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1.
碳水化合物结合组件(carbohydrate—binding module,CBM)是一些糖基水解酶分子上的结构域,它在纤维素酶降解不可溶纤维素中起着重要的作用。本研究的目的是检测一个新的内切葡聚糖酶Umcel5N(GenBank登录号为ACH67609)加上一个碳水化合物结合组件后得到的融合酶是否获得降解结晶纤维素的能力。本文将编码内切葡聚糖酶Umcel5N的催化结构域(catalytic工能力domain,CD)的序列与编码Umcel6A的CBM序列通过接头序列进行基因融合,得到融合基因umCel5N-CBM,并实现了融合基因在大肠杆菌BL21(DE3)pLysS中的表达。研究结果表明,融合酶Umcel5N-CBM与结晶纤维素(avicel)以及滤纸粉末的结合能力比原始酶Umcel5N提高了约一倍,但未显示出降解结晶纤维素的新活性,说明在结晶纤维素的降解过程中,纤维素酶的催化功能域起到关键作用。  相似文献   

2.
纤维素资源是地球上最丰富的可再生资源,纤维素酶的酶活力和生产成本制约着纤维素的利用.本研究从多年户外放置的朽木(Populus bonatii)中分离出一株真菌G-1,经18S rDNA序列分析鉴定为绿色木霉(Trichoderma viride).从G-1中分离出一种纤维素内切酶基因(endoglucanase,EG;GenBank登录号:HM116999.1).通过重叠引物延伸法对EG进行定点突变,得到一个突变序列EG-mut,将EG和EG-mut分别插入分泌型表达载体pPIC9K,并导入巴斯德毕赤酵母(Pichia pastoris),筛选到两个菌株,经刚果红染色和DNS法对酶活性测定,显示其内切酶的活性分别达到20.915 U/mL和24.110 U/mL,酶活力高于本实验中得到的其它重组菌株.结果表明某些定点突变可以揭示影响酶活的重要功能域.  相似文献   

3.
本研究从哈茨木霉(rrichoderma harzianMm)A25—2总RNA中利用RT—PCR的方法扩增到其纤维二糖水解酶I基因的cDNA序列,并对该基因编码的氨基酸序列进行分析,得到cbhI基因中编码催化功能域的序列。将催化功能域编码序列克隆到表达载体pCP—GH中,用PEG—CaCl2介导的原生质体转化方法将重组质粒转化到绿色木霉(Trichoderma viride)HP35—3中,筛选得到12个转化子。以P-NPC为底物,测定了该12个转化子的酶活力,获得比活力最高的转化子Tv/CDHI-CBM-5,其纤维二糖水解酶活力是HP35—3的3.8倍。SDS—PAGE分析表明,绿色木霉表达了导入的含A25—2纤维二糖水解酶I催化功能域的编码序列。  相似文献   

4.
化学预处理提高酒糟生物质酶解糖化效果   总被引:1,自引:2,他引:1  
为促进酒糟生物质的酶解糖化,筛选适宜的预处理方法,以脱除木质素,提高综纤维素(纤维素和半纤维素之和)保留率为目标,研究比较了酸-超声波耦合(ultrasound-assisted acid pretreatment,UAAP)、液氨(pretreatment by soaking in aqueous ammonia,PSAA)、碱性双氧水(alkaline hydrogen peroxide pretreatment,AHPP)和酸性亚硫酸氢盐(bisulfite pretreatment,BP)4种预处理法对酒糟化学组分、结构特性和酶解得率的影响。结果表明,与其余3种方法相比,BP法处理后酒糟的纤维素和半纤维素保留率最高,分别为84.59%和84.87%,即综纤维素保留率为84.68%。与未处理酒糟(unpretreatment,UP)相比,4种方法预处理后酒糟的综纤维素酶解得率分别提高了49.12%(酸-超声波,UAAP)、55.48%(液氨,PASS)、92.79%(碱性双氧水,AHPP)和99.15%(酸性亚硫酸氢盐,BP),其中BP法对酒糟酶解糖化的促进作用最有效。扫描电镜(scanning electron microscope,SEM)和X-衍射(X-ray differaction,XRD)结果显示,酒糟经不同方法预处理后表观结构发生了明显变化,木质纤维网络结构遭到破坏,表面呈现无规则或形状各异的膨松状态,沟壑明显,孔隙率增加,比表面积增大,有利于提高水解酶的可及性。化学组分和结构特性的变化说明酒糟的酶解得率与综纤维素的保留、木质素的去除、表面微观形貌变化以及纤维素结晶度等因素直接相关。总之,酸性亚硫酸氢盐(BP)法是适用于酒糟生物质糖化预处理的一种有效可行方法。  相似文献   

5.
本碱性羧基末端结构域(basic C-terminal domain,BTD)是瘤胃细菌的碳水化合物活性酶(carbohydrateactive enzymes)中未知功能的一个结构域。BTD都位于酶或蛋白质的羧基最末端,大小为30~80个氨基酸(aa),含有较多的碱性氨基酸,一般该结构域等电点都大于10。本工作构建了来源于水牛瘤胃未培养微生物的内切葡聚糖酶C5614-1的BTD缺失酶C5614-1RBTD57(缺失C5614-1羧基末端的57个aa)和C5614-1RBTD40(缺失C5614-1羧基末端的40个aa)以及C67-1的BTD缺失酶C67-1ΔBTD(缺失C67-1羧基末端的42个aa),酶学特性分析发现BTD缺失酶与野生酶对pH和温度的稳定性是相似的,说明BTD结构域对酶的酶学特性方面贡献不大。尽管缺失了C5614-1的羧基末端57个氨基酸后,缺失酶C5614-1RBTD57对温度和pH的稳定性明显降低,但同时缺失酶对Avicel的结合能力也显著下降,说明该缺失酶是因为该酶的碳水化合物结合组件(carbohydrate binding module,CBM)的部分缺失而导致了酶的稳定性的改变。本文还对BTD可能的功能做了预测。  相似文献   

6.
β-1,4内切葡聚糖酶是植物寄生线虫食道腺分泌的一类细胞壁降解酶,在植物线虫的侵染过程中具有重要的作用.以马铃薯腐烂茎线虫(Ditylenchus destructor)为材料,用RT-PCR和RACE方法,获得了β-1,4-内切葡聚糖酶基因的cDNA全长,并将该基因命名为Dd-eng-1b(GenBank登录号为FJ430142).此cDNA全长序列为1 640 bp,包括1个1 443 bp的完整ORF,编码1含481个氨基酸的蛋白,其理论分子量为50.89 kD,等电点pI为6.94.序列比对分析表明,含有糖基水解酶的保守结构域,属于纤维素酶第五家族成员,N端具有19个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域(CBD Ⅱ).cDNA与基因组DNA重叠分析表明,此基因包含4个内含子,长度分别为36、76、187和344bp,切割点符合5'-GT……AG-3'的规律.系统进化树分析表明,该基因与细菌Bacillus subtilis和Erwina carotovora分泌的纤维素酶属于同一支,推测该基因可能来源于细菌的水平基因转移.  相似文献   

7.
采用发表的引物对,以猪链球菌2型(Streptococcus suis type 2)江苏分离株HA9801的基因组DNA为模板,采用PCR方法扩增纤连蛋白/血纤蛋白原结合蛋白基因fbps(GenBank登录号为AY565303)克隆于pMD-T18载体构建成载体pMD-T-fbps.经内切酶酶切和测序鉴定后,将由pMD-T-fbps内切酶切下的片段定向克隆于表达载体pET-32a(+),得到重组质粒pfbps.将重组质粒pfbps转化大肠杆菌(Escherichia coli)BL21株,经IPTG诱导,可高水平地表达相对分子量为83 kD的融合蛋白.配体印迹结合试验表明,表达的融合蛋白可与人纤连蛋白结合.  相似文献   

8.
将经过纯化的鼠李糖脂二糖脂添加于纤维素酶酶水解试验中,以稻草、竹叶为底物,分析水解过程中纤维素酶酶活(以FPA计)及还原糖浓度的变化特征,探讨和分析鼠李糖脂二糖脂对稻草和竹叶中木质纤维素水解产还原糖能力、纤维素酶活的稳定性、发酵液表面张力和pH值的影响作用。结果表明,添加鼠李糖脂二糖脂对木质纤维素类底物酶水解过程中还原糖浓度的增加、酶活稳定性的提高有明显的促进作用,并且其促进作用随着表面活性剂添加量的适量增加而增强,当添加量为0.24%时,稻草和竹叶还原糖的产量分别提高了17.19%和27.68%。此外,水解反应结束后,加入鼠李糖脂二糖脂的水解液表面张力值显著降低,且随着添加量的增高而降低,当添加量为0.24%时,可分别降至63.4和60.8mN·m^-1左右,而pH值的变化微小。  相似文献   

9.
为了实现棉秆的高效资源化利用,该文提出一种碱液浸泡-蒸汽闪爆-漆酶/介体联合处理棉秆皮制备纺织纤维的新方法。研究了浸泡NaOH用量对棉秆皮分离效果的影响以及漆酶/介体处理介体种类及用量、漆酶用量对木质素去除的影响。采用扫描电子显微镜、X射线衍射、热稳定性分析等方法,对比研究了蒸汽闪爆、漆酶/介体处理后棉秆皮纤维的化学成分、结构与性能。研究结果表明,碱液浸泡-蒸汽闪爆-漆酶/介体处理(NaOH用量10 g/L,介体ABTS用量为棉秆皮纤维干质量的1%,漆酶用量为600 U/g)能制得表面洁净、热稳定性好的棉秆皮纤维,其长度为55.7 mm,细度为28 dtex,长径比为1 139,断裂强度为2.97 cN/dtex,纤维素质量分数为78%,结晶指数为67.5,得率为40%,可用于纺织,研究结果为木质纤维素纤维的制备提供参考。  相似文献   

10.
本研究从金钱树(Zamioculcas zamiifolia)白绢病(Southern blight)病株上分离到一株产纤维素酶菌株SM,经形态观察和rDNA—ITS序列分析鉴定为齐整小核菌(Sclerotium rolfsii Sacc),经以羧甲基纤维素钠为唯一碳源的培养基培养和刚果红染色测定,证明SM菌株可产生高活性纤维素降解酶。本文对SM菌株产纤维素降解酶的液态发酵条件进行了研究,结果表明:在起始pH6.0,无机氮:有机氮:纤维素碳源之比为0.07g:1.4g:1.6g,温度为30℃,摇床转速为160r/min,发酵培养时间为5d的条件下,该菌株发酵液的CMC酶活性达到11.7U/mL,滤纸酶活性达到2.156U/mL,β-葡萄糖苷酶活性达到3.911U/mL。  相似文献   

11.
Preparation of three novel dietary fibers (DFs) from mushroom sclerotia, namely, Pleurotus tuberregium, Polyporous rhinocerus, and Wolfiporia cocos, by a scale-up modified AOAC procedure using industrial enzymes was investigated. A remarkably high level of total dietary fiber (TDF) ranging from 81.7 to 96.3% sample dry matter (DM), in which a content of nonstarch polysaccharide (NSP) ranging from 86.6 to 94.3% sclerotial TDF DM, was obtained from the three sclerotia. All sclerotial DFs were rich in beta-glucan (the glucose residue ranged from 89.7 to 94.5% NSP DM) with a very low level of resistant glycogen (ranged from 3.77 to 3.94% sclerotial TDF DM). All three novel sclerotial DFs also exhibited similar, if not better, physicochemical and functional properties (pH, color, water binding capacity, oil holding capacity, and emulsifying properties) as those of barely DF control and commercial DF-rich ingredients. The potential use of the three mushroom sclerotial DFs as a new beta-glucan type DF-rich ingredient in the food industry was discussed.  相似文献   

12.
Two malting hulled varieties (Sebastian, Malz) and one nonmalting hull-less variety (AF Lucius) were used to assess vitamins C and E in the green biomass of young plants of spring barley ( Hordeum vulgare L.) in three stages of growth and development (BBCH 29, 31, 32-33). The samples from sampling I (BBCH 29) had statistically significantly higher vitamin C content and vitamin E activity compared to sampling I (BBCH 31). The highest average vitamin content was determined in the malting variety Sebastian (vitamin C, 520 mg 100 g(-1) DM; vitamin E, 73.06 mg kg(-1) DM) compared to the varieties Malz (501 mg 100 g(-1) DM; 61.84 mg kg(-1) DM) and AF Lucius (508 mg 100 g(-1) DM; 67.81 mg 100 g(-1) DM). The locality Krome?r?i?z? (Czech Republic, CR), with vitamin C and E contents of 524 mg 100 g(-1) DM and 68.74 mg kg(-1) DM, respectively, proved to be more suitable for growing green biomass compared to the locality Z?abc?ice (CR) (content of vitamins C and E, 477 mg 100 g(-1) DM and 66.39 mg kg(-1) DM, respectively). During the research period (2005-2007), it was determined that the green mass of young plants of spring barley was a significant source of vitamins C and E in the growth stage BBCH 29; in later samplings (BBCH 32-33) the vitamin levels dropped (by as much as 48%). These vitamins are important antioxidants for human health. Therefore, "green barley" can be recommended for the preparation of natural dietary supplements and is preferred to synthetic vitamin preparations.  相似文献   

13.
The proximate composition of sclerotia of Pleurotus tuber-regium, Polyporus rhinoceros, and Wolfiporia cocos, together with the yield, purity, monosaccharide profile, and microstructure of their insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) fractions prepared from AOAC enzymatic-gravimetric methods were investigated and compared. All three sclerotia were typical carbohydrate rich sclerotia [ranging from 90.5 to 98.1% dry matter (DM)] with an exceptionally low amount of crude lipid content (ranging from 0.02 to 0.14% DM). Besides, all three sclerotia possessed substantial amounts of IDF (ranging from 77.4 to 94.6% DM) with notably high levels of nonstarch polysaccharides (NSP) (89.9-92.2% DM) in which glucose was the predominant sugar residue (90.6-97.2% of NSP DM). On the contrary, both the yield (only ranging from 1.45 to 2.50% DM) and the amount of NSP (ranging from 22.4 to 29.6% DM) of the three sclerotial SDF fractions were very low. Scanning electron micrographs showed fragments of interwoven hyphae and insoluble materials in the three sclerotial IDF fractions, but only an amorphous structure of soluble materials was observed in the SDF fractions. The potential use of these fiber preparations was discussed.  相似文献   

14.
The purposes of this study were evaluate the effects of biofertilizers inoculation on growth indices, yield and radiation use efficiency (RUE) in black cumin under Mashhad climatic conditions. This field experiment was carried out based on a randomized complete block design with three replications. This experiment was performed at Agricultural Research Station, Ferdowsi University of Mashhad, Iran, during two growing seasons 2007–2008 and 2008–2009. Treatments were (A) Azotobacter paspali, (B) Azospirillum brasilense, (C) Rhizophagus irregularis, A + C, B + C, A + B, A + B + C and control. In all treatments, except control, 100 g of seeds were inoculated with15 mg of each biofertilizer. Results indicated that leaf area index (LAI) and accumulative dry matter (DM) showed an increasing trend up to 1863°Cd and a short declining trend afterwards. The relation between accumulative DM and absorbed photosynthesis active radiation (PARa) was linear. Radiation use efficiency (RUE) fluctuated from 0.55 gMJ?1 (control) to 0.89 gMJ?1 (B+C). Inoculation with biofertilizers enhanced root development and hence availability of moisture and nutrients, particularly nitrogen and phosphorus. Since these ecological fertilizers produce many growth regulators for the plant which promote LAI and accumulative DM and therefore yield, PARa and RUE enhanced.  相似文献   

15.
Abstract

A three-year experiment was carried out at three different sites in northern Germany to investigate the effects of combined sulphur (S, up to 50 kg S ha?1 year?1) and nitrogen (N, up to 300 kg N ha?1 year?1) fertilization on dry matter (DM) yield and forage quality. There was an interaction effect of site, year, S and N fertilization. The greatest DM yield increment relative to yield at the start of the experiment (1997) with no S and N applied was 10.2 t DM ha?1 at Ostenfeld (arable grassland). Cattle slurry when applied to provide 50 kg N ha?1 and 10 kg S ha?1 did not noticeably increase yield. The S content in forage decreased significantly over the years without S fertilization. At 300 kg N ha?1 and 0 kg S ha?1, crude protein (CP) contents achieved 173 g kg?1 DM and were diluted due to higher DM yields with S fertilization. The true protein content (TP% of CP) differed significantly at 300 kg N ha?1. TP achieved 93% with 50 and 87% with 0 kg S ha?1 year?1, respectively. In conclusion, with N fertilizer intensities in the range of 300 kg N ha?1, it is necessary to apply 25 kg S ha?1 to improve forage yield and quality. On the other hand, with N fertilization levels below 300 kg N ha?1, S fertilization could be omitted.  相似文献   

16.
Cattle dung contributes to hot‐spot inputs of nutrients to grassland systems, but not much is known about its organic P (Po) composition and fate in the grassland soils. We used 31Phosphorus (P)–Nuclear Magnetic–Resonance (NMR) spectroscopy of alkaline soil extracts to examine potentials for tracing of different functional Po forms into a temperate grassland soil amended with dung. The proportion of monoester, DNA‐diester, and phospholipid+teichoic acid P were comparable in dung extracts, but the soil was dominated by monoester P. The temporal trends in the DNA‐diester P–to–monoester P (DDNAM) and diester P–to–monoester P (DM) ratio of dung, native soil, and soil amended with dung were monitored in the 70 d field experiment. The DDNAM and DM ratio in the dung‐amended soil (0–1 and 1–5 cm depth) were always intermediate between the dung and (unamended) control soil. Clearly, extracted soil P was a mixture of incorporated dung‐derived P and native soil P. The dung‐P contribution in the 0–1 cm samples peaked at 47% of the total extracted P at day 70 and at 15% after 42 d in the 1–5 cm soil depth (based on the DM ratio). The proportions of dung‐derived P and C in the soil were positively correlated with: 1) topsoil, using the DDNAM ratio (r2 = 0.975), and 2) top‐ and subsoil, using the DM ratio (r2 = 0.656). We concluded that our DDNAM and DM‐P ratios approach (obtained from solution‐31P NMR) did trace successfully the short‐term dynamics and fate of dung Po in soil. It indicated that dung‐derived Po varied as rapidly in soil as the dung‐derived C.  相似文献   

17.
18.
Dmrt 基因编码的是一类新的具有锌指结构的转录因子,它们均含有一个DM(Double sex 和Mab - 3) 结构域,在性别分化发育和体节分化等过程中起着重要调控作用。尽管已在多种动物中克隆了Dmrt基因的多个成员,但在贝类中未见任何有关Dmrt基因的报道。本研究通过简并PCR克隆技术,从马氏珠母贝(Pinctada matensii)基因组中克隆到3个不同的DM序列(pmDmrt2,pmDmrt3 pmDmrt4)。证实了在马氏珠母贝基因组中也存在Dmrt基因家族的多个成员。序列分析表明,pmDmrt2编码的氨基酸序列与斑马鱼、爪蟾、红原鸡、家鼠、人等的Dmrt2的一致性为95 %;pmDmrt3编码的氨基酸序列与斑马鱼、青鳉、红原鸡、家鼠、人的Dmrt3的一致性为85%;pmDmrt4编码的氨基酸序列与青鳉、红鳍多纪鲀的Dmrt4一致性高达97%,进一步证明该基因家族在无脊椎动物和脊椎动物都具有高度的进化保守性。  相似文献   

19.
Uptake and utilization of phosphorus by three inbred lines of Lolium multiflorum L. and their hybrids Three Lolium multiflorum inbred lines A, B and C and their hybrids (AB, AC, BC) were tested for their shoot dry matter (DM) production, P uptake (mg P pot?1), P utilization (mg DM · mg P?1) and P influx (P uptake rate per cm root length) by growing in plastic pots for a maximum period of 92 days at two P levels in a loess loam soil. The acid phosphatase activity of roots of inbreds A and C grown in nutrient solutions with 1.10 and 100 μmol P · L?1 was also determined using the hydrolysis of Nitrophenyl phospate (NPP). Compared to the average of the inbreds DM production of the hybrids was 8 % higher at high and 12 % higher at low P level. With an increase of 14% and 25% DM respectively hybrid BC had the highest heterosis effect. The biomass production corresponded with the P uptake. Therefore, the P utilization efficiency of the hybrids was nearly the same as that of the inbreds. The increase in P uptake of the hybrids was related to the increase of root length. This means that the P uptake efficiency, i.e. the influx per unit of root remained unchanged between the inbreds and their hybrids. It can thus be concluded that the heterosis effect corresponds to an increase of P uptake caused by an increase of the size of the root system. The activity of the root phosphatases increased with declining P status of shoots and being absolutely higher in inbred A than C. Both inbreds responded within 3 days to a decreased P supply (from 100 to 1 μmol P · L?1).  相似文献   

20.
Dry matter (DM) partitioning into root, leaf, stem, shoot dry weight plant?1 response in four cool season C3-cereals viz. wheat (Triticum aestivum L.), rye (Secale cereale L.), barley (Hordeum vulgare L.) and oats (Avena sativa L.) was investigated at 30, 60 and 90 days after emergence (DAE) under eight nitrogen, phosphorus and potassium (NPK) sources: S1 = 20-20-20, S2 = 20-27-5, S3 = 7-22-8, S4 = 10-10-10-20S, S5 = 11-15-11, S6 = 31-11-11, S7 = 24-8-16, and S8 = 19-6-12 in pot experiment at Dryland Agriculture Institute, West Texas A&;M University, Canyon, Texas, USA during winter 2009-10. A considerable variation in DM partitioning into various plant parts was observed in the four crop species at different growth stages and NPK source. At 30 DAE, 27% of the total DM per plant (TDMPP) was partitioned into roots and 73% into shoots (19% stems + 54% leaf). Only16 % of the TDMPP was partitioned into roots and 84% into shoots (18 % stem + 66 % leaf) at 60 DAE. At 90 DAE, 29% of TDMPP was partitioned into roots and 71 % into shoots (33 % stems + 38 % leaf) at 90 DAE. Percent DM partitioning into stems ranked first (33%) at 90 DAE > at 30 DAE (19%) > at 60 DAE (18 %). With advancement in crops age, DM partitioning into various crop parts increased. The root DM plant?1 (RDMPP) increased from 11.5–722 mg plant?1; stem DM plant?1 (STDMPP) from 8.3–889.0 mg plant?1; leaf DM plant?1 (LDMPP) from 23.1–1031.0 mg plant?1; shoot DM plant?1 (SHDMPP) from 31.3–1921 mg plant?1, and TDMPP increased from 42.9–2693.0 mg plant?1 at 30 and 90 DAE, respectively. Because of the higher N contents in S7 (24:8:16) and S6 (31:11:11) reduced the DM partitioning into various plants parts as well as TDMPP at all three growth stages. The adverse effects of S6 and S7 on DM partitioning was more on oats > rye > wheat > barley. The S4 with 10:10:10 (NPK) and :20S was not toxic at 30 DAE, but at 60 and 90 DAE it became toxic that adversely affected the DM partitioning as well as TDMPP probably may be due its high sulfur (20%) content which lacking in other NPK sources. The DM partitioning to various parts of barley and wheat was more than oats and rye at different growth stages (barley > wheat > rye > oats). Since the DM portioning values were determined on the average of five plants in pot experiment under organic soil at field capacity; in case of field experiments more research is needed on various crop species/varieties under different environmental conditions particularly under moisture stress condition.  相似文献   

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