黑曲霉产木聚糖酶发酵条件的研究

将经过诱变选育高产木聚糖酶并具有Nystatin抗性的黑曲霉,分别在不同条件下进行固体发酵培养,探讨最佳产酶条件。结果显示:在以质量分数1%木糖为附加碳源,以质量分数2%NH4NO3为氮源,无机盐为质量分数1%NaCl,加水比例为1:1.3,接种量为1.5%,装料量为5g/300m l三角瓶,培养温度为30℃,培养周期为72 h的培养条件下,菌株产木聚糖酶活力提高至7285.4 IU.g-1。比出发菌株提高了20%。酶活力测定采用3,5-二硝基水杨酸(DNS)法。     

Alkalitalea saponilacus产木聚糖酶的碳源优化及酶学特性

【目的】确定厌氧盐碱细菌Alkalitalea saponilacus产木聚糖酶所需的碳源,优化木聚糖粗酶的提取条件并分析酶学性质。【方法】应用GC技术分析A.saponilacus发酵木聚糖的主要产物;利用二硝基水杨酸法(DNS)测定木聚糖酶活力以获得最优的碳源、提取粗酶的最佳条件及其酶学特性。【结果】A.saponilacus以不同来源木聚糖为底物时,发酵产生的主要产物丙酸含量都在80%以上。若以0.4%(W/V)蔗糖+0.1%(W/V)桦木木聚糖为复合碳源时,木聚糖酶活力是以桦木木聚糖或者蔗糖为单一碳源时的3.2倍。木聚糖酶的酶活力在盐度2%–6%、pH 7.0和55°C达到最佳且在该条件下的酶活力为590 IU/mg。此外,该酶活力在0.2%Tween 20存在时增加,而在5 mmol/L Mg~(2+)和0.2%Triton X-100存在时无显著影响,但在Cu~(2+)、Fe3+和Ni~(2+)等金属离子存在时则被显著抑制。【结论】A.saponilacus发酵主产物丙酸以及生物合成的木聚糖酶在工业生产中具有广泛的应用前景。     

链霉菌Strz-2胞外木聚糖酶的纯化和固定化研究

为探讨木聚糖酶被固定化后的酶活力变化 ,采用盐析、离子交换和分子筛层析方法对链霉菌胞外木聚糖酶进行了纯化 ,并采用DNS方法对固定化酶的性质进行了研究。结果如下 :粗酶液被纯化了 30 .5倍 ,比活力达 4 5 7.5 ,活力回收 4 2 .6 %。纯化后的酶固定在戊二醛交联的壳聚糖上 ,残活力为 4 1.8%。固定化酶的最适pH为 6 .0 ,最适温度为 5 5℃ ,且固定化酶在 6 5 -75℃活力都较高。该酶的耐热性比较强 ,固定化酶热稳定性优于原酶 ;以木聚糖为底物 ,固定化酶的表观米氏常数为 0 .83× 10 -2g/L。因此 ,固定化的木聚糖酶优于原酶     

应用正交旋转组合设计构建测定条件对木聚糖酶活力影响的数学模型

通过正交旋转试验,探讨了反应温度（X1）、pH值（X2）、反应时间（X3）、底物浓度（X4）、DNS用量（X5）5个测定条件对木聚糖酶活力（Y）测定结果的影响,并构建了测定条件对木聚糖酶活力影响的数学模型：Y=10．2950＋1．6563X1-0．0704X2＋0．3179X3＋1．7004X4-1．3413X5＋0．0669X1X2＋0．2094X1X3＋0．7631X1X4＋0．3301X1X5＋0．1256X2X3-0．0881X2X4＋0．1544X2X5＋0．2596X3X4＋0．1469X3X5-0．2594X4X5-0．4121X1^2-0．1258X2^2-0．2233X3^2-0．8358X1^2＋0．5217X3^2．实验结果表明：反应温度、底物浓度和DNS用量对木聚糖酶活力测定结果有极显著影响．     

一株产木聚糖酶的黑曲霉固态发酵产酶性质的研究

目的：选育产木聚糖酶活力高的黑曲霉菌株,对其产酶条件进行优化,并研究其酶学性质。方法：通过木聚糖酶解木聚糖产生透明圈的方法,筛选产木聚糖酶菌株,测定固体发酵培养基中玉米芯与麸皮的比例、培养温度、培养时间、添加氮源对产酶的影响。进行了作用温度、pH值、金属离子对酶活力的影响试验,以及酶不同温度下的热稳定性的试验。结果：从自然界筛选得到一株产木聚糖酶的黑曲霉菌株,通过对固态发酵培养条件优化,最终产酶水平达到了5500u／g固体干曲。酶的最适作用温度是45℃、最适作用pH值4．8,是一种偏酸性酶。该酶在45℃以上的温度保存会使酶活力迅速丧失,Mg^2＋、Zn^2＋对该酶有激活作用,而Mn^2＋、Cu^2＋、Hg^2＋则完全抑制酶的活性。结论：选育的黑曲霉菌株产木聚糖酶活力较高,培养条件简单。     

高活力5′-磷酸二酯酶制备及水解RNA的研究

目的:获得高活力5′-磷酸二酯酶液,提高核酸RNA酶解效率。方法:采用超滤和盐析技术对从麦芽根浸提液中纯化5′-磷酸二酯酶工艺进行研究,采用单因子试验法优化酶解工艺条件。结果:浸提液依次经过5万Da超滤膜浓缩、40%饱和度硫酸铵盐析、5万Da超滤膜脱盐后,酶活力可达1 500U/ml;第1次超滤膜透过液可作为浸提液循环使用,酶活力是水浸提的1.15倍;第2次超滤膜透过液浓缩5倍后,可回收56.46%硫酸铵,浓缩母液可按1∶2比例循环使用;在底物浓度5.8%、酶用量8%、反应时间2h条件下,RNA酶解率可达95%。结论:初步建立了适合工业化规模的核苷酸生产新工艺。     

两株保加利亚乳杆菌产β-半乳糖苷酶条件的优化及酶活力测定

目的优化2株保加利亚乳杆菌产β-半乳糖苷酶的培养条件,测定其在最适于产酶条件下的酶活力,并初步观察酶活力稳定性。方法测定2株保加利亚乳杆菌——来源于酸奶的wch9901和标准菌株1．1480在不同培养时间、培养基碳源、摇床转速、气体环境下产生的β-半乳糖苷酶的活力,优化产酶条件。测定2株菌在其最适产酶条件下的酶活力。将制得的1．1480粗酶液分别置冰浴和20℃水浴,每隔1h测定一次粗酶液中β-半乳糖苷酶活力,观察酶活力稳定性。结果2株保加利亚乳杆菌的最适产酶条件分别为：1．1480于30℃有氧静止培养36h;wch9901于37℃厌氧静止培养18h,培养基含乳糖。在最适产酶条件下,1．1480的酶活力为0．321NLU／ml粗酶液,比酶活为2．469NLU／mg蛋白;wch9901的酶活力为0．401NLU／ml粗酶液,比酶活为6．169NLU／mg蛋白。1．1480粗酶液于冰浴可稳定保存6h,于20℃水浴可稳定保存5h。结论wch9901与1．1480的最适产酶条件有所差异,前者产酶迅速,产酶量多。     

饲用型青霉α-半乳糖苷酶活力测定的研究

研究了比色法测定饲用α-半乳糖苷酶活力,以对硝基苯酚-α-D-吡喃半乳糖苷为底物和对硝基苯酚作标准产物。酶活力的测定条件受很多因素的影响,如酶浸提液的pH、酶液稀释倍数、反应温度、作用时间。其中稀释倍数和pH的影响较为显著。稀释酶液的酶活测定值在0．02～0．07U／mL内较为合适。考虑到动物饲用酶制剂的特殊性,测定α-半乳糖苷酶活力时建议规定pH为5．5,反应温度为40℃。根据研究结果,反应时间采用10min,比色波长405nm为宜。     

牦牛源木聚糖酶产生菌的筛选和鉴定

目的以牦牛粪便为样本,筛选并鉴定产木聚糖酶菌株。方法利用碱提取法从玉米芯中提取木聚糖,以自制木聚糖为唯一碳源,从牦牛牛粪中筛选产木聚糖酶细菌,利用16S rDNA基因序列分析鉴定菌种,3,5-二硝基水杨酸法(DNS)测定其产酶能力并分析所产酶的酶学特性。结果筛选获得牦牛源产木聚糖酶类芽胞杆菌,所产木聚糖酶的最适反应条件为50℃、pH 8.0,在pH值为7.0或8.0以及温度50℃条件下,表现出较好的稳定性,Mn~(2+)对酶活力具有显著抑制作用,该菌最佳发酵时间为12 h,酶活最高达到1.2 U/mL。结论该菌所产木聚糖酶能够针对性地降解玉米芯木聚糖,在畜牧业和工业上有一定的应用价值。     

1株褐藻胶降解菌的筛选、鉴定及产酶条件优化

为获得可产生褐藻胶裂解酶并高效降解褐藻胶的菌株,以海藻酸钠为唯一碳源配制培养基,以透明圈法进行初筛,DNS法复筛,从海洋生物中筛选得到1株高酶活力褐藻胶降解菌株B12,经16S rDNA序列分析、生理生化试验、电镜观察,确定该菌为弧菌属(Vibrio sp.)。通过单因素试验及响应面优化试验对影响菌株生长和产酶条件的5个因素(发酵初始pH值、发酵温度、NaCl质量浓度、接种量和装液量)进行优化。得到该菌株最佳产酶条件:pH 6.52,发酵温度28.2℃,NaCl质量浓度20.1 g/L,接种量2.1%,装液量59.5 mL。在最佳发酵条件下,B12菌株酶活力可达91.68 U/mL,相比于优化前提高了38.5%。菌株开始产酶时间提前6 h, 4℃冷藏酶活力稳定性较好。     

草菇不同菌株木聚糖酶xyn基因的表达及其酶活性分析

木聚糖酶是半纤维素酶系的重要组成部分,能够分解水稻、小麦等农作物秸秆中的半纤维素。研究探讨木聚糖酶相关基因及其功能,为进一步探讨木聚糖酶与草菇生物转化率之间的关系提供理论依据。首先通过生物信息学手段构建了草菇2个木聚糖酶基因xyn1和xynII编码氨基酸序列的系统进化树,然后从生物转化率不同的草菇菌株分别提取各自的RNA,反转录为cDNA后,采用实时荧光定量PCR技术分析了xyn1和xynII基因的转录表达情况;最后应用DNS法对这些菌株中的木聚糖酶活性进行了测定。研究结果表明,xyn1编码的氨基酸序列与草腐菌相似性较高,而xynII编码的氨基酸序列与木腐菌遗传差异较小。在木聚糖酶活性测定和实时荧光定量PCR结果中,不同菌株的木聚糖酶活性趋势与xynII的转录表达趋势相似,均呈现依次递减。草菇的木聚糖酶活性与其生物转化率之间存在正相关性,推测草菇不同菌株木聚糖酶活性差异可能表现在转录水平上的差异。     

有机复合物H对纤维素CMC酶活的影响

采用3,5-二硝基水杨酸(DNS)为显色剂,CMC为底物,测定纤维素酶的酶活。考察了在不同的酶促反应温度、pH值、底物浓度、反应时间等反应条件下,有机复合物H对纤维素酶CMC酶活(CMCA)的影响。结果表明,有机复合物H对纤维素酶的CMCA活性有明显促进作用,且在不同条件下的促进效果有较大差异。     

中度嗜盐菌产木聚糖酶发酵条件的研究

中度嗜盐菌在盐碱环境下生长繁殖,其产生的木聚糖酶也同样具有在盐碱环境下发挥作用的特性。本文对一株中度嗜盐菌的产木聚糖酶活性进行了初步研究。研究包括氮源、液体种子接种量、培养温度、pH值、培养时间等因素对该菌株产木聚糖酶能力的影响。结果表明,最佳培养氮源为蛋白胨;最佳产生木聚糖酶的发酵条件是液体种子接种量为6%,温度为35℃,pH值7,培养时间为4 d。     

Purification and some properties of high-molecular-weight xylanases, the xylanases 4 and 5 of Aeromonas caviae W-61

Aeromonas caviae W-61 produces multiple extracellular xylanases, the xylanases 1, 2, 3, 4, and 5 [Nguyen, V. D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993)]. Here we purified and characterized high-molecular-weight xylanases, the xylanases 4 and 5 from the culture fluids of the bacterium. The purified xylanases 4 and 5, which had molecular masses of 120 and 140 kDa, respectively, were endo-beta-1,4-xylanases with similar enzymatic properties except for trans-xylosidase activity. The xylanase 4 showed a prominent transxylosidase activity when xylotriose and xylotetraose were used as the substrates, while the xylanase 5 had little transxylosidase activity under the same conditions. Protein sequencing indicated that the xylanase 4 was a C-terminally-truncated xylanase 5, suggesting that the C-terminal truncation of the xylanase 5 may endow the enzyme with transxylosidase activity.     

产木聚糖酶白地霉培养特性及部分纯化的酶学特性

本文对白地霉Ref1的培养特性、产酶条件和酶学特性进行了初步研究。结果表明:该菌为低温型菌株,其最佳生长条件为pH6、20℃和酵母膏作为氮源;最佳产酶条件为pH3-7、15℃及以酵母膏氮源;条件优化后产酶可达118.7U/mL,可溶蛋白含量可达到60μg/mL,酶溶液的比活可达到1250U/mg蛋白质;该木聚糖酶的最适反应温度和pH分别为50℃和5,金属离子Mg2+、Na+和8mmol/L的Fe2+、Cu2+、Zn2+等对木聚糖酶的活性有抑制作用,而Ca2+、4mmol/L的Fe2+、Cu2+、Zn2+和8mmol/L的Mn2+等对该酶反应则有促进作用;该木聚糖酶在保温2h后在15-40℃范围内能保持80%以上的酶活性,在50℃时能保持68%的酶活性;用lineweaver-Burk作图法(双倒数作图法)求得该酶的最大反应速度Vmax和Km值分别为163.38mmol/mg/min和0.75mg/mL。     

来源于橄榄绿链霉菌A1的高比活木聚糖酶XYNB的高效表达及性质的比较分析

高效表达高比活木聚糖酶是进一步提高木聚糖酶发酵效价、降低其生产成本的有效途径。将橄榄绿链霉菌(Streptomyces olivaceoviridis) A1的高比活木聚糖酶成熟蛋白编码基因xynB克隆到毕赤酵母表达载体pPIC9中,转化毕赤酵母得到重组酵母,在重组酵母中木聚糖酶基因得到了高效分泌表达,且表达产物具有生物学活性。在3L发酵罐中蛋白表达量约14mg/mL, 酶活性(效价)为1200IU/mL。SDSPAGE分析表明,表达的木聚糖酶XYNBa为糖基化蛋白, 分子量为31kD, 经脱糖基化处理得到21kD 的XYNBb, 与橄榄绿链霉菌A1所产原酶XYNB大小一致。通过对XYNB、XYNBa及XYNBb酶学性质的比较发现：三者在比活性、Vmax及热稳定性方面有较大差异。该酶对不同木聚糖的酶解产物的糖份分析表明：酶解产物的主要成分为木二糖、木三糖和木四糖,占总糖含量的95%以上。     

Hydrolytic potential of Trichoderma sp. strains evaluated by microplate-based screening followed by switchgrass saccharification

Bioconversion of lignocellulosic biomass to fuel requires a hydrolysis step to obtain fermentable sugars, generally accomplished by fungal enzymes. Large-scale screening of different microbial strains would provide optimal enzyme cocktails for any target feedstock. The aim of this study was to screen a large collection of Trichoderma sp. strains for the hydrolytic potential towards switchgrass (Panicum virgatum L.). Strains were cultivated in a small-scale system and assayed in micro-plates for xylanase and cellulase activities. The population distributions of these traits are reported after growth on switchgrass in comparison with cellulose. The distribution profiles suggest that the growth on switchgrass strongly promotes xylanase production. The IK4 strain displayed the highest xylanase activity after growth on switchgrass (133U/mL). Enzymes (10FPU/g substrate) from IK4 were compared with those from 2 cellulolytic Trichoderma strains and a commercial enzyme in saccharification time-course experiments on untreated and pretreated switchgrass and on an artificial substrate. Samples were analysed by DNS assay and by an oxygraphic method for sugar equivalent or glucose concentration. On the untreated substrate, IK4 enzymes even outperformed a 5-fold load of commercial enzyme, suggesting that xylanase or accessory enzymes are a limiting factor on this type of recalcitrant substrate. On the other substrates, IK4 preparations showed intermediate behaviour if compared with the commercial enzyme at 10FPU/g substrate and at 5-fold load. IK4 also nearly halved the time to release 50% of the hydrolysable sugar equivalents (T(50%)), with respect to the other preparations at the same enzymatic load. DNS assay and oxygraphic method gave highly correlated results for the 3 saccharified substrates. The study suggests that accessory enzymes like xylanase play a key role in improving the performance of cellulase preparations on herbaceous lignocellulosic feedstocks like switchgrass.     

Isolation, purification and characterization of xylanasefrom Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp

A haloalkalophilic Staphylococcus sp. SG-13 produced an alkalistable xylanase in wheat bran medium. A 12-fold purification was achieved by using standard purification techniques. The purified xylanase exhibited a dual pH optima of 7.5 and 9.2. The optimum temperature for enzyme activity was 50 degrees C. The enzyme was stable at 50 degrees C for more than 4 h. The xylanase exhibited Km and Vmax values of 4 mg ml-1, 90 micromol min-1 per mg for birchwood xylan and 7 mg ml-1, 55 micromol min-1 per mg for oatspelt xylan, respectively. The substrate binding affinity of xylanase was more for oatspelt xylan but birchwood xylan was hydrolysed more rapidly. The xylanase activity was stimulated by Fe2+, Ni2+, Cu2+ and dithiothreitol up to 60% and was strongly inhibited in the presence of Co2+, Hg2+, Pb2+, phenyl methane sulphonyl fluoride, ethylenediaminetetraacetic acid, and acetic anhydride up to 100%. The xylanase dose of 1.8 U g-1 moisture free pulp, exhibited bleach boosting of kraft pulps optimally at pH 9.5-10.0 and 50 degrees C after 4 h of reaction time. Pretreatment of pulp with xylanase and its subsequent treatment with 8% hypochlorite, reduced the kappa number by 30%, enhanced the brightness and viscosity by 11% and 1.8%, respectively, and improved the paper properties such as tensile strength and burst factor up to 10% and 17%, respectively.     

有机复合物H对纤维素酶促反应动力学特性的影响

采用3,5-二硝基水杨酸(DNS)为显色剂,滤纸和CMC为底物,测定纤维素酶的酶活.考察了在不同温度、pH值、反应时间、底物浓度等反应条件下,有机复合物H对纤维素酶滤纸酶活(FPA)和CMC酶活(CMCA)的影响.结果表明,有机复合物H对纤维素酶的FPA活性和CMCA活性均有一定的促进作用,且在不同条件下的促进效果有较大差异.     

Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs

The initial moisture content, cultivation time, inoculum size and concentration of basal medium were optimized in solid state fermentation (SSF) for the production of xylanase by an Aspergillus niger mutant using statistical experimental designs. The cultivation time and concentration of basal medium were the most important factors affecting xylanase activity. An inoculum size of 5 x 10(5) spores/g, initial moisture content of 65%, cultivation time of 5 days and 10 times concentration of basal medium containing 50 times concentration of corn steep liquor were optimum for xylanase production in SSF. Under the optimized conditions, the activity and productivity of xylanase obtained after 5 days of fermentation were 5,071 IU/g of rice straw and 14,790 IU l(-1) h(-1), respectively. The xylanase activity predicted by a polynomial model was 5,484 IU/g of rice straw.