一株毛栓菌静置液体发酵产纤维素酶条件研究

考察一株新分离的毛栓菌静置产纤维素酶的条件,通过单因素试验和正交设计试验分别对培养基初始pH、培养基中的碳源、氮源及诱导物进行研究。利用DNS（3,5-二硝基水杨酸）法测定CMC-Na（羧甲基纤维素钠）纤维素酶活。结果表明：该毛栓菌静置发酵产纤维素酶的最适pH为5.0,最适碳源、氮源和诱导物分别是淀粉、酵母粉和干麸皮,且质量浓度分别为淀粉1%、酵母粉0.4%、干麸皮1.5%,在此条件下,进行静置发酵3d,可获得CMC-Na（羧甲基纤维素钠）纤维素酶活力最高为56.62U/mL。此研究开创了纤维素酶生产菌的新发现,与以往所报道的好氧产纤维素酶菌种黑曲霉、木霉等相比,具有发酵时间短、耗能小等优点,具有一定的潜在应用价值。     

紫外线和60Co-γ联合诱变褐色高温单孢菌及产酶特性研究

本研究以褐色高温单孢菌(Thermomonospora fusca)为出发菌株,通过紫外线和60Co-γ射线联合诱变,获得了一株纤维素酶高产菌株AV8,CMC酶活力达到0.679 IU/mL,与出发菌株相比,其产酶能力提高3.53倍。通过对AV8产纤维素酶的培养条件进行测定,结果显示:产纤维素酶最适应温度为55℃;该菌的最适产CMC酶初始PH值为7.0,最适产FPA酶初始PH值为8.0;当培养到第7天时,产CMC酶达到高峰。当培养到第8天时产FPA酶达到高峰。     

甜菜渣发酵制备蛋白饲料的研究

以甜菜渣为原料,对固态发酵制备菌体蛋白饲料进行了研究。将纤维素酶水解法替代常规的黑曲霉发酵法进行原料的预处理,其最适酶解条件为:纤维素酶添加量为25 u.g-1,酶解时间为16 h。以面包酵母B188和产朊假丝酵母B204为菌种进行混合发酵,在最适发酵条件下,50 h粗白质质量分数达到21%,蛋白质净增量为14%。     

一株产纤维素酶菌株的分离、鉴定及产酶特性

【目的】筛选并鉴定一株产纤维素酶的菌株,初步探究该菌的产酶特性,为综合利用纤维素筛选菌源。【方法】在常温条件下,采用滤纸培养基对菌种富集,采用CMC-Na初筛纤维素降解菌,采用LB培养基分离纯化菌株,经形态学、生理生化特征试验、16S r RNA基因序列测定等分析筛选菌株的系统分类地位。单因素试验确定培养时间、培养温度、初始p H及Na Cl浓度对筛选菌株产酶活力的影响。【结果】从腐烂的玉米秸秆中分离出一株在常温下产纤维素酶细菌KZ-2,根据菌落形态特征、生理生化特征鉴定以及16S r RNA基因序列分析,初步鉴定KZ-2为肠杆菌(Enterobacter sp.),为潜在新种。产酶条件实验显示:该菌使用产酶发酵培养基120 h产酶量达到最大值,在25–35°C、初始p H 4.5–5.5、Na Cl浓度1.0%–2.0%范围内为最佳产酶条件,在最适条件下酶活可达80.93 U/m L。该菌株所产纤维素酶最适反应p H为7.0,最适反应温度为50°C。【结论】KZ-2是一株具有降解纤维素能力的细菌,在常温下即可分泌纤维素酶,并且该菌株为潜在新种,具有潜在的开发价值。     

黄绿木霉原生质体诱变育种研究

利用正交实验方法研究了影响黄绿木霉(Trichoderma aureoviride)原生质体形成的因素,并利用紫外线、硫酸二乙酯、氯化锂几种因素复合诱变原生质体筛选高产纤维素酶菌株。影响原生质体形成的因子顺序是酶系统>菌龄>酶解时间>酶解温度,原生质体形成的最佳条件是0.5%蜗牛酶 0.5%溶菌酶 1.0%纤维素酶,菌龄为18h,酶解时间为3.0h,酶解温度为32℃;在该条件下原生质体产量可达到4.25×106个mL-1。Ca2 和PEG对提高原生质体再生率的作用明显;复合诱变后得到酶活显著提高、遗传性能稳定的诱变株T-14,其EG酶活与BG酶活分别提高了58.43%、44.48%。     

一株产纤维素酶细菌的筛选、鉴定及产酶条件优化

目的：筛选1株产纤维素酶的细菌。方法：通过对从腐烂朽木及其附近土壤中得到的样品进行富集培养、分离纯化得到16株纤维素分解菌,经刚果红染色鉴定和液体发酵培养后对其进行了菌种初步鉴定及产酶条件的初步优化。结果：获得1株纤维素酶分泌量较高的细菌LT3。结论：LT3为革兰氏阳性菌,菌体成杆状,经发酵优化培养后,较适产酶条件为甘蔗渣20g/L,pH7.0、30℃培养120h,CMC酶活为71.17U/mL,滤纸酶活为33.37U/mL。通过克隆其16S rDNA序列,对其进行系统进化分析,鉴定为蜡状芽孢杆菌。     

不同产纤维素酶菌种特定底物培养产酶活力比较

纤维素酶是由几种不同酶组成的复合酶系,由于酶催化反应底物的复杂性,从不同霉菌和不同底物发酵分离得到的纤维素酶组分和酶活力有着很大差别.本论文的主要目的主要是针对不同底物和不同霉菌进行产纤维素酶活力比较评价.实验选用CGMCC3.3002 和CICC13048 两种菌,使用液体发酵法在微晶纤维素和糠醛渣两种底物上进行产酶培养,在特定的时间测定其产的纤维素酶的纤维二糖酶活力、内切葡聚糖酶活力、外切葡聚糖酶活力以及滤纸酶活.产滤纸酶活比较高的菌株CGMCC3.3002,以糠醛渣为特定底物的滤纸酶活要高于微晶纤维素特定底物,且CGMCC3.3002 在微晶纤维素和糠醛渣底物的酶活力差异较大,该菌株可通过紫外诱变使其酶活力更高,有望用于糠醛渣生产燃料乙醇的过程中.     

粗糙脉孢菌(Neurospora crassa)纤维素酶液体发酵培养基的优化

粗糙脉孢菌是天然纤维素降解真菌,具有产纤维素酶能力,国内外对其纤维素降解机理和发酵产酶有一定的研究,但对其产酶的条件优化研究得不多,其产酶潜力需要进一步挖掘。以粗糙脉孢菌基因组测序菌株FGSC 2489为对象,采用响应面分析法对Neurospora crassa摇瓶发酵产纤维素酶进行培养基优化。采用Plackett-Burman(PB)实验设计考察发酵培养基中关键参数对产酶条件的影响,进而采用最陡爬坡实验逼近最大响应区域,并结合中心组合实验(central composite design,CCD)和响应面分析法对两个显著因素进行分析。PB实验结果显示:Peptone、Yeast extract对产纤维素酶有显著影响。通过响应面分析得到一元二次方程,对方程求解得到Peptone 7.27g/L、Yeast extract 5.51g/L。采用该优化培养基,最大纤维素酶活可达1.27FPU/ml,较优化前提高了2.03倍;CMC酶活14.15IU/ml,比优化前提高1.88倍;木聚糖酶活24.13IU/ml,比优化前提高1.86倍;葡萄糖苷酶酶活1.22IU/ml比优化前提高2.08倍。     

一株纤维素降解真菌的筛选、鉴定及酶学性质分析

通过对富含枯枝败叶的土壤样品进行富集培养,利用刚果红纤维素培养基初筛和酶活测定复筛得到产纤维素酶的一株真菌,将其命名为GC2-2,并对该菌株进行鉴定及酶学性质研究。结果表明该菌株是一株耐高温、碱性纤维素酶的真菌GC2-2。通过18S rDNA分子克隆测定,该菌为球孢枝孢菌,其滤纸酶的活力优于CMC酶的活力。该菌所产酶的最适反应条件为温度35°C,最适pH值7.5。     

一株耐冷玫红假裸囊菌HD1031的鉴定及其所产纤维素酶的研究

[目的]以纤维素为唯一碳源,从四川省阿坝自治州黄龙沟的高山低温环境中分离筛选产纤维素酶的耐冷菌,并研究菌株的产酶特征.[方法]根据菌株的ITS序列分析及形态特征,对菌株进行鉴定.利用DNS法测定纤维素酶酶活性.[结果]从四川省阿坝自治州黄龙沟的高山腐殖土中筛选出一株产纤维素酶的耐冷菌HD1031,经鉴定该菌为玫红假裸囊菌(Pseudogymnoascus roseus).该菌可在4℃-25℃生长,最适生长温度为16℃-17℃.该菌在以微晶纤维素和玉米芯粉为碳源、硫酸铵和Tryptone为氮源的培养基中,17℃、160 r/min摇瓶发酵8d后产生纤维素酶,其中内切葡聚糖酶酶活为366.67 U/mL,滤纸酶酶活87.6 U/mL,β-葡萄糖苷酶酶活90.8 U/mL,酶最适反应pH为6.0,最适反应温度为50℃.[结论]筛选获得一株产纤维素酶的耐冷菌HD1031,此菌株所产纤维素酶在20℃-40℃下活性较高,对热敏感,具有低温纤维素酶的特点.     

Laboratory evolution and multi-platform genome re-sequencing of the cellulolytic actinobacterium Thermobifida fusca

Biological utilization of cellulose is a complex process involving the coordinated expression of different cellulases, often in a synergistic manner. One possible means of inducing an organism-level change in cellulase activity is to use laboratory adaptive evolution. In this study, evolved strains of the cellulolytic actinobacterium, Thermobifida fusca, were generated for two different scenarios: continuous exposure to cellobiose (strain muC) or alternating exposure to cellobiose and glucose (strain muS). These environmental conditions produced a phenotype specialized for growth on cellobiose (muC) and an adaptable, generalist phenotype (muS). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains. Phenotypically, the muC strain showed decreased cell yield over the course of evolution concurrent with decreased cellulase activity, increased intracellular ATP concentrations, and higher end-product secretions. The muS strain increased its cell yield for growth on glucose and exhibited a more generalist phenotype with higher cellulase activity and growth capabilities on different substrates. Whole genome re-sequencing identified 48 errors in the reference genome and 18 and 14 point mutations in the muC and muS strains, respectively. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. By conducting and characterizing evolution experiments on Thermobifida fusca, we were able to show that evolutionary changes balance ATP energetic considerations with cellulase activity. Increased cellulase activity is achieved in stress environments (switching carbon sources), otherwise cellulase activity is minimized to conserve ATP.     

黄瓜膨胀素的重组表达及活性分析

目的:提高纤维素的酶水解效率和开发高效的纤维素酶水解过程。方法:采用RT-PCR方法从黄瓜胚轴细胞中分离了膨胀素S1的cDNA,并使之与毕赤酵母表达质粒pPICZ(A连接,形成重组质粒pPICZ(A-exs1。通过电转化方法,用质粒pPICZ(A-exs1转化巴氏毕赤酵母GS115,得到重组菌株P.pastoris-exs1。在该重组菌株中,膨胀素的基因通过同源重组整合在毕赤酵母的染色体上,并处于毕赤酵母甲醇氧化酶启动子的下游。重组菌株P.pastoris-exs1在甲醇诱导下可合成并分泌膨胀素。结果:培养上清液没有纤维素酶活性,但具有破坏滤纸纤维素结晶结构的能力。培养上清液与里氏木霉纤维素酶等量混合后,可使纤维素酶的滤纸酶活力提高50%。结论:采用巴氏毕赤酵母GS115重组成功表达了黄瓜膨胀素,其表达产物可以促进纤维素酶对滤纸的水解。     

利用红色荧光蛋白分析里氏木霉合成纤维素酶的机理

以红色荧光蛋白作为报告蛋白研究了里氏木霉的纤维素酶合成机理。构建了里氏木霉的表达盒,通过该表达盒使红色荧光蛋白的基因整合到里氏木霉的基因组DNA上,并受纤维二糖水解酶基因启动子的调控,得到重组菌株T.reeseiTR2。在不同的条件下培养T.reeseiTR2,红色荧光蛋白的表达情况可以反映在不同条件下里氏木霉合成纤维素酶的情况。在诱导的情况下,红色荧光蛋白随时间变化的情况与培养液中纤维素酶活性的变化相似,培养至36h后可以观察到荧光,并且不断增强,到菌丝自溶时荧光减弱。另一方面,诱导后里氏木霉菌丝的各个部位均可以观察到荧光,而且分布均匀,表明菌丝的各个部位在纤维素酶合成过程中所起的作用相同。在非诱导的情况下,培养时间较长时也可以观察到较弱的荧光,表明在此条件下里氏木霉仍可以合成少量的纤维素酶,这一结果为解释纤维素诱导里氏木霉合成纤维素酶的机理提供了另一个试验依据。     

High-yield cellulase production by Trichoderma reesei ZU-02 on corn cob residue

Cellulase production using corn cob residue from xylose manufacture as substrate was carried out by Trichoderma reesei ZU-02. It was found that on the same cellulose basis, the cellulase activity and yield produced on corn cob residue were comparable with that on purified cellulose. Under batch process, the optimum concentration of substrate was 40 g/l and the optimum C/N ratio was 8.0. In 500 ml flasks, cellulase activity reached 5.25 IU/ml (213.4 IU/g cellulose) after seven days' cultivation. In a 30 m(3) stirred fermenter for large scale production, cellulase and cellobiase activity were 5.48 IU/ml (222.8 IU/g cellulase) and 0.25 IU/ml (10.2 IU/g cellulose), respectively, after four days' submerged fermentation. The produced cellulase could effectively hydrolyze the corn cob residue, and the yield of enzymatic hydrolysis reached 90.4% on 10% corn cob residue (w/v) when the cellulase dosage was 20 IU/g substrate.     

低温纤维素酶菌株CNY086选育及发酵培养基优化(Ⅱ)

自渤海湾海泥中分离21株低温纤维素酶产生菌。其中菌株CNY01为绿色木霉(Trichoderma viride), 酶活力为67.30 U/mL。以该菌株为出发菌株, 经UV、DES等诱变, 选育出高产突变菌株CNY086, 酶活力为92.17 U/mL。该突变菌株低温纤维素酶发酵具有遗传稳定性。通过单因素和正交实验确定突变菌株CNY086低温纤维素酶发酵最适培养基: 秸秆粉1.20%、麸皮0.70%、硫酸铵0.50%、磷酸二氢钾0.55%, 上述条件下CNY086菌株酶活力达到108.55 U/mL。     

液态发酵条件下拟康宁木霉8985产纤维素酶能力初探

液态发酵条件下,以微晶纤维素为唯一碳源,比较了拟康宁木霉Trichoderma koningiopsis 8985和里氏木霉T. reesei QM9414产纤维素酶的能力。8985发酵12 h开始产生纤维素酶,36 h时酶活达到产酶峰值的50%,此时QM9414尚未诱导产酶。测定8985发酵84 h时上清液中滤纸纤维素酶、羧甲基纤维素酶、β-葡萄糖苷酶和木聚糖酶的酶活分别为1.06、3.62、1.80和6.67 IU/mL,分别是QM9414上述酶活的1.72、1.70、6.35和1.12倍。8985滤纸纤维素酶酶活的最适反应条件为pH 4.5,反应温度50 ℃,在Fe³⁺ (≤ 4 mmol/L)和Cu²⁺ (0-10 mmol/L)存在条件下酶活稳定。     

The impact of a single-nucleotide mutation of <Emphasis Type="Italic">bgl2</Emphasis> on cellulase induction in a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> mutant

Background

The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results

To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion

We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

     

大熊猫肠道纤维素分解菌的分离鉴定及产酶性质

【目的】从健康大熊猫新鲜粪便中分离具有纤维素酶活性的菌株,并对其进行菌种鉴定及产酶性质研究。【方法】利用羧甲基纤维素钠培养基分离纯化具有较高纤维素酶活性的菌株,根据形态学特征、生理生化特性以及16S rDNA分析对其进行分类鉴定,研究影响该菌株纤维素酶的产酶条件,以及对不同纤维素底物的降解情况。【结果】分离得到一株纤维素酶产生菌株P2,该菌株为好氧的革兰氏阳性细菌,生长温度范围20-50℃(最适温度37℃),pH范围6.0-9.0(最适pH7.0),NaCl浓度范围0%-15%(最适2%NaCl),培养24h达到产酶高峰。16S rDNA基因序列分析显示,菌株P2与解淀粉芽胞杆菌(Bacillusamyloliquefaciens)NBRC15535相似性为99.66%。该菌株对四种纤维素底物(滤纸、脱脂棉、秸秆、竹纤维)均有不同程度的降解,内切葡聚糖酶、外切葡聚糖酶、β-葡萄糖苷酶和总酶活具有不同的酶活变化。【结论】本研究首次从大熊猫粪便中分离出了好氧纤维素分解菌,并鉴定为解淀粉芽胞杆菌,对上述四种纤维结构均有一定的破坏和分解作用,为进一步研究大熊猫竹纤维消化机制提供了菌源。     

Cellulase production by a thermophilic clostridium species

Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 μm) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C₁ cellulase (degrades native cellulose) and C_x cellulase (β-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl₂ precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C_x cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature.