鸡肠源芽胞杆菌的分离、鉴定和抗菌活性

从人工饲养的健康三黄肉鸡的盲肠和大肠中分离出10株芽胞杆菌,对其进行菌落形态观察、革兰染色、芽胞染色和生理生化特性鉴定,确定为枯草芽胞杆菌、地衣芽胞杆菌、蜡状芽胞杆菌、巨大芽胞杆菌、球形芽胞杆菌、短小芽胞杆菌、凝结芽胞杆菌。同时测定了它们对动物病原性大肠埃希菌和链球菌的抑菌活性。其中有9株芽胞杆菌对大肠埃希菌有抑制作用,8株芽胞杆菌对链球菌有抑制作用。结果表明芽胞杆菌分离株Y2、Y3、Y4、Y5、Y6、Y7、Y8具有作为益生菌开发的潜力。     

水产源芽胞杆菌的分离鉴定及生物学功能分析

筛选具有较好生物学功能的芽胞杆菌(Bacillus),用以改善池塘养殖过程饲料等有机物降解、抑制水体中的病原菌,对从健康养殖鱼虾塘水体中分离的菌株,进行生化试验和16S rDNA序列分子鉴定;通过产酶、耐酸、耐高温试验,抑菌试验以及安全性试验研究分离菌株的生物学功能。试验共筛选出8株细菌,经生化试验及16S rDNA序列的分子鉴定,确定菌株ZHX17、ZHX18、ZHX31为贝莱斯芽胞杆菌（Bacillus velezensis）,菌株ZHX14、ZHX15为地衣芽胞杆菌（Bacillus licheniformis）、菌株NSX4、NSX7、NSX9为枯草芽胞杆菌（Bacillus subtilis）。试验结果表明,8株芽胞杆菌均具有较强的耐酸、耐高温特性,其中3株贝莱斯芽胞杆菌具有较强的分泌淀粉酶、纤维素酶、蛋白酶的能力,抑菌效果好于其他5株芽胞杆菌。安全性试验结果表明,8株芽胞杆菌对草鱼、罗非鱼相对安全。8株芽胞杆菌同时具备分泌淀粉酶、纤维素酶和蛋白酶3种胞外酶的能力,其中贝莱斯芽胞杆菌具有较强的病原菌抑菌能力,可以作为病原拮抗益生菌做进一步研究。     

鸡源益生芽胞杆菌的筛选

根据菌体对低pH和高胆盐的耐受性、产酶特性和对病原菌的抑菌效果,对家鸡肠道中的芽胞杆菌进行了筛选。结果表明,有4株芽胞杆菌(A2、C5、C19和D8)能抑制金黄色葡萄球菌(Staphylococcus aureus)的生长。这4株菌在pH 3.0和高胆盐条件下的存活率都达到60%以上,产淀粉酶和蛋白酶的能力也较好,表明这些菌株具有作为益生菌的开发潜力。经形态学和生理生化反应鉴定,初步确定A2和D8为地衣芽胞杆菌(Bacillus licheniform is),C5和C19为蜡样芽胞杆菌(Bacillus cereus)。     

渤海湾仿刺参养殖圈内贝莱斯芽胞杆菌B-VE的分离鉴定及其产抗菌脂肽的研究

为分离筛选具有广谱抑菌作用的细菌并研究其抗菌物质,实验利用琼脂扩散法和牛津杯法,以溶壁微球菌、金黄色葡萄球菌、铜绿假单胞杆菌、大肠埃希菌为指示菌,从渤海湾大连海域仿刺参养殖圈中分离筛选出9株活性菌,2株具有广谱抑菌效果,其中1株抑菌效果最强.通过形态学和分子生物学检测分析,鉴定该菌株为贝莱斯芽胞杆菌(Bacillus ...     

拮抗Bacillus subtilis的筛选及发酵条件对拮抗能力的影响

筛选了3株对大肠埃希菌具有拮抗活性的枯草芽胞杆菌,并对这3个菌株的拮抗能力最强的Bf菌株的发酵条件进行了初步研究。先将不同枯草芽胞杆菌菌株与株大肠埃希菌进行拮抗试验,根据抑菌率,筛选出对大肠埃希菌拮抗效果较好的枯草芽胞杆菌Bf。然后,在不同pH值、温度以及不同的培养时间下观察记录Bf对大肠埃希菌的拮抗作用。根据实验结果分析得到:筛选到的枯草芽胞杆菌在pH值为7.0,37℃下培养40 h后抑菌效果最好。     

普通卷甲虫肠道可培养细菌的分离、鉴定及产消化酶活性

目的分离培养普通卷甲虫肠道中的可培养细菌,筛选有产消化酶活性的细菌,推测其在协助普通卷甲虫消化食物中的作用。方法通过传统分离培养法分离普通卷甲虫肠道中的可培养细菌,利用平板透明圈法筛选产淀粉酶、蛋白酶、纤维素酶和脂肪酶活性的细菌,利用水解圈与菌落直径的比值,比较不同细菌的产消化酶活性。利用SPSS 20.0软件进行统计学分析,组间比较采用单因素方差分析。结果在普通卷甲虫肠道中分离出4个属9种细菌,其中气单胞菌属3种,假芽胞杆菌属和柠檬酸杆菌属各2种,芽胞杆菌属和假单胞菌属各1种。9种细菌中弗氏柠檬酸杆菌、豚鼠气单胞菌、南海假芽胞杆菌等3种细菌可产蛋白酶,嗜水气单胞菌、波特卡伦柠檬酸杆菌、水生气单胞菌、豚鼠气单胞菌和南海假芽胞杆菌等5种细菌可产纤维素酶,嗜水气单胞菌、波特卡伦柠檬酸杆菌、印度芽胞杆菌、水生气单胞菌、嗜盐假芽胞杆菌、豚鼠气单胞菌和南海假芽胞杆菌等7种细菌可产淀粉酶,未筛选到产脂肪酶细菌。统计学分析表明,3种产蛋白酶细菌和5种产纤维素酶细菌的产酶活性差异无统计学意义。而7种产淀粉酶细菌产酶的活力间差异有统计学意义,水生气单胞菌的产淀粉酶活性能力最强。结论普通卷甲虫肠道可培养细菌结构简单,但有消化酶活性的细菌种类多,5种细菌有产2种以上消化酶功能,说明肠道细菌可能在普通卷甲虫食物消化中起着重要作用。     

野生东亚钳蝎消化道可培养细菌的分离鉴定、耐药性及产消化酶活性

目的分离东亚钳蝎消化道中的可培养细菌,并研究其产消化酶活性和耐药性,探讨消化道细菌对东亚钳蝎消化食物的影响。方法野生东亚钳蝎采用传统细菌分离培养方法分离其消化道内可培养细菌,利用16S rRNA序列进行细菌的分子鉴定;利用平板透明圈法测定可培养细菌产淀粉酶、蛋白酶、脂肪酶和纤维素酶等消化酶的活性;采用药敏纸片扩散法进行消化道可培养细菌的药敏试验。结果在东亚钳蝎消化道中共分离到4个属10种细菌,其中芽胞杆菌属7种,分别为蛋白水解芽胞杆菌、解淀粉芽胞杆菌、巨大芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌、婴儿芽胞杆菌和长形赖氨酸芽胞杆菌;葡萄球菌属、纤维微菌属和短波单胞菌属各1株,分别为松鼠葡萄球菌、纤维化纤维微细菌和泡囊短波单胞菌。对10种细菌的产消化酶活性分析表明,8种细菌有产消化酶活性,占细菌总数的80%;5种细菌有产蛋白酶活性,分别为蛋白水解芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌、松鼠葡萄球菌和纤维化纤维微细菌;7种细菌有产淀粉酶活性,分别为蛋白水解芽胞杆菌、解淀粉芽胞杆菌、巨大芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌、松鼠葡萄球菌和婴儿芽胞杆菌;未筛选到产纤维素酶和脂肪酶活性的细菌;蛋白水解芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌和松鼠葡萄球菌可同时产2种消化酶。在药敏试验中,8种细菌对多粘菌素B耐药,5种细菌对四环素耐药,3种细菌对万古霉素耐药。结论东亚钳蝎消化道中可培养细菌的组成相对单一,多数细菌有分泌淀粉酶和蛋白酶的功能,说明东亚钳蝎消化道中的细菌可能有协助其进行食物消化的功能。消化道细菌对抗生素多表现为敏感,推测其生存环境中抗生素的污染较少,同时在对东亚钳蝎人工饲养的场所进行消毒时,要谨慎选择抗生素,防止抗生素使用不当致使东亚钳蝎肠道菌群紊乱引起疾病。     

五株乳酸菌和三株芽孢杆菌的生物学特性和功能

【背景】乳酸菌和芽孢杆菌是应用于生产最多的益生菌,但不同菌株间的生长特性均不相同,因此了解菌株的生物学特性具有重要意义。【目的】研究菌株的生物学特性,能合理地开发和利用菌株,以保证菌株生产应用的安全性。【方法】活化后鉴定5株乳酸菌和3株芽孢杆菌并对其形态进行观察,探究菌株的生长曲线、产酸能力及最适生长条件,测定菌株的抑菌活性和产酶性能,同时探究菌株的益生性和安全性。【结果】五株乳酸菌分别编号鉴定为干酪乳杆菌R1、副干酪乳杆菌R2、香肠乳杆菌R3、福莱乳杆菌R4和唾液乳杆菌R5;3株芽孢杆菌分别编号命名为贝莱斯芽孢杆菌Y1、枯草芽孢杆菌Y2和地衣芽孢杆菌Y3。八株菌形态结构均不相同但都为杆状,均在2–10 h为对数生长期,18–24 h为稳定期,培养24 h时乳酸菌和芽孢杆菌的活菌数均保持在10⁹和10⁸ CFU/mL,最适生长温度为37.0℃。乳酸菌具有较强的产酸能力和抑菌活性,芽孢杆菌有较强的产酶性,在人工胃液中都有较强的耐受性。八株菌都无溶血活性、无毒力基因、对抗生素都保持中度敏感以上;其中唾液乳杆菌有四环素耐药基因,但对四环素抗性为中度敏感。【结论】八株菌生长繁殖速度快,乳酸菌产酸能力和抑菌活性较强,芽孢杆菌具有较强的产酶性能,在体外具有较好的益生性和安全性,可应用于生产实践。     

一株产淀粉酶芽胞杆菌SY200的鉴定及其对动物病原菌的生物拮抗试验

目的对一株产淀粉酶芽胞杆菌SY200进行鉴定及其对动物病原菌的生物拮抗试验。方法提取芽胞杆菌SY200基因组DNA,采用细菌16S rRNA通用引物进行PCR扩增及对扩增到的目标片段的测序,将测序结果与NCBI上已知菌种的16S rRNA序列进行BLAST对比,并构建系统进化树进行分析。采用滤纸片法和牛津杯法分别研究该芽胞杆菌的全菌液及培养物上清液对3株病原菌的生物拮抗。结果结合细菌形态观察及生理生化特性鉴定,最终确定菌株SY200为甲基营养型芽胞杆菌(Bacillus methylotrophicus);芽胞杆菌SY200全菌培养液和培养上清液对产肠毒素大肠埃希菌、鸡白痢沙门菌、金黄色葡萄球菌均有较强的生物拮抗作用,抑菌物质主要为细菌的代谢产物。结论芽胞杆菌SY200被鉴定为甲基营养型芽胞杆菌,该菌株对3株动物性病原菌具有较强的生物拮抗作用。     

西洋参内生真菌抑菌活性的初步研究

以金黄色葡萄球菌、蜡状芽胞杆菌、枯草芽胞杆菌、大肠杆菌、鼠伤沙门氏菌及肠炎沙门氏菌为测试菌种,对从西洋参(Panax quinquefolium L.)的茎和叶中分离得到的36株内生真菌进行抑菌活性试验,并测定活性菌株的代谢产物和不同极性物质对测试病菌的抑菌活性。试验筛选出2株具有明显抑菌活性的内生真菌,它们的代谢产物和不同极性物质对金黄色葡萄球菌和蜡状芽胞杆菌的抑菌效果最佳,其中胞外多糖和正丁醇提取物的抑菌效果较好。     

秸秆纤维素分解菌的酶活力测定

目的:测定秸秆纤维素分解菌的酶活力。方法:从土壤中分离出具有分解纤维素能力的菌株,采用刚果红染色法进行粗选,得到7株透明圈较大的菌株。将这7株菌株液体发酵培养6d,再分别用滤纸分解度观察、羧甲基纤维素酶活法(CMC)、滤纸酶活法(FPA)和天然纤维素酶活法测定其酶活力。结果:在7株菌株中,F-1、F-2、F-3、F-5的酶活力测定结果与其溶解圈的测定结果、滤纸分解结果基本相同。且天然纤维素酶活力高的菌株,其CMC酶活、FPA酶活也高,滤纸分解效果也比较明显。结论:CMC法、FPA法和天然纤维素酶活法适于测定秸秆纤维素分解菌的酶活力。     

从海水环境分离筛选甘蔗渣纤维素降解菌

【目的】筛选海水环境高效甘蔗渣纤维素降解菌,并研究不同菌株间的混合发酵对甘蔗渣纤维素酶活力的影响,为纤维素降解菌在海水养殖中的应用提供理论基础。【方法】采用刚果红染色法进行菌株初筛,利用DNS法测定各菌株胞外纤维素酶活力及不同菌株间的混合酶液与混合发酵酶液的纤维素酶活力。【结果】筛选得到两株具有较强纤维素分解能力的细菌菌株Z4和S5,经16S rRNA基因序列分析,初步鉴定为地衣芽孢杆菌(Bacillus licheniformis)。菌株S5具有最高的全酶活和甘蔗渣纤维素酶活,分别为1.16 U/mL和2.80 U/mL。菌株Z4与S5间混合发酵能明显提高菌株的纤维素酶活力,比S5单独发酵时全酶活、甘蔗渣纤维素酶活分别提高40.60%、14.21%。同时菌株S5与芽孢杆菌BZ5混合发酵也能提高其纤维素酶活力,比S5单独发酵时全酶活、甘蔗渣纤维素酶活分别提高6.23%、25.92%。【结论】筛选得到两株酶系较全且酶活较高的纤维素降解菌Z4、S5,适宜的混合发酵可明显提高纤维素降解能力,在海水养殖中有较大的应用前景。     

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。     

Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae

Recombinant plasmids for expression of bovine cytochrome P450c21 (pA gamma 2), both P450c21 and yeast NADPH-cytochrome P450 reductase (pAR gamma 1), P450c21/yeast reductase fused enzymes (pAF gamma R1, pAF gamma R2, and pAF gamma R20), and yeast reductase/P450c21 fused enzymes (pAFR gamma 1 and pAFR gamma 2) were constructed by using expression vector pAAH5. The plasmids were each introduced into the yeast Saccharomyces cerevisiae AH22 cells. The recombinant yeast strains AH22/pA gamma 2 (Y21) and AH22/pAR gamma 1 (Y21R) produced 2-3 X 10(3) molecules of P450c21 per cell. The cultures of both strains converted progesterone and 17 alpha-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively. The 21-hydroxylase activity per cell of the strain Y21R was about three times higher than that of the strain Y21, probably due to overproduction of yeast reductase. The recombinant yeast strains AH22/pAF gamma R1 (Y21RF1), AH22/pAF gamma R2 (Y21RF2), and AH22/pAF gamma R20 (Y21RF20) produced about 1.1-2.0 X 10(4) molecules per cell of the corresponding P450c21/yeast reductase fused enzymes. The specific 21-hydroxylase activity toward 17 alpha-hydroxyprogesterone per cell of the strains Y21RF1, Y21RF2, and Y21RF20 was about 21, 28, and 49 times higher than that of the strain Y21, respectively. Thus, the fused enzymes were superior to P450c21 in the specific activity and in the expression level in the yeast. The Km values for 17 alpha-hydroxyprogesterone of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 0.29, 0.30, 0.67, and 0.65 microM, respectively. The Vmax values of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 28, 124, 151, and 222 moles/min.mole P450c21 or fused enzyme, respectively. These results indicated that the fused enzymes showed lower affinity for the substrate, probably due to structural modification and higher reaction rates through efficient intramolecular electron transfer as compared with those of P450c21. While the strain AH22/pAFR gamma 2 (YR21F2) produced about 3 X 10(4) molecules per cell of the reductase/P450c21 fused enzyme, the specific 21-hydroxylase activity of the fused enzyme toward 17 alpha-hydroxyprogesterone was extremely low, suggesting that the structure of the fused enzyme might not be suited for electron transfer in yeast microsomes.     

Polysaccharide-hydrolyzing enzymes ofFrankia (Actinomycetales)

Studies were made of the polysaccharide-hydrolyzing activity inFrankia (Actinomycetales) grown in synthetic media using modifications of three standard assay procedures. In screening five different strains ofFrankia for cellulase activity, based on the method of utilization of cellulose in liquid culture, only one strain, CcI3, degraded filter paper cellulose to complete disintegration and only under very specific conditions of pH and primary carbon source. When carboxymethylcellulose (CMC) at 1% was used as substrate, all five strains showed the capacity to produce reducing sugars as hydrolytic products. Microcystalline cellulose, xylans and gum arabic were hydrolyzed to a lesser extent. Optimum activity depended upon pH and primary carbon source with pH 5.0 and pyruvate or propionate producing highest activities. In fractionation studies of culturedFrankia, assays for hydrolysis of 1% CMC in liquid medium showed that highest activity was in the enzyme preparation supernatant with lesser activity in the cell-free extract and cell wall fractions.Frankia strain CpI1 showed the greatest total hydrolytic activity against CMC after 2 weeks of culture. Strains ArI3 and CcI3 also showed good activity. The agar plate method for direct dye-polysaccharide interaction proved to be the least sensitive assay method with only ArI3 showing significant activity using CMC as substrate. It appears that theFranka strains grown in synthetic media all showed hydrolytic activity but the degree of hydrolysis of polysaccharides to reducing sugars depends upon strain of bacteria and very specific cultural conditions.     

Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis.

Cellulase production was examined in two strains of Neocallimastix frontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 X 10(3) to 10.4 X 10(4) U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-1. The range for PN-2 was 8.1 X 10(3) to 11 X 10(4) U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-1) to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (less than 20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (less than 2 U ml-1) and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis

Cellulase production was examined in two strains of Neocallimastix frontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 X 10(3) to 10.4 X 10(4) U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-1. The range for PN-2 was 8.1 X 10(3) to 11 X 10(4) U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-1) to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (less than 20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (less than 2 U ml-1) and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response surface optimization of cellulase production from Aneurinibacillus aneurinilyticus BKT-9: An isolate of urban Himalayan freshwater

Due to their vast industrial potential, cellulases have been regarded as the potential biocatalysts by both the academicians and the industrial research groups. In the present study, culturable bacterial strains of Himalayan Urban freshwater lake were investigated for cellulose degrading activities. Initially, a total of 140 bacterial strains were isolated and only 45 isolates were found to possess cellulose degrading property. On the basis of preliminary screening involving cellulase activity assay on CMC agar (with clear zone of hydrolysis) and biosafety assessment testing, only single isolate named as BKT-9 was selected for the cellulase production studies. Strain BKT-9 was characterized at the molecular level using rRNA gene sequencing and its sequence homology analysis revealed its identity as Aneurinibacillus aneurinilyticus. Further, various physico-chemical parameters and culture conditions were optimized using one factor approach to enhance cellulase production levels in the strain BKT-9. Subsequently, RSM based statistical optimization led to formulation of cellulase production medium, wherein the bacterial strain exhibited ~60 folds increase in enzyme activity as compared to un-optimized culture medium. Further studies are being suggested to scale up cellulase production in A. aneurinilyticus strain BKT-9 so that it can be utilized for biomass saccharification at an industrial level.     

Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions

Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 h. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given times was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration.