里氏木霉LW1固态发酵纤维素酶条件的研究

采用里氏木霉LW 1(Trichoderma.Reesei)固体发酵生产纤维素酶,研究了秸杆粉和麦麸用量、料水比、起始pH值、发酵温度和发酵时间对该菌株产纤维素酶活力的影响。试验结果表明,里氏木霉LW 1的适宜发酵条件为:在秸秆∶麦麸=1∶1,料水比为1∶2的前提条件下,培养温度28℃,发酵周期为72h,起始pH5.5时产酶活力最高。浸出液中FPA酶活为119.417u/g干物质,CMC酶活为452.433u/g干物质。     

里氏木霉与黑曲霉混合发酵产纤维素酶及其水解特性

研究了利用里氏木霉和黑曲霉混合培养产纤维素酶,以黑曲霉孢子悬浮液的不同活化浓度及不同的活化时间来寻找2个菌种发挥最大协同作用的结合点以及所产纤维素酶的水解特性。以里氏木霉单一培养和黑曲霉单一培养为参照进行对比研究。底物为农林废弃物之一的玉米秸秆,经过蒸气爆破预处理后,用作产酶C源。结果表明:黑曲霉孢子悬浮液活化浓度为10个/mL,活化时间为12 h时,滤纸酶比酶活最高,达3.32 U/mL,高于里氏木霉单一培养的2.25 U/mL,β-葡萄糖苷酶比酶活达1.32 U/mL,高于里氏木霉单一培养的0.57 U/mL。为进一步验证混合菌产纤维素酶的水解效果,利用混合菌产纤维酶的酶液及里氏木霉产纤维素酶的酶液进行酶水解实验,当酶用量为20 U/g绝干纤维素,底物质量浓度为100 g/L条件下水解48 h,混合菌所产酶液酶解得率达70.00%,高于里氏木霉所产酶液的酶解得率63.05%。实验表明里氏木霉与黑曲霉混合培养产酶是可行的,并优于单一菌种培养。     

甘蔗渣固态发酵产纤维素酶

以甘蔗渣和麸皮混合作为固态发酵产酶培养基,采用单因素优化实验对里氏木霉固态发酵产纤维素酶进行优化。结果表明,在50 m L体系培养基中,在底物绝干原料5.2 g、甘蔗渣与麸皮质量比7∶3、氮源((NH4)2SO4)7.5 g/L、产酶诱导物1.6 g/L、表面活性剂(聚乙二醇PEG6000)0.1 g、发酵起始p H 4.4、培养基中里氏木霉孢子接入量5×105个的条件下,温度30℃时发酵120 h,里氏木霉固态发酵产纤维素酶的酶活达76.39 IU/g,是起始优化前20.29 IU/g的3.76倍。     

里氏木霉液体发酵产纤维素酶的研究

在摇瓶试验基础上,采用里氏木霉(Trichoderma reesei)HC-415菌株进行5L自控罐产纤维素酶深层发酵试验。在通气量为 0.2—0.6vvm、搅拌速度为 400r/min、发酵液pH控制在5.8—6.1的条件下,发酵液的羧甲基纤维素(CMC)酶酶活最高为325.0mg糖/ml,滤纸糖酶(FPA)酶活最高达17.9mg糖/ml。发酵周期为108h。所得冻干纤维素酶粉CMC酶活最高3111IU/g,FPA最高135IU/g ,对发酵液得率平均6.7g/L。酶活总收率CMC酶活平均78.2％,FPA酶活平均73.5％。     

利用木霉与根霉两步发酵秸秆制备L-乳酸研究

以秸秆为原料进行生物转化大量制备有机酸意义重大.在秸秆汽爆法预处理的基础上,以绿色木霉为菌种转化制备秸秆糖,对降解单糖接种米根霉进行二次发酵制备L-乳酸.试验结果表明,第一步绿色木霉固态培养制备纤维素酶时,控温30℃、通气0.12L/(L.min)、发酵40h后制备干曲,后按10g干曲/L汽爆液的配比进行55℃酶解36h,五、六碳糖累积浓度达到86g/L.第二步米根霉发酵时,控制温度32℃、通气0.4L/(L·min)、转速450r/min,发酵48h,最终产L-乳酸累积浓度为81.6g/L.秸秆制备L-乳酸的两步发酵法发酵工艺具有推广价值.     

三菌混合固态发酵产纤维素酶

以稻草粉和麸皮为主要原料,对白腐菌(White-rot fungi) NS75、黑曲霉(Aspergillus niger)NS83和絮凝酵母(Saccharomyces cerevisiae)SP5混合菌固态发酵产纤维素酶进行研究.实验结果显示,在白腐菌和黑曲霉双菌混合培养2d后接入絮凝酵母,培养到第7d产酶达到峰值;三菌混合发酵产纤维素酶酶活明显高于白腐菌和黑曲霉双菌混合培养,其β-葡萄糖苷酶(β-G)和羧甲基纤维素酶(CMCase)酶活比白腐菌(White-rot fungi) NS75和黑曲霉(Aspergillus niger)NS83双菌发酵产酶分别提高了143.3％和68.2％.单因素实验和正交实验结果表明,当稻草粉麸皮质量比为8∶2,料水比为1∶2,白腐菌NS75、黑曲霉NS83和絮凝酵母SP5的接种比例为1:2∶1.5 (v/v/v)时,于30℃培养7d,固态发酵基中β-G和CMCase酶活分别达到62305 U/g和30241 U/g.     

沉默里氏木霉组蛋白赖氨酸甲基转移酶基因对纤维素酶表达的影响

【背景】里氏木霉(Trichoderma reesei)是一种比其他真菌小很多的多细胞真核微生物,在工业上受到广泛应用,而里氏木霉QM9414是目前研究最多基因产纤维素酶丰富的突变菌株。【目的】构建里氏木霉中组蛋白赖氨酸甲基化酶(Histone Lysine Methyltransferase)基因hkmt的siRNA沉默载体和过表达载体来降低或者增强hkmt在里氏木霉QM9414中的表达量,以分析其对里氏木霉纤维素代谢的调控作用。【方法】根据里氏木霉hkmt序列设计siRNA沉默片段并用反转录的方法获得过表达hkmt片段。将沉默片段和过表达片段克隆至里氏木霉组成型表达载体中,构建沉默hkmt的载体和过表达hkmt的载体,并将其转化里氏木霉QM9414。通过荧光显微镜观察重组菌的菌丝生长情况,此外对各重组菌进行纤维素酶的滤纸酶活性(Filter Paper Enzyme Activity,FPA)和羧甲基纤维素钠酶活性(Carboxymethyl Cellulose Enzyme Activity,CMCA)的测试;利用荧光定量PCR的方法检测hkmt、纤维素酶基因cbh1、egl1及木聚糖酶激活因子xyr1的表达量变化。【结果】通过使用荧光显微镜观察,发现沉默、过表达hkmt重组菌的菌丝形态均与出发菌株无明显差异。荧光定量PCR测定结果表明,沉默载体和过表达载体可以分别沉默和促进hkmt的表达。沉默hkmt重组菌株中FPA和CMC酶活力相比出发菌平均升高2.5倍。此外,纤维素酶相关基因和激活因子在沉默hkmt重组菌中的表达量均有所增加,但是在过表达hkmt重组菌株中以上相应指标均呈现相反的趋势。【结论】组蛋白赖氨酸甲基转移酶基因表达产物负调控里氏木霉产纤维素酶基因的表达,这为提高里氏木霉产纤维素酶水平提供了参考,并为里氏木霉产纤维素酶的表观遗传调控研究提供了新的证据。     

黑曲霉与里氏木霉固态混合发酵产β-葡萄糖苷酶

对黑曲霉NL02与里氏木霉RUT-C30固态混合发酵产β-葡萄糖苷酶的发酵培养基进行优化,研究培养基含水率、C源、N源、接种量、温度和2种菌种不同延长接种时间与接种比例对β-葡萄糖苷酶活力的影响。研究表明：麸皮17.5 g、玉米芯7.5 g、（NH4）2SO4 0.40 g、尿素0.37 g、黑曲霉孢子接入量为107个接种到250 mL三角瓶中,温度30 ℃、摇床转速100 r/min时,里氏木霉以105个孢子与黑曲霉同时接入,每克干曲所得β-葡萄糖苷酶的活力为132.45 IU,较黑曲霉单独培养时的104.35 IU提高了26.94%。     

碱与微波联合处理蔗渣对里氏木霉发酵产纤维素酶的影响

以蔗渣为原料,采用碱和微波辐射联合处理后用于里氏木霉纤维素酶的液态发酵。采用单因素试验与正交试验确定了最佳的处理条件为:0.30 mol/L的NaOH溶液浸泡,微波功率160 W,处理5 min。在此条件下得到的单位能耗的酶活净增值最高。后续发酵结束后,酶活较未经处理的蔗渣发酵后所得酶活有显著提高。其中,β-葡萄糖苷酶活、滤纸酶(FPase)活、羧甲基纤维素酶(CMCase)活分别提高了81.3%,88.2%,154.5%。     

里氏木霉及其纤维素酶高产菌株的研究进展

随着纤维素在能源、材料及化工等领域的广泛开发和应用,里氏木霉作为一种重要的产纤维素酶工业用菌种,越来越受到人们的广泛关注.为了提高其酶活,人们做了大量的工作,获得了一些相当好的突变株.对里氏木霉及其突变株的基因组进行研究,有助于人们理解其高效产酶的机制,同时也有利于构建其基因工程菌.介绍里氏木霉Trichoderma reesei 的背景及其部分高产纤维素酶突变株,并阐述近些年来对其突变株的基因组的研究进展.     

Optimization of ellagic acid production from ellagitannins by co-culture and correlation between its yield and activities of relevant enzymes

Aspergillus oryzae was co-cultured with Trichoderma reesei using acorn cups extract containing up to 62% ellagitannins as substrate to produce ellagic acid with relatively high levels of ellagitannin acyl hydrolase, cellulase and xylanase. Ellagitannins concentration, initial pH, T. reesei and A. oryzae during the fermentation were identified as important process parameters effecting ellagic acid accumulation and the enzymes syntheses. These parameters were optimized by uniformity design to determine the optimum condition for ellagic acid production. Under optimum operational condition, ellagic acid yield could be arrived at 24%, when the fermentation run lasted 96h with an initial pH of 4.5, an ellagitannins concentration of 4gl(-1), T. reesei of 3ml and A. oryzae of 3ml. Meanwhile, it was found that the three enzymes activities correlated very well with ellagic acid yield, resulting in model with high coefficient of determination (R(2)=0.98). The results indicate that the mixed culture of T. reesei and A. oryzae is an effective approach to produce an enzyme system of degrading ellagitannins for ellagic acid production.     

泊沙康唑对临床来源接合菌的体外抗菌活性研究

目的研究泊沙康唑对临床来源的接合菌体外抗菌活性。方法对临床来源的43株接合菌采用ITS区序列分析进行准确鉴定,采用E-test纸片法研究泊沙康唑对43株菌的体外药物敏感性,观察其MIC值。结果经ITS序列分析鉴定,43株临床来源接合菌中最为常见的是小孢根霉和不规则毛霉(各12株),其次为米根霉(10株),ramosa(3株)。其他菌种分别为ornata、总状共头霉、微小根毛霉、卷曲毛霉、印度毛霉和冻土毛霉各1株。E-test纸片法测得泊沙康唑对毛霉属、根霉属和Lichtheimia spp.的MIC值范围分别为0.19～>32μg/mL、0.19～3μg/mL、0.002～0.38μg/mL,对总状共头霉和微小根毛霉的MIC值分别为1μg/mL和0.19μg/mL。结论泊沙康唑对大部分接合菌有效,不同种属MIC值范围不同,毛霉属真菌的MIC值差异较大。     

重组巴斯德毕赤酵母产米根霉α-淀粉酶发酵条件的研究

为了提高重组菌的淀粉酶表达量,以可分泌表达米根霉α-淀粉酶的甲醇快速利用型巴斯德毕赤酵母重组菌为基础,采用摇瓶发酵方式对影响重组菌表达淀粉酶的多个因素进行了研究和优化。摇瓶发酵条件确定为:温度为30℃,pH值为6.0,接种量为2.0(OD_(600)),甲醇补加方式采用前72 h发酵时间内每隔12h添加至终浓度为1.0%,72 h以后每隔24 h添加至终浓度为1.0%,在此条件下获得的淀粉酶最高表达量为47.5 U/mL,且在无机盐培养基中和有机氮源培养基中获得的淀粉酶发酵单位相当。以摇瓶发酵数据为基础确定15 L发酵罐放大实验条件为:无机盐培养基,温度为30℃,pH值为6.0,接种量为10%,甲醇流加方式采用DO—Start法控制,在此发酵条件下获得的淀粉酶表达量为440 U/mL,约为摇瓶发酵方式获得的淀粉酶表达量的9倍。     

Addendum to Issue 1 - ENZITEC 2012Simultaneous biosynthesis of biomass-degrading enzymes using co-cultivation of Aspergillus niger and Trichoderma reesei

Abstract

The biotransformation of lignocellulosic materials into biofuels and chemicals requires the simultaneous action of multiple enzymes. Since the cost of producing an efficient enzyme system maybe high, mixed cultures of microorganisms maybe an alternative to increase enzymatic production and consequently reduce costs. This study investigated the effects of different inoculum ratios and inoculation delays on the biosynthesis of cellulases and xylanases during co-cultivation of Aspergillus niger and Trichoderma reesei under solid-state fermentation (SSF). While the monoculture of T. reesei was more efficient for CMCase production than the co-cultivation of A. niger and T. reesei, a significant increase in β-glucosidase and xylanase production was achieved by co-cultivation of both species. The maximum CMCase activity of 153.91 IU/g was obtained with T. reesei after 48 h of cultivation, while the highest β-glucosidase activity of 119.71 IU/g (after 120 h) was obtained by co-cultivation of A. niger and T. reesei with a 3:1 inoculum ratio (A. niger: T. reesei). The greatest xylanase activity observed was 589.39 IU/g after 72 h of mixed culturing of A. niger and T. Reesei with a 1:1 inoculum ratio. This is the first study where the effects of inoculum ratio and inoculation delay in mixed culture of T. reesei and A. niger under SSF have been systematically assessed, and it indicates co-cultivation as a feasible alternative to increase enzymatic production.     

Evaluation of culture conditions for cellulase production by two Trichoderma reesei mutants under solid-state fermentation conditions

Response surface methodology (RSM) was used to evaluate the effects of fermentation parameters for cellulase production by Trichoderma reesei QM9414 and T. reesei MCG77 in solid-state fermentation using rice bran as substrate. Initial pH, moisture content and temperature were optimized using filter paper activity (FPA) as response. Statistical analysis of the results for T. reesei QM9414 showed that only moisture content had significant effect on cellulase activity and had a linear effect on enzyme activity (maximum enzyme activities were obtained at 70% moisture content). The results for T. reesei MCG77 showed that temperature and moisture content were the most significant parameters for cellulase activity. The optimum cellulase production was in the temperature range of 25-30 degrees C and moisture content between 55% and 70%. After the optimization, the FPA in T. reesei MCG77 was increased by 2.5 folds compared to that of T. reesei QM9414.     

Effect of culture phasing and a polysaccharide on production of xylanase by mixed culture of Trichoderma reesei D1-6 and Aspergillus wentii Pt 2804

A significant increase in extracellular xylanase activity was observed in the mixed culture fermentation of Trichoderma reesei D1-6 and Aspergillus wentii Pt 2804 when A. wentii inoculation was phased by 15 h. A. wentii produced a polysaccharide, chiefly consisting of glucose monomeric units, which was required for expression of maximum xylanase activity. Expression of high activity of xylanase in the A. wentii phased mixed culture compared to that in either T. reesei or A. wentii single cultures appeared to be controlled by the combined action of a polysaccharide produced by A. wentii and the relative growth of the two fungi in the mixed culture.     

固态混合发酵提高木聚糖酶和纤维素酶活力的研究

研究了接种比例、接种时间、碳源、氮源等因素对木霉和黑曲霉混合发酵产木聚糖酶和纤维素酶的影响。试验结果表明,当木霉和黑曲霉按4:6同时接种,以玉米芯3．75g、麸皮3．75g、葡萄糖37．5mg为混合碳源,Mandels营养盐11．5mL、添加NH_4NO_37．5mg为氮源,在84h产纤维素酶活力达到230IU/g干物质,木聚糖酶活力达到1308IU/g干物质,与两菌纯培养相比,纤维素酶活力提高163％,木聚糖酶活力提高79．5％。     

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。     

Changes in lipid, fatty acids and phospholipids composition of whole rice bran after solid-state fungal fermentation

The aim of this study was to evaluate fermented rice bran phospholipids, lipids and fatty acid content in a fermentation solid system with Rhizopus oryzae fungus. For this, aliquots were withdrawn every 24h over 120 h. The content of phospholipids was determined by colorimetric method. Esterified fatty acids were separated by gas chromatography, then identified and quantified. The total lipids from fermented rice bran (FB) decreased from 20.4% to 11.2% in the range between 0 h and 120 h of fermentation while phospholipid contents were increased up to 2.4 mg P g(lipid)(-1). In fermented bran, oleic, palmitic and linoleic acids prevailed, with a decrease in saturated fatty acids (20%) and increase in the unsaturated ones (5%). This study showed that rice bran fermentation with R. oryzae can be applied to the production of phospholipids altering the saturated to unsaturated fatty acid ratio.