高产纤维素酶的木霉菌株筛选及发酵条件优化

纤维素是由β-1,4糖苷键链接而成的葡萄糖单体聚合物,广泛分布于自然界,是一类易获取且廉价的可再生资源。自然界含纤维素材料的利用和处理是目前亟待解决的问题,筛选产纤维素酶的优良菌株并实现纤维素的有效利用具有重要意义。利用纤维素酶降解纤维素是绿色、高效和可持续的利用方式,广泛应用于工业和农业领域(养殖、能源、纺织等)。因此,实现高效低成本的纤维素酶生产成为研究和发展的目标。木霉属真菌是重要的纤维素酶产生菌,具有产酶量大、酶系全和酶活性高等优点,分泌的胞外酶易于工业生产中的分离提纯。本研究以课题组前期积累和保存的25种木霉150个菌株为材料,通过刚果红平板法筛选、滤纸酶活测定和天然纤维素降解等测试,筛选出14株产纤维素酶能力较强的菌株(优于对照菌株里氏木霉QM9414),从中选取3株进行最佳产酶条件探索,如接种量、吐温80添加量、发酵培养基pH值等。初步测试结果表明,贵州木霉菌株8705在20℃条件下、摇瓶发酵9 d、在优化后的发酵培养基中产酶效果最佳,其上清液酶活可达到6.63 IU/mL。通过优化发酵温度、发酵时间和培养基成分配比等发酵条件,为资源利用打下物质基础并提供理论依据。     

表面活性剂对木霉菌产纤维素酶的影响

研究了TW80和“一枝花”洗衣粉作为表面活性剂对木霉菌纤维表酶发酵的影响。结果表明：当TW80在培养基中用量为0.2-0.5%时,对FPA最大提高率为29.6%,对木聚糖酶（Xylanase)最大提高率为31.1%,洗衣粉在培养基中用量为0.5%时,对促进木霉菌产Xylanase有较好的效果。最大提高率达34.4%。     

一株近玫色锁掷酵母的分离鉴定及其产胞外乳糖酶性质的研究

从广州徐闻农垦丰收农场土壤样品中分离到一株产乳糖酶菌株,结合菌株形态特征和ITS基因序列同源性分析结果,表明该菌株为Saccharomycetes sp.的未定种,其系统分类学关系与近玫色锁掷酵母Sporidiobolus pararoseus strain AUMC 7791(JQ425362.1)最近,故命名为:近玫色锁掷酵母XWSP1(Sporidiobolus pararoseus XWSP1),该菌种保藏号为CCTCC NO:M2019119。该菌发酵液能水解邻硝基酚-β-D-半乳糖苷生成黄色的邻硝基酚,具有产乳糖酶性能。优化了该菌株发酵培养条件,结果表明:该菌株在蛋白胨15 g/L、酵母粉20 g/L、半乳糖20 g/L和初始pH 7.0的培养基中,以1×106 CFU/mL接种浓度、4%接种比例,28℃180 r/min恒温振荡培养54 h时,菌株分泌的胞外乳糖酶具有最高的活力。酶学性质初步研究结果表明,该胞外乳糖酶在pH 7.0的反应条件下酶活力最高,Ca^2+、Mn^2+、Mg^2+和Cu^2+对酶活有不同程度抑制作用,其中Cu^2+对酶活抑制作用最强。     

长梗木霉纤维素酶基因的克隆及序列分析

从富含纤维素环境筛选获得一株纤维素降解菌株FU05,通过形态学特征及ITS序列分析确定其为长梗木霉(Trichoderma longibrachiatum)。PCR扩增获得该菌株的bgl2、cbh2和eg1。序列分析表明,这3种纤维素酶基因与GenBank上其他木霉同种纤维素酶基因具有较高同源性:bgl2基因与里氏木霉bgl2基因(AB003110)同源性达91%;cbh2基因与康宁木霉cbh2基因(DQ504304)同源性达99%;eg1基因与长梗木霉eg1基因(X60652)同源性达95%。3种纤维素酶基因编码的相应氨基酸序列与其他木霉纤维素酶的氨基酸序列相似性也非常高。对上述纤维素酶基因编码的相应蛋白进行PROSITE motif search,对其N端糖基化位点、纤维素结合区、糖基水解酶家族特征结构区等进行了定位。     

三种霉菌产纤维素酶能力分析与培养条件优化

对实验室现有3种真菌产纤维素酶能力的分析及培养条件优化。比较了3种菌在刚果红培养基上的透明圈大小、并分析产纤维素酶酶活;通过单因素与响应面分析的方法优化毛酶产纤维素酶的培养条件。通过试验得出3种真菌均能产纤维素酶,毛霉能产较多的纤维素酶。毛霉产纤维素酶的最佳条件为:pH 5.0,转速220 r/min,发酵时间47 h,发酵温度35℃,纤维素酶活为6.99U/mL。毛霉、青霉、曲霉均产纤维素酶,毛霉能降解玉米芯纤维素。     

青霉菌m8产半纤维素酶条件及酶性质研究

半纤维素酶可用于造纸工业的生物制浆和废水处理。通过碳、氮源对产胞外半纤维素酶的影响确定了青霉菌m8的适宜培养基 ,即 4 .5 %麦草粉 ,0 .5 0 % (NH4 ) 2 SO4 ,0 .1%KH2 PO4 ,0 .0 5 %MgSO4 ·7H2 O ,0 .0 3%NaCl,0 .33%Tween80 ,0 .15 %CaCO3。在上述培养基中 ,2 8℃恒温振荡 (12 0r min)培养 4 - 6d ,半纤维素酶活力可达 80u ml左右。用DNS法研究了该酶的性质。结果表明 ,其最适pH值为 4 .5 ,最适反应温度为 5 5℃ ;表观Km值为 4 .6× 10 - 2 g L ;该酶的耐热性比较强 ,可被K+ 、Ca2 + 、Mg2 + 离子激活 ,而被Ag+ 、Fe3+ 和Cu2 + 离子抑制。     

绿色木霉ZY-1固态发酵产纤维素酶

利用筛选的绿色木霉ZY-1（Trichoderma viride ZY-1）固态发酵产纤维素酶,采用稻草和麸皮为底物,考察稻草与麸皮比例随发酵时间对产酶的影响。结果表明：底物中,在m（稻草）：m（麸皮）为0：5和1：4时,发酵48h,pH保持4．5左右,还原糖量急剧上升,胞外蛋白产量最低;仅以稻草作底物时,整个发酵过程中pH约为7,还原糖量最低,胞外蛋白产量较高而滤纸酶活、羧甲基纤维素酶（CMCase）和β-葡萄糖苷酶（β-Gase）酶活均较低;在m（稻草）：m（麸皮）为3：2时,发酵96h,滤纸酶活达最大值5．01U／g干曲;m（稻草）：m（麸皮）为1：4时,发酵96h,β-Gase酶活达最大值4．6U／g干曲;m（稻草）：m（麸皮）为4：1时,发酵72h,CMCase酶活达最大值6．01U／g干曲。因此,底物中存在适量的稻草和麸皮有利于Trichoderma viride ZY—1产纤维素酶。     

产纤维素酶菌株宇佐美曲霉Y-11产酶条件及酶性质的研究

通过正交试验,对宇佐美曲霉纤维素酶固态发酵条件进行了研究,其较适培养基为：麸皮与稻草配比为２：３,氮源：尿素,含水量：２００％（液固比）,ｐＨ：５．５。２８－３０℃培养７２小时,ＣＭＣ酶活力为５．０５（ｕ／ｍｌ）,滤纸酶活力达到１．０４（ｕ／ｍｌ）。ＣＭＣ酶及滤纸酶最适ｐＨ分别为４．４１、６．０６;最适温度分别为６５℃、５５℃。酶的热稳定性与ｐＨ稳定性较高。     

黄绿木霉菌产纤维素酶条件优化

利用正交设计方法研究了温度、接种量、pH值、装液量等不同发酵条件对黄绿木霉菌产纤维素酶的影响,研究结果表明,在这些因素中影响该菌株产纤维素酶的最主要因素为温度,而其他三个因素对该菌株产纤维素酶影响比较小.研究中得出该菌株最适产酶条件为发酵5d,28℃、初始pH为6、接种量为8%、装液量为40 mL(150 mL三角瓶),摇床转数为170 r/min.     

长梗木霉纤维素酶的产生及提取

对长梗木霉(Trichoderma Longibrachiatum)ANU_3-958纤维素酶的产生及提取进行了研究。结果表明,固体曲培养144小时,固体曲与浸提液比例为1:7(W/V),采用硫酸铵分级沉淀法时所得纤维素酶活力最高。所得冻干纤维素酶粉经测定:羧甲基纤维素(CMC)酶活最高为2788.89IU/g,滤纸糖酶活(FPA)最高为79.44IU/g。相对固体曲得率平均为13.28%。     

Effect of cobalt and nickel on growth and carboxymethyl cellulase activity ofCellulomonas spp

ACellulomonas spp isolated from soil produced carboxymethyl cellulase (CMCase) and xylanase enzymes. Cobalt (0.1mm) and nickel (0.1mm) decreased the growth rate ofCellulomonas spp. These metal ions activated CMCase activity but not xylanase activity; cobalt being the greater stimulatory ion than nickel. A predominant long lag phase was observed in adapted cells when compared with the non-adapted cells. However, the growth level of control cells was never obtained by the adapted cells.     

pH and thermal stability studies of carboxymethyl cellulase from intergeneric fusants of Trichoderma reesei/Saccharomyces cerevisiae

The combined effect of pH and temperature on carboxymethyl cellulase from two intergeneric fusants (M 14 and M 62) of Trichoderma reesei QM 9414/Saccharomyces cerevisiae NCIM 3288 was studied using response surface methodology. A central composite design for two variables was employed for the optimization studies. This study was compared with similar studies carried out with Trichoderma reesei QM 9414. The optimal pH and temperature for the enzymes derived from these organisms were: for the fusant M 14—pH 5.7 and 41.7°C, for the fusant M 62—pH 5.3 and 43°C, and for Trichoderma reesei QM 9414—pH 4.31 and 38.3°C. Received 5 May 1997/ Accepted in revised form 17 July 1998     

黑曲霉纤维素酶的纯化及酶学性质研究

黑曲霉(Aspergillusniger)固态发酵后粗酶液经硫酸铵盐析,2次SephadexG-200柱层析后可提纯8倍左右.CMC酶最适作用温度为60℃,最适作用pH为3.5,30℃～70℃区间酶活力较稳定,在pH3.0～5.0范围内,50℃保温30min能保持80%的酶活力.CMC酶的Km、Vmax值分别为7.69%CMCg/ml、0.33mg/ml·     

嗜热毛壳菌(Chaetomium thermophile)液体发酵产生纤维素酶及酶学性质的研究

探讨了嗜热真菌Chaetomium thermophile产生纤维素酶的液体发酵条件及滤纸酶(FPA)的特性。采用液体发酵培养法,通过对碳源、氮源、培养时间、培养基的起始pH值及产酶过程中pH值和蛋白质含量变化的研究发现：在2％纤维素、1％可溶性淀粉为碳源;2．0％KNO3 0．2％酵母粉为氮源;起始pH值为6．5,50℃下培养9d后,各种酶活最高。发酵过程中,pH值和蛋白质的含量均在前3d下降,后升高。FPA的反应最适温度和pH值分别为60℃和5．5～6．0;且具有较高的热稳定性和DH稳定性。     

碱性纤维素酶及其应用的研究进展

碱性纤维素酶在碱性条件下具有内切β-1,4葡萄糖苷酶活力,在加酶洗涤剂等行业具有重要的应用价值。目前已发现由芽孢杆菌和链霉菌产生的多种碱性纤维素酶,其中大部分的基因已被克隆、测序和表达,其催化反应特性、反应机理和应用也得到了较深入的研究。从碱性纤维素酶的产生菌、酶反应特性和反应机理、碱性纤维素酶的基因克隆和表达,以及碱性纤维素酶的应用等几个方面,介绍了有关碱性纤维素酶的最新研究进展。     

Application and characterization of magnetic chitosan microspheres for enhanced immobilization of cellulase

In this study, a unique carrier magnetic chitosan microspheres (MCTS) was simply synthesized by anchoring Fe₃O₄ onto chitosan for direct immobilization of cellulases cross-linked by gluteraldehye. The structure and morphology were characterized using FT-IR, TGA, VSM and SEM. The optimum immobilization conditions were investigated: immobilized pH 7.0, amount of enzyme 15?mL (0.1?mg/mL), immobilization temperature 30?°C, immobilization time 5?h. At optimum conditions, MCTS achieved maximum enzyme solid loading rate of 73.5?mg/g, while recovery of enzyme activity approached to 71.6%. In the recycle test, immobilized cellulases operated without significant loss in its initial performances after 3 cycles, which indicated that immobilized cellulases can be regenerated and reused. The immobilized enzyme has better values of thermal and storage stability than that of free enzyme. Therefore, MCTS may be considered as a candidate with potential value of application in large-scale operations for cellulases immobilization.     

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。     

耐碱性真菌纤维素酶生产菌的筛选及酶学性质的初步研究

从造纸厂碱性土样中分离到２４株能够产生纤维素酶的真菌,经过摇瓶昨筛,选出一株耐碱性纤维素酶产生菌Ｓ６０７。经过鉴定。该菌株为黑色葡萄装穗霉（Ｓｔａｃｈｙ－ｂｏｔｒｙｓ ａｔｒａ）。通过对其粗酶液的性质进行测定,该菌所产纤维素酶粗酶液中的ＦＰＡ、ＣＭＣａｓｅ和β－葡萄糖苷酶的最适ｐＨ分别为６．０,６．０和６．５,其中内切酶在ｐＨ９．０时仍能保持最高酶活力的５０％以上,实验表明该菌株所产纤维素酶具有较     

Ultrasound mediated enzymatic hydrolysis of cellulose and carboxymethyl cellulose

A recombinant Trichoderma reesei cellulase was used for the ultrasound‐mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4–11.8 W cm^?2 sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis‐Menten kinetics. The Michaelis‐Menten parameter of the maximum reaction rate V_max was enhanced by sonication relative to controls, but the value of the saturation constant K_m was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm^?2. Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm^?2 power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1448–1457, 2013