检索问题
关于葡聚糖的酶催化聚合体系，有哪些酶可以催化反应，我希望这些酶已经商业化，或者可以被原核表达系统表达，或者说已有研究体系发现它们的聚合模式可以得到可控的分子量。
葡聚糖的酶催化聚合体系涉及多种酶，这些酶能够催化葡聚糖的合成，并且在商业化、原核表达或分子量可控的聚合模式方面已有研究。
1. 葡聚糖蔗糖酶 (Dextransucrase, EC 2.4.1.5)
葡聚糖蔗糖酶是广泛研究和应用的一类酶，主要由乳酸菌（如明串珠菌属、链球菌属和乳杆菌属）产生 。
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催化反应与聚合模式：
- 葡聚糖蔗糖酶从蔗糖中合成葡聚糖，其主要通过 α-(1,6) 糖苷键连接葡萄糖单元，并可能形成 α-(1,3) 糖苷分支 。
- 例如，来自 Leuconostoc mesenteroides NRRL B-1299 的葡聚糖蔗糖酶 (dsrA) 产生的葡聚糖由 85% 的 α-(1,6) 键和 15% 的 α-(1,3) 键组成，在麦芽糖存在下合成的寡糖主要由 α-(1,6) 键组成 。
- Leuconostoc citreum JZ-002 菌株提取的葡聚糖蔗糖酶 DsrB 可以合成分子量为 2.45 × 10 Da 的葡聚糖，其中 64% 为主链 α-(1,6) 糖苷键，34% 为支链 α-(1,3) 糖苷键 。
- 在麦芽糖作为受体存在时，葡聚糖蔗糖酶的反应会转向寡糖形成，但也会出现副产物葡聚糖 。麦芽糖作为受体对葡聚糖合成的抑制作用强于乳糖和葡萄糖 。然而，也有研究表明麦芽糖不能控制和降低葡聚糖的分子量分布，有或无麦芽糖的合成葡聚糖具有相同的分子量分布特征 。
- 葡聚糖蔗糖酶的活性受多种因素影响，例如，Ca²⁺ 离子可以刺激葡聚糖蔗糖酶的产生并恢复 EDTA 引起的活性下降 。Mn²⁺ 离子可以轻微激活重组和天然葡聚糖蔗糖酶的活性，而 Cu²⁺ 或 Al³⁺ 离子则强烈抑制其活性 。Zn、Cd、Pb、Hg 和 Cu 离子在不同程度上具有抑制作用 。
- 葡聚糖蔗糖酶的活性位点大小已被发现比之前报道的短 49 个氨基酸 。
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原核表达系统表达：
- 多种葡聚糖蔗糖酶基因已成功克隆到 Escherichia coli (大肠杆菌) 等原核表达系统中进行表达。
- 来自 Leuconostoc mesenteroides CGMCC 1.544 的葡聚糖蔗糖酶基因 dsrX 已克隆到 pET-28a(+) 载体中，并在 E. coli BL21(DE3) 中表达为 N-末端 His₆ 标签融合蛋白，表达量达到总细胞蛋白的 24.5% 。
- 来自 Leuconostoc mesenteroides B-1299 的截短葡聚糖蔗糖酶 DsrE563ΔCD2ΔGBD (DsrE563-1) 和 DsrE563ΔCD2ΔVR (DsrE563-2) 已在 E. coli 中表达为活性酶 。
- 来自 Leuconostoc mesenteroides B-512 FMC 的葡聚糖蔗糖酶经过截短并与谷胱甘肽 S-转移酶 (GST) 融合，在 E. coli BL21 (DE3) 中表达，使其生物合成量增加了 35 倍 。
- 来自 Leuconostoc citreum KM20 的重组葡聚糖蔗糖酶 (DexT) 在 E. coli 表达系统中通过改变菌株/诱导剂组合，其活性显著提高了 12 倍 。
- Leuconostoc citreum JZ-002 的葡聚糖蔗糖酶 DsrB 成功克隆到 pET-28a-AcmA 载体中并实现了重组表达 。
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分子量可控的聚合模式：
- 通过控制酶浓度和蔗糖浓度的比例，可以酶法合成具有不同分子量的葡聚糖 。例如，在蔗糖浓度为 100-2000 mM、酶浓度为 320-1280 U 的条件下，合成的葡聚糖分子量范围可从 4680 Da 扩展到 1,320,000 Da 。
- 截短和融合的葡聚糖蔗糖酶 fTDSR-S 可以有效地进行葡聚糖生物合成和受体反应，表明其在生产具有特定性质的葡聚糖方面具有潜力 。
- 临床型葡聚糖（分子量 70 kDa）具有很高的商业价值和科学兴趣 。近年来，通过酶和生物工艺工程以及分析技术的进步，显著增强了控制葡聚糖分子量和分支模式的能力 。
2. 葡聚糖糊精酶 (Dextran dextrinase, DDase, EC 2.4.1.2)
葡聚糖糊精酶是一种转葡糖苷酶，由 Gluconobacter oxydans 等菌株产生 。
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催化反应与聚合模式：
- DDase 催化将 α-1,4 连接的葡糖基单元从供体转移到受体分子，形成 α-1,6 连接，连续的葡糖基转移导致从麦芽糊精形成高分子量葡聚糖 。
- DDase 催化麦芽糊精转化为 (寡) 葡聚糖 。
- 根据营养发酵条件，G. oxydans 会产生两种形式的 DDase：细胞内和细胞外形式 。
- DDase 的应用包括生产 (寡) 葡聚糖、转葡糖基化产物和特种寡糖 。
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商业化和研究进展：
- 20 世纪 40 年代以来，Gluconobacter oxydans 的某些菌株就被发现能产生 DDase 。
- 日本 K. Yamamoto 团队在 20 世纪 90 年代初期重新开展了 DDase 的研究，重点关注 G. oxydans ATCC 11894 产生的细胞内 DDase 的纯化和表征 。
- Y. Suzuki 及其同事进一步研究了同一菌株形成的细胞外 DDase 的性质和动力学 。
- 研究团队进一步阐述了优化 DDase 生产和葡聚糖形成的发酵过程，DDase 的表征及其作为生物催化剂的使用，以及细胞内和细胞外 DDase 之间的生理联系 。
3. 淀粉麦芽糖酶 (Amylomaltase, 4-α-glucanotransferase, EC 2.4.1.25)
淀粉麦芽糖酶能够进行双步催化过程，产生转糖基化反应 。
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催化反应与聚合模式：
- 它们水解 α-1,4'-D-葡聚糖的糖苷键，并将葡聚糖部分与新可用的异头碳转移到 α-1,4'-D-葡聚糖受体的 4'-位置 。
- 分子内反应产生环状 α-1,4'-葡聚糖 。
- 淀粉麦芽糖酶在原核生物中发现，参与糖原降解和麦芽糖代谢 。
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商业化和研究进展：
- 淀粉麦芽糖酶正被研究用于生物技术应用，如生产糖替代品、环淀粉（比环糊精大的分子，可作为疏水分子载体和封装剂，以及有效的蛋白质伴侣）和热可逆淀粉凝胶（可用作非动物明胶替代品） 。
- 极端嗜热原核生物已被研究用于鉴定淀粉麦芽糖酶，以用于需要高温或极端条件的淀粉改性过程 。
4. 葡聚糖酶 (Dextranase)
葡聚糖酶通常用于降解葡聚糖，但其与葡聚糖蔗糖酶的联合使用可以调控葡聚糖的合成和产物组成 。
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催化反应与聚合模式：
- 葡聚糖酶特异性地降解葡聚糖的 α-1,6 糖苷键 。
- 通过控制葡聚糖酶的活性、底物浓度和时间，可以获得具有良好均一性的特定分子量葡聚糖 。例如，当葡聚糖产物的分子量约为 5 kDa 时，它在溶液中呈紧凑的球形链构象 。
- 在双酶（葡聚糖蔗糖酶和葡聚糖酶）反应中加入麦芽糖受体，足够量的葡聚糖酶优先降解葡聚糖而不是受体产物，从而产生异麦芽寡糖 (IMO) 。这种方法是促进高质量功能性 IMO 高效合成的简单有效方法 。
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原核表达与商业化：
- 来自 Penicillium cyclopium CICC-4022 的葡聚糖酶已被纯化和表征，其分子量为 77 kDa，最适 pH 为 5.0，最适温度为 55 °C 。Na⁺、K⁺ 和 NH⁺ 离子可以有效提高葡聚糖酶活性，而 Cu²⁺ 和 Pb²⁺ 离子则强烈抑制其活性 。
5. 糖基转移酶 (Glycosyltransferases, GTFs) 和磷酸化酶 (Phosphorylases)
这些酶在更广泛的 α-葡聚糖合成中发挥作用。
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糖基转移酶：
- 糖基转移酶是合成多种 α-葡聚糖（如葡聚糖、变聚糖、再聚糖和交替聚糖）的有效工具 。
- 它们利用可再生且低成本的蔗糖和淀粉作为底物，合成具有各种大小、连接组成和分支程度的 α-葡聚糖 。
- GH70 家族葡聚糖蔗糖酶在从蔗糖合成具有各种结构组织的 α-葡聚糖方面已被广泛表征 。
- 糖基转移酶和 β-1,4- 和 β-1,3-葡聚糖磷酸化酶在葡聚糖合成中显示出高潜力和多功能性，无论是在工业还是学术背景下 。
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磷酸化酶 (Cellodextrin phosphorylase, CDP)
- CDP 催化的酶促反应在宏观分子拥挤条件下用于合成纤维素寡聚体水凝胶 。
- 虽然主要用于纤维素寡聚体，但磷酸化酶是通用 D-葡聚糖聚合的酶之一，可以在自下而上的合成方法中实现更精确的聚合物结构控制和功能多样化 。
总结：
在葡聚糖的酶催化聚合体系中，葡聚糖蔗糖酶是最受关注和研究最深入的酶，因为它能够直接从蔗糖合成葡聚糖，并且在原核表达、分子量控制和商业应用方面都有显著进展 。葡聚糖糊精酶虽然主要用于降解，但通过与葡聚糖蔗糖酶的联合作用，也能实现对产物组成和分子量的调控 。淀粉麦芽糖酶则在生产环淀粉等其他葡聚糖衍生物方面具有潜力 。糖基转移酶和磷酸化酶则代表了更广泛的 α-葡聚糖合成酶，尤其是在自下而上精确控制聚合物结构方面具有重要意义 。这些酶的发现和工程化为葡聚糖及其衍生物在食品、医药、化妆品和材料科学等领域的应用提供了广阔前景 .
References
1Amylomaltases in Extremophilic Microorganisms.PubMed
Claudia Leoni, Bruno A R Gattulli, Graziano Pesole, et al.
Amylomaltases (4-α-glucanotransferases, E.C. 2.4.1.25) are enzymes which can perform a double-step catalytic process, resulting in a transglycosylation reaction. They hydrolyse glucosidic bonds of α-1,4'-d-glucans and transfer the glucan portion with the newly available anomeric carbon to the 4'-position of an α-1,4'-d-glucan acceptor. The intramolecular reaction produces a cyclic α-1,4'-glucan. Amylomaltases can be found only in prokaryotes, where they are involved in glycogen degradation and maltose metabolism. These enzymes are being studied for possible biotechnological applications, such as the production of (i) sugar substitutes; (ii) cycloamyloses (molecules larger than cyclodextrins), which could potentially be useful as carriers and encapsulating agents for hydrophobic molecules and also as effective protein chaperons; and (iii) thermoreversible starch gels, which could be used as non-animal gelatin substitutes. Extremophilic prokaryotes have been investigated for the identification of amylomaltases to be used in the starch modifying processes, which require high temperatures or extreme conditions. The aim of this article is to present an updated overview of studies on amylomaltases from extremophilic and , including data about their distribution, activity, potential industrial application and structure.
2Expression and characterization of dextransucrase gene dsrX from Leuconostoc mesenteroides in Escherichia coli.PubMed
Yang Yalin, Luo Jin, Wang Jianhua, et al.
The dextransucrase gene dsrX from Leuconostoc mesenteroides CGMCC 1.544 was cloned into the vector pET-28a(+) and expressed as a N-terminal His(6)-tag fusion protein of 167.57 kDa in Escherichia coli BL21(DE3). DsrX with the high volumetric activity of 8.8 U ml(-1) culture and the specific activity of 97.37 U mg(-1) crude enzyme extracts was measured in the optimized recombinant expression system. The resultant expression level of the fusion protein amounted to 24.5% of the total cell proteins. The results of affinity chromatography and western blotting indicated that the three sensitive sites of proteolysis existed in the N-terminal catalytic domain of DsrX. Both the recombinant and native enzyme activity were slightly activated by 1 mmol l(-1) Mn(2+) and strongly inhibited by 1 mmol l(-1) Cu(2+) or Al(3+), and their optimum pH values were 5.4. The optimum temperature of the recombinant enzyme for dextran synthesis was 30 degrees C, which was 10 degrees C less than that of the native one. The transglucosylation products of two enzymes were studied by using thin layer chromatography and high-performance anion exchange chromatography. It could be concluded that the better sample-pretreatment temperature in SDS-PAGE was 37 degrees C, which significantly improved the detection of thermal instable enzyme than that of 100 degrees C.
3Synthesis of dextran of different molecular weights by recombinant dextransucrase DsrB.PubMed
Yuanhao Pu, Kaige Peng, Jilu Sun, et al.
Leuconostoc citreum JZ-002 was extracted from artisanal orange wine. This strain was used to synthesize dextran with a purification extraction of 27.9 g/L. The resulting dextran had a molecular weight of 2.45 × 10 Da. A significant portion, amounting to 64 % of the structure, is constituted by the main chain, with α-(1,6) glycosidic bonds acting as the linkages. In contrast, the branched chain, comprising 34 % of the entire molecule, is characterized by the presence of α-(1,3) glycosidic bonds. The dextransucrase DsrB, believed to be accountable for the formation of the dextran backbone, was successfully cloned into the pET-28a-AcmA vector. The recombinant expression of the enzyme was achieved. Purified recombinant enzymes and immobilized in a single go using the gram-positive enhancer matrix (GEM). The maximum yield of dextran produced by suchimmobilized enzyme was 191.9 g/L. The composition featured a dextran connected via α-(1,6) glycosidic linkages. Molecular weight controlled synthesis was achieved with sucrose concentrations of 100-2000 mM and enzyme concentrations of 320-1280 U. The Mw of the synthesized dextran extended from 4680 to 1,320,000 Da. By controlling the ratio between enzyme concentration and sucrose concentration, dextrans with diverse Mw can be enzymatically generated.
4Production, purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.PubMed
J F Robyt, T F Walseth
The production of dextransucrase from Leuconostoc mesenteroides NRRL B-512F was stimulated 2-fold by the addition of 0.005% of calcium chloride to the medium; levansucrase levels were unaffected. Dextransucrase was purified by concentration and dialysis of the culture supernatant with a Bio-Fiber 80 miniplant, and by treatment with dextranase followed by chromatography on Bio-Gel A-Fm. A 240-fold purification, with a specific activity of 53 U/mg, was obtained. Contaminating enzyme activities of levansucrase, invertase, dextranase, glucosidase, and sucrose phosphorylase were decreased to non-detectable levels. Poly(acrylamide)-gel electrophoresis of the purified enzyme showed only two protein bands, both of which had dextransucrase activity. These bands also gave a carbohydrate stain, indicating that the dextransucrase could be a glycoprotein. Acid hydrolysis, followed by paper chromatography, of the purified enzyme showed that the major carbohydrate was mannose. Concanavalin A completely removed dextransucrase activity from solution, confirming the mannoglycoprotein character of the enzyme. Dextransucrase activity was not altered by the addition of 0.008-4 mg/ml of dextran, but its storage stability was increased by the addition of 4 mg/ml of dextran. As previously shown by others, the activity of dextransucrase was decreased by EDTA, and was restored by the addition of calcium ions. Zinc, cadmium, lead, mercury, and copper ions were inhibitory to various degrees.
5Recent advances in enzymatic synthesis of β-glucan and cellulose.PubMed
Gregory S Bulmer, Peterson de Andrade, Robert A Field, et al.
Bottom-up synthesis of β-glucans such as callose, fungal β-(1,3)(1,6)-glucan and cellulose, can create the defined compounds that are needed to perform fundamental studies on glucan properties and develop applications. With the importance of β-glucans and cellulose in high-profile fields such as nutrition, renewables-based biotechnology and materials science, the enzymatic synthesis of such relevant carbohydrates and their derivatives has attracted much attention. Here we review recent developments in enzymatic synthesis of β-glucans and cellulose, with a focus on progress made over the last five years. We cover the different types of biocatalysts employed, their incorporation in cascades, the exploitation of enzyme promiscuity and their engineering, and reaction conditions affecting the production as well as in situ self-assembly of (non)functionalised glucans. The recent achievements in the application of glycosyl transferases and β-1,4- and β-1,3-glucan phosphorylases demonstrate the high potential and versatility of these biocatalysts in glucan synthesis in both industrial and academic contexts.
6Molecular cloning and characterization of active truncated dextransucrase from Leuconostoc mesenteroides B-1299CB4.PubMed
Hee-Kyoung Kang, Eun-Ah Ko, Jong-Ho Kim, et al.
The open reading frame of dsrE563, a dextransucrase gene obtained from a constitutive mutant (CB4-BF563) of Leuconostoc mesenteroides B-1299, consists of 8,511 bp encoding 2,836 amino acid residues. DsrE563 contains two catalytic domains (CD1 and CD2). Two truncated derivative mutants DsrE563ΔCD2ΔGBD (DsrE563-1) and DsrE563ΔCD2ΔVR (DsrE563-2) of DsrE563 were constructed and expressed using the pRSETC vector in Escherichia coli. The derivatives DsrE563-1 (deletion of 1,620 amino acids from the C-terminus) and DsrE563-2 (deletion of 1,258 amino acids from the C-terminus and 349 amino acids from the N-terminus) were expressed as active enzymes. Both enzymes synthesized less-soluble dextran, mainly containing α-1,6 glucosidic linkage. The synthesized less-soluble dextran also had a branched α-1,3 linkage. DsrE563-2 showed 4.5-fold higher dextransucrase activity than that of DsrE563-1 and showed higher acceptor reaction efficiency than that of dextransucrase from L. mesenteroides 512 FMCM when various mono or disaccharides were used as acceptors. Thus, the glucan-binding domain was important for both enzyme expression and dextransucrase activity.
7Enzymatic synthesis of non-digestible oligosaccharide catalyzed by dextransucrase and dextranase from maltose acceptor reaction.PubMed
Shuang-Xia Huang, Dian-Zhi Hou, Peng-Xiang Qi, et al.
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.
8Designing of a novel dextransucrase efficient in acceptor reactions.PubMed
Mahmut Parlak, Duran Ustek, Aziz Tanriseven
Dextransucrase is produced by Leuconostoc, Streptococcus and Lactobacillus Species. The enzyme synthesizes dextran and acceptor products some of which act as prebiotics that are increasingly used in such industries as food, medicine, and cosmetics. B-512F Leuconostoc mesenteroides dextransucrase (DSR-S) is the preferred enzyme in commercial production of dextran and prebiotics. In the present work, a novel dextransucrase which is efficient in prebiotics production was designed. The enzyme was produced at optimal conditions in Escherichia coli by truncation and fusion to glutathione S-transferase (GST) in the gene from Leuconostoc mesenteroides B-512 FMC. The novel enzyme (MW: 119 kDa) was active and carried out dextran biosynthesis and acceptor reactions effectively. The novel dextransucrase (fTDSR-S) was produced by truncating signal, variable, and the glucan-binding regions in the gene and fusion of gst gene at the 5' end. fTDSR-S was characterized in detail and compared to the DSR-S. Truncation and fusion resulted in an increase in fTDSR-S biosynthesis in E. coli BL21 (DE3) by 35 fold. fTDSR-S leads to production of dextran as well as increased acceptor reactions. Due to GST fusion, it was possible to immobilize fTDSR-S covalently onto Eupergit C successfully. It was also found that the size of the active site of dextransucrase is 49 amino acids shorter than that reported previously in the literature.
9Enzymatic Synthesis of Cellulose Oligomer Hydrogels Composed of Crystalline Nanoribbon Networks under Macromolecular Crowding Conditions.PubMed
Yuuki Hata, Tomoya Kojima, Taro Koizumi, et al.
Macromolecular crowding, a solution state with high macromolecular concentrations, was used to promote the crystallization-driven self-assembly of enzymatically synthesized cellulose oligomers. Cellulose oligomers were synthesized via cellodextrin phosphorylase-catalyzed enzymatic reactions in the concentrated solutions of water-soluble polymers, such as dextran, poly(ethylene glycol), and poly(-vinylpyrrolidone). The reaction mixtures were transformed into cellulose oligomer hydrogels composed of well-grown crystalline nanoribbon networks irrespective of the polymer species. This method was successfully applied in the one-pot preparation of double network hydrogels composed of the nanoribbons and physically cross-linked gelatin molecules through the simple control of reaction temperatures, demonstrating the superior mechanical properties of the composite hydrogels. Our concept that promotes the growth of self-assembled architectures under macromolecular crowding conditions demonstrates a new avenue into developing novel hydrogel materials.
10Dextran dextrinase and dextran of Gluconobacter oxydans.PubMed
Myriam Naessens, An Cerdobbel, Wim Soetaert, et al.
Certain strains of Gluconobacter oxydans have been known since the 1940s to produce the enzyme dextran dextrinase (DDase; EC2.4.1.2)-a transglucosidase converting maltodextrins into (oligo)dextran. The enzyme catalyses the transfer of an alpha1,4 linked glucosyl unit from a donor to an acceptor molecule, forming an alpha1,6 linkage: consecutive glucosyl transfers result in the formation of high molecular weight dextran from maltodextrins. In the early 1990s, the group of K. Yamamoto in Japan revived research on DDase, focussing on the purification and characterisation of the intracellular DDase produced by G. oxydans ATCC 11894. More recently, this was taken further by Y. Suzuki and coworkers, who investigated the properties and kinetics of the extracellular DDase formed by the same strain. Our group further elaborated on fermentation processes to optimise DDase production and dextran formation, DDase characterisation and its use as a biocatalyst, and the physiological link between intracellular and extracellular DDase. Here, we present a condensed overview of the current scientific status and the application potential of G. oxydans DDase and its products, (oligo)dextrans. The production of DDase as well as of dextran is first described via optimised fermentation processes. Specific assays for measuring DDase activity are also outlined. The general characteristics, substrate specificity, and mode of action of DDase as a transglucosidase are described in detail. Two forms of DDase are produced by G. oxydans depending on nutritional fermentation conditions: an intracellular and an extracellular form. The relationship between the two enzyme forms is also discussed. Furthermore, applications of DDase, e.g. production of (oligo)dextran, transglucosylated products and speciality oligosaccharides, are summarized.
11Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only alpha (1-6) and alpha (1-3) linkages.PubMed
V Monchois, R M Willemot, M Remaud-Simeon, et al.
The coding region for a Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrA) was isolated and sequenced. Using a pair of primers designed on the basis of two highly conserved amino-acid (aa) sequences in L. mesenteroides NRRL B-512F dextransucrase and streptococcal glucosyltransferases (GTFs), a fragment of dsrA was amplified by the polymerase chain reaction (PCR). This PCR product was used as an hybridization probe to isolate a 1.8-kb fragment identified as the central region of dsrA. Cleavage by Sac I of this fragment allowed two probes to be obtained to isolate the 5' and the 3' ends of dsrA. The nucleotide sequence of the dsrA gene was determined and found to consist of an open reading frame (ORF) of 4870 base pairs (bp) coding for a 1290-aa protein with an M(r) of 145590. The aa sequence exhibited a high similarity with other GTFs. The two domains previously described in GTFs are conserved in DSRA: an N-terminal conserved domain and a C-terminal domain composed of a series of repeats. Surprisingly, the expected signal peptide was not detected. The entire gene was reconstructed and the activity of DSRA was investigated. The dextran produced appeared to be composed of 85% alpha (1-6) and 15% alpha (1-3) linkages and the oligosaccharides synthesized in the presence of maltose were mainly composed of alpha (1-6) linkages. This enzyme is a novel dextransucrase from L. mesenteroides NRRL B-1299 producing no alpha (1-2) linkages and is the first glucosyltransferase having no signal peptide described.
12The effect of maltose on dextran yield and molecular weight distribution.PubMed
Sueli Rodrigues, Liliane M F Lona, Telma T Franco
Dextran synthesis has been studied since the Second World War, when it was used as blood plasma expander. This polysaccharide composed of glucose units is linked by an alpha-1,6-glucosidic bond. Dextransucrase is a bacterial extra cellular enzyme, which promotes the dextran synthesis from sucrose. When, besides sucrose, another substrate (acceptor) is also present in the reactor, oligosaccharides are produced and part of the glucosyl moieties from glucose is consumed to form these acceptor products, decreasing the dextran yield. Although dextran enzymatic synthesis has been extensively studied, there are few published studies regarding its molecular weight distribution. In this work, the effect of maltose on yield and dextran molecular weight synthesized using dextransucrase from Leuconostoc mesenteroides B512F, was investigated. According to the obtained results, maltose is not able to control and reduce dextran molecular weight distribution and synthesis carried out with or without maltose presented the same molecular weight distribution profile.
13Polymeric iminosugars improve the activity of carbohydrate-processing enzymes.PubMed
Yoan Brissonnet, Simon Ladevèze, David Tezé, et al.
Multivalent iminosugars have recently emerged as powerful tools to inhibit the activities of specific glycosidases. In this work, biocompatible dextrans were coated with iminosugars to form linear and ramified polymers with unprecedently high valencies (from 20 to 900) to probe the evolution of the multivalent inhibition as a function of ligand valency. This study led to the discovery that polyvalent iminosugars can also significantly enhance, not only inhibit, the enzymatic activity of specific glycoside-hydrolase, as observed on two galactosidases, a fucosidase, and a bacterial mannoside phosphorylase for which an impressive 70-fold activation was even reached. The concept of glycosidase activation is largely unexplored, with a unique recent example of small-molecules activators of a bacterial O-GlcNAc hydrolase. The possibility of using these polymers as "artificial enzyme effectors" may therefore open up new perspectives in therapeutics and biocatalysis.
14Lactic acid bacteria-derived α-glucans: From enzymatic synthesis to miscellaneous applications.PubMed
Ziwei Chen, Dawei Ni, Wenli Zhang, et al.
Lactic acid bacteria (LAB) are capable of producing a variety of exopolysaccharide α-glucans, such as dextran, mutan, reuteran, and alternan. Their structural diversity allows LAB-derived α-glucans to hold vast commercial value and application potential in the food, cosmetic, medical, and biotechnology fields, garnering much attention in recent years. Glycoside Hydrolase 70 family (GH70) enzymes are efficient tools for the biosynthesis of α-glucans with various sizes, linkage compositions, and degrees of branching, using renewable and low-cost sucrose and starch as substrates. To date, plenty of various LAB-derived GH70 glucansucrases (especially dextransucrase) have been biochemically characterized to synthesize α-glucans from sucrose with a variety of structural organizations. This review mainly aimed at the biotechnological synthesis of α-glucans using GH70 family enzymes and their diverse (potential) applications. The purification, structural analysis and physicochemical properties of α-glucan polysaccharides were reviewed in detail. Synchronously, some new insights and future perspectives of LAB-derived α-glucans enzymatic synthesis and applications were also discussed. To expand the range of applications, the physicochemical properties and bioactivities of LAB-derived α-glucans, other than dextran, should be further explored. Additionally, screening novel GH70 subfamily starch-acting enzymes is conducive to expanding the repertoire of α-glucans.
15Large increase in Leuconostoc citreum KM20 dextransucrase activity achieved by changing the strain/inducer combination in an E. coli expression system.PubMed
Jin-A Ko, Hyung Jae Jeong, Young Bae Ryu, et al.
A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction (OD₆₀₀) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of 17 degrees C. These results may effectively apply to the heterologous expression of other large DexT genes.
16Purification and characterization of dextranase from Penicillium cyclopium CICC-4022 and its degradation of dextran.PubMed
Xuejiao Wang, Yirui Zhang, Mei Li, et al.
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 NH can effectively improve the dextranase activity, Cu and Pb 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.
17Enzymatic Synthesis of α-Glucan Microparticles Using Amylosucrases from Species and Its Physicochemical Properties.PubMed
Ye-Jin Kim, Yun-Sang So, Moo-Yeol Baik, et al.
α-Glucan microparticles (GMPs) have significant potential as high-value biomaterials in various industries. This study proposes a bottom-up approach for producing GMPs using four amylosucrases from sp. (BASs). The physicochemical characteristics of these GMPs were analyzed, and the results showed that the properties of the GMPs varied depending on the type of enzymes used in their synthesis. As common properties, all GMPs exhibited typical B-type crystal patterns and poor colloidal dispersion stability. Interestingly, differences in the physicochemical properties of GMPs were generated depending on the synthesis rate of linear α-glucan by the enzymes and the degree of polymerization (DP) distribution. Consequently, we found differences in the properties of GMPs depending on the DP distribution of linear glucans prepared with four BASs. Furthermore, we suggest that precise control of the type and characteristics of the enzymes provides the possibility of producing GMPs with tailored physicochemical properties for various industrial applications.
18Cloning, expression and characterization of an extracellular enolase from Leuconostoc mesenteroides.PubMed
Jin-Ha Lee, Hee-Kyoung Kang, Young-Hwan Moon, et al.
Enolase on the surface of streptococci putatively facilitates pathogenic invasion of the host organisms. The related Leuconostoc mesenteroides 512FMCM is nonpathogenic, but it too has an extracellular enolase. Purified isolates of extracellular dextransucrase from cultures of L. mesenteroides contain minute amounts of enolase, which separate as small crystals. Expression of L. mesenteroides enolase in Escherichia coli provides a protein (calculated subunit mass of 47 546 Da) catalyzing the conversion of 2-phsopho-D-glycerate to phosphoenolpyruvate. The pH optimum is 6.8, with Km and kcat values of 2.61 mM and 27.5 s(-1), respectively. At phosphate concentrations of 1 mM and below, fluoride is a noncompetitive inhibitor with respect to 2-phospho-D-glycerate, but in the presence of 20 mM phosphate, fluoride becomes a competitive inhibitor. Recombinant enolase significantly inhibits the activity of purified dextransucrase, and does not bind human plasminogen. Results here suggest that in some organisms enolase may participate in protein interactions that have no direct relevance to pathogenic invasion.
19Clinical-type dextran: A review on process and enzyme engineering strategies to control molecular weight distribution.PubMed
Pia Lanvers, Jannis Broeker, Jochen Schmid
Dextrans are α-(1 → 6)-linked, mostly branched α-d-glucopyranosyl polysaccharides synthesized from sucrose by lactic acid bacteria. The unique properties of dextrans, their biocompatibility, biodegradability, and non-immunogenicity support a wide range of medical and industrial applications. Key characteristics of dextran, including solubility, viscosity, and molecular weight, highly depend on the bacterial strain and the conditions under which it is synthesized. The structural diversity within the family of dextran-synthesizing enzymes contributes to dextran diversity. Conventionally, dextran with low molecular weight is obtained by fermentative production of high molecular weight dextran, followed by partial hydrolysis and a precise fractionation procedure, which is a costly and hazardous process. However, recent advances in enzyme- and bioprocess engineering in combination with analytical techniques have significantly enhanced the ability to control the molecular weight and branching patterns of dextran. Thereby improving its effectiveness and broadening its applications in both industrial and clinical settings. This review focuses on the production of dextrans with defined molecular weight and the enzymatic mechanisms underlying their synthesis, which have been increasingly studied in recent years. In particular, clinical-type dextran with a molecular weight of 70 kDa is of high commercial value and scientific interest.
20Bottom-Up Synthesized Glucan Materials: Opportunities from Applied Biocatalysis.PubMed
Chao Zhong, Bernd Nidetzky
Linear d-glucans are natural polysaccharides of simple chemical structure. They are comprised of d-glucosyl units linked by a single type of glycosidic bond. Noncovalent interactions within, and between, the d-glucan chains give rise to a broad variety of macromolecular nanostructures that can assemble into crystalline-organized materials of tunable morphology. Structure design and functionalization of d-glucans for diverse material applications largely relies on top-down processing and chemical derivatization of naturally derived starting materials. The top-down approach encounters critical limitations in efficiency, selectivity, and flexibility. Bottom-up approaches of d-glucan synthesis offer different, and often more precise, ways of polymer structure control and provide means of functional diversification widely inaccessible to top-down routes of polysaccharide material processing. Here the natural and engineered enzymes (glycosyltransferases, glycoside hydrolases and phosphorylases, glycosynthases) for d-glucan polymerization are described and the use of applied biocatalysis for the bottom-up assembly of specific d-glucan structures is shown. Advanced material applications of the resulting polymeric products are further shown and their important role in the development of sustainable macromolecular materials in a bio-based circular economy is discussed.