Acremonium纤维素酶在玉米芯糖化中的应用

玉米芯作为一种木质纤维素类农业废弃物,同时也是生产生物乙醇的潜在原料。在玉米芯糖化过程中,纤维素酶的作用是十分关键的。本研究比较了里氏木霉纤维素酶、绿色木霉纤维素酶和Acremonium纤维素酶各相关酶活。其中Acremonium纤维素酶的滤纸酶活约是里氏木霉纤维素酶的6倍,是绿色木霉纤维素酶的8倍。其羧甲基纤维素酶活和绿色木霉纤维素酶基本相等。Acremonium纤维素酶的β-葡萄糖苷酶酶活是里氏木霉纤维素酶的38倍,以及绿色木霉纤维素酶的41倍。而Acremonium纤维素酶的木聚糖酶活只相当于绿色木霉纤维素酶的70%。这说明Acremonium纤维素酶降解纤维素的能力可能强于另两种纤维素酶,而降解半纤维素类物质的能力要弱于绿色木霉纤维素酶。在玉米芯糖化实验中,使用Acremonium纤维素酶的糖化液中产生的最高葡萄糖浓度比里氏木霉纤维素酶的高14%,比绿色木霉纤维素酶的高58%。Acremonium纤维素酶用量在10 FPU/g时,反应16 h就基本可以达到最佳效果,而另两种酶用量则需达到30 FPU/g,反应48h才能达到最佳效果。使用Acremonium纤维素酶的糖化液中产生的最高木糖浓度与里氏木霉纤维素酶相等,比绿色木霉纤维素酶低42%。而同时使用Acremonium纤维素酶及绿色木霉纤维素酶时,其糖化液中最高木糖浓度有所提高,比绿色木霉纤维素酶的高31%。Acremonium纤维素酶可以有效地应用于玉米芯糖化,为玉米芯的资源化提供一种可能的方案。     

里氏木霉产纤维素酶研究进展

木质纤维素类生物质被认为是重要且可持续的可再生能源,其主要组成部分是纤维素.纤维素酶是一种能将纤维素分解为葡萄糖的复合酶,能有效地降解木质纤维素生物质.真菌、细菌、放线菌、酵母等多种微生物均可以产生纤维素酶,其中里氏木霉具有完整的纤维素酶系结构,常作为生物技术领域中一个重要菌株,广泛应用于纤维素酶的商业生产.介绍了纤维...     

里氏木霉中纤维素酶的合成诱导及调控*

随着绿色化学的兴起,天然纤维素原料转化和利用的研究受到了高度重视和广泛应用。利用纤维素酶降解纤维素为燃料乙醇、生物柴油的生产铺设了道路。但纤维素酶的生产成本较高,限制了纤维素酶产业化应用。里氏木霉生产的纤维素酶组分丰富,是纤维素酶高产菌株,深入研究里氏木霉的纤维素酶诱导及表达调控机制,有助于提高其纤维素酶产率。近年来人们对里氏木霉的纤维素酶诱导过程和调控机制有了一定研究进展,综述了里氏木霉纤维素酶诱导和基因表达调控,首先介绍了纤维素、纤维二糖、槐糖、乳糖等几种诱导物及诱导物的转运蛋白,进一步综述了几种转录因子的调控作用,同时介绍了染色体调控、信号通路和光条件对纤维素酶诱导的影响。最后展望了未来里氏木霉纤维素酶诱导表达的研究方向,包括探明诱导物的本质及其具体过程、揭示转录因子之间的联系及转录调控网络、寻找信号转导关键功能蛋白及研究环境因素对纤维素酶的诱导作用等。     

筛选最佳生物质材料诱导里氏木霉生产纤维素酶

本研究用小麦、芒草、水稻这三种低木质素突变株材料作为新型诱导物筛选的对象,对三种木质纤维素材料进行成分分析,然后利用它们分别作为诱导物诱导里氏木霉生产纤维素酶,对其诱导产生的酶活力,胞外蛋白含量,糖化能力进行比较。结果表明,对里氏木霉产纤维素酶诱导效果最好的是水稻H*14、芒草W56、小麦Q142。相比于玉米秸秆作为诱导物,水稻H*14单独诱导里氏木霉β-葡萄糖苷酶效果最好,β-葡萄糖苷酶酶活提高了75.2%,滤纸酶活提高了86.6%。相比玉米秸秆作为诱导物,芒草W56单独诱导里氏木霉木聚糖酶的效果最好,木聚糖酶酶活力提高了9.93%,内切葡聚糖酶酶活也提高了30.8%。相比玉米秸秆作为诱导物,小麦Q142诱导里氏木霉的外切葡聚糖酶酶活效果最好,里氏木霉的外切葡聚糖酶酶活提高了88.6%。本研究发现低木质素的木质纤维素材料作为诱导物对里氏木霉诱导产酶效果较好,并且促进真菌胞外蛋白的分泌,诱导纤维素酶酶系平衡分泌,使得纤维素酶糖化水解能力的提高。该研究为今后纤维素酶工业化生产提供参考和帮助。     

里氏木霉GH61家族糖苷酶的高效表达及酶学特性研究

【目的】GH61家族糖苷水解酶具有葡聚糖氧化酶活性,通过对葡聚糖链的随机氧化而破坏木质纤维素的结晶结构,从而使木质纤维素容易被纤维素酶降解。重组表达、纯化获得里氏木霉的GH61家族糖苷水解酶(TrGH61,原名为EGⅣ),并研究其在纤维素酶水解木质纤维素中的作用。【方法】通过Overlap PCR将里氏木霉丙酮酸脱羧酶的启动子、纤维二糖水解酶cbh1的信号肽、EGⅣ基因和PDC终止子依次连接构建了里氏木霉的表达盒,通过该表达盒使TrGH61蛋白基因整合到里氏木霉的基因组DNA上进行同源表达。研究表达产物TrGH61的水解活性、与纤维素酶水解协同效应,以及TrGH61作为金属氧化酶的特性研究。【结果】在PDC启动子的作用下,TrGH61得到高效表达,摇瓶培养的表达量达到2.33 g/L。TrGH61有微弱的内切葡萄糖苷酶活性,比活力为0.02 IU/mg,但能显著提高纤维素酶水解稻草粉的活性,协同度最高可达1.998。低浓度的金属离子Cu2+、Co2+和还原性电子供体还原型谷胱甘肽、L-抗坏血酸、焦性没食子酸均能显著促进其水解效应。TrGH61能够降低稻草粉纤维素聚合度和结晶度。【结论】通过PDC启动子可以实现TrGH61蛋白高效组成型表达,TrGH61作为纤维素酶活性促进因子,通过破坏纤维素结晶结构作用机制协同增强纤维素酶水解木质纤维素。     

芒草木质素含量对里氏木霉产木质纤维素酶的影响

[目的]研究芒草木质素含量对里氏木霉产木质纤维素酶的影响。[方法]选择了2组木质素含量差异显著(p0.01,n=3)、纤维素和半纤维素含量相近的芒草材料作底物来培养3株里氏木霉(野生型QM6a、突变型QM9414和Rut C30),对诱导产生的木质纤维素酶的酶活和降解木质纤维素的效率进行分析。[结果]在第Ⅰ组中,Mfl40木质素含量比Msa02低36%(p0.01,n=3),其诱导3株菌生产的木质纤维素酶体积比酶活高于Msa02组1.2~1.8倍(p0.05或者p0.01,n=3);在第Ⅱ组中,Mfl37木质素含量比Mlu13低48%(p0.01,n=3),其生产的木质纤维素酶体积比酶活高于Mlu13材料1.2~3.3倍(p0.05或者p0.01,n=3)。[结论]芒草细胞壁中木质素含量对里氏木霉产木质纤维素酶量有显著负影响。     

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。     

人工锌指蛋白介导调控的里氏木霉纤维素酶生产

里氏木霉Trichoderma reesei Rut-C30是目前研究最广泛的纤维素酶生产菌,选育高产纤维素酶的里氏木霉菌株有助于提高木质纤维素资源生物炼制的经济性。利用人工锌指蛋白文库转化T.reeseiRut-C30,筛选获得了两株高产纤维素酶的突变株T. reesei M1和M2,与出发菌株比较,突变株M1和M2滤纸酶活分别提高100%和53%,且M1突变株外泌蛋白量提高69%,M2内切纤维素酶活提高64%。实时定量PCR分析结果表明,与对照菌株相比,突变株M1和M2中主要纤维素酶基因转录均上调,但不同酶基因在两株菌中有不同的变化特征。此外,纤维素酶抑制转录因子基因ace1在两株突变株中都转录下调,而纤维素酶正调控转录因子基因xyr1仅在M1突变株中上调。以上结果表明,不同人工锌指蛋白对纤维素酶活性的影响具有多样性。对这些突变体中人工锌指蛋白靶基因进行深入分析,为进一步深入探究里氏木霉纤维素酶合成调控的机理,以及利用代谢工程选育更高效的产酶菌株提供了基础。     

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.     

高效低成本纤维素酶诱导物的制备、筛选及应用

本研究利用玉米芯、甘蔗渣、脱木素木糖渣及粗纤维诱导里氏木霉产纤维素酶,对4种材料进行成份测定,然后以逐步添加的方式与微晶纤维素混合诱导里氏木霉产纤维素酶,和使用微晶纤维素诱导产酶对比,玉米芯含有的纤维素代替总纤维素的50%时,酶活力降低2个单位,蛋白减少0.8 g左右,其酶水解能力降低0.4%,对其产纤维素酶的水解能力没产生不利影响。甘蔗渣纤维素替代量可以达到30%,酶活力有1个单位的降低,蛋白分泌降低0.5 g左右,酶的水解能力提高7%左右。脱木素木糖渣纤维素替代量也可达到50%,酶活力和蛋白降低分别达到0.5个单位和0.2 g左右,酶水解能力降低了4.45%。粗纤维的利用可以达到100%替代,对里氏木霉产酶的酶活力影响有0.3个单位之差,水解能力降低1.625%。这说明这几种物质可以部分替代或者完全替代微晶纤维素,诱导里氏木霉发酵产纤维素酶,特别是由玉米芯和甘蔗渣制备的脱木素木糖渣和粗纤维有着较高的应用前景。该研究对降低纤维素酶的生产成本及其工业化应用具有重要意义。     

Repeat-induced point (RIP) mutation in the industrial workhorse fungus Trichoderma reesei

Trichoderma reesei (syn. Hypocrea jecorina) is a filamentous ascomycete. Due to its capability of producing large amounts of lignocellulolytic enzymes and various heterologous proteins, this fungus has been widely used for industrial applications for over 70 years. It is also a model organism for lignocellulosic biomass degradation and metabolic engineering. Recently, we experimentally and computationally demonstrated that Trichoderma reesei exhibits high homology pairing and repeat-induced point (RIP) mutation activities at a premeiotic stage, i.e., between fertilization and karyogamy or premeiotic DNA replication. The discovery of RIP in Trichoderma reesei not only reveals significant impacts of sexual reproduction on evolution and chromosome architecture but also provides intriguing perspectives for industrial strain improvement. This review emphasizes two major points about RIP and RIP-like processes in Pezizomycotina fungi. First, the molecular mechanisms of RIP and RIP-like processes in Trichoderma reesei and other Pezizomycotina fungi are apparently distinct from those originally described in the model fungus Neurospora crassa. Second, orthologs of the rid1 (deficient in RIP-1) DNA methyltransferase gene were shown to be essential for sexual development in at least four Pezizomycotina fungi, including Trichoderma reesei. In contrast, rid1 is dispensable for Neurospora crassa sexual development. We suggest that the rid1-like gene products and/or their DNA methyltransferase activities play critical roles in promoting fungal sexual development. The Neurospora crassa rid1 gene might have lost this evolutionarily conserved function.

     

A copper-responsive promoter replacement system to investigate gene functions in Trichoderma reesei: a case study in characterizing SAGA genes

Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The molecular mechanism underlying the regulation of cellulase production as well as other physiological processes in T. reesei is still insufficiently understood. We constructed a P _tcu1 -based promoter substitution cassette that allowed one-step replacement of the endogenous promoter for controlling the target gene expression with copper. We then showed that copper repression of the histone acetyltransferase gene gcn5 phenocopied the gcn5 deletion strain. Using the same strategy, we further characterized the function of another putative Spt-Ada-Gcn5 acetyltransferase (SAGA) complex subunit encoding gene, ada2, in T. reesei. Similar to the repression of gcn5, the addition of copper to the P _tcu1 -ada2 strain not only drastically reduced the vegetative growth and conidiation in T. reesei but also severely compromised the induced cellulase gene expression. The developed strategy will thus be potentially useful to probe the biological function of the large fraction of T. reesei genes with unknown functions including those essential genes in the genome to expand its extraordinary biotechnological potential.

     

Enhanced cellulase production using Solka Floc in a fed-batch fermentation

Summary Use of a fed-batch mode of cultivation of T. reesei has permitted high concentrations of substrate to be consumed. This has resulted in the production of high titre cellulase preparations around 30 FPU/ml at high volumetric productivities (177 IU/L.hr).Perhaps the most obvious area for major improvement in the process of cellulose utilization is the production of cellulase enzyme for hydrolysis of wood and agricultural residues. It has been estimated that some 50% of the cost of producing glucose from cellulosic material is attributable to enzyme production alone (Perez, et al., 1980). Improvements in the area would therefore have a dramatic impact, and are of paramount importance if economical hydrolysis processes are to be realized. The first major thrust in the area has been the development of improved mutant strains of T. reesei, free from catabolite repression and capable of constitutive cellulase production (Montenecourt and Eveleigh, 1977; Gallo, 1982).While this effort continues to develop further high yielding mutants, improvement must also come from developments in fermentation techniques. A major advance is the use of fed-batch cultivation, which provides a means of avoiding the agitation and aeration difficulties, as well as repression effects encountered with high substrate concentration batch fermentation. This report briefly compares batch and fed-batch operation over a range of substrate concentrations.     

Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2

The Trichoderma reesei xln2 gene coding for the pI 9.0 endoxylanase was isolated from the wild-type strain QM6a. The gene contains one intron of 108 nucleotides and codes for a protein of 223 amino acids in which two putative N-glycosylation target sites were found. Three different T. reesei strains were transformed by targeting a construct composed of the xln2 gene, including its promoter, to the endogenous cbh1 locus. Highest overall production levels of xylanase were obtained using T. reesei ALK02721, a genetically engineered strain, as a host. Integration into the cbh1 locus was not required for enhanced expression under control of the xln2 promoter.