Understanding the molecular organization of amino polysaccharide networks is essential for designing functional hydrocolloids. Here, we combine solid-state NMR spectroscopy and molecular dynamics simulations to characterize the native chitin-chitosan matrix from Mucor racemosus. NMR spectra reveal a clear separation of rigid and mobile domains, with the rigid core dominated by polymorphic chitin and partially deacetylated chitosan. Intermolecular contact analysis identifies 125 cross peaks, consistent with a densely interconnected carbohydrate network stabilized primarily by hydrogen bonding. Notably, no (3-1,3-glucan signal is detected in the rigid domain, indicating a structural organization distinct from glucan-rich fungi and driven mainly by direct chitinchitosan associations. Simulations support a cohesive yet dynamic network in which hydrogen bonds rearrange under thermal motion. The mobile fraction contains galactose, fucose, and glucuronic acid residues that are predicted to enhance hydration and interfacial interactions. Together, these results define a hierarchical architecture that integrates mechanical stability with hydration functionality and suggest routes for exploiting chitin and chitosan in food hydrocolloid and biomaterial applications.

Millions of tons of shrimp and crab waste, rich in chitin, are produced annually worldwide. To efficiently utilize this resource and address the contamination caused by traditional chitin treatment, a high-chitinase-producing strain, GXMU-J23.1, was isolated from the marine environment and identified as Bacillus paralicheniformis. Genome sequencing revealed several chitinolytic enzymes, such as chitinase, chitin deacetylase, and polysaccharide monooxygenases. Under optimal conditions, the chitinase activity increased 9.1-fold to 356.32 +/- 1.21 U/mL. The purified chitinase Chi23 exhibited optimal activity at 50 degrees C and pH 5.0, degrading various chitin substrates. Metal ions such as Ca2+ and reagents such as EDTA increased the activity, whereas Fe2+ and Zn2+ inhibited the activity. Chi23, an endochitinase, converts chitin into chitotriose and diacetylchitobiose. Based on the structural reconstruction and molecular docking of Chi23, the potential enzyme-substrate mode of action was elucidated, which will support subsequent enzyme modification and in-depth development of enzyme systems assisting in chitin degradation.

Ganoderma lucidum is a medicinal mushroom long esteemed in Asian traditional medicine for its health-promoting and longevity-enhancing properties. It contains over 400 bioactive compounds, among which its polysaccharides (GLPs) are especially significant due to their diverse biological functions. However, the molecular arrangement of GLPs within the cell-wall matrix remains poorly understood. To address this, we applied solid-state NMR for non-destructive structural characterization. Our analysis reveals a rigid core scaffold composed predominantly of β-1,3-glucan, chitin and chitosan, encased by a more mobile outer layer rich in β-1,4-glucan, α-1,3-glucans, α-1,4-glucan, arabinose, galactofuranose and α-mannose. Proton-driven spin-diffusion (PDSD) experiments identified 39 intermolecular contacts, especially between β-1,3-glucan and chitin, as well as among chitin chains. Within the core, chitosan displays nanosecond-to-microsecond mobility, suggesting a dynamically adaptable network. Hydration-kinetics measurements further distinguish these components, underscoring the hydrophilicity of β-1,3-glucan versus the hydrophobicity of chitin. Altogether, these findings support a model in which β-1,3-glucan-chitin interactions confer mechanical strength, while chitosan provides flexibility to enable adaptive cell-wall remodeling.

The expression of polysialic acid (polySia) on the neuronal cell adhesion molecule (NCAM) is called NCAM-polysialylation, which is strongly related to the migration and invasion of tumor cells and aggressive clinical status. During the NCAM polysialylation process, polysialyltransferases (polySTs), such as polysialyltransferase IV (ST8SIA4) or polysialyltransferase II (ST8SIA2), can catalyze the addition of CMP-sialic acid (CMP-Sia) to the NCAM to form polysialic acid (polySia). In this study, the docking models of polysialyltransferase IV (ST8Sia4) protein and different ligands were predicted using Alphafold 3 and DiffDock servers, and the prediction accuracy was further verified using the NMR experimental spectra of the interactions between polysialyltransferase domain (PSTD), a crucial peptide domain in ST8Sia4, and a different ligand. This combination strategy provides new insights into a quick and effective screening for inhibitors of tumor cell migration.

Efficient bioconversion of lignocellulose to valuable products can be performed by bacteria that are both capable of degrading biomass and producing platform chemicals. This study reported a newly isolated lignocellulolytic strain, Raoultella ornithinolytica TH-21 showing great potential as an ideal biocatalyst for producing 2,3-butanediol (2,3-BDO). Strain TH-21 exhibited the capability of assimilation of multiple carbon sources for 2,3-BDO production. It could directly convert 50 g/L of xylose, mannitol, cellobiose and xylan into 2,3-BDO with the maximum production of 20.04 g/L, 16.56 g/L, 20.06 g/L and 11.25 g/L, respectively. In addition, the lignin, cellulose, and hemicellulose present in sugarcane bagasse were degraded by strain TH-21 with a rate of 26.96%, 24.6%, and 33% after 7 days of treatment, respectively. Furthermore, FTIR, XRD and SEM analysis revealed that strain TH-21 can effectively depolymerize the lignin and holocellulose in sugarcane bagasse through its efficient secretion of lignocellulolytic enzymes. Thus, these findings indicated that strain TH-21 might be a promising biocatalyst for the effective bioconversion of lignocellulose to high-value chemicals.

The overexpression of polysialic acid (polySia) on neural cell adhesion molecules (NCAM) promotes hypersialylation, and thus benefits cancer cell migration and invasion. It has been proposed that the binding between the polysialyltransferase domain (PSTD) and CMP-Sia needs to be inhibited in order to block the effects of hypersialylation. In this study, CMP was confirmed to be a competitive inhibitor of polysialyltransferases (polySTs) in the presence of CMP-Sia and triSia (oligosialic acid trimer) based on the interactional features between molecules. The further NMR analysis suggested that polysialylation could be partially inhibited when CMP-Sia and polySia co-exist in solution. In addition, an unexpecting finding is that CMP-Sia plays a role in reducing the gathering extent of polySia chains on the PSTD, and may benefit for the inhibition of polysialylation. The findings in this study may provide new insight into the optimal design of the drug and inhibitor for cancer treatment.

IntroductionChitin, abundant in marine environments, presents significant challenges in terms of transformation and utilization. A strain, T22.7.1T, with notable chitin deacetylation capabilities, was isolated from the rhizosphere of Acanthus ebracteatus in the North Sea of China. Comparative 16S rDNA sequence analysis showed that the new isolate had the highest sequence similarity (99.79%) with Rhodococcus indonesiensis CSLK01-03T, followed by R. ruber DSM 43338T, R. electrodiphilus JC435T, and R. aetherivorans 10bc312T (98.97%, 98.81%, and 98.83%, respectively). Subsequent genome sequencing and phylogenetic analysis confirmed that strain T22.7.1T belongs to the R. indonesiensis species. However, additional taxonomic characterization identified strain T22.7.1T as a novel type strain of R. indonesiensis distinct from CSLK01-03T.MethodsThis study refines the taxonomic description of R. indonesiensis and investigates its application in converting chitin into chitosan. The chitin deacetylase (RiCDA) activity of strain T22.7.1T was optimized, and the enzyme was isolated and purified from the fermentation products.ResultsThrough optimization, the RiCDA activity of strain T22.7.1T reached 287.02 U/mL, which is 34.88 times greater than the original enzyme’s activity (8.0 U/mL). The natural CDA enzyme was purified with a purification factor of 31.83, and the specific activity of the enzyme solution reached 1200.33 U/mg. RiCDA exhibited good pH and temperature adaptability and stability, along with a wide range of substrate adaptabilities, effectively deacetylating chitin, chitooligosaccharides, N-acetylglucosamine, and other substrates.DiscussionProduct analysis revealed that RiCDA treatment increased the deacetylation degree (DD) of natural chitin to 83%, surpassing that of commercial chitosan. Therefore, RiCDA demonstrates significant potential as an efficient deacetylation tool for natural chitin and chitooligosaccharides, highlighting its applicability in the biorefining of natural polysaccharides.

The expression of polysialic acid (polySia) on the neuronal cell adhesion molecule (NCAM) is called NCAM-polysialylation, which is strongly related to the migration and invasion of tumor cells and aggressive clinical status. Thus, it is important to select a proper drug to block tumor cell migration during clinical treatment. In this study, we proposed that lactoferrin (LFcinB11) may be a better candidate for inhibiting NCAM polysialylation when compared with CMP and low-molecular-weight heparin (LMWH), which were determined based on our NMR studies. Furthermore, neutrophil extracellular traps (NETs) represent the most dramatic stage in the cell death process, and the release of NETs is related to the pathogenesis of autoimmune and inflammatory disorders, with proposed involvement in glomerulonephritis, chronic lung disease, sepsis, and vascular disorders. In this study, the molecular mechanisms involved in the inhibition of NET release using LFcinB11 as an inhibitor were also determined. Based on these results, LFcinB11 is proposed as being a bifunctional inhibitor for inhibiting both NCAM polysialylation and the release of NETs.

为提高低品位锰矿的利用,实现有机废水对锰矿物粉的浸提,通过改造微生物燃料电池(MFC),在阴极反应池中增加电解室及矿物质室,构建用于锰矿湿法浸提的MFC反应器,实现有机废水与锰矿物粉的还原氧化反应的分离.结果显示,以乙酸钠为碳源,以低品位锰矿粉为阴极的MFC输出电压最高可达0.81 V,是以K3[Fe(CN)6]为阴极液(0.631 V)的1.23倍.在相同条件下,矿物浸提MFC对COD的去除率可达90.9％,高于对照组(87.5％),而降解时间为3 d,显著少于对照组(5.2 d).在降解COD能力的提升上,可实现对低品位锰矿粉中MnO2的还原,其锰含量由原矿粉的23％降至0.98％,浸提率可达95.7％.进一步通过高通量测序对阳极端菌群结构进行分析发现,其主要菌群为地杆菌属(Geobacter),相对于对照组的85％,矿物浸提MFC中该属比例占总菌群的95％.结果表明,矿物浸提MFC可有效地将有机降解与矿物浸提耦合,避免两者直接混合反应带来的二次废水污染及浸提产物硫酸锰的净化问题,在提高MFC产电效率、处理废水的同时,实现对低品位锰矿的浸提.

目的 以胶体几丁质为唯一碳源的培养基,从广西茅尾海红树林桐花树根际土壤中分离几丁质降解菌株,并应用复筛培养基筛选产几丁质酶菌株,结合平板对峙法挖掘能高效抑制植物病原真菌生长的活性菌株,为红树林来源微生物农用生物防治药物的研发奠定基础.方法 采用平板稀释涂布法和划线法分离纯化菌株,并应用复筛培养基通过透明圈观察法检测菌株几丁质酶活;采用Chelex-100法提取菌株基因组DNA,通过PCR扩增菌株的16S rRNA基因序列,上传至EzBioCloud数据库进行在线比对;采用平板对峙法筛选抗植物病原真菌的活性菌株;采用PKS和NRPS基因的扩增引物分别检测基因组聚酮合酶基因与非核糖体肽合成酶基因.结果 从桐花树根际土壤中分离获得28株几丁质降解菌,隶属于10目10科17属;其中5株几丁质降解菌对4种植物病原真菌均显示出抑菌活性,抑菌活性阳性率为17.86％;并在17株几丁质降解菌中检测到抗生素生物合成基因.结论 广西茅尾海红树林桐花树根际土壤来源的细菌及放线菌是重要的几丁质酶菌种资源,它们在抗植物病原真菌方面表现出良好的应用潜力,在新型绿色海洋生物农药、新颖结构海洋药物及其他高赋值产品开发和利用方面具有应用前景.

The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. It has been proposed that NCAM polysialylation could be inhibited by two types of heparin inhibitors, low molecular heparin (LMWH) and heparin tetrasaccharide (DP4). This review summarizes how the interactions between Polysialyltransferase Domain (PSTD) in ST8SiaIV and CMP-Sia, and between the PSTD and polySia take place, and how these interactions are inhibited by LMWH and DP4. Our NMR studies indicate that LMWH is a more effective inhibitor than DP4 for inhibition of NCAM polysialylation. The NMR identification of heparin-binding sites in the PSTD may provide insight into the design of specific inhibitors of polysialylation.

Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of α2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction, the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.

Profiling the transcriptome changes involved in xylose metabolism by the fungus Trichoderma reesei allows for the identification of potential targets for ethanol production processing. In the present study, the transcriptome of T. reesei HJ-48 grown on xylose versus glucose was analyzed using nextgeneration sequencing technology. During xylose fermentation, numerous genes related to central metabolic pathways, including xylose reductase (XR) and xylitol dehydrogenase (XDH), were expressed at higher levels in T. reesei HJ-48. Notably, growth on xylose did not fully repress the genes encoding enzymes of the tricarboxylic acid and respiratory pathways. In addition, increased expression of several sugar transporters was observed during xylose fermentation. This study provides a valuable dataset for further investigation of xylose fermentation and provides a deeper insight into the various genes involved in this process.

采用毛细管差示扫描量热法,分析Ca2+对嗜热菌A noxybacillus sp.来源的α-淀粉酶AGXA的热稳定性的影响及其机制.脱气后的蛋白样品和缓冲液,分别加入对应的样品池和缓冲液池中,测量温度为10℃～120℃,升温速率为1℃/min.测量结果中,纵坐标为Cp,横坐标为温度,去折叠变化曲线峰最高点对应的横坐标为蛋白质去折叠温度Tm,去折叠变化曲线与对应温度的积分值为热焓变化值△H,范特霍夫焓变化值△Hv由去折叠变化曲线峰形状决定.根据改进的吉布斯-亥姆霍兹方程计算AGXA的自由能变化值△G,绘制AGXA自由能变化与温度关系的稳定性曲线.结果 表明:有Ca2+和无Ca2+存在时,AGXA的△Hv与△H的比值都约等于1.0;有Ca2+时,AGXA的Tm、△H和△Cp值,分别为77.8℃、292.5 kcal/mol和2.76 kcal·mol-1·℃-1;无Ca2+时,AGXA的Tm、△H和△Cp值,则分别为67.3℃、238.3 kcal/mol和3.81 kcal·mol-1·℃-1;有Ca2+时的AGXA,其Tm、△H值分别比无Ca2+时的高,△Cp值则比无Ca2+时的低.有Ca2+和无Ca2+的α-淀粉酶AGXA的热变性过程皆为不可逆的双态模式,有Ca2+的AGXA,通过增大△H和降低△Cp的方式提高其热稳定性.研究揭示了Ca2+提高AGXA的热稳定性机制,为进一步扩大该酶的应用提供了理论和实践支撑.

: ST8Sia II (STX) is a highly homologous mammalian polysialyltransferase (polyST), which is a validated tumor-target in the treatment of cancer metastasis reliant on tumor cell polysialylation. PolyST catalyzes the synthesis of α2,8-polysialic acid (polySia) glycans by carrying out the activated CMP-Neu5Ac (Sia) to N- and O-linked oligosaccharide chains on acceptor glycoproteins. In this review article, we summarized the recent studies about intrinsic correlation of two polybasic domains, Polysialyltransferase domain (PSTD) and Polybasic region (PBR) within ST8Sia II molecule, and suggested that the critical amino acid residues within the PSTD and PBR motifs of ST8Sia II for polysialylation of Neural cell adhesion molecules (NCAM) are related to ST8Sia II activity. In addition, the conformational changes of the PSTD domain due to point mutations in the PBR or PSTD domain verified an intramolecular interaction between the PBR and the PSTD. These findings have been incorporated into Zhou’s NCAM polysialylation/cell migration model, which will provide new perspectives on drug research and development related to the tumor-target ST8Sia II.

红树蚬(Polymesoda erosa)作为一种主要栖息于潮间带的沼泽地或红树林的双壳贝类,近年来很多研究都证明其具有做为海洋污染监测指示物种的潜力.红树蚬受六溴环十二烷(HBCD)不同浓度(0、0.086、0.860、8.600μg/dm3)及不同天数(1、3、11、15、22 d)胁迫后,从转录组上调文库中挑取6个线粒体编码基因:细胞色素c氧化酶亚基Ⅰ、Ⅲ(COX Ⅰ、COX Ⅲ),NADH脱氢酶亚基Ⅰ、Ⅲ(ND Ⅰ、ND Ⅲ),细胞色素b(Cyt b),无机焦磷酸酶(PPase),并用实时荧光定量PCR研究各基因在红树蚬各组织内的表达情况,因为基因不稳定性,利用β-actin作为内参对各目的基因所得数据进行均一化处理.结果发现:6个基因在HBCD胁迫后都有转录且差异表达.经生物统计学分析,胁迫后鳃及肝胰腺组织中各基因的表达量较对照组中有显著变化,总体上随着胁迫天数及浓度的增加呈增加趋势,但当胁迫浓度过高时,因线粒体产生的应激能力有限,表达量反而降低.最后从酶复合物在呼吸链、ROS生成、ATP合成中的作用等各方面分析了HBCD胁迫导致线粒体基因表达变化的原因,为进一步开展分子毒理研究和开发分子生物标志物在海洋监测中的应用奠定了基础.

BACKGROUND:Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed.OBJECTIVE:In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively).METHODS:Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package.RESULTS:According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and β-sheet, β-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme.CONCLUSION:This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

Background:The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies.Objective:To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes?Methods:Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4.Results:The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4.Conclusion:The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

Background: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotech-nology. Objective: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. Methods: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (half-life, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template. Results: With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly high-er than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α-helical contents increasing, and the transition from α-helices to β-sheet structures, which was obviously dif-ferent in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure. Conclusion: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

Bifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate.