紫外线诱变提高细菌产纤维素酶活力的研究

从南京红山动物园土样及食草动物的粪便中分离出14种产纤维素酶菌株,其中F1表现出较高的酶活力.以F1为出发菌株,通过紫外线诱变处理,采用透明圈法初筛和摇瓶培养复筛,获得了10株高产纤维素酶的突变株Q1～Q10.经紫外线诱变处理的Q3突变株产酶活力最高,与出发菌株相比酶活力提高了15.8倍.     

^60Co-γ射线诱变褐色高温单孢菌的研究

以褐色高温单孢菌(Thermomonospora fusca)为出发菌株,通过60Co-γ射线诱变孢子悬液,采用透明圈法初筛和摇瓶培养复筛的方法,获得了一株纤维素酶高产菌株AV5,与出发菌株相比,其产酶能力提高1.8倍.     

~(60)Co―γ射线诱变褐色高温单孢菌的研究

以褐色高温单孢菌(Thermomonospora fusca)为出发菌株,通过60Co―γ射线诱变孢子悬液,采用透明圈法初筛和摇瓶培养复筛的方法,获得了一株纤维素酶高产菌株AV5,与出发菌株相比,其产酶能力提高1.8倍。     

5-氟尿嘧啶与甲基磺酸乙酯复合诱变选育高产弹性蛋白酶菌株

目的以弗氏链酶菌为出发菌,使用化学诱变剂甲基磺酸乙酯(Ethylmethylsulfone,EMS)和5-氟尿嘧啶(5-Fluorouracil,5-FU)复合诱变,选育高产弹性蛋白酶突变菌株。方法用高氏培养基培养弗氏链霉菌,收集菌悬液经摇瓶发酵培养后,加入6 mg/ml 5-FU进行诱变处理,收集处理液,经10倍系列稀释(10-1~10-7)后,涂布初筛培养基平板,选取透明圈出现较早和HC(透明圈直径/菌落直径)值较大的菌株培养后,加入2%EMS,37℃分别作用0、15、30、45、60 min,涂布牛奶培养基,计数菌落数,计算菌悬液存活率,选择合适条件进行复合诱变选育高产弹性蛋白酶突变菌株,选取HC值较大的5个菌株,进行摇瓶发酵培养,采用Sacher法检测弹性蛋白酶活力。结果弗氏链霉菌菌液经0.6 mg/ml 5-FU处理后,经2%EMS作用30 min进行复合诱变,孢子存活率为16.1%;经初筛、复筛,获得高产生弹性蛋白酶菌株,酶活力达35.98 U/ml,比出发菌株提高了35.6%。结论选择5-FU浓度为0.6 mg/ml,EMS浓度为2%作用30 min时,对弗氏链酶菌进行复合诱变,其诱变效率高,可获得高酶活的突变株。     

抗药性基因突变筛选高产菌株

本文通过洁霉素对盐酸林可霉素产生菌 98-1菌株孢子的致死浓度测定 ,采用诱变剂EMS的四种不同诱变剂量对菌株孢子进行诱变处理 ,诱变处理的孢子涂布在含洁霉素 ( 2 0 0 0 0ug/ml)的高氏平板上 ,获得了大量的洁霉素抗性基因突变株 ,然后从 90 0株洁霉素抗性基因突变株中通过初筛获得高于诱变出发菌株产素能力的菌株 5 6株 ,再进一步通过摇瓶复筛 ,并结合菌丝生长及摇瓶代谢情况 ,获得优于出发菌株的诱变菌株 4株。将这 4个菌株连同出发菌株连续三批次进行摇瓶发酵 ,结果 4个突变株的产素能力 (产量 )及摇瓶代谢和菌丝生长情况均优于出发菌株 ,试验筛选出最优菌株 0 2 -0 3 -40 2。将 0 2 -0 3 -40 2进行罐上发酵生产试验 ,结果试验罐比对照罐平均效价提高 1 7.5 6% ,平均产量提高 1 9.3 4%。本文建立了林可霉素高产菌株的洁霉素抗性基因突变诱变快速高效的筛选方法     

磷脂酶D的紫外诱变选育及发酵条件的优化

磷脂酶D在催化磷脂酰基交换反应中具有重要的应用价值。本文对产磷脂酶D野生链霉菌株进行紫外诱变,筛选得到一株产酶活力提高42.5%的变异株。通过摇瓶培养,对发酵条件进行探究。确定的最佳培养基组成为：葡萄糖10.0 g/L,牛肉膏和蛋白胨各5.0 g/L,MgSO4?7H2O 1.0 g/L,CaCl2 3.0 g/L,NaCl 2.0 g/L;表面活性剂Tween80对产酶有促进作用,其适宜浓度为0.60 g/L。在上述条件下,摇瓶发酵产酶活力达3.23 U/mL。     

应用~(60)C_0辐射诱变选育赤霉酸GA_3高产菌株的研究

通过藤仓赤霉菌原生质体对多种抗生素敏感性测定,采用诱变剂60C0不同诱变剂量对菌株原生质体进行诱变处理,筛选抗多粘菌素的变株。经多轮次诱变摇瓶初复筛,获得抗性突变株1130#的摇瓶发酵单位比对照提高13.3%,取得了较好诱变效果。     

微波诱变选育木糖醇发酵高产菌株

研究了使用微波诱变筛选木糖醇发酵菌株的处理方法和适宜的微波诱变时间。结果表明,微波处理时间为40 s时,致死率达90.2%,获得一支突变菌株M-11,300 mL摇瓶发酵最终转化率高达83.3%。     

亚硝基胍-紫外复合诱变选育高产衣康酸菌株

为提高土曲霉KYK-031发酵生产衣康酸能力,对出发菌株开展诱变育种和高产能力选育研究。采用亚硝基胍（NTG）和紫外（UV）复合诱变方法,通过平板初筛和摇瓶复筛,筛选衣康酸高产突变株。结果表明,亚硝基胍在500μg/mL浓度下的最佳诱变时间为40 min,15 W紫外诱变的最佳诱变时间为2 min,亚硝基胍-紫外复合诱变后筛选得到一株衣康酸高产菌株KYK-1035,摇瓶发酵产量为73.7 g/L,比出发菌株（41.8 g/L）提高了76.3%,糖酸转化率为63.4%。采用亚硝基胍-紫外复合诱变,提高了出发菌株诱变的正突变率,为衣康酸发酵继续放大和工业化生产奠定了基础。     

稀土和硫酸二乙酯联合诱变米曲霉产氨基酰化酶的研究

为获得高产氨基酰化酶的米曲霉菌株,选用稀土硝酸镧和硫酸二乙酯对米曲霉菌株CICC-2339进行联合诱变,经摇瓶发酵筛选到氨基酰化酶高产菌株SDESLa,其酶活为39.63 μmol·g-1·h-1,较出发菌株提高了39.50%,诱变效果显著.     

Pretreatment of Rice Hulls for Cellulase Production by Solid Substrate Fermentation

Abstract

Rice hulls via pretreatment served as a solid substrate for cellulase production. Different pretreatment methods, including microwave irradiation, organosolv process catalyzed by acid or alkali were compared. With rice hulls pretreated by microwave irradiation coupled with alkaline pretreatment and rice hulls pretreated with organosolv process catalyzed with acid or alkali, the maxima of filter paper activity (FPA) obtained during the fermentation were 18%, 21%, 31% higher, and maxima of the carboxy‐methyl cellulase (CMCase) were 7.7%, 14%, 16% higher than those obtained with the rice hulls pretreated only with alkali, respectively. The ratios of cellulose degradation were also estimated and they showed similar tendency with the maxima of FPA detected in the fermentation processes.     

应用里氏木霉发酵制备纤维素酶固体曲的研究

应用本实验室筛选得到的纤维素酶高产菌株里氏木霉（Trichoderma reesei）150-1-1发酵制备纤维素酶固体曲,通过优化固态发酵培养基组成及发酵条件,制备得到纤维素酶活力较高的固体曲及粗酶液。实验结果表明,在原料量为10 g,麸皮与秸杆粉质量比为4：1,料液比为1：2.5,发酵时间为120 h,发酵温度为31℃,发酵起始pH值为5.5的条件下,应用里氏木霉150-1-1发酵得到的纤维素酶固体曲酶活力达到423.6 U/g。     

Production of cellulases from coconut coir pith in solid state fermentation

Coconut coir pith, available in abundance especially in tropical countries, was studied as a substrate for the production of cellulase[1,4(1,3;1,4)-β-D -glucan 4-glucanohydrolase, EC 3.2.1.4] and β-D -glucosidase(β-D -glucoside glucohydrolase, EC 3.2.1.21) in solid state fermentation. The effects of fermentation time, nutrient level, substrate particle size and inoculum size have been examined for optimal production of these enzymes by the fungal strain Aspergillus niger NCIM 1005. The highest filter paper activity (FPA) of 4.11 IU g^?1, carboxyl methyl cellulose (CMCase) activity of 15·55 IU g^?1 and cellobiase activity of 9·31 IU g^?1 were obtained after 7 to 8 days of fermentation. Reese and Mandel's mineral solution in the substrate to mineral solution ratio of 1:10 (w/v) supported high cellulase and cellobiase activities. An inoculum size of 20–50% (v/v) based on the volume of mineral medium and substrate average particle size of 375 μm were optimum for enzyme production.     

粗纤维素酶液对微晶纤维素酶解的影响因素

栗生灰黑孔菌多被用于产漆酶的研究,很少有利用其纤维素酶的报道。为了降低在纤维素水解中纤维素酶的使用成本,利用栗生灰黑孔菌发酵制备的粗纤维素酶液,以微晶纤维素为底物模型,研究粗纤维素酶液水解微晶纤维素的最佳pH、温度和最佳表面活性剂助剂种类及浓度,并对不同表面活性剂存在条件下的纤维素酶解动力学、紫外和荧光光谱进行了研究。结果表明,粗纤维素酶水解微晶纤维素的最佳条件为pH 4.8,温度50℃,最佳表面活性剂助剂为吐温80,添加剂量为1.12mg/g底物;吐温80的添加可提高粗纤维素酶解的最大反应速度常数V_max,降低米氏常数K_m;表面活性剂改变了纤维素酶的紫外和荧光最大吸收峰,酰胺Ⅰ带和酰胺Ⅲ带的谱峰,可能通过与纤维素酶中的氨基酸残基发生反应影响了纤维素酶的结构,进而影响了微晶纤维素的水解反应。该研究为进一步降低纤维素水解成本提供了理论指导。     

Effect of pretreatment of cellulosic wastes on the production of cellulase enzymes by Trichoderma reesei

The cellulosic wastes, bagasse and sawdust, were treated with different acidified solvents: chloroform, dioxan, ethanol, ethylacetate, dimethyl sulfoxide and dimethyl formamide; for increasing periods up to 36 h. The treated materials were delignified, dried and introduced in the fermentation media as inducing carbon source for Trichoderma reesei. The untreated materials and pure cellulose powder were used individually in control and reference media, respectively. The fermentation media containing bagasse treated for 8 h in chloroform or dioxan gave a promising cellulase activity, nearly the same as that from the reference medium. The untreated bagasse gave a markedly low cellulase activity. The media with treated sawdust gave a relatively higher enzyme activity but far below the reference medium. The sawdust treatment with the solvents mentioned resulted in a very mild effect because of its high content of admixtures, lighin and hemicellulose bonding cellulose.     

降解秸秆的纤维素酶产生菌的筛选及产酶条件研究

从土壤、霉变的农产品和实验室保存的真菌中筛选到15株产纤维素酶的菌株,其中绿色木霉T206产酶能力最强。利用液体发酵,研究了碳源、氮源、接种量、起始pH值、培养时间等对菌株产酶的影响,以及该菌株所产纤维素酶的种类。在最适条件下菌株培养96h后,羧甲基纤维素酶活(CMCA)最高达到7654.33U,是优化前酶活的1.7倍,滤纸酶活(FPA)达到1675.12U。     

牛粪中高温纤维素降解菌的性能研究

对从牛粪中分离出来的高温单孢菌(Thermomonospora sp.)SQ3菌株的生物学特征进行了研究,包括菌种生化鉴定培养和产纤维素酶及其培养条件等的研究。降解纤维素结果显示SQ3在纤维素刚果红培养基上4天可见明显的分解纤维素透明圈。产酶条件研究显示:SQ3在发酵时间5天,发酵温度55℃,pH7.0～8.0,尿素氮源,玉米秸秆碳源的条件下产CMC酶性能最佳,在发酵8天时,产FPA酶达到最高峰值。     

黑曲霉纤维二糖酶基因的克隆及其在里氏木霉中的表达

里氏木霉(Trichodema reesei)是目前常用的纤维素酶生产菌种,其分泌的纤维素酶是一个多组分的复合体系,主要包括5种内切型-β-葡聚糖酶(endo-J3-glucanase,EG,EC3.2.1.4),两种外切型β-葡聚糖酶(exo-β-glucanase,EC3.2.1.91,也称纤维二糖水解酶cellobiohydrolase,CBH)和两种纤维二糖酶(cellobiase,EC3.2.1.21,也称β-葡萄糖苷酶β-glucosidase)[1-3].在里氏木霉的纤维素酶蛋白中,纤维二糖水解酶I(CBHI)受强启动子控制,其蛋白质比例最高,约占50%～60%[1],而纤维二糖酶(CB)的比例最低,只占1%左右[4].     

Cooperative Action of Cellulase Enzyme and Carboxymethyl Cellulose on Cotton Fabric Cleanability from a Topographical Standpoint

In this study, the effect of cotton treatment with cellulose and carboxymethyl cellulose on soil release of three different types of fabric: woven plain, woven twill and knitted were systematically studied. A recent study of the effect of a cleaning cellulase enzyme on cellulose films has proven that this substance selectively attacks amorphous cellulose regions, consisting of small hills in a matrix of flat crystalline regions. According to our previous investigations, where carboxymethyl cellulose is present in the formula, the enzyme seems to drive soil release performance. However, the mechanism has not yet been sufficiently studied from the topographical standpoint. In the present study, topographical changes caused by the treatment with cleaning cellulase enzyme and carboxymethyl cellulose on the fabrics by conditioning while washing were analysed on three different length scales in order to interpret their cooperation on water and oil absorption mechanisms and, hence, on cleanability of cotton fabrics stained with liquid–solid, liquid and solid soils.