Purification and characterization of extracellular chitinase from a novel strain Aspergillus fumigatus CS-01

Twelve samples derived from different locations in south central area of China are treated by enrichment and spread-plate technique for initial screening. Seven chitinase-producing strains are isolated. The chitinase present in the culture supernatant of strain CS-01 possesses the maximum activity of 0.118 U/mL. Analysis of the morphological feature and the ITS rDNA sequence reveals that strain CS-01 belongs to Aspergillus fumigatus. Production of the chitinase is regulated by a inducible way and the maximum activity appears at 36 h in colloidal chitin culture. Purification of the chitinase is carried out by salting out, gel filtrate chromatography and anion exchange chromatography sequentially. Native-PAGE and SDS-PAGE indicate that the chitinase from A. fumigatus CS-01 is a monomer with the relative molecular mass estimated to be 4.50×10⁴. Its maximum activity appears at pH 5 and 55 °C. The chitinase is stable at pH 4.0–7.5 and below 45 °C. Foundation item: Projects(50621063, 50674101) supported by the National Natural Science Foundation of China     

Production and characterization of alkaline extracellular lipase from newly isolated strain Aspergillus awamori HB-03

A strain HB-03 to produce alkaline extracellular lipase was isolated from oil-rich soil samples and identified as Aspergillus awamori.The growth conditions and nutritional factors for lipase production by strain HB-03 were optimized,and the maximum lipase production of (45.9±2.3) U/mL was obtained at 30 °C and pH 7.0 after 36 h using olive oil (1%) and sucrose (0.5%) as carbon sources and combination of peptone (2%),yeast extract (0.5%) and ammonium sulfate (0.1%) as nitrogen sources.The lipase was purified...     

黑曲霉葡萄糖氧化酶的分离制备及其应用研究

本文研究了用110树脂层析(Ⅰ)、硫酸铵盐析(Ⅱ)和硫酸铵盐析—DEAE—纤维素层析(Ⅲ)等三种方法分离制备黑曲霉葡萄糖氧化酶(GOD),特别对方法Ⅰ作了较详细的研究。试验表明,方法Ⅰ最为简便有效,适用于工业上生产GOD酶制剂。菌体破碎后得到的粗酶液经方法Ⅰ提纯,GOD可回收85％以上,提纯倍数3至5倍;经方法Ⅱ和Ⅲ提纯,GOD回收率分别为67％左右和53％左右,提纯倍数分别为3倍左右和4倍左右。 最适培养条件下,每升培养液可收获黑曲霉菌体7克左右,其内含有的GOD经110树脂层析分离得到大约4000单位淡黄色液体GOD酶制剂产品。产品中含有一定量的过氧化氢酶(CAT),能满足蛋品脱糖需要;用于鸡蛋蛋白脱糖,可在5小时内把蛋白中的葡萄糖由0.45％脱除到0.01％以下。小白鼠急性毒性试验表明,产品不引起小白鼠发生急性中毒。产品在0—4℃保存5个月,GOD酶活力基本无损失。     

海洋来源真菌烟曲霉(Aspergillus fumigatus)次级代谢产物研究(Ⅰ):生物碱

对一株海洋来源的微生物中的次级代谢产物进行了研究,从中分离并鉴别了5个喹唑啉生物碱类化合物(1~5),分别为fumiquinazoline G(1),fumiquinazoline F(2),fumiquinazoline A(3),fumiquinazoline C(4)和fumiquinazoline D(5)。     

哈茨木霉几丁质酶基因的cDNA克隆及表达

为了研究全新几丁质酶基因Chit37(DQ647957)的特性与功能,将该基因在大肠杆菌中进行了外源表达.通过构建哈茨木霉(Trichoderma harzianum)菌丝生长期的cDNA文库及对部分表达序列标签序列的测定与生物信息学分析,成功获得了基因Chit37的全长cDNA序列.该基因的编码框长度为1032bp,编码343个氨基酸,理论分子量为36.6kDa.采用PCR扩增的方法克隆该基因并将其构建到原核表达载体pET28(a+)上,构建了原核表达载体pET-Chit37并转化宿主菌BL21(DE3),菌落PCR及酶切鉴定均证实转化子的正确性,重组蛋白经IPTG诱导后获得表达.优化的表达条件为终浓度0.1mM IPTG诱导培养4h.     

球孢白僵菌几丁质酶基因chit1真核表达

采用PCR方法从球孢白僵菌的DNA中克隆出几丁质酶基因chit1,并将该序列构建到酿酒酵母(Saccharomyces cerevisiae)诱导型表达载体pYES2上,转化到酿酒酵母H158菌株中,通过Northern检验确定该基因在酿酒酵母中成功表达.经过酶活检测该转化子在培养48 h达到酶活高峰,达0.47 U/mL.     

海参肠中溶菌酶的分离纯化及其酶学性质

从海参肠中分离纯化溶菌酶,并测定其酶学性质及抑菌作用。在4℃下将海参肠打浆,用Tris-HCl缓冲液抽提。上清液经浊点萃取法(CPE)浓缩、阳离子交换柱(CM52纤维素)层析和SephadexG-75凝胶过滤层析分步纯化,得到电泳纯的溶菌酶。该酶的比活力达到3 026.1 U/mg,纯化倍数为18.7,活力回收为46.0%。对纯化的溶菌酶性质研究表明,该酶相对分子质量约为16 000,对溶壁微球菌的最适作用温度为35℃,最适作用pH 6.5,在45℃以下和pH 4.0~8.0都有较好的稳定性,并与常见金属离子和化学试剂有良好的配伍性。与通常从蛋清中提取的溶菌酶相比较,从海参中分离纯化的溶菌酶具有广谱杀菌功能,即对常见的革兰氏阴性菌和阳性菌细胞壁均有溶解作用。     

海参肠中溶菌酶的分离纯化及其酶学性质

从海参肠中分离纯化溶菌酶,并测定其酶学性质及抑菌作用。在4℃下将海参肠打浆,用Tris-HCl缓冲液抽提。上清液经浊点萃取法（CPE）浓缩、阳离子交换柱（CM52纤维素）层析和SephadexG-75凝胶过滤层析分步纯化,得到电泳纯的溶菌酶。该酶的比活力达到3026．1U／mg,纯化倍数为18．7,活力回收为46．0％。对纯化的溶菌酶性质研究表明,该酶相对分子质量约为16000,对溶壁微球菌的最适作用温度为35℃,最适作用pH6．5,在45℃以下和pH4．O～8．0都有较好的稳定性,并与常见金属离子和化学试剂有良好的配伍性。与通常从蛋清中提取的溶菌酶相比较,从海参中分离纯化的溶菌酶具有广谱杀菌功能,即对常见的革兰氏阴性菌和阳性菌细胞壁均有溶解作用。     

米曲霉HDF-7蛋白酶的分离纯化及酶学性质研究

对米曲霉 HDF-7 所产蛋白酶进行分离纯化,经硫酸铵沉淀和凝胶过滤层析得到电泳纯的蛋白酶,经SDS-PAGE 电泳测定其相对分子质量约为30 KDa.该酶的最适反应温度为50℃,最适作用pH值为7.0,该酶在20℃时具有良好的热稳定性,在pH6.0～8.0的条件下酶是相对稳定的,Mn2+对该蛋白酶有明显的激活作用,Cu2+对该蛋白酶有强烈的抑制作用,Km值为78μg/mL,最大反应速度V max为6.29μg/min.     

芦丁鼠李糖苷酶水解反应液中鼠李糖的分离纯化

利用微生物Absidiasp.R9g产的芦丁-α-鼠李糖苷酶(RhaR),水解芦丁制备鼠李糖。24.0g芦丁经酶解后,采用大孔吸附树脂对产物进行分离,得到含有4.18g鼠李糖的粗糖液,其得率为17.4%。结合结晶法对鼠李糖粗糖液进行精制,当鼠李糖水溶液质量浓度达到0.4g/mL时,鼠李糖可达到过饱和状态而开始结晶,所得晶体颗粒饱满,无色透明,结晶得率为27.5%。通过高效液相色谱检测,纯度达到99.0%。     

Isolation and characterization of YNTC- 1, a novel Alicyclobacillus sendaiensis strain

A heterotrophic acidothermophilic bacterial strain, YNTC-1, was isolated from an acidic hot spring in Tengchong, Yunan, China. YNTC-1 grows at pH value of 1.5-8.0 and temperature of 40-70℃, with optimal pH and temperature at 3.0 and 55℃,respectively. The cells of the strain are in shape of short rod, with 1.0-1.2 μm in length and 0.7-0.8 μm in diameter, and with distinct spores at both poles of each cell. The predominant fatty acids in cellular membrane of the strain are C18:1ω7c. 16s rRNA gene analysis reveals that this strain is closely related to Alicyclobacillus sendaiensis, with over 99% sequence similarity. Based on phenotypie and genotypic analyses, YNTC-1 is identified as a member ofA. sendaiensis. Considering some important morphological and biochemical differences between strain YNTC-1 and A. sendaiensis ATCC 27009T, YNTC-1 may be proposed to be a novel subspecies of A. sendaiensis. However, this viewpoint has to be confirmed by further studies. Co-bioleaching of pyrite and chalcopyrite with strain YN22, Sulfobacillus thermosulfidooxidans, shows that strain YNTC-1 has no evident influence on bioleaching rates of these two sulphide minerals.     

盐藻水溶性多糖的提取和纯化

采用热水浸提法提取盐藻中的水溶性多糖,通过正交试验,对影响多糖得率的参数如温度、时间和水料比进行优化,确定了盐藻多糖提取的最佳工艺条件为：提取温度85℃,时间300min,水料比20∶1,在此条件下多糖得率为13.09%。利用DEAE-52纤维素柱层析纯化经脱蛋白和透析处理的多糖,得到1种中性多糖和2种酸性多糖,经Sephadex G-100柱洗脱后仍显示为单一峰。 更多还原     

萝卜CBPs的分离及部分酶学特性研究

通过脱氨再生几丁质凝胶亲和层析和CM-纤维素离子交换层析从萝卜中分离出一组CBPs，并对CBP1和CBP2溶菌酶酶学特性作了研究。活力分析表明：CBP1和CBP2均为溶菌酶／几丁质酶双功能酶；CBP3和CBP4只有几丁质酶活力。SDS—PAGE纯度分析表明：CBP1和CBP2已达到电泳纯，相对分子质量分别为26900和24800；CBP3仍具有两个组分，CBP4则由于含量过低，在凝胶上无蛋白带。CBP1和CBP2部分酶学特性分析表明：CBP1的最适反应条件为PH5．8，55C，0．4g／L溶壁微球菌；CBP2的最适反应条件为pH6．8，45℃，0．4g／L溶壁微球菌；CBP1在pH3．4～10．6，0～55℃较稳定，CBP2在pH2．0～10．6，0～60℃较稳定。     

不同米曲霉降解草甘膦能力及其甘氨酸氧化酶的生物信息学分析

采用高效液相色谱法测定6株米曲霉菌株发酵液中的草甘膦降解情况,结果表明米曲霉(Aspergillus oryzae)A-FEC04的草甘膦降解率最高,为12.5%。提取6株米曲霉菌株的基因组DNA,PCR扩增后得到甘氨酸氧化酶序列,测序结果发现只有米曲霉A-FEC04发生部分基因突变。米曲霉(Aspergillus oryzae)A-FEC04的ORF DNA序列全长1 263 bp,编码一个由420个氨基酸组成的蛋白质,其理论分子量为46 965.5 Da,理论等电点为5.85,为弱酸性蛋白。对该氨基酸序列进行结构域架构分析,结果表明该序列具有明显的D-氨基酸氧化酶结构域。更多还原     

黄绿木霉纤维素酶提纯及其性质研究

通过(NH4)2SO4分级沉淀、Sephadex G-100分子筛层析和DEAE Sephadex A-50离子交换层析等步骤,分离纯化出木霉T2纤维素酶系中达到电泳纯的3种内切葡聚糖酶(EGⅠ、EGⅡ、EGⅢ)和2种β-葡萄糖苷酶(BGⅠ、BGⅡ).通过SDS-PAGE和IEF电泳测得5个酶组分的相对分子质量分别为62.3 ku、71.9 ku、52.6 ku、85.3 ku和78.3 ku,等电点分别为5.4、4.8、5.0、5.6和5.8.此酶系均为糖蛋白,含糖量分别为17.7%、11.7%、15.6%、17.8%、19.3%.5个酶组分均属酸性纤维素酶,最适pH都是5.0;3种内切葡聚糖酶的最适温度皆为60℃,2种β-葡萄糖苷酶的最适温度都是55℃.Mn2 、Fe2 、Zn2 对5种纤维素酶都有一定的激活作用,尤其是Mn2 对内切葡聚糖酶的激活作用极为显著;Mg2 、Cu2 、脲对5种纤维素酶都具有不同程度的抑制作用.酶学动力学分析表明5种纤维素酶组分的Km值分别为0.093、0.083 8、0.079 0、0.085 1和0.077 8 mg/mL,Kcat值为101.7、119.6、11.4、111.4和162.1 s-1.内切葡聚糖酶对底物CMC-Na亲和力的大小与酶的催化效率之间并无相关性,BG对底物水杨苷亲和力的大小与酶的催化效率之间有一定的相关性.β-葡萄糖苷酶有较高的底物专一性,只对水杨苷有活力;内切葡聚糖酶在对羧甲基纤维素钠有活力的同时,对滤纸和秸秆粉也有微弱的活力.     

Isolation and characterization of YNTC-1, a novel Alicyclobacillus sendaiensis strain

A heterotrophic acidothermophilic bacterial strain, YNTC-1, was isolated from an acidic hot spring in Tengchong, Yunan, China. YNTC-1 grows at pH value of 1.5-8.0 and temperature of 40-70 ℃, with optimal pH and temperature at 3.0 and 55 ℃, respectively. The cells of the strain are in shape of short rod, with 1.0-1.2 μm in length and 0.7-0.8 μm in diameter, and with distinct spores at both poles of each cell. The predominant fatty acids in cellular membrane of the strain are C18：1 ω7c. 16s rRNA gene analysis reveals that this strain is closely related to Alicyclobacillus sendaiensis, with over 99% sequence similarity. Based on phenotypic and genotypic analyses, YNTC-1 is identified as a member ofA. sendaiensis. Considering some important morphological and biochemical differences between strain YNTC-1 and A. sendaiensis ATCC 27009T, YNTC-1 may be proposed to be a novel subspecies of A. sendaiensis. However, this viewpoint has to be confirmed by further studies. Co-bioleaching of pyrite and chalcopyrite with strain YN22, Sulfobacillus thermosulfidooxidans, shows that strain YNTC-1 has no evident influence on bioleaching rates of these two sulphide minerals.     

淫羊藿黄酮苷酶解产物分离纯化的研究

以得到淫羊藿黄酮苷酶解产物中的未知次生苷为目的,利用大孔吸附树脂SP207对淫羊藿黄酮苷酶解产物进行分离.结果表明,用70%的乙醇洗脱,可大量富集目标产物;目标产物经硅胶柱层析,最终得到目标产物单体0.161 g,经高效液相色谱测定,纯度达到99%.     

淫羊藿黄酮苷酶解产物分离纯化的研究

以得到淫羊藿黄酮苷酶解产物中的未知次生苷为目的,利用大孔吸附树脂SP207对淫羊藿黄酮苷酶解产物进行分离.结果表明,用70%的乙醇洗脱,可大量富集目标产物;目标产物经硅胶柱层析,最终得到目标产物单体0.161 g,经高效液相色谱测定,纯度达到99%.