酶促反应制备壳寡糖条件的研究

通过膜分离法从Microbacterium sp.OU01的发酵液中分离到内切壳聚糖酶ChiN，并对其酶解制备壳寡糖条件进行了优化，获得的酶解产物数均分子量为1200。经薄层层析与HPLC分析，降解产物中不含单糖，初步说明降解产物为壳寡糖。酶解制备壳寡糖反应条件温和，操作简便，为实现壳寡糖产业化奠定了基础。     

海洋寡糖工程药物——壳寡糖制备分离新工艺及其抗癌活性研究

以海洋甲壳多糖为原料,研究了酶法解制备壳寡糖的方法,通过对专一性和非专一性除解用酶的筛选,确定以壳聚糖酶或6036要降为降解酶;考察了底物浓度、酶量、温度、pH、溶解介质等因素对降解反应的影响;提出了反应与分离相耦合制备壳寡糖的新构思,在此基础上进行了扩大试验,确认了过程实现工业化生产的可行性,对所生产壳寡糖的抑瘤活性研究证明一定铁壳寡糖具有很好的生物活性,具有抗肿瘤药物前景。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度、pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解。结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%。     

壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响

目的检测壳寡糖对人肝癌SMMC-7721细胞的抑制效果及对凋亡调控蛋白Bcl-2和Caspase-3的影响。方法采用噻唑蓝（MTT）法检测不同浓度壳寡糖对肝癌细胞SMMC-7721细胞增殖的抑制作用,并利用荧光Hoechst33258染色法检测细胞凋亡状况。最后通过免疫细胞化学方法研究壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响。结果壳寡糖能够抑制SMMC-7721细胞增殖,并且促进SMMC-7721细胞的凋亡,并且壳寡糖能够上调促凋亡蛋白Caspase-3的表达和降低抑制凋亡蛋白Bcl-2的表达。结论壳寡糖对人肝癌SMMC-7721细胞的增殖有抑制作用,此作用可能是通过促进Caspase-3的表达和抑制Bcl-2的表达来实现的。     

Fe^2＋-H2O2体系降解壳聚糖

Fe2 -H2O2体系能够有效地降解壳聚糖,反应介质的pH值、反应时间、反应温度、Fe2 浓度及H2O2浓度等实验因素对壳聚糖的降解效果都有程度不同的影响,其中以反应介质的pH值和H2O2浓度对降解反应的影响为最大.在pH值为3～5时Fe2 -H2O2体系降解壳聚糖的活性最高.适当增大H2O2的用量可以增大壳聚糖的降解程度,但当其用量增大至一定程度后,壳聚糖降解产物分子量的下降趋势明显变缓.合理的Fe2 -H2O2体系降解壳聚糖的实验条件为:介质pH值3～5;温度,室温;时间60～90 min;壳聚糖:H2O2:Fe2 =240:12～24:1～2(摩尔比).     

里氏木霉几丁质酶表达及其水解产物组成与结构分析

优化并全合成里氏木霉几丁质酶基因,在毕赤酵母中实现分泌表达。产物几丁质酶的蛋白浓度达0. 17mg/ml,最适pH为5. 6,最适温度为65℃,酶活为0. 52U/ml。该酶在50℃及以下较稳定。利用该酶水解低脱乙酰度壳聚糖并对产物的组成及结构进行分析。超高效液相色谱-四极杆飞行时间质谱(ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry,UPLC-QTOF MS)检测及分析结果显示,酶解产物中包含至少41种聚合度2～18,不同脱乙酰度的壳寡糖组分;核磁共振(nuclear magnetic resonance,NMR)检测及分析结果显示,产物壳寡糖的还原端主要为N-乙酰氨基葡萄糖,非还原端则同时含有N-乙酰氨基葡萄糖及氨基葡萄糖。相关结果可为壳寡糖的结构与功能关系研究提供参考。     

小鼠肠道对壳寡糖的吸收率及吸收成分的影响

目的通过分析壳寡糖在小鼠肠道内的吸收率以及吸收成分,了解肠道对壳寡糖代谢的影响,初步判断壳寡糖的有效生物成分,为壳寡糖生物活性的进一步研究提供必要的实验材料和线索。方法首先用蜗牛酶将壳聚糖水解成聚合度不同的壳寡糖,并通过聚丙烯酰胺凝胶层析柱（Bio-Gel P4凝胶）将不同聚合度的壳寡糖分开,分别收集聚合度为1-3,8-11的壳寡糖,并用异硫氰酸荧光素（FITC）标记,再通过聚丙烯酰胺凝胶层析柱（Bio-Gel P-2/P4）将游离的FITC除去,随后对两组禁食24 h的小鼠分别用FITC标记的大分子量和小分子量的壳寡糖灌胃。1 h后取血清和小肠,经分离水溶成分后,通过荧光分光光度计检测血清样品和肠溶物样品中荧光的强度,进而确定壳寡糖的吸收率和吸收成分。结果通过改变酶解时间,蜗牛酶可以将壳聚糖水解成不同聚合度的壳寡糖。利用Bio-Gel PA聚丙烯酰胺凝胶层析柱可以将不同聚合度的壳寡糖分离成具有一定聚合度范围的壳寡糖。用F1TC标记的大、小分子量的壳寡糖给小鼠灌胃,从血清和肠溶物中均检测到荧光强度,两者比值平均值分别为5.68 ： 1和9.84 ： 1。结论壳寡糖在肠道的吸收率随分子量的减小而增大,除小分子壳寡糖外,吸收成分也包括部分大分子量壳寡糖。     

一种源于蜗牛的壳聚糖水解酶的纯化和定性

目的研究属于蜗牛的壳聚糖水解酶的纯化方法,得到壳聚糖水解酶的纯品,从而为氨基酸序列分析、基因克隆及工业菌制备奠定前期基础。方法建立检测蜗牛壳聚糖水解酶活性的手段并考察影响酶活性的各种因素,比较现有层析方法纯化蜗牛壳聚糖水解酶的实际效果,确定纯化的最佳条件,从而设计出最合理的纯化方案。结果经苯基琼脂糖柱层析,DEAE-Sepharose离子交换层析和Sephacryl S-300凝胶过滤分离,得到高纯高活性蛋白质,在SDS-PAGE上用银染的方法呈单一蛋白质条带,比活性提高33.333倍,纯化倍数为18.272,得率为0.15。结论实验建立了1种从蜗牛中分离高效高纯度壳聚糖水解酶的方法,为壳寡糖的酶解工业生产提供了新思路、新方法。     

木瓜蛋白酶水解壳聚糖的工艺研究

本文通过正交试验对木瓜蛋白酶水解壳聚糖的工艺进行优化,并对降解过程中粘度、还原糖等一些指标的动态变化进行研究。结果显示,木瓜蛋白酶在反应温度45℃下降解壳聚糖的最佳工艺条件为壳聚糖的脱乙酰度70%,pH4.0,底物浓度1%,壳聚糖与酶的比例为25∶1(w/w)。其中底物脱乙酰度对酶解效果影响呈极显著水平,pH值影响呈显著水平。木瓜蛋白酶可较为有效地降解脱乙酰度为70%的壳聚糖,在其最适条件下对壳聚糖水解约60min,可控制产物平均分子量在1万以内。木瓜蛋白酶起始降解速率很快,20min后VDP变化趋于平稳,60min后基本维持在93～94%上下。反应进行60min后产物的平均分子量约为9000。还原糖含量在反应进行150min之后,还原糖的生成速度基本趋于平稳。     

甲壳胺诱导人肝癌HepG2细胞凋亡的实验研究

目的:通过体外实验探讨甲壳胺是否对人肝癌细胞HepG2具有生长抑制及诱导凋亡作用.方法:在HepG2细胞培养液中加入不同浓度的甲壳胺,培养48 h,于倒置相差显微镜下观察甲壳胺处理组及对照组细胞形态学变化;用流武细胞术(FCM)检测HepG2细胞的凋亡率,Western印迹检测甲壳胺处理组及对照组Bcl-2和p53蛋白...     

绿色木霉粗纤维素酶液水解壳聚糖的研究

利用自制绿色木霉粗纤维素酶液降解壳聚糖制备低聚壳聚糖.采用粘度法、乙酰丙酮法和还原糖浓度分析,研究了温度、pH值及反应时间等因素对壳聚糖水解程度和产物相对分子质量的影响,并采用质谱法对水解产物进行定性分析.结果表明,粗纤维素酶液水解壳聚糖作用的最适pH为5.0、最适反应温度为50 ℃、最适反应时间为12 h.粗纤维素酶...     

Fermentation conditions and properties of a chitosanase from Acinetobacter sp. C-17

A species of bacterium with high chitosanase activity was isolated from soil samples in Haiyan City, China, and identified as an Acinetobacter species. This strain, named Acinetobacter sp. strain C-17, produced a chitosanase that was inducible and secreted into the medium. The optimal conditions for enzyme production were cells used to inoculate a medium containing 1% chitosan (pH 7.0) followed by culture at 30 degrees C. The chitosanase activity reached 1.7 U/ml when strain C-17 was incubated in a 250-ml flask under the optimal conditions for 24 h, and reached 2.8 U/ml when cells were incubated in a 3-l fermentor. The optimal pH and temperature for hydrolysis of chitosanase were 7.0 and 36 degrees C, respectively. The chitosanase activity was stable in the pH range of 5-8 and temperature range of 30-40 degrees C. The chitosanase of the strain was extracted by zinc acetate and ammonium sulfate precipitation. The molecular mass was estimated to be 35.4 kDa by SDS-PAGE.     

The use of chitosan solutions for defatting and clarification of protein hydrolysates]

The possibility of the use of small amounts of chitosan for defatting and clarification of protein solutions prepared by enzymatic hydrolysis was tested. The following treatment conditions were shown to be optimal: chitosan concentration range, from 1.0 to 1.5 g per kg raw weight; pH of precipitation medium, from 8.0 to 8.5; and duration of incubation of protein hydrolysate solution with chitosan, less than 1 h. The hydrolysate defatting grade was found to depend on the degree of chitosan deacetylation. A possible mechanism of the chitosan-induced effects was suggested. The use of chitosan allows the mass fraction of enzyme protein hydrolysates to be reduced fourfold to fivefold.     

Defatting and Clarification of Protein Hydrolysates by Using Chitosan Solutions

The possibility of the use of small amounts of chitosan for defatting and clarification of protein solutions prepared by enzymatic hydrolysis was tested. The following treatment conditions were shown to be optimal: a chitosan concentration range, from 1.0 to 1.5 gram per kilogram raw weight; pH of the precipitation medium from 8.0 to 8.5; and duration of the incubation of the protein hydrolysate solution with chitosan, less than 1 h. The hydrolysate defatting grade was found to depend on the degree of chitosan deacetylation. A possible mechanism of the chitosan-induced effects was suggested. The use of chitosan allows the mass fraction of enzyme protein hydrolysates to be reduced fourfold to fivefold.     

The potential of enzyme recycling during the hydrolysis of a mixed softwood feedstock

Despite recent improvement in cellulase enzymes properties, the high cost associated with the hydrolysis step remains a major impediment to the commercialization of full-scale lignocellulose-to-ethanol bioconversion process. As part of a research effort to develop a commercial process for bioconversion of softwood residues, we have examined the potential for recycling enzymes during the hydrolysis of mixed softwood substrate pretreated by organosolv process. We have used response surface methodology to determine the optimal temperature, pH, ionic strength, and surfactant (Tween 80) concentration for maximizing the recovery of bound protein and enzyme activity from the residual substrates after hydrolysis. Data analysis showed that the temperature, pH and surfactant concentration were the major factors governing enzyme desorption from residual substrate. The optimized conditions were temperature 44.4 °C, pH 5.3 and 0.5% Tween 80. The optimal conditions significantly increased the hydrolysis yield by 25% after three rounds of hydrolysis. This bound enzyme desorption combining with free enzyme re-adsorption is a potential method to recover cellulase enzymes and reduce the cost of enzymatic hydrolysis.     

Purification and Characterization of Three Chitosanase Activities from Bacillus megaterium P1

Bacillus megaterium P1, a bacterial strain capable of hydrolyzing chitosan, was isolated from soil samples. Chitosan-degrading activity was induced by chitosan but not by its constituent d-glucosamine. Extracellular secretion of chitosanase reached levels corresponding to 1 U/ml under optimal conditions. Three chitosan-degrading proteins (chitosanases A, B, and C) were purified to homogeneity. Chitosanase A (43 kilodaltons) was highly specific for chitosan and represented the major chitosan-hydrolyzing species. Chitosanases B (39.5 kilodaltons) and C (22 kilodaltons) corresponded to minor activities and possessed comparable specific activities toward chitosan, chitin, and cellulose. Chitosanase A was active from pH 4.5 to 6.5 and was stable on the basis of activity up to 45 degrees C. The optimum temperature for enzymatic chitosan hydrolysis was 50 degrees C. Kinetic studies on chitosanase A suggest that the enzyme is substrate inhibited. The apparent K(m) and V(max) determined at 22 degrees C and pH 5.6 were 0.8 mg/ml and 280 U/mg, respectively. End products of chitosan hydrolysis by each of the three chitosanases were identified as glucosamine oligomers, similar to those obtained for previously reported chitosanase digestions.     

High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes--cellulase, pepsin and lipase A--were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers released from chitosan by the other enzymes after a 20h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 degrees C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1h.     

影响纳豆激酶酶促反应速度因素的研究

利用分光光度法对由纳豆菌发酵产生的纳豆激酶 (NK)进行了动力学性质的研究。以双倒数作图法 (L -B作图法 )求取Km。采用单因素试验法和正交试验法研究了底物浓度、酶浓度、温度、pH值对酶促反应速度的影响。结果表明该纳豆激酶的Km值为 3.4 98× 1 0 -6g·mL-1 ,当水解时间为 1 0min时 ,最适底物浓度为 1 6mg·mL-1 ,最适温度为 6 0℃ ,最适 pH为 8.0。     

Purification and characterization of an intracellular beta-glucosidase from the protoplast fusant of Aspergillus oryzae and Aspergillus niger

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4.000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular beta-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme had an optimum pH of 5.4 and temperature of 65 degrees C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0-6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product ofglucoside hydrolysis. The K(m) and V(max) values against salicin as substrate were 0.035 mM and 1.7215 micromol min(-1), respectively.