顶青霉木聚糖酶的分离纯化与鉴定

使用硫酸铵分级沉淀、ＳｅｐｈａｄｅｘＧ－２５凝胶色谱脱盐、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５和ＣＭ－ＳｅｐｈａｄｅｘＣ－５０离子交换色谱等分离纯化技术,从顶青霉（Ｐｅｎｃｉｌｉｕｍｃｏｒｙｌｏｐｈｉｌｕｍ）培养液中分离到３种木聚糖酶组分,分别称为ＰａｒｔＡ、ＰａｒｔＢ和ＰａｒｔＣ．ＰａｒｔＢ和ＰａｒｔＣ经ＳｅｐｈａｃｒｙｌＳ－２００凝胶过滤色谱和ＣｏｎＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和色谱进一步分离纯化,分别得到２个纯木聚糖酶组分．ＰａｒｔＢ经ＳＤＳ－ＰＡＧＥ鉴定为单带,相对分子质量为２４２００;ＰａｒｔＣ经ＳＤＳ－ＰＡＧＥ鉴定基本为单带,相对分子质量为４８３００     

稻米中普鲁兰酶的纯化与性质的研究

稻米的普鲁兰酶（ＰｕｌｌｕｌａｎａｓｅＥ．Ｃ．３．２．１．４１）用ＤＥＡＥ－纤维素吸附，ＳｅｐｈａｄｅｘＧ－１００凝胶过滤，聚丙烯酰胺盘状凝胶电泳纯化，得到了聚丙烯酰胺凝胶电泳均一的纯酶。纯酶作用的最适温度为５０℃，最适ｐＨ为５．５。以普鲁兰（Ｐｕｌｌｕｌａｎ）为底物酶的米氏常数Ｋｍ为０．０１２５％，以支链淀粉为底物，酶的米氏常数Ｋｍ为２．５％，用ＳｅｐｈａｄｅｘＧ－１００凝胶层析法测得酶的分子量为７００００。     

黑曲霉β-D甘露聚糖酶的纯化及基本性质

设计了利用硫酸铵分级沉淀，凝胶过滤层析和羟基磷灰石吸附层析相结合及二次羟基磷灰石吸附层析二种分离方案，使一种黑曲霉β－Ｄ甘露聚糖酶得到纯化，纯化酶经聚丙烯酰胺凝胶电泳显示基本为单一蛋白带。凝胶过滤法测的全酶相对分子质量为４９０００，ＳＤＳ－ＰＡＧＥ法测得相对分子质量为５１０００．聚丙烯酰胺等电聚焦测得此纯化酶等电点为３．８．     

蚯蚓血纤蛋白溶酶提取工艺研究

以新鲜蚯蚓为材料,先采用硫酸铵沉淀出粗酶,再用Ｓｅｐｈａｄｅｘ－５０,ＤＥＡＥ－纤维素层析方法从蚯蚓粗酶中分离纯化出血纤蛋白溶酶,为开发、生产纤溶酶提供参考。     

蚯蚓血纤蛋白溶酶提取工艺研究

以新鲜蚯蚓为材料,先采用硫酸铵沉淀出粗酶,再用ＳｅｐｈａｄｅｘＧ－５０,ＤＥＡＥ－纤维素层析方法从蚯蚓粗酶中分离纯化出血纤蛋白溶酶,为开发、生产纤溶酶提供参考。     

广东芝麻香蕉加工中的酶褐变研究（Ⅰ）──多酚氧化酶的提取、分离及其特性研究

由于香蕉加工中的酶褐变主要是多酚氧化酶引起的，因此本文研究了多酚氧化酶的提取及其特性和交联葡聚糖Ｇ－７５（ＳＥＰＨＡＤＥＸＧ－７５）分离纯化；比较了加入ＰＶＰ（聚乙烯吡咯酮）和ＰＥＧ（聚乙二醇）的效果，发现对酶特性有一定的影响。通过差热扫描分析，由ＳＥＰＨＡＤＥＳＧ－７５分离的两种不同形式的多酚氧化酶蛋白的变性温度分别为９３．０３℃和８９．４２℃；最适ｐＨ分别为６．８和５．９．多酚氧化酶与邻－二酚反应产物在λ＝４１４ｎｍ下有最高吸收。     

平菇多糖的分离纯化及其对超氧自由基的效应

平菇经热水抽提,乙醇沉淀,Ｓｅｖａｇ法去蛋白,乙醇分级分离,再经ＤＥＡＥ－纤维素柱层析得纯化多糖ＰＳＩ和ＰＳⅡ,分别经聚丙烯酰胺凝胶电泳分析都呈一第带。在体外产生超氧阴离子自由基Ｏ＾－的反应体系中,平菇多糖组分ＰＳⅠ和ＰＳⅡ在较低浓度下都有清除Ｏ＾－２的作用,而在较高浓度下作用不明显。     

酶法改变甘草苷糖醛酸基提高其甜度的研究(Ⅱ)——能水解甘草苷糖醛酸基酶的提纯与酶性质研究

以酶法改变甘草苷糖醛酸基提高其甜度为目的,对新分离筛选的甘Ｍ－２和甘Ｍ－６霉菌产的β－葡萄糖醛酸苷酶进行了分离提纯和酶学方面的研究。结果表明,两株菌产的β－葡萄糖醛酸苷酶被６０％饱和硫酸铵沉淀较好,经ＤＥＡＥ－ＣｅｌｕｏｓｅＤＥ５２离子交换层析柱梯度洗脱,得到纯酶,提纯倍数分别为１０．６７和６．１５倍,收率分别为３３．０％和２４．２％,其中甘Ｍ－２菌产β－葡萄糖醛酸苷酶只能将甘草苷水解成甘草次酸（ＧＡ）;甘Ｍ－６菌产β－葡萄糖醛酸苷酶水解甘草苷主要变成甜度很高的单葡萄糖醛酸基甘草苷（ＧＡＭＧ）;两种酶在ＳＤＳ－聚丙烯酰胺凝胶电泳都得到电泳单点,其酶蛋白相对分子质量分别为６００００和４２０００;酶反应最适ｐＨ分别为５．０和６．０,最适温度均为４０℃,均在ｐＨ４．０～８．０和２０～７０℃范围内相对稳定。     

烟草内切多聚半乳糖醛酸酶抑制蛋白分离纯化研究

首次报道烤烟中存在ＰＧＩＰ，并以烤烟苗为材料通过硫酸铵沉淀、离子交换层析和分子筛凝胶过滤等步骤分离得到了ＰＧＩＰ，经高ｐＨＤｉｓｃ－ＰＡＧＥ和ＳＤＳ－Ｄｉｓｃ－ＰＡＧＥ分析为电泳纯，紫外吸收为典型的蛋白质吸收光谱。烤烟ＰＧＩＰ是一种糖蛋白，含糖量为６．０３％，等电点为６．４和６．１。     

螺旋藻多糖提取新工艺的研究

提取螺旋藻多糖的传统工艺存在着流程长,操作复杂,有机溶剂消耗大等问题。采用三氯乙酸（ＴＣＡ）法代替Ｓｅｖａｇ法去除蛋白质,使改进后的提取工艺中蛋白质去除率增加,多糖损失率降低,流程缩短。通过用ＤＥＡＥ－Ｓｐｅｈａｒｏｓｅ和Ｓｅｐｈａｄｅｘ－Ｇ５０柱的分离纯化得到２个多糖组分。经测定,其相对分子质量分别为Ｍｒ１＝１２６００,Ｍｒ２＝１６６００。组分１的单糖组成：Ｄ－葡萄糖、Ｄ－甘露糖、Ｄ－半乳糖和葡萄糖醛酸;组分２的单糖组成：Ｄ－葡萄糖、Ｄ－木糖、Ｌ－鼠李糖和葡萄糖醛酸。     

黑曲霉N5-5单宁酶的纯化及酶学性质测定

采用中空纤维膜超滤和葡聚糖凝胶层析相结合的方法对黑曲霉N5-5单宁酶进行纯化,然后对纯酶性质进行测定。结果显示,黑曲霉N5-5单宁酶用该方法纯化后,可纯化近20倍,酶活力可回收23.30%。对纯酶作十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,可知黑曲霉N5-5单宁酶为分子质量64.2 k D的单肽链蛋白。纯酶的酶促反应最适温度为45℃,且在25~45℃范围内热稳定性良好;酶促反应最适p H值为5.0,且在p H 5.0~5.5范围内酸碱稳定性良好。另外,反应动力学测定结果表明,该酶对底物没食子酸丙酯的米氏常数K_m为0.916 mmol/L,最大反应速率vmax为0.877 mmol/(L·min)。     

黑曲霉N5-5产单宁酶的酶学性质与固定化

本实验室自主分离的黑曲霉N5-5所产单宁酶已发现对没食子酸丙酯具有良好酶解效果。为了单宁酶的工业化应用,本次研究大批量培养单宁酶,探究其对单宁酸的酶解效果及酶的固定化效果。发酵采用先液体扩培后固体发酵的形式,提酶后利用陶瓷膜过滤技术纯化、浓缩酶液,并对比冷冻干燥和喷雾干燥两种不同干燥方式,还使用树脂载体对酶进行固定化。实验表明,纯化后单宁酶酶活达258.53 U/mL,可水解10%至50%浓度的单宁酸,其中30%以下底物浓度酶解效果较好;冷冻干燥对酶活影响不大而喷雾干燥使酶活降为174.02 U/mL;另外,单宁酶通过树脂载体固定化后,在实验条件下可重复使用至少4次。研究为提高黑曲霉N5-5发酵所产单宁酶的媒介效率、降低酶解反应成本、实现商业化生产建立了理论基础。     

Bacillus sphaericus: the highest bacterial tannase producer with potential for gallic acid synthesis

An indigenously isolated strain of Bacillus sphaericus was found to produce 1.21 IU/ml of tannase under unoptimized conditions. Optimizing the process one variable at a time resulted in the production of 7.6 IU/ml of tannase in 48 h in the presence of 1.5% tannic acid. A 9.26-fold increase in tannase production was achieved upon further optimization using response surface methodology (RSM), a statistical approach. This increase led to a production level of 11.2I U/ml in medium containing 2.0% tannic acid, 2.5% galactose, 0.25% ammonium chloride, and 0.1% MgSO(4) pH 6.0 incubated at 37°C and 100 rpm for 48 h with a 2.0% inoculum level. Scaling up tannase production in a 30-l bioreactor resulted in the production of 16.54 IU/ml after 36 h. Thus far, this tannase production is the highest reported in this bacterial strain. Partially purified tannase exhibited an optimum pH of 5.0 with activity in the pH range of 3 to 8; 50°C was the optimal temperature for activity. Efficient conversion of tannic acid to purified gallic acid (90.80%) was achieved through crystallization.     

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.     

Purification and Biochemical Characterization of Tannase from a Newly Isolated Strain of Paecilomyces Variotii

An extracellular tannase was isolated from Paecilomyces variotii and partially purified using ammonium sulphate precipitation followed by DEAE-Sepharose ion exchange chromatography. Paecilomyces variotii is a newly isolated strain obtained in São Paulo, Brazil, from the screening of 500 fungi evaluated for their production of tannase. The tannase was separated into two peaks. SDS-PAGE analysis indicated that the purified enzyme migrated as a single protein band corresponding to molecular mass of 87.3 kDa (major peak) and 71.5 kDa (minor peak). The peaks eluted very close together between 150 and 250 mM NaCl. DEAE-Sepharose column chromatography led to an overall purification of 19.3 fold. The K_m was found to be 0.61 μmol and the V_max?=?0.55 U.mL^?1. Temperatures from 40 to 65°C and pH values from 4.5 to 6.5 were optimum for tannase activity and stability. This tannase could find potential use in the food-processing industry.     

薤白多糖的分离纯化及组成分析

采用DEAE-纤维素离子交换柱层析和SephadexG-150凝胶柱层析,对薤白粗多糖(PAM)进行了分离纯化;利用醋酸纤维素薄膜电泳和凝胶过滤法鉴定精制薤白多糖纯度;硅胶薄层层析分析精制多糖的单糖组成。结果表明:DEAE-纤维素柱分别用水、0.1mol.L-1NaCl、0.5mol.L-1Na0H洗脱分得三种级分,即水洗级分(PAM-I)、盐洗级分(PAM-II)和碱洗级分(PAM-III);这三种级分再经过SephadexG-150柱进一步分离纯化,得三个主要级分PAM-Ib、PAM-IIa及PAM-III’;经醋酸纤维素薄膜电泳和凝胶过滤法鉴定,它们为均一的多糖;硅胶薄层层析分析显示,PAM-Ib主要由半乳糖和葡萄糖组成,PAM-IIa主要由半乳糖、葡萄糖、果糖、木糖和鼠李糖组成,PAM-III’主要由半乳糖、葡萄糖和木糖组成。     

罗汉果蛋白酶的分离纯化和部分性质研究

对罗汉果蛋白酶的分离纯化和部分性质进行研究.利用硫酸铵盐析、离子交换和凝胶过滤柱层析对罗汉果蛋白酶进行了分离纯化,对纯化所得的酶进行了分子量和等电点测定.结果表明,纯化酶为电泳纯,回收率为4.2％,纯化倍数为31.1,凝胶过滤层析和SDS-PAGE电泳测得酶的分子量分别为40.3 ku和39.0 ku,等电聚焦电泳测得其等电点为8.55.     

ISOLATION OF MUCOR MIEHEI AND M. PUSILLUS ASPARTIC PROTEINASES FROM PARTIALLY PURIFIED SOURCES USING PREPARATIVE ISOELECTRIC FOCUSING

Dialysis, preparative isoelectric focusing (IEF) and gel filtration were employed to obtain final enrichments of 2.2-fold and 1.4-fold for Mucor miehei (CBS 360.75) proteinase (MMP) and M. pusillus (var. Lindt) proteinase (MPP), respectively, from partially purified sources. Recovery of total activity (percent yield) of MMP was 38% following preparative IEF and 6.7% after gel filtration. Percent yield of MPP was 61% following preparative IEF and 15% after gel filtration. Preparative IEF fractionation with a lowpH (3–5) ampholyte produced most of the increase, and partitioned brown pigments into fractions of pH 2.0 to 3.5. The protocol produced proteinases of high purity as indicated by SDS-PAGE. The isoelectric point, molecular weight, extinction coefficient and amino acid composition of MMP and MPP proteinases were in agreement with previously determined values. Secondary structure analysis confirmed previous findings that these enzymes contain a high proportion of β-sheet structure. Purifications starting with 4 to 5 g of crude partially purified MMP preparation (at least 50% NaCl) yielded between 18 and 28 mg of purified protein.     

小球藻锌结合类金属硫蛋白的提取和分离

小球藻经60μmol/L的锌胁迫培养5天后,离心收集藻泥,经超声波细胞破碎后,离心得到上清液即蛋白质粗提液。粗提液经葡聚糖凝胶层析柱G-75及脱盐柱G-25分离、纯化后,收集的蛋白组分经真空冷冻干燥后获得小球藻锌结合蛋白。实验测定了在不同pH值下小球藻锌结合蛋白的紫外光谱特征,发现该蛋白质的紫外光谱特征与兔肝金属硫蛋白(Zn-MTs)十分相似。经阴离子交换层析柱(DEAE Sephadex A-25)及反相液相色谱分离表明,小球藻锌结合蛋白的色谱行为与兔肝Zn-MTs有一定的相似性。此外,经Tricine-SDS-PAGE法测定该蛋白质的分子量约为8.2kDa;蛋白质分子中半胱氨酸含量达15%以上。由此可见,小球藻在锌胁迫下可诱导生成与兔肝Zn-MTs相似的锌结合蛋白质,即类金属硫蛋白(Zn-MT-like)。每克鲜藻可诱导Zn-MT-like蛋白达0.2g以上。     

Purification and characterization of a cysteine protease in Vitis vinifera L. grapes (Macabeo variety)

Protease from Vitis vinifera L. (Macabeo variety) was partially purified (15-fold) by PVPP and Sephadex gel G-100 filtration and characterized afterwards by gel electrophoresis. The enzyme had an optimum pH of 2.50 and a Km value of 0.32 mM for haemoglobin. The results of applying diagnostic inhibitors indicated that the enzyme studied was a cysteine-protease.