Study on Cellulose Degradation Enzymes and Gene Expression of Hypocrea orientalis EU7-22

随着石油资源的日渐枯竭以及人类对能源需求的增长，寻求廉价的、清洁的可再生能源已成为世界各国共同目标。纤维素生物质是地球上最重要的可再生资源，全球每年通过植物光合作用合成的生物质高达2000亿吨以上。纤维素酶和半纤维素酶是一种生物催化剂，它能有效地将纤维素生物质水解成糖类物质，通过进一步转化加工成人类所必需的生物基燃料、生物基化学品和生物基材料。纤维素生物质的利用与转化对于解决环境污染以及能源危机等问题具有十分重要的意义。纤维素酶与半纤维素酶主要由微生物生物合成，如何提高微生物分泌表达酶蛋白的能力，已成为科学界重要的研究热点问题。 东方肉座菌EU7-22是本实验室对东方肉座菌XC-9进行复合诱...With the depletion of oil resources and the concerns of increasing energy demands, seeking a cheap, clean and renewable energy has become a common goal of all countries in the world. Cellulosic biomass is the most abundant renewable resource in the biosphere, and the total biomass formed by plant photosynthesis reached more than 200 billion tons every year. Cellulase and hemicellulase, as the effi...学位：理学博士院系专业：能源研究院_能源化学学号：3242010015380

Screening, Identifying of Cellulose-Decomposing Strain L-06 and Its Enzyme-Producing Conditions

从用于堆肥的水稻秸秆中初筛出一株高效纤维素降解菌L-06,根据18SrRNA基因序列和菌株形态分析,初步鉴定该菌为斜卧青霉(Penicillium decumbens)。研究了液体发酵培养基中氮源、碳源、表面活性剂、培养温度、起始pH以及接种量对该菌株各纤维素酶活力的影响。在最适条件下,该菌的β-葡萄糖苷酶(BGL)、外切纤维素酶(CBH)于培养第3天酶活力分别达到1662u/mL和2770u/mL;内切纤维素酶(EG)、滤纸糖化力(FPase)于培养第4天酶活力分别达到18064u/mL和4035u/mL。在产酶优化实验中,该菌的EG和CBH在pH10的培养条件下分别保持了70%和43%的酶活性;在50oC培养条件下EG和CBH分别保持了68%和59%的酶活性。表现出了耐热,耐碱的特性。Cellulases are relatively costly enzymes that are sold in large volumes for use in different industrial applications, and a significant reduction in cost will be important for their commercial use in biorefineries. The production of cellulase is a major factor in the hydrolysis of cellulosic materials. Hence it is essential to make the process economically viable. A strain (L-06) with high cellulase activity was screened from rice straw compost and classified as Penicillium decumbens by the analysis of its morphology and 18S rRNA gene sequences. Different conditions of liquid fermentation medium including nitrogen source, carbon source, surfactant, temperature, initial pH, inoculation quantity for the production of cellulase had been studied. The maximal β-1, 4-glucosidase(BGL) activity was 1662 u/mL which is 1.49 times of the previous and the maximal exo-β-1, 4-glucanases(CBH) activity was 2770 u/mL which is 1.36 times of the previous, cultured in the optimal condition for three days. And the maximal endo-β-1, 4-glucanases (EG) activity was 18064 u/mL which is 1.87 times of the previous and the maximal filter paper enzyme(FPase) activity was 4035 u/mL which is 1.47 times of the previous , cultured in the optimal condition for four days. In the optimization experiments, the EG and CBH in the culture condition (pH10) maintained 70% and 43% activity. In the culture condition(50oC) EG and CBH maintained 59% and 68% activity, which showed heat and alkali resistant characteristics.国家高技术研究与发展项目(No.2006AA05Z111);; 广东省自然科学基金(No.06300998);; 福建省科技平台项目(No.2006H0091)资助~

Heterologous Expression of the β-Glucosidase and Its Synergistic Hydrolysis of Bamboo with Cellulase

在毕赤酵母gS115中表达东方肉座菌Eu7-22的β-葡萄糖苷酶基因(bgl),获得基因工程菌株bP17。优化bP17収酵产酶条件后,重组β-葡萄糖苷酶活力达121 Iu/Ml。酶学性质研究表明,该酶最适反应温度为70℃,在60℃以下有较好的热稳定性;最适催化PH为5.0,在PH 3.0~8.0之间有较好的稳定性。将异源表达的β-葡萄糖苷酶添加到东方肉座菌的纤维素酶液中协同降解经过预处理的竹纤维,当纤维素酶添加量为fPA 20 Iu/g底物,β-葡萄糖苷酶添加量为bg 6 Iu/g底物时,纤维二糖浓度显著下降,酶解得率达到83.03%,表明重组β-葡萄糖苷酶的加入更有利于纤维素的酶解糖化。The β-glucosidase gene(bglI) from Hypocrea orientalis EU7-22 was cloned and effectively expressed in Pichia pastoris GS115.The-glucosidase activity expressed by recombinant strain BP17 reached 121 IU/mL.The expressed-glucosidase exhibited the optimum catalytic activity at 70°C and pH 5.0.The enzyme exhibited good stability at pH 3.0 ~ 8.0 and remained 65% of its original activity after 1 h at 60°C.The pretreated bamboo cellulose was synergistic hydrolyzed by the cellulases from Hypocrea orientalis EU7-22 and the recombinant β-glucosidase from strain BP17.Supplementing recombinant β-glucosidase greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.03% with enhanced β-glucosidase activity of 6 IU/g substrate.The results indicated the recombinant β-glucosidase significantly boost the efficiency of saccharification.国家重点基础研究发展计划(2010CB732201); 国家自然科学基金(31170067); 中央高校基本科研业务费专项资金(201112G026

Gene cloning and bioinformatics analysis of Xylanases and Xylosidase from Hypocrea orientalis

东方肉座菌Eu7-22具有高产半纤维素酶的能力。根据已报道的同属里氏木霉及绿色木霉木聚糖酶,木糖苷酶相关基因序列,设计PCr引物扩增出东方肉座菌内切木聚糖酶(XynⅠ,XynⅡ)及β-木糖苷酶(β-bXl)基因。序列经nCbIblAST分析:东方肉座菌XynⅠ基因与里氏木霉Xyn1基因(X69573.1)的同源性最高达到91%;XynⅡ基因与绿色木霉Xyn2基因(Ef079061)同源性最高达到93%;β-bXl基因与里氏木霉β-bXl1基因(z69257.1)的同源性最高达到94%。生物信息学分析表明内切木聚糖酶Ⅰ和Ⅱ均属于糖基水解酶家族11,n末端前19个氨基酸均为信号肽。内切木聚糖酶Ⅰ分子量为24.13kd,等电点为7.87,含有3个糖基化位点;内切木聚糖酶Ⅱ分子量为24.44kd,等电点为4.86,含有1个糖基化位点;β-木糖苷酶属于糖基水解酶家族3,分子量为87.27kd,等电点为5.49,n末端前20个氨基酸为信号肽,含有8个糖基化位点。利用SWISS-MOdEl对木聚糖酶,木糖苷酶蛋白质三级结构进行了预测和模拟。对木聚糖酶和木糖苷酶基因及其编码蛋白质的生物信息学分析,为进一步研究这些基因的表达与调控、构建高效利用纤维素组份的工程菌株奠定基础。Hypocrea orientalis EU7-22 had a high potential to yield hemicellulase.Two endo-xylanases genes (xyn Ⅰ, xyn Ⅱ) and one β-xylosidase gene (β-bxl) were successfully cloned by PCR, according to the reported xylanases and β-xylosidase gene sequences of Trichoderma reesei and Trichoderma viride.The xyn I and β-bxl gene from H.orientalis showed the highest nucleotide homology of 91% and 94% with the corresponding gene from T.reesei, respectively.While the xyn II gene from H.orientalis showed the highest of 93% nucleotide homology with the corresponding gene from T.viride.The bioinformatics analysis indicated that the enzyme XYN I and XYN II belonged to the glycosyl hydrolase family 11 with the molecular weight of 24.13 kD and 24.44 kD, respectively.The first 19 AA of N-terminal of XYN I and XYN II were the signal peptide sequence.The enzyme XYN I and XYN II contained three and one N-glycosylation site, respectively.The isoelectric point of enzyme XYN I and XYN II were identified as 7.87 and 4.86, respectively.The enzyme β-BXL belonged to glycosyl hydrolase family 3 with molecular weight of 87.27 kD and isoelectric point of 5.49.The first 20 AA of N-terminal of β-BXL belonged to signal peptide sequence.The enzyme contained 8 N-glycosylation sites.By using SWISS-Model, the tertiary structure of the three enzyme proteins were predicted and simulated.These genes cloning and bioinformatics analysis would help to the further research on mechanism of expression and regulation of hemicellulase.国家重点基础研究发展计划(973计划)(NO.2010CB732201); 中央高校基本科研业务费专项资金(NO.201112G026); 国家自然科学基金(NO.31170067

Cloning and Sequence Analysis of Cellulase Genes from Hypocrea orientalis EU7-22

作者简介: 龙传南, 男, 博士研究生, 研究方向: 生物能源; E-mail: [email protected] 通讯作者: 龙敏南, 男, 教授, 研究方向: 生物质资源利用; E-mail: [email protected][中文文摘]东方肉座菌EU7-22与XC-9、里氏木霉、康宁木霉、黑曲霉、斜卧青霉进行产纤维素酶比较,结果表明菌株EU7-22具有较高的产纤维素酶能力及完整的纤维素酶系。根据里氏木霉和绿色木霉的外切葡聚糖酶,内切葡聚糖酶及β-葡萄糖苷酶相关基因序列,设计引物PCR扩增出菌株EU7-22 cbhⅠ、cbhⅡ、egⅠ、egⅡ及bglⅠ。基因序列经NCBI Blast分析表明,cbhⅠ与绿色木霉cbh1基因(FJ871063)同源性最高达99%;cbhⅡ与康宁木霉cbh2基因(DQ504304)同源性最高达99%;egⅠ与长枝木霉eg1基因(GU144298)同源性最高达99%;egⅡ与绿色木霉eg2基因(EF602036)同源性最高达99%;bglⅠ与菌株Trichodermasp.SSLbgl基因(FJ040193)同源性最高达100%。5种纤维素酶基因编码的相应氨基酸序列与其他木霉纤维素酶的氨基酸序列相似性也非常高。对上述纤维素酶基因编码的相应蛋白的分子量、等电点、N-糖基化位点、信号肽序列进行分析;对纤维素结合区及糖基水解酶家族特征结构区进行了定位;用SWISS-Model模拟了酶蛋白的三级结构。[英文文摘]The fungi of Hypocrea orientalis EU7-22, Hypocrea orientalis XC-9, Trichoderma reesei, Trichoderma koningii, Aspergillus niger and Penicillium decumbens were investigated to produce cellulase. The results indicated that the strain EU7-22 had highly cellulase activities in comparison with other fungi, and possessed of integrated cellulase system. According to the reported cellobiohydrolase, endoglucanase and β-glucosidase gene sequences of T. reesei and Trichoderma viride, the primers were designed and five cellulase genes（cbhⅠ, cbhⅡ, egⅠ, egⅡ, bgl Ⅰ）were successfully cloned by PCR. By conducting sequence alignment analysis with NCBI Blast, it was found that the homology of same cellulase genes between strain EU7-22 and other Trichoderma were: 99% to cbh1（FJ871063）from T. viride；99% to cbh2（DQ504304）from T. koninqii ；99% to eg1（GU144298）from Trichoderma longibrachiatum ；99% to eg2（EF602036）from T. viride ；100% to bgl（FJ040193）from Trichoderma sp. SSL. Furthermore, the corresponding amino acid sequences were also quite similar. The molecular weight, isoelectric point, N-glycosylation sites and signal peptide sequence of these cellulase genes encoding the corresponding protein were analyzed. The cellulosebinding domain and conserved domains of glycosyl hydrolases family were confirmed. By using SWISS-Model, the tertiary structure of cellulase proteins were predicted and simulated.国家重点基础研究发展计划(“973”计划)(2010CB732201);中央高校基本科研业务费专项资金项目(201112G026);国家自然科学基金项目(31170067

Research on pretreatment by white-rot Fungi for the contribution to hydrolysis of bagasse

[中文文摘]利用一株产木质素降解酶的白腐菌Ceripriopsis sp.,以固态发酵形式对甘蔗渣进行预处理,选择性降解其中的木质素,从而提高其水解效率。甘蔗渣固态发酵过程中,发酵温度、培养基成分、含水量和初始pH值对酶活和糖收率都有影响。含水量是对糖收率影响最大的因素,其次是麸皮添加量,再次是温度,而初始pH值影响最小。甘蔗渣固态发酵的最佳条件为:培养基中含水量为80%,麸皮含量为5%,初始pH=4.0时28℃发酵14d,获得的糖产率最大,达20.6%。[英文文摘]The research is focused on pretreatment on bagasse by solid-state fermentation with selective white-rot fungus Ceripriopsis sp.which degrades of lignin selectively,thereby enhancing the hydrolysis efficiency.The fermentation temperature,medium composition,moisture content and initial pH have an impact on enzyme activity and sugar yield in solid-state fermentation process.The moisture is the most influential factor on the sugar yield,followed by wheat bran added,temperature,and the initial pH with minimal impact.The best conditions for the solid-state fermentation of bagasse are menium moisture of 80%,5% of wheat bran,initial pH 4.0 at 28℃ for 14 days to obtain the sugar yield of 20.6%

Isolation and Properties of a High Rate H_2-producing Thermoduric Bacteria Strain

从高温环境中分离得到具有高产氢活力的微生物菌株,经16SrRNA基因序列分析及生理生化特性分析,鉴定该菌株为Proteussp.该菌株能以葡萄糖、蔗糖、麦芽糖、乳糖和淀粉等多种有机物为底物催化产氢.该菌株利用葡萄糖产氢的最适条件为:葡萄糖浓度0.1mol/L;菌体细胞密度OD600=0.60;反应系统起始pH值7.0;反应温度40℃.在该条件下菌株的最高产氢活性可达8.05μmolH2/h·mgprot.A thermoduric bacteria strain with high H2producing activity was isolated from high temperature environment. It was identified to be Proteus sp. by the analysis of sequence of 16S rRNA gene and its physiological properties. The thermoduric bacteria Proteus sp. could use different organic substrates such as glucose, sucrose, maltose, lactose and soluble starch for H2production under fermentative conditions. And glucose was the favorite substrates for H2production. The optimum conditions for Proteus sp. H2production were: substrate concentration: 0.1 mol/L glucose; cell density: OD600=0.6; starting pH:7.0; reaction temperature:40℃. Under the optimum conditions, the maximum H2production activity reached 80.5 μmol H2/h·mg prot.The results suggest that Proteus sp. is an ideal material for biological H2production.国家863项目(2001AA515040);; 国际科技合作重点项目(2002AA515030);; 国家自然科学基金(302703228)资

Primary Studies on Purification and Properties of Soluble Hydrogenase from Klebsiella oxytoca HP1

采用硫酸铵沉淀和柱层析(DEAE-Sepharose F.F.,Sephacryl S-200,TSK-DEAE)初步分离纯化了产酸克雷伯氏菌(Klebsiella oxytoca)HP1可溶性氢酶,研究了温度、pH值、电子载体等对氢酶催化放氢活性的影响.研究表明,可溶性氢酶催化放氢的最适温度为30℃,催化放氢的最适pH值为7.5,甲基紫晶(MV)是氢酶催化放氢的最适人工电子载体,氧对氢酶催化活性有较大的抑制作用.The hydrogenase-containing crude extraction of Klebsiella oxytoca HP1 was firstly precipitated by 20% and 60% of ammonium sulfate solution in water,respectively.It was subsequently purified by three steps of column chromatography(DEAE-Sepharose F.F.,Sephacryl S-200 and TSK-DEAE).After three steps of purification,the soluble hydrogense of K.oxytoca HP1 was purified about 200 folds with a yield of 15.3% and a specific activity of 17.68 μmol H_2/min·mg prot.The catalytic property of soluble hydrogenase was studied.The optimal conditions for hydrogen production were obtained as follow: temperature 30℃,pH 7.5.Oxygen was an inhibitor for the hydrogen production of soluble hydrogenase.MV was the optimal electron carrier for the soluble hydrogenase′s activity.国家自然科学基金(30470395);; 厦门大学新世纪优秀人才支持计划(X07114)资