康氏木霉纤维素酶系中C_x酶多分子型的分离纯化及某些性质

从康氐木霉(Trichoderma k(?)ningii)白色变异株As 3.4001的粗酶制剂中,获得了纤维素酶系中的一组C_x酶(C_(x1) C_(x2) C_(x3) C_(x4))。分离步骤包括Sephadex G-75凝胶过滤,DEAESephadex A-50离子交换层析,ConA-Sepharose亲合层析,SE-Sephadex C-50离子交换层析及聚丙烯酰胺凝胶电泳。C_(x1)与C_(x2)的分子量不同而所带电荷相同,它们的分子量各自为44,500和34,000。C_(x2)—C_(x4)的分子量相同而所带电荷不同。纯化的C_(x1)—C_(x4)经聚丙烯酰胺凝胶电泳鉴定为单带。比较它们对羧甲基纤维素钠(CMC-Na)的糖化力及液化力表明在作用方式的随机性上C_(x2)>C_(x3)>C_(x1)>C_(x4)。     

特异腐质霉纤维素酶的研究

由腐植土中分离到一株嗜热真菌,经鉴定为特异腐质霉(Humicola insolens Cooney etEmerson)。研究了这株菌纤维素酶的产生条件和一般性质。菌在含麦麸5％、NaNO0.3％的液体培养基(灭菌前pH7.5,灭菌后pH7.2)中,于45℃培养4天,以羧甲基纤维素钠为底物,每ml滤液酶活力为20个单位。酶作用的最适条件为:pH6.0,温度为65—70℃。该纤维素酶是一种耐热酶,热稳定性较强,70℃保温5分钟后,酶活力剩余88％。底物对该酶的热钝化有较强的保护作用,无底物存在条件下,70℃保温6小时后,酶活力仅剩余1％,而在同样的处理温度和时间,在有底物存在条件下,酶活力可剩余30％。该酶在45℃保温15小时的条件下,pH稳定范围为6.0—9.0。     

浙江省仓贮稻谷大米真菌区系的研究

在浙江省采集仓贮稻谷及大米样品,共分离到真菌17属48种(不包括酵母菌)。其中:曲霉属(Aspergillus)18种,青霉属(Penicillium)10种,镰孢霉属(Eusarium)4种。研究表明:在浙江省地区之间,贮粮真菌种类无明显差异。稻谷加工成大米后,籽粒表面带菌量及内部带菌率均明显下降,其真菌区系以贮藏真菌为主。稻谷在贮藏前以田间真菌为主。贮藏1—4个月的稻谷因贮藏真菌的种类、数量明显增长,而田间真菌的种类和数量仍保持在较高的水平上,其表面带菌量及谷粒带菌率处于高峰期;贮藏1年以上的稻谷,其主要带菌种类为贮藏真菌,带菌量及带菌率明显下降。早籼谷和晚粳谷在相同贮藏条件下其带菌种类和数量基本一致。     

斜卧青霉转录调控因子BglR的缺失对纤维素酶生产的影响

【目的】斜卧青霉(Penicillium decumbens)作为高效分泌纤维素酶的重要丝状真菌,其纤维素酶的合成与分泌在转录水平上被调控。进一步研究纤维素酶基因表达的转录调控,构建高效高产纤维素酶的工业菌株。【方法】根据斜卧青霉114-2在不同碳源生长条件下基因组表达谱的差异,发现新的转录调控因子BglR(PDE-01706),该蛋白与产黄青霉(Penicillium chrysogenum)Pc20g04780的锌指结构蛋白具有59%同源性。通过基因同源双交换,得到BglR缺失突变株ΔbglR-1,对突变株ΔbglR-1的表型、营养生长、产纤维素酶活、蛋白分泌能力及发酵液pH变化进行研究。【结果】转录调控因子BglR的缺失可导致突变株ΔbglR-1的β-葡萄糖苷酶活力提高40%,并造成其滤纸酶活、内切葡聚糖酶及木聚糖酶活明显降低。【结论】结果表明转录调控因子BglR对于斜卧青霉纤维素酶的调控有重要作用。     

康氏木霉纤维素酶系中Cx酶多分子型的分离纯化及某些性质

从康氐木霉(Trichoderma kkoningii) 白色变异株 AS 3．4001的粗酶制剂中,获得了纤维素酶系中的一组C_x酶(C_xt C_xz C_z3 C_z4)。分离步骤包括Sephadcx G-75凝胶过滤,DEAE-Sephadex A-50离子交换层析,ConA-Sepharnse亲合层析,SE—Sephadcx C-50离子交换层析及聚丙烯酰胺凝胶电泳。C_xt 与C_xt 的分子量不同而所带电荷相同,它们的分子量各自为44,500和34,000。C_xz—C_x4 的分子量相同而所带电荷不同。纯化的C_xt—C_z4“经聚丙烯酰胺凝胶电泳鉴定为单带。比较它们对羧甲基纤维素钠(CMC—Na)的糖化力及液化力表明在作用方式的随机性上C_xz>C_z3>C_z1>C_x4。     

里氏木霉纤维素酶的分离纯化及酶学性质

通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting凝胶色谱脱盐、Source 15 Q阴离子交换色谱技术,里氏木霉(Rut C-30)纤维素酶主要组分得以初步分开,再经过Source 15 S阳离子交换色谱、HiPrep Sephacryl S-100 HR凝胶过滤色谱、Superdex 75 PrepGrade凝胶过滤色谱进一步分离纯化,得到2个纯化的内切葡聚糖酶组分EGⅡ、EGⅠ和一个外切葡聚糖酶组分CBHⅠ;经过SDS-PAGE电泳鉴定为电泳纯,测得相对分子质量分别为5.22×104,5.62×104和6.90×104。EGⅡ的最适反应pH是5.6,最适反应温度为65℃;EGⅠ的最适反应pH是4.4,最适反应温度为55℃;以羧甲基纤维素(CMC)为底物时,EGⅠ、EGⅡ的米氏常数(Km)分别为2.20 mg/mL、3.38 mg/mL。CBHⅠ的最适反应pH是5.8,最适反应温度为60℃,以对硝基苯基-β-D-纤维二糖苷(PNPC)为底物时,米氏常数(Km)为0.12 mg/mL。     

康宁木霉QF-02纤维素酶酶学性质的研究

对康宁木霉QF-02生产的纤维素酶的一般酶学性质进行了研究。该纤维素酶系中滤纸酶、羧甲基纤维素酶、微晶纤维素酶、β-葡萄糖苷酶的最适作用温度分别为55℃、65℃、50℃和70℃,最适作用pH为4.0-5.0;在40-50℃范围内热稳定性较好,24 h保温后的残留酶活在48.5%以上;在pH3.0-8.0范围内比较稳定,4℃保存24h后的残留酶活在75.7%以上。与几种商品纤维素酶相比,该纤维素酶对未处理和碱预处理稻草都表现出较强的糖化能力。     

脱墨用棘孢曲霉SM-L22纤维素酶系中内切酶的纯化及性质

通过Bio GelP 60分子筛和DEAE 与Q sepharose离子交换层析等手段 ,分离纯化了棘孢曲霉SM L2 2纤维素酶系中五种内切酶组分EGⅡ 1、EGⅡ 2、EGⅢ 1、EGⅢ 2和EGⅣ ,并且对这五种内切酶组分的基本性质进行了研究。通过SDS PAGE和IEF电泳测得其分子量分别为 38 7,34 4,31 4,36 9和 2 3 7kD ,等电点分别为pH <3 5,<3 5,4 9,4 5和 5 0。 5个酶组分均属酸性纤维素酶 ,最适pH在 3 5～ 4 0之间 ;最适温度分别为 55℃、60℃、( 60～ 70 )℃、( 60～70 )℃和 60℃。各酶组分有较宽的pH稳定性 ;温度稳定性表现为EGⅡ 1 >EGⅡ 2 >EGⅢ 1>EGⅢ 2 >EGⅣ。EGⅡ 1和EGⅡ 2有较高的底物专一性 ,而EGⅢ 1、EGⅢ 2和EGⅣ对木聚糖有交叉活性。Fe2 +对除EGⅣ以外的四种酶组分都有激活作用 ,尤其是对EGⅢ 2有强烈的激活作用。动力学分析表明各纤维素酶组分对底物亲和力的大小与酶的催化率之间并无相关性。     

培养条件对棘孢曲霉SM-L22所产纤维素酶系组成的影响*

本文研究了影响棘孢曲霉SM-L22纤维素酶系组成的培养条件。研究结果表明,碳源、氮源和初始pH对棘孢曲霉所产生纤维素酶的内、外切酶组分的比例有明显的影响。在2%麸皮,1%CF11,0.5%尿素或含尿素的复合氮源为氮源,初始pH为4.5时,28℃培养120h后,内、外切酶的比值最大,内切酶活可达到3.1 IU/ml,FPA为0.105 IU/ml,CMCase/FPase的比值为30.6。     

里氏木霉纤维素酶产生条件的研究

通过对培养基、水份含量、氮源、培养时间、培养基的起始PH以及培养温度的研究,测定TrichodermareeseiGAB纤维素酶的C₁酶和Cx酶的酶活,找到了一个最佳的条件,即：稻草粉：花生壳=4：1,物料：水份=2：3,以NH₄Cl、（NH₄）₂SO₄、NH₄H₂PO₄为氮源,起始pH为自然pH（约5．8）,在28℃     

Biosorption of Mercury from Industrial Effluent by Fungal Consortia

Mercury is present in different types of industrial effluents that cause environmental pollution. Conventional methods such as precipitation, oxidation/reduction, ion exchange, filtration, membranes, and evaporation are extremely expensive or inefficient for the removal of mercury from diluted solutions. In this context, the biosorption process has recently been shown to be an effective and economical method. The present work describes the mercury biosorption ability of three fungi, i.e., Aspergillus niger, Trichoderma viride, and Humicola insolens. Monocultures of these strains and 10 different combinations were investigated. The consortium of 24-h-old H. insolens and 48-h-old of A. niger and T. viride in equal ratio was found to be compatible. This consortium decreased the residual mercury from 2.02 to 0.001 μ g/L after 7 days of incubation, and caused a significant reduction in chemical oxygen demand (COD) (92.6%) from an initial level of 21 mg/L.     

Soil fungi of some low-altitude desert cotton fields and ability of their extracts to inhibit Aspergillus flavus

Soil is presumed to be a major source of inoculum for Aspergillus flavus which contaminates cottonseed and produces the potent carcinogen, aflatoxin. Little is known about the mycoflora of the low desert soils of cotton fields where aflatoxin is a chronic problem. In this study, soils from cotton fields in southwestern Arizona and southeastern California were assayed for filamentous fungi. Forty-two taxa, predominantly in the genera Aspergillus, Penicillium and Fusarium, were isolated. To determine whether or not compounds produced by these fungi could be potential inhibitors of A. flavus, extracts of strains of each taxon were tested for their ability to inhibit growth of A. flavus. Twelve taxa produced compounds inhibitory to A. flavus, including several strains of Fusarium solani, Penicillium vinaceum and Aspergillus auricomus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mycoflora and naturally occurring mycotoxins in poultry feeds in Argentina

The purpose of this work was to determine the mycoflora and mycotoxins natural incidence in poultry feeds from 2 factories in Río Cuarto, Córdoba. One hundred and thirty samples were taken from May/1996 to May/1997. The most dominant species isolated of poultry feed samples belonged to the genera Aspergillus spp 85% and Fusarium spp 70%. From Aspergillus genus eleven species were identified and A. flavus was the most frequent. Nine species were identified from the Fusarium genus and the predominant was F. moniliforme. Penicillium ranked third in the number of isolated cases. From this genus twelve species were collected of which P. brevicompactum (15%), P. restrictum (14%) and P. purpurogenum (12%) were the most common.The most significant mycotoxin from poultry feeds was aflatoxin B₁ (AFB₁) found in 48% of the samples, with levels ranging from 10 to 123 ng/g. For zearalenone (ZEA) the levels were 327 to 5,850 ng/g and DON was not detected from the samples. Due to the fact that in Argentina there is little information about this topic, these data on poultry feeds in our region would be of worldwide interest.This revised version was published online in October 2005 with corrections to the Cover Date.     

黑曲霉β-葡萄糖苷酶产酶条件的优化

本研究对Aspergillus niger Glu05生产β-葡萄糖苷酶的培养基组分及培养条件进行了优化.优化后的培养基组成和培养条件分别为:麸皮4％,tryptone 4％,1μmol MnSO4,1μmol NaCl,KH2PO40.2％,oH自然,摇床转速250 r/min,培养温度30℃,培养周期5d.优化后发酵液中酶活力达到44.11 IU/mL,与初始的产酶水平32.87 IU/mL相比,提高了36％.     

Fungi associated with sorghum grain from Argentina

Sorghum (Sorghum bicolor (L.) Moench) is an important cereal produced in and exported from Argentina. The risk of contamination by mycotoxins is related to the mycoflora associated with the sorghum grain. This paper reports on the identification of internal mycoflora of sorghum grain harvested in Argentina in 1991, 1992 and 1993, years with different total rainfall levels. A mycological survey was carried out on sorghum samples, from a location at the humid Argentinian pampa, using a Fusarium/dematiaceous fungi selective medium. The relative density of the prevalent fungal genera were statistically compared. Genus Fusarium was the most prevalent component of the internal seedborne mycoflora in the three harvest seasons. Genera Alternaria, Phoma, Penicillium and Aspergillus were also isolated. The predominant Fusarium was F. moniliforme and the most frequently isolated species of Alternaria, Phoma, Penicillium and Aspergillus were Alt. alternata, Pho. sorghima, Pen. funiculosum and Asp. flavus, respectively. This is the first report of the isolation of Fusarium napiforme in sorghum grain in Argentina. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and characterization of two novel β-glucosidases from Penicillium oxalicum and their application in bioactive ginsenoside production

Abstract

The fungus Penicillium oxalicum is able to selectively metabolize the 20(S)-protopanaxadiol ginsenosides Rb1, Rb2 and Rc to the bioactive ginsenoside compound K using extracellular glycosidases. In this study, two novel extracellular ginsenoside-hydrolyzing enzymes GH3-1 and GH3-2 were purified and characterized from P. oxalicum culture. Using ginsenosides as substrates, GH3-1 and GH3-2 synergistically catalyzed the hydrolysis of Rb1, Rb2 and Rc to yield the final product Compound K (C-K). The hydrolysis pathways were determined to be: Rb1→Rd→F2→C-K, Rb2→CO→CY→C-K and Rc→Mb→Mc→C-K for GH3-1 and GH3-2, respectively. The two enzymes differ, especially in composition, molecular weight, stability and substrate specificity, from GH1, a glycosidase previously purified from the same fungus. These enzymes could be of interest in glycoside degradation, especially in the production of minor ginsenosides.     

黑曲霉与里氏木霉固态混合发酵产β-葡萄糖苷酶

对黑曲霉NL02与里氏木霉RUT-C30固态混合发酵产β-葡萄糖苷酶的发酵培养基进行优化,研究培养基含水率、C源、N源、接种量、温度和2种菌种不同延长接种时间与接种比例对β-葡萄糖苷酶活力的影响。研究表明：麸皮17.5 g、玉米芯7.5 g、（NH4）2SO4 0.40 g、尿素0.37 g、黑曲霉孢子接入量为107个接种到250 mL三角瓶中,温度30 ℃、摇床转速100 r/min时,里氏木霉以105个孢子与黑曲霉同时接入,每克干曲所得β-葡萄糖苷酶的活力为132.45 IU,较黑曲霉单独培养时的104.35 IU提高了26.94%。