, for in vitro and in vivo preparation of chitosan oligosaccharides (COSs), focusing on advances in the analysis of the AP and structural modification of chitosan to tune its functions. By ‘mapping’ current knowledge on chitosan’s in vitro and in vivo activity with its MW and AP, this work could pave the way for future studies in the field.     

A comprehensive review on application of bioreactor for industrial wastewater treatment

In the recent past, wastewater treatment processes performed a pivotal role in accordance with maintaining the sustainable environment and health of mankind at a proper hygiene level. It has been proved indispensable by government regulations throughout the world on account of the importance of preserving freshwater bodies. Human activities, predominantly from industrial sectors, generate an immeasurable amount of industrial wastewater loaded with toxic chemicals, which not only cause dreadful environmental problems, but also leave harmful impacts on public health. Hence, industrial wastewater effluent must be treated before being released into the environment to restrain the problems related to industrial wastewater discharged to the environment. Nowadays, biological wastewater treatment methods have been considered an excellent approach for industrial wastewater treatment process because of their cost-effectiveness in the treatment, high efficiency and their potential to counteract the drawbacks of conventional wastewater treatment methods. Recently, the treatment of industrial effluent through bioreactor has been proved as one of the best methods from the presently available methods. Reactors are the principal part of any biotechnology-based method for microbial or enzymatic biodegradation, biotransformation and bioremediation. This review aims to explore and compile the assessment of the most appropriate reactors such as packed bed reactor, membrane bioreactor, rotating biological contactor, up-flow anaerobic sludge blanket reactor, photobioreactor, biological fluidized bed reactor and continuous stirred tank bioreactor that are extensively used for distinct industrial wastewater treatment.     

细胞膜糖蛋白及其寡糖链分析方法的研究进展

细胞膜与细胞的各种生命活动如细胞识别粘着、运动迁移、免疫应答、物质运输、信息传导、细胞分裂、细胞分化、衰老及病变、癌变都有密切的关系。细胞表面糖蛋白是细胞膜的主要成分,而糖蛋白的性质及功能又和糖链的结构有关,因此细胞膜糖蛋白中寡糖链的结构及作用机制研究成为生物学基础理论的课题之一。主要综述了近年来细胞膜糖蛋白研究中细胞膜的类别、细胞膜的制备方法、细胞膜糖蛋白的提取与分离纯化、寡糖链的结构分析方法。     

Structural characterization of N-linked oligosaccharides of laminin from rat kidney: changes during diabetes and modulation by dietary fiber and butyric acid

Carbohydrates of laminin, a family of large multidomain glycoproteins, have been implicated in various cellular activities including maintaining the protein structure, its function and also basement membrane integrity. During the course of our investigation, we observed that purified laminin from kidneys of control, diabetic, and dietary fiber- and butyric acid-treated diabetic rats showed differences in binding to extracellular matrix components. This prompted us to determine whether there are structural changes in laminin oligosaccharides. In this study, we have characterized a few major N-linked oligosaccharides isolated from purified laminin in various experimental groups, viz. normal, diabetic and diabetic rats fed with dietary fiber and butyric acid. Sugar composition, as identified by GLC, revealed the presence of mannose, galactose and N-acetylglucosamine. In order to study fine structures of the oligosaccharides, N-linked oligosaccharides of laminin were released by Peptide-N-glycosidase F digestion, end-labeled with 2-anthranilic acid and fractionated by lectin affinity chromatography. Furthermore, structural elucidation carried out by MALDI-TOF MS/MS analysis showed variations in the oligosaccharide sequence of laminin during diabetes which were altered by the feeding of dietary fiber and butyric acid.     

Effects of chitooligosaccharides on human red blood cell morphology and membrane protein structure

Recent studies of chitosan have increased the interest in its conversion to chitooligosaccharides (COSs) because these compounds are water-soluble and have potential use in several biomedical applications. Furthermore, such oligomers may be more advantageous than chitosans because of their much higher absorption profiles at the intestinal level, which permit their facilitated access to systemic circulation and potential distribution throughout the entire human body. In that perspective, it is important to clarify their effect on blood further, namely, on human red blood cells (RBCs). The aim of this work was thus to study the effect of two COS mixtures with different molecular weight (MW) ranges, <3 and <5 kDa, at various concentrations (5.0-0.005 mg/mL) on human RBCs. The interactions of these two mixtures with RBC membrane proteins and with hemoglobin were assessed, and the RBC morphology and surface structure were analyzed by optical microscopy (OM) and atomic force microscopy (AFM). In the presence of either COS mixture, no significant hemolysis was observed; however, at COS concentrations >0.1 mg/mL, changes in membrane binding hemoglobin were observed. Membrane protein changes were also observed with increasing COS concentration, including a reduction in both alpha- and beta-spectrin and in band 3 protein, and the development of three new protein bands: peroxiredoxin 2, calmodulin, and hemoglobin chains. Morphologic evaluation by OM showed that at high concentrations COSs interact with RBCs, leading to RBC adhesion, aggregation, or both. An increase in the roughness of the RBC surface with increasing COS concentration was observed by AFM. Overall, these findings suggest that COS damage to RBCs was dependent on the COS MW and concentration, and significant damage resulted from either a higher MW or a greater concentration (>0.1 mg/mL).     

Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides

Mammalian cell surfaces are all covered with bioactive oligosaccharides which play an important role in molecular recognition events such as immune recognition, cell-cell communication and initiation of microbial pathogenesis. Consequently, bioactive oligosaccharides have been recognized as a medicinally relevant class of biomolecules for which the interest is growing. For the preparation of complex and highly pure oligosaccharides, methods based on the application of glycosyltransferases are currently recognized as being the most effective. The present paper reviews the potential of glycosyltransferases as synthetic tools in oligosaccharide synthesis. Reaction mechanisms and selected characteristics of these enzymes are described in relation to the stereochemistry of the transfer reaction and the requirements of sugar nucleotide donors. For the application of glycosyltransferases, accepted substrate profiles are summarized and the whole-cell approach versus isolated enzyme methodology is compared. Sialyltransferase-catalyzed syntheses of gangliosides and other sialylated oligosaccharides are described in more detail in view of the prominent role of these compounds in biological recognition.     

虫蛹活性多肽的制备、分离和生物活性研究进展

我国拥有悠久的蜂、蚕养殖历史,其虫蛹富含各种营养物质,其中蛋白质的含量最高。现代营养学研究发现,通过酶解等生物手段从虫蛹蛋白中分离出的生物活性肽,具有多种生物活性,如抗氧化、抗肿瘤、降血压和增强免疫等,同时可有效地提高虫蛹的利用率。基于此,就近年来国内外对虫蛹多肽的制备、分离纯化的方法以及生物活性的研究进展作一综述,以期为虫蛹多肽的深入研究与开发提供参考。     

Sulfated chitooligosaccharides as prolyl endopeptidase inhibitor

Prolyl endopeptidase (PEP, EC 3.4.21.26) is a proline-specific endopeptidase with a serine-type mechanism, which digests small peptide-like hormones, neuroactive peptides, and various cellular factors. PEP has been involved in neurodegenerative disorders, therefore, the discovery of PEP inhibitors can revert memory loss caused by amnesic compounds. In this study, we prepared hetero-chitooligosaccharides (COSs) with different molecular sizes using ultrafiltration (UF) membrane reactor system from hetero-chitosan with different degrees of deacetylation (DD; 90%, 75% and 50% deacetylation), and synthesized sulfated COSs (SCOSs). PEP inhibitory activities of SCOSs were evaluated and the results showed that 50% deacetylated SCOSs (50-SCOSs) exhibited higher inhibitory activities than those of 90% and 75% deacetylated SCOSs (90-SCOSs and 75-SCOSs). Among the 50-SCOSs (50-SCOS I, 5000–10,000 Da; 50-SCOS II, 1000–5000 Da; 50-SCOS III, below 1000 Da), 50-SCOS II possessed the highest inhibitory activity and IC₅₀ value was 0.38 mg/ml. Kinetics studies with 50-SCOS II indicated a competitive enzyme inhibition with a K_i value of 0.78 mg/ml. It was concluded that the 50-SCOS II may be useful for PEP inhibitor and for developing a new type PEP inhibitor from carbohydrate based materials.     

Monolith enzymatic microreactor at the frontier of glycomic toward a new route for the production of bioactive oligosaccharides

Recent research in the area of bioactive carbohydrates has shown the efficiency of oligosaccharides as signal molecules in a lot of biological activities. Newly observed functions of oligosaccharides and their abilities to act as specific regulatory molecules on various organisms have been more and more described. A successful development of these bioactive molecules in future needs efficient processes for specific oligosaccharides production. To exploit them for putative industrial scale up processes, two main strategies are currently investigated: the synthesis (chemical or bioconversion processes) and the polysaccharide cleavage (chemical, physical or biological processes). Nevertheless, if new manufacturing biotechnologies have considerably increased the development of these functional molecules, the main drawback limiting their biological applications is the complexity to engender specific glycosidic structures for specific activities. In the recent years, new enzymatic reactors have been developed, allowing the automatic synthesis of oligosaccharide structures. This review focuses on the knowledge in the area of bioactive oligosaccharides and gives the main processes employed to generate them for industrial applications with challenges of monolith microreactors.     

Purification and properties of the membrane form of variant surface glycoproteins (VSGs) from Trypanosoma brucei

Variant surface glycoproteins (VSG) of Trypanosoma brucei are released in a water soluble form on impairment of membrane integrity. We have previously shown that this release is the result of an enzyme-mediated event which converts the hydrophobic membrane form VSG into the hydrophilic water-soluble form. We now present further details of the methods by which membrane form VSG ( mfVSG ) may be isolated, uncontaminated by water-soluble VSG ( sVSG ). The sensitivity to different metal ions of the enzyme that mediated the conversion event is discussed, and some biochemical characteristics of different mfVSG preparations are presented.     

海洋寡糖及其衍生物活性的研究进展

海洋寡糖由海洋多糖经弱酸或糖苷酶等方法降解制备,具有来源丰富、结构新颖、分子量小、类型多样等特点,表现出抗肿瘤、抗氧化、免疫调节、抗病毒等多种生物活性,在食品、医药、保健品及化妆品等领域有着广泛的应用,具有巨大的开发潜力。为了及时了解海洋寡糖的研究进展,促进海洋寡糖的应用与开发,就海洋寡糖及其衍生物的免疫调节、抗肿瘤、抑菌、抗病毒、抗氧化、抗辐射、抗凝血、抗糖尿病以及调节肠道微生物等方面的活性研究进展进行了简要总结,以期为海洋寡糖及其衍生物的深入研究和深度开发提供参考。     

Purification and Properties of the Membrane Form of Variant Surface Glycoproteins (VSGs) from Trypanosoma brucei1,2,3

Variant surface glycoproteins (VSG) of Trypanosoma brucei are released in a water soluble form on impairment of membrane integrity. We have previously shown that this release is the result of an enzyme-mediated event which converts the hydrophobic membrane form VSG into the hydrophilic water-soluble form. We now present further details of the methods by which membrane form VSG (mfVSG) may be isolated, uncontaminated by water-soluble VSG (sVSG). The sensitivity to different metal ions of the enzyme that mediated the conversion event is discussed, and some biochemical characteristics of different mfVSG preparations are presented.     

膜生物反应器的研究进展

膜生物反应器是近年来发展的废水处理新技术,具有活性污泥浓度高、污泥龄长、占地面积小、投资省的特点。利用膜生物反应器进行污水处理不仅可以大大节约水资源,还可以大大节约能源,节省设备和运行费用,已成为二十一世纪研究热点。膜生物反应器是通过高效膜分离技术与活性污泥相结合,增大污泥中的特效菌来加快生化反应速率,提高废水处理效果。目前处理对象已从生活污水扩展到高浓度的有机废水和难降解的工业废水。本文综述了膜生物反应器在废水中的应用研究情况,并分析比较了各种膜材质的特点、适用范围以及膜的污染因素和清洗方法,展望了膜生物反应器的应用前景及进一步研究方向。     

甘露寡糖分离纯化研究进展*

甘露寡糖具有润肠通便、降血脂、抗结肠炎症、增强免疫及调节肠道菌群等生理功能,在食品药品等领域具有广阔的应用前景。水解法制得的甘露寡糖含有单糖、未分解聚糖、不同聚合度寡糖及盐离子等杂质,还需要进一步分离纯化。综述了近年来国内外甘露寡糖的主要纯化方法包括柱层析法、膜分离法、乙醇沉淀法和微生物发酵法等。总结了各种方法的原理、应用范围和应用实例,并对不同方法的优缺点进行了分析。     

Selective and stable production of physiologically active chitosan oligosaccharides using an enzymatic membrane bioreactor

We investigated the production of chitosan oligosaccharides by continuous hydrolysis of chitosan in an enzyme membrane bioreactor, with the goal of improving the yield of physiologically active oligosaccharides (pentamers and hexamers) and achieving operational stability. The bioreactor was a continuous-flow stirred-tank reactor equipped with an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and the hydrolysis was accomplished with chitosanase from Bacillus pumilus. After optimization of the reaction parameters, such as the amount of enzyme, the yield of the target oligosaccharides produced in the membrane bioreactor with free chitosanase reached 52% on the basis of the fed concentration of chitosan. An immobilized chitosanase prepared by the multipoint attachment method was used to improve the operational stability of the membrane bioreactor. Under the optimized conditions, pentameric and hexameric chitosan oligosaccharides were steadily produced at 2.3 g/L (46% yield) for a month. The half-life of the productivity of the reactor was estimated to be 50 d under the conditions examined.     

Renin inhibition activity by chitooligosaccharides

Six kinds of chitooligosaccharides (COSs) with different molecular weight (MW) and degree of deacetylation (DD) were prepared using ultrafiltration membrane reactor, and their renin inhibition modes were evaluated. All the COSs showed the renin-inhibitory activities with dose-dependent manner, and 90-COSs had the potent renin-inhibitory activity than that of 50-COSs. Among them, 90-MMWCOS (1000-5000Da) exhibits the highest activity with IC(50) value of 0.51mg/mL and acts as competitive inhibitor with K(i) value of 0.28mg/mL by Lineweaver-Burk and Dixon plots. These results indicated that DD value and MW of COSs are important factors affecting renin-inhibitory activity.