体外模拟猪胃条件下木聚糖酶活力稳定性研究

木聚糖酶是饲用复合酶制剂的主要酶系之一,本实验对某木聚糖酶在体外模拟猪胃条件下酶活力的稳定性进行了研究.结果表明:实验所用酶最适温度为50℃左右,最适pH为5.5左右;酶的耐热性能比较好,pH稳定性范围为5～8,体外模拟猪胃条件下木聚糖酶稳定性实验表明,酶活损失较大,经不同方法处理木聚糖酶,酶活损失百分率大小顺序为:胃蛋白酶<新鲜猪胃液<胃蛋白酶+金属离子.     

一株耐热纤维素酶产生菌的筛选及酶学特性

从辽宁鞍山汤岗子温泉附近土样中分离得到能产生纤维素酶的真菌,通过形态观察和18S rRNA序列分析,该菌株为蒙昧的散囊菌纲（Uncultured Eurotiomycetes）。实验中对酶学性质进行了检验,测定得出该菌株产生的纤维素酶最适温度为65℃,在温度高达75℃仍能保持70%的酶活力,它的最适pH值为6.5,pH在5～8的范围内酶活力保持稳定。实验表明该菌株所产纤维素酶具有较高的pH稳定性和温度稳定性,值得对该酶进行进一步的研究。     

海洋细菌Bacillus sp．HN07的分离鉴定及所产碱性纤维素酶性质研究

从渤海海域（121°30′00″E,40°25′00″N）海泥中筛选到一株产碱性纤维素酶的海洋细菌。通过形态观察、生理生化特性及16SrDNA序列分析,鉴定该菌株属于芽孢杆菌属（Bacillus）,命名为Bacillussp．HN07。研究表明：HN07最适生长温度30℃,适宜在pH7．0～8．0、含2．0％～3．0％NaCl的培养条件下生长和产酶,并且具有较好的遗传稳定性。酶学性质初步研究显示,HN07所产碱性纤维素酶最适反应温度为45℃,最适pH为8．0,在碱性条件下具有较好的稳定性,具有潜在的工业应用价值。     

爱尔兰帚霉产低温纤维素酶的酶学性质和发酵工艺

本文旨在对爱尔兰帚霉E71702M菌株产低温纤维素酶的酶学性质进行初步研究,并获得其最优发酵条件,为低温纤维素酶开发利用提供参考。通过单因素试验确定温度、pH及金属离子对纤维素酶活力的影响;以Plackett-Burman试验设计从8个培养条件中筛选出影响产酶的3个主要因素,即麸皮含量、装瓶量和起始pH;运用Box-Behnken试验设计及响应面分析法确定这3个主要因素的最优发酵条件。对酶学性质的初步研究表明,该酶最适pH为3.0,最适反应温度为30℃,在0℃时残余酶活力为最适反应温度时的56.3%。响应面分析得到最优发酵条件为:麸皮含量8.79g/L、装瓶量40.93m L、初始pH 4.01。通过优化,纤维素酶活力由0.6338IU/m L提高到1.7386IU/m L,提高了174%。     

碱性纤维素酶革兰氏阴性菌株筛选及酶学性质研究

用CMC平板筛选方法,从造纸厂碱性淤泥中获得透明圈直径大于30mm的产碱性纤维素酶革兰氏阴性菌H8005。液体摇瓶培养产生碱性CMC酶活力高达4．2IU／mL。酶学性质初步研究显示,H8005产生的CMC酶反应的pH值以8．0左右为适;在碱性条件下具有较高的酶活和一定的稳定性;反应温度以55℃左右为宜;且具有较好的温度稳定性。Mn^2＋与Fe^3＋对酶反应有促进作用,Cu^2＋和Pb^2＋对酶反应有抑制作用。该菌产生的纤维素酶在棉织品的水洗整理及洗涤剂工业中具有非常良好的应用前景。     

黑曲霉GD-6纤维素酶液体发酵条件的研究

采用黑曲霉 (Aspergillusniger)GD 6液体发酵生产纤维素酶 ,研究了碳源、氮源、培养基起始 pH值、接种量、摇床转速、通气量对该菌株产纤维素酶活力的影响。结果表明 ,GD 6的最适发酵温度为 2 8～ 3 0℃ ,产酶pH为 5 .5～ 6.0 ,摇床最适转速为 1 5 0r/min ,最佳接种量为 1 0 %。在以 6.0 %稻草粉为碳源、1 %豆饼粉为氮源时产酶活力最高。在最适培养条件下 ,发酵周期为 1 2 0h,发酵液中CMC酶活为 1 88.6U/mL ,FP酶活为 2 7.0U/mL。     

一步法发酵菊芋产乙醇菌种的筛选及产酶条件、酶学性质的研究

从腐烂的菊芋及实验室保存的菌种中,选育到一株发酵菊芋产乙醇的菌株克鲁维酵母Kluyveromyces marxianus Y1。利用正交实验法对克鲁维酵母产菊粉酶的培养基组成及培养条件进行优化,确定培养基组成（g/L）为：菊粉40,酵母粉4,蛋白胨4,尿素1;初始pH5．0,温度30℃,150r／min条件下培养达到最佳产酶效果（57U／mL）。该菌株所产菊粉酶的性质测定结果表明：以菊粉为底物,该菊粉酶最适反应温度为55℃,在60℃以下稳定性很好,高于60℃时酶迅速失活;最适pH为5．0,pH4．6—5．2范围内酶稳定性很好;该酶属于外切型菊粉酶,体积分数为8％的乙醇对酶活力基本没有影响。     

家蚕肠道枯草杆菌产蛋白酶的发酵条件与酶学性质

肠道微生物分泌的蛋白酶可促进家蚕对桑叶养分的消化吸收,枯草芽孢杆菌是家蚕肠道内一种重要的产蛋白酶菌株。为提高枯草芽孢杆菌蛋白酶的高效利用,对该菌株适宜发酵条件及酶学性质进行了研究。结果表明：各因素对枯草芽孢杆菌产酶活性影响的大小顺序依次为：pH值〉培养温度〉培养时间〉装液量;最适的产酶条件为：pH=7,培养温度：30 ℃,培养时间：36 h;对枯草芽孢杆菌产蛋白酶进行初步提纯后并研究得出该酶反应的最适pH 10.0,最适反应温度为：60 ℃;该酶为碱性蛋白酶、不耐高温、不耐酸,但在35 ℃条件下热稳定性较好。     

烟曲霉A-16产纤维素酶工艺优化及酶学特性北大核心CSCD

分离筛选高效降解稻草的菌株,研究菌株产纤维素酶工艺条件及酶学性质。采用刚果红染色法从腐败木质下的土壤中分离筛选到一株产纤维素酶菌株,结合菌株的形态特征和18S rDNA序列同源性比较进行鉴定;通过单因素试验和响应面分析法确定菌株最适产酶条件,并对纤维素酶的稳定性进行研究。分离纯化得到的菌株命名为烟曲霉(Aspergillus fumigatus A-16);响应面实验结果表明,最优产纤维素酶工艺参数为:稻草粉添加量7 g/100 mL,pH 6.0,温度65℃,发酵时间5 d;在此最优条件下,该菌产生的羧甲基纤维素酶(CMCase)和滤纸酶(FPA)活力分别为2 954.76 U/mL和1 086.37 U/mL;其总活力较优化前提高了26.4%。该纤维素酶的适宜反应温度为70℃,适宜pH 6.0。在80℃热处理90 min条件下酶活力可保持在80%以上,说明该酶热稳定性较好。同时,在pH 5.0-7.0范围内比较稳定,放置1 d后可保持70%以上的酶活力。该研究可为利用富含纤维素的生物质原料开发洁净能源及食品级葡萄糖资源提供了基础支撑。     

纤维素酶水解糠醛渣生产酵母I．纤维素酶的形成和酶解糠醛渣

本实验研究了康氏术霉AS．3．4290(白色突变株)纤维素酶产生和糠醛渣酶解的适宜条件。在稻草粉固体培养基上,纤维素酶产生的最适条件是：稻草粉细度为0．35厘米筛孔的筛下物;培养基含水量67％左右;pH 6.0—6.5;添加氪源是必要的,其中以磷酸氢二铵(4％)和硫酸铵(2％)为佳;培养温度28℃;时间为3天;葡萄糖在浓度低时,对纤维素酶产生有好处,而浓度过高时(大于6％),反而对酶形成有抑制作用。上述培养物直接用于糠醛渣的酶水解,其酶作用的最适条件是：pH5．0,温度50℃,作用时间48小时。葡萄糖和单宁对纤维素酶有抑制作用。     

pH-dependent stability of EGX, a multi-functional cellulase from mollusca, Ampullaria crossean

Cellulose and hemicellulose are the most abundant andmajor constituents of plants and the potential rawmaterials for enzyme hydrolysis related biotechnologicalprocesses. The cellulase and hemicellulase have beenconsidered as two of the most important enzymes inbiotechnology market which can hydrolyze cellulosic ma-terials to fermentable sugars for various biotechnologicalpurposes [1,2], and can also be used in textile- and paper-making industry [3]. It is important to use these enzymesunder re…     

Expression of <Emphasis Type="Italic">Trichoderma</Emphasis><Emphasis Type="Italic">viride</Emphasis> endoglucanase III in the larvae of silkworm, <Emphasis Type="Italic">Bombyx mori L.</Emphasis> and characteristic analysis of the recombinant protein

Endoglucanase is a part of cellulase which hydrolyzes cellulose into glucose. In this study, we cloned endoglucanase III (EG III) gene from Trichoderma viride strain AS 3.3711 using a PCR-based exon splicing method, and expressed EG III recombinant protein in both silkworm BmN cell line and silkworm larvae with an improved Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the chiA and v-cath genes of Bombyx mori nucleopolyhedrovirus (BmNPV). The result showed that around 45 kDa protein was visualized in BmN cells at 48 h after the second generation recombinant mBacmid/BmNPV/EG III baculovirus infection. The enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 8.0 and temperature 50°C, and increased 20.94 and 19.13% compared with that from blank mBacmid/BmNPV baculoviruses infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 9.0 and at temperature range from 40 to 60°C. It provided a possibility to generate transgenic silkworms expressing bio-active cellulase, which can catabolize dietary fibers more efficiently, and it might be of great significance for sericulture industry.     

麝鼠肠道纤维素分解菌的分离鉴定及其酶学特性分析

本研究旨在从麝鼠(Ondatra zibethicus)肠道中分离出高效分解纤维素的菌株,为开发纤维素分解菌微生物制剂提供菌种资源。本研究利用以羧甲基纤维素钠(CMC-Na)为单一碳源的培养基,从麝鼠盲肠内分离出--株高效分解纤维素的菌株WJ-3,并对该菌株进行形态鉴定、生理生化鉴定和16S.rDNA分子鉴定。对菌株WJ-3所产羧甲基纤维素酶(CMCase)进行酶学特性实验,分析此纤维素酶的最佳反应pH和最佳反应温度,以及此纤维素酶对不同温度和不同酸碱度的耐受性。结果表明,菌株WJ-3属于空气芽孢杆菌(Bacillus aerius),并将其命名为Bacillus aerius WJ-3。菌株WJ-3所产羧甲基纤维素酶在pH 4.0~6.0的范围内反应时,酶活性随pH值升高而增加,其最佳反应pH为6.0,且此纤维素酶在pH4.0~8.0范围内保存30min后均能保持80%以上的相对酶活性:菌株WJ-3所产羧甲基纤维素酶在温度30~50 ℃范围内反应时,随温度上升酶活性逐渐增加,在50 ℃时酶活性最高,之后随温度的升高酶活性逐渐下降,且纤维素酶在此温度范围内保存30 min后均能保持较高的酶活性。综上所述,菌株Bacillus aerius WJ-3所产羧甲基纤维素酶的酶活性较高,并且此纤维素酶的耐酸碱性及热稳定性良好,是具有一定利用价值的菌种资源。     

Purification and characterization of an organic-solvent-tolerant cellulase from a halotolerant isolate, Bacillus sp. L1

A halotolerant isolate Bacillus sp. L1 producing extracellular cellulase was isolated from Yuncheng, China. Production of the enzyme started from mid-exponential phase of bacterial growth and reached a maximum level during the post-stationary phase. The cellulase was purified to homogeneity with molecular mass of 45 kDa. Substrate specificity test indicated that it was an endoglucanase for soluble cellulose. Optimal enzyme activity was found to be at 60 °C, pH 8.0, and 7.5 % NaCl. Furthermore, it was highly active and stable over broad ranges of temperature (30-80 °C), pH (7.0-9.0), and NaCl concentration (2.5-15 %), thus showing its excellent thermostable, alkali-stable, and halotolerant nature. The cellulase activity was greatly inhibited by ethylenediaminetetraacetic acid, indicating that it was a metalloenzyme. Significant inhibition by phenylmethylsulfonyl fluoride and phenylarsine oxide revealed that serine and cysteine residues were essential for the enzyme catalysis. Moreover, the cellulase was highly active in the presence of surfactants, and it showed high stability in the presence of water-insoluble organic solvents with log P (ow)at least 0.88. Results from this study indicate that the purified cellulase from isolate L1 may have considerable potential for industrial application owing to its useful properties.     

一株低温玉米秸秆降解真菌的筛选、鉴定及降解特性

【背景】在我国北方地区玉米秸秆还田时期地温低、秸秆降解慢,如何加速玉米秸秆低温腐解成为研究热点。【目的】从冷凉地区土壤中筛选具有高效降解纤维素能力的低温菌株,为秸秆的有效利用奠定基础。【方法】在低温培养条件下,采用稀释涂布平板法、羧甲基纤维素钠(sodium carboxymethyl cellulose,CMC-Na)水解圈测定法、胞外酶活测定法、秸秆失重法进行低温秸秆降解菌株的初筛、复筛和秸秆降解性能的测定;根据菌株形态学特征及ITSrDNA序列分析对筛选菌株进行鉴定;利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid,DNS)法和秸秆失重法对菌株在不同接种量、培养基初始pH、温度情况下的纤维素酶活力和玉米秸秆降解能力进行研究。【结果】以16°C为筛选温度,获得一株在刚果红-羧甲基纤维素钠平板上D/d值为2.17、CMC酶活力为703 U/mL的高产纤维素酶低温真菌SDF-25;该菌株在4°C可以生长,10-16°C为最适生长温度,37°C条件下仍能生长;综合菌株的形态学和分子生物学测定结果,菌株SDF-25为草酸青霉菌(Penicillium oxalicum);该菌株最佳产纤维素酶的培养条件为接种量2%、初始pH为7.0、培养温度为10°C,在该培养条件下菌株SDF-25的CMC酶活为993.3 U/mL。失重法测定接种SDF-25于10°C培养15 d时秸秆降解率为39.5%,16°C时为44.9%。【结论】草酸青霉菌SDF-25可在低温条件下生长并具有较强的纤维素酶生产能力,在秸秆还田方面具有良好的应用前景。     

Purification and properties of a cellulase from Aspergillus niger.

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.     

Cellulase production in a new mutant strain of Penicillium decumbens ML-017 by solid state fermentation with rice bran

To produce cellulolytic enzyme efficiently, Penicillium decumbens strain L-06 was used to prepare mutants with ethyl methane sulfonate (EMS) and UV-irradiation. A mutant strain ML-017 is shown to have a higher cellulase activity than others. Box-Behnken's design (BBD) and response surface methodology (RSM) were adopted to optimize the conditions of cellulase (filter paper activity, FPA) production in strain ML-017 by solid-state fermentation (SSF) with rice bran as the substrate. And the result shows that the initial pH, moisture content and culture temperature all have significant effect on the production of cellulase. The optimized condition shall be initial pH 5.7, moisture content 72% and culture temperature 30°C. The maximum cellulase (FPA) production was obtained under the optimized condition, which is 5.76 IU g(-1), increased by 44.12% to its original strain. It corresponded well with the calculated results (5.15 IU g(-1)) by model prediction. The result shows that both BBD and RSM are the cellulase optimization methods with good prospects.     

Characterization of a commercial cellulase for hydrolysis of agroindustrial substrates

This work is focused on the characterization of a commercial cellulase in terms of optimum pH and temperature, stability to pH and temperature and affinity of this enzyme to several substrates, determining the Michaelis-Menten parameters. Maximum activity of cellulase was obtained for the temperature range from 40 to 50 °C and pH from 5.2 to 5.5. Enzyme activity decreased only 15% after 150 h of reaction at temperatures between 30 and 50 °C. No loss of activity was observed at pH 5.0 and 5.5. The cellulase showed satisfactory results in the hydrolysis of agroindustrial substrates, since similar activity was verified on filter paper and other agroindustrial substrates.     

Purification and characterization of a carboxymethyl cellulase from Artemia salina

Brine shrimp (Artemia salina) belong to a group of crustaceans that feed on microalgae and require a cellulase enzyme that can be used in ethanol production from marine algae. Protein with potential cellulase activity was purified and the activity analyzed under different conditions. After initial identification of cellulase activity by CMC cellulase, surface sterilization and PCR using 16s rRNA primers was conducted to confirm that the cellulase activity was not produced from contaminating bacteria. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. After the final purification, a 70-fold increase in specific enzyme activity was observed. SDS–PAGE results revealed that the cellulase enzyme had a molecular mass of 96 kDa. Temperature, pH, and salinity values were found to be optimal at 55 °C, pH 8.0, and 600 mM NaCl, respectively. Specifically, the enzyme showed a fivefold increase in enzyme activity in seawater compared to 600 mM NaCl in phosphate buffer. Further analysis of the purified enzyme by molecular spectrometry showed no match to known cellulases, indicating this enzyme could be a novel halophilic cellulase that can be used for the production of bioethanol from marine macroalgae.