Purification and properties of extracellular chitinases from the parasitic fungus Isaria japonica

Two chitinases (P-1 and P-2) induced with colloidal chitin were purified from the culture supernatant of Isaria japonica by chromatography on DEAE Bio-Gel, chromatofocusing and gel filtration with Superdex 75 pg. The enzymes were electrophoretically homogeneous and estimated to have a molecular mass of 43,273 (±5) for P-1 and 31,134 (±6) for P-2 by MALDI-MS. The optimum pH and temperature was 3.5–4.0 and 50°C for P-1 and 4.0–4.5 and 40°C for P-2. P-1 acted against chitosan 7B (degree of deacetylation, 65–74%) = glycol chitin> colloidal CHITIN = chitosan 10B (degree of deacetylation, above 99%) and P-2 against chitosan 7B> glycol CHITIN = chitosan 10B> colloidal chitin in order of activity. The products of hydrolysis of chitin and chitosan hexamer were analyzed by MALDI-MS. The products from the chitin hexamer obtained with P-1 were almost all dimers with only a small amount of trimer whereas those obtained with P-2 were mainly trimers with some dimer and tetramer. No hydrolysis of chitosan hexamer was observed. High homology in the amino-terminal sequence for chitinase P-1 was exhibited by chitinases from Trichoderma harzianum, Candida albicans and Saccharomyces cerevisiae in the range of 48–39%. The highest homology for Chitinase P-2 was shown by an endochitinase from Metarhizium anisopliae of 66%, while 44% homology was exhibited by chitinases of Leguminosae plants.     

Purification and properties of an extracellular inulinase from Rhizopus sp. strain TN-96

A filamentous fungus, Rhizopus sp. strain TN-96, was isolated from rhizosphere soil samples. An extracellular inulinase was purified from the culture filtrate of strain TN-96 grown on inulin by DEAE-Cellulofine A-500 and Sephacryl S-200 HP chromatographies. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent M(r) of 83 kDa. The purified enzyme had specific activities of 17 U/mg toward inulin (I) and 0.32 U/mg toward sucrose (S) (I/S ratio, 53). Inulinase activity was optimal at pH 5.5 and 40 degrees C. The inulinase exhibited an apparent K(m) value of 9.0 mM for inulin. The enzyme also hydrolyzed raffinose, but not bacterial levan.     

海带岩藻多糖的分离纯化及结构特性的初步研究

采用稀酸浸提和乙醇沉淀的方法提取海带岩藻多糖(Laminaria japonica Fucoidan,LJF),经DEAE-52分离纯化后,以LJF-2为研究对象,用SephadexG-200凝胶柱色谱、凝胶渗透色谱法和比旋度法对LJF-2的纯度进行鉴定,结果表明LJF-2为均一组分,其分子量为69518。红外光谱仪分析LJF-2有一般糖类物质的特征吸收峰,并且有S=O的吸收峰;X-射线表明LJF-2几乎不含结晶;波谱扫描实验暗示LJF-2在水溶液中可能不具有三股螺旋状构象,LJF-2的特性粘度值较大,表明此多糖在水溶液中具有较高的刚性。     

嗜热链球菌胞外多糖的分离纯化及理化性质研究

以一株从西藏灵菇中分离的高产胞外多糖嗜热链球菌为研究对象,利用IRIE培养基进行发酵培养,通过离心、除蛋白、醇沉、透析等分离手段获得嗜热链球菌胞外多糖,并通过DEAE纤维素52和DEAE Sepharose CL-6B柱进一步分离纯化。高效液相色谱证实该多糖为纯品。差热扫描量热(DSC)研究显示该多糖有两个熔点分别为120.83℃和212.89℃。热重分析(TGA)表明该多糖第一步热分解温度为264.88℃,第二步热分解温度为441.82℃。红外光谱分析揭示该多糖具有较长分子链,有β-糖苷键构型特征。     

薇菜多糖的分离纯化及体外抗氧化活性

通过水提醇沉法制备薇菜粗多糖（water-soluble polysaccharide of Osmunda japonica,WOJP）,并用二乙氨乙基（diethylaminoethyl,DEAE）-纤维素层析法对其进行分离纯化,获得2?个多糖组分薇菜中性糖（neutral WOJP,WOJP-N）和薇菜酸性糖（acidic WOJP,WOJP-A）。WOJP-N的分子质量为31.8?kDa,由鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖和阿拉伯糖组成,对应各成分物质的量比为9.1∶5.7∶13.3∶37.6∶5.6∶11.8;WOJP-A的分子质量为15.7?kDa,由鼠李糖、半乳糖醛酸、半乳糖和阿拉伯糖组成,对应各成分物质的量比为7.0∶56.4∶26.1∶5.2。傅里叶变换红外光谱分析结果表明,WOJP-N主要由β-构型的半乳糖组成;WOJP-A主要由吡喃半乳糖醛酸组成,且存在部分酯化修饰。体外抗氧化活性实验结果表明,WOJP的Fe3＋还原能力、1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）清除能力和超氧阴离子清除能力与VC相当,具有较好的抗氧化活性,其中WOJP-N和WOJP-A在WOJP发挥抗氧化活性时具有协同作用,两者均是WOJP发挥抗氧化活性的多糖组分。本研究结果可为进一步研究薇菜多糖的构效关系和开发功能性食品提供依据,并为薇菜的应用提供理论参考。     

Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus

Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.     

Purification and properties of pectinesterase from papaya

Pectinesterase (PE) was partially purified from papaya pulp, and its biochemical properties were studied. The enzyme was eluted in a single peak after DEAE-cellulose and Sephadex G-100 chromatography. The PE had a molecular weight of 53000 and showed an optimum pH of 8.0. Its activity was dependent on an NaCl concentration of 0.2M . The enzyme was heat stable: approximately 80% of the original activity remained after 60 min of heating at 50°C but completely inactivated by incubation at 80°C for 1 min. The activity was linear with time and protein concentration. The maximum reaction in 3 min was found at 60°C and the initial rate increased 9-fold from 20 to 60°C. The estimated K_m was 0.12g litre^?1 with citrus pectin as the substrate. The kinetic study revealed that polygalactur-onic acid is a competitive inhibitor, and a K_i value of 0.07 g litre^?1 was determined. On the basis of this study, papaya PE properties resembled those of pectinesterase from other sources.     

Detoxification of bisphenol A and nonylphenol by purified extracellular laccase from a fungus isolated from soil

Purified laccase from a fungus (family Chaetomiaceae) was used for the enzymatic oxidation of bisphenol A and nonylphenol, endocrine-disrupting chemicals. It rapidly oxidized both chemicals in the absence of mediators and within 24 h their estrogenic activities were completely removed.     

猫棒束孢真菌多糖提取工艺研究

为优化猫棒束孢真菌多糖提取工艺,采用液体发酵法培养猫棒束孢菌丝体,采用水溶醇沉法提取猫棒束孢真菌多糖,采用苯酚-硫酸比色法测定多糖含量,通过单因素实验和L9（33）正交实验,确定猫棒束孢真菌多糖的最佳提取工艺条件为:水料比6:1,提取液浓缩至原料重量的2倍,加入浓缩液4倍的无水乙醇,在此条件下多糖的提取率为16.24%。      

大孔吸附树脂法纯化槐米总黄酮的研究

对大孔吸附树脂纯化槐米总黄酮的工艺进行了研究.方法是采用优选树脂纯化槐米总黄酮,以分光光度法测定槐米总黄酮含量.结果表明AB-8树脂吸附与纯化槐米总黄酮的效果较好,其工艺参数为:原液上样质量浓度为2.802mg/mL,最大吸附量为30.8mg/g,吸附速度为1 BV/h,上样2次,吸附时间为1 h,洗脱剂为45%乙醇,用量为3BV,洗脱速度为4BV/h,且树脂可重复使用3次.     

海带多糖纯化及清除自由基活性研究

海带多糖经S-300凝胶层析分离后得到纯化组分WPS-1-1。采用高效液相色谱、红外光谱对WPS-1-1结构进行解析,结果表明,WPS-1-1分子量为112ku,主要由岩藻糖、木糖、半乳糖和甘露糖构成,其摩尔比为6.9∶3.3∶1.5∶1。WPS-1-1含有β-吡喃糖苷键和硫酸基。体外自由基清除实验结果表明,WPS-1-1具有超氧阴离子自由基和羟自由基清除活性,其IC50分别为0.35、1.0mg/mL,可作为天然的功能性自由基高效清除剂。      

海带内生真菌的分离鉴定及其抑制乙酰胆碱酯酶活性的发酵工艺优化

本文以青岛海域的海带为原料,对其进行内生真菌的分离纯化,共得到7株内生真菌,采用改良Ellman法对其乙酰胆碱酯酶（ACh E）抑制活性进行筛选,并对其中一株活性最高内生真菌ML-X进行形态学和18S rDNA分子生物学鉴定,通过单因素实验和正交实验筛选出最适合该菌进行液体生长发酵的培养条件。结果表明,分离获得的菌株ML-X为白地霉属,序列号为KY977411,与遗传距离最近的Galactomyces geotrichum strain LMA-21株（JQ668739）同源性达99%。最佳发酵条件为:PS培养基,培养基的初始p H6.0,接种量5%,装瓶量150 m L/500 m L,培养温度31℃,发酵8 d,当发酵液粗浸膏为5 mg/m L时,对乙酰胆碱酯酶的抑制率可达28.91%±0.25%。      

Purification and characterisation of two extracellular acid proteinases from Monascus pilosus

Genus Monascus is one of the most important microorganisms in the fermentation industry in Asia. However, only a little attention has been paid to the proteinases produced by this fungus and their role in the fermentation process. The main objective of this study was to purify and characterise acid proteinases produced by Monascus pilosus. Two acid proteinases (MpiAP1 and MpiAP2) were purified to homogeneity. Both purified enzymes, MpiAP1 and MpiAP2, were monomeric structures with molecular masses of around 43 and 58 kDa, respectively. The former was an acidic non-glycoprotein, whereas the latter was an acidic glycoprotein with 27% carbohydrate content. Although amino-terminal amino acid sequence analysis of both enzymes (MpiAP1 and MpiAP2) of 20 amino acid length showed over 90% similarity, their amino-terminal amino acids were different from each other. Both enzymes were optimally active at 55 °C and at pH 2.5–3.0 against casein or human haemoglobin. The T_1/2 values of MpiAP1 and MpiAP2 were 65 and 70 °C, respectively. Both of the enzymes were completely inhibited by pepstatin A, and markedly by SDS. MoO₃ also showed a partial inhibition of both enzymes. Milk casein and haemoglobin were good substrates for these enzymes. Eleven cleavages were detected using the oxidised insulin B-chain as a peptide for the proteolytic specificity test of MpiAP1, while seven cleavages were detected for MpiAP2.     

蝉花菌质的营养及脂质成分分析

本研究以蝉花菌质为试材,采用常规分析法测定一般营养成分;氯仿-甲醇改良法提取总脂肪,硅胶柱层析法分离总脂肪中的极性、中性脂质,TLC分析鉴定其功能性脂质成分;高效液相色谱法和气相色谱法分别测定氨基酸及脂肪酸组成。结果表明:蝉花菌质干物质中的粗蛋白、总脂肪、灰分和粗纤维含量分别为15.22%、1.47%、1.33%和5.54%。钙和磷以及维生素A和维生素E含量分别为0.27%、0.13%、266.59 mg/100 g、62.98 mg/100 g。TLC分析表明脂肪成分中含有鞘磷脂(SM)、卵磷脂(PC)、脑磷脂(PE)和甘油三酯(TG)等功能性脂肪。蝉花菌质富含多不饱和脂肪酸,占脂肪酸总量的48.06%,其中亚油酸含量高达47.94%。蝉花菌质含17种氨基酸,总量为88.27mg/g,其中必需氨基酸占氨基酸总量的53.11%。综上结果,蝉花菌质富含各类营养素,可作为食品或动物饲料添加剂开发利用。     

Purification and characterization of an extracellular beta-agarase from Bacillus sp. MK03

A new beta-agarase was purified from an agarolytic bacterium, Bacillus sp. MK03. The enzyme was purified 129-fold from the culture supernatant by ammonium sulfate precipitation, anion exchange and gel filtration column chromatographic methods. The purified enzyme appeared as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Estimation of the molecular mass by SDS-PAGE and gel filtration gave values of 92 kDa and 113 kDa, respectively. The N-terminal amino acid sequence of the enzyme showed no homology to those of other known agarases. The optimum pH and temperature for this enzyme were 7.6 and 40 degrees C, respectively. The predominant hydrolysis product of agarose by this enzyme was neoagarotetraose, indicating the cleavage of beta-1,4 linkage. This enzyme could hydrolyze neoagarohexaose to produce neoagarotetraose and neoagarobiose; it could not hydrolyze these products. The enzyme digested agarose by endo-type hydrolysis.     

金银花过氧化物酶的三相分离纯化及酶学性质

采用三相分离法提取纯化金银花中过氧化物酶,结果表明最优纯化条件为pH?5.60、硫酸铵质量浓度39.49?g/100?mL、提取液与叔丁醇体积比1∶1.38,在该条件下纯化倍数为5.849,回收率为87.64%。金银花过氧化物酶比活力为1 021.6 U/mg,色素清除率为92%;酶学性质研究表明:金银花过氧化物酶最适温度为30℃,热稳定范围为10~40℃;最适pH值为5,pH值稳定范围为4~7。在金银花过氧化物酶催化的双底物酶促反应中,当H_2O_2浓度一定时,酶对愈创木酚的Km值为8.12?mmol/L,vmax值为1.71?mmol/(min·L)。当愈创木酚浓度一定时,H_2O_2的Km值为0.822?mmol/L,vmax值为1.38?mmol/(min·L)。金银花过氧化物酶与底物的亲和力由强到弱依次是邻苯三酚、邻苯二酚、联甲氧基苯胺、愈创木酚。Ca~(2+)、Cu~(2+)、Zn~(2+)对金银花过氧化物酶有激活作用,Mg~(2+)、Mn~(2+)、柠檬酸、抗坏血酸、L-半胱氨酸、亚硫酸钠、偏重亚硫酸钠、十二烷基磺酸钠对金银花过氧化物酶有不同程度的抑制作用。