DETERMINATION OF SPARTINA ANGLICA STOCK COMPONENTS BY NREL METHOD

采用美国国家可再生能源实验室(NREL)方法定量大米草原料中纤维素、半纤维素及木质素。72%浓硫酸水解1 h、4%稀硫酸水解1 h可将大米草的纤维素、半纤维素降解为可用HPLC定量的单糖,适宜的样品添加量为0.3 g。同时,NREL法测定大米草纤维素、半纤维素及木质素的含量分别为32.92%、27.65%和24.2%。这三个组分的含量是评价大米草预处理、酶解及发酵工艺条件的重要依据。Cellulose, hemicellulose and lignin of Spartina anglica were quantified by National Renewable Energy Lab(NREL) method. It was showed that the cellulose and semicellulose of S. anglica could be degraded to mono-sugars which could be quantified by HPLC after it was hydrolyzed with 72% concentrated sulphuric acid for 1 h and 4% dilute sulphuric acid for 1h with the appropriate additive amount of 0.3 g sample. Meanwhile, the contents of cellulose, hemicellulose and lignin of S. anglica determined by NREL method were 32.92%, 27.65%and 24.2% respectively. The contents of these three fractions are important basis for the evaluation of the pre-processing, enzymatic hydrolysis and fermentation conditions of S. anglica.闽海高新[2014]25号“滩涂大米草高值化综合利用关键技术与示范”项目;; 福建省生物医药产业中试与检测技术公共服务平台;; 海洋生物制品福建省高校应用技术工程中

几种不同生物质的快速热解

在一流化床连续热解装置中(生物质处理能力1 kg/H),对松树、杨树、芒草、和甜高粱等几种典型的生物质进行了快速热解研究。考察了热解温度、停留时间、进料颗粒大小等对生物油产率与组成的影响,获得了这几种生物质的最佳热解工艺条件。研究结果表明,在相同的操作条件下,木质类生物质具有相对较高的生物油产率;而芒草和甜高粱等草本类生物质由于含灰分较多,热解后所得固体产物——生物焦的产率较高,但生物油的产率相对较低,不过其生物油组成中含氧量也相对较低

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

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

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

Optimization and characterization of sodium carboxymethyl cellulose with a high degree of substitution prepared from bamboo shavings

从竹材加工剩余物竹屑中提取纤维素,然后制备羧甲基纤维素钠。采用PlACkETT-burMAn(Pb)设计与中心组合法对影响竹纤维羧甲基纤维素钠取代度的6个相关因素进行了研究,结果表明,关键因子为氢氧化钠与纤维素的质量比、氯乙酸钠与纤维素的质量比和醚化时间;建立此三因素对羧甲基纤维素钠取代度影响的二次回归模型,优化得到最佳制备条件是:氢氧化钠与纤维素的质量比为0.8、氯乙酸钠与纤维素的质量比为1.2,醚化时间为2 H。制得羧甲基纤维素钠的黏度为136 M PA·S,取代度为0.93。利用扫描电镜、红外光谱、X射线衍射和热重分析等对制得样品进行表征。结果表明,利用廉价的竹屑可以制备高取代度的羧甲基纤维素钠。The preparation of carboxymethyl cellulose( CMC) with a high degree of substitution( DS) using cellulose from bamboo residues is investigated.The factors affecting DS of CMC are optimized by Plackett-Burman Design( PB) and Central Composite Design.The results of PB indicate that the mass ratio of Na OH and cellulose,the mass ratio of Cl CH2 COONa and cellulose,and the etherification time are the key factors.The quadratic regression model of these three factors is established.The optimal conditions are shown as follows: 0.8 of the mass ratio of Na OH and cellulose,1.2of the mass ratio of Cl CH2 COONa and cellulose,2 hours of the etherification time.Under this condition,CMC with a DS of 0.94 and viscosity of 136 m Pa·s is obtained.The CMC is characterized by SEM,FT-IR,XRD,and TGA.It indicates that cheap bamboo shavings could be used to prepare CMC with a high DS.国家自然科学基金(21303142;31170067); 福建省中青年教师教育科研项目(JA14010); 厦门市海洋经济发展专项资金项目(14GZP59HJ29

Purification and Properties of β-glucosidase from Aspergillus glaucus EU7-22

目的:利用灰绿曲霉Eu7-22发酵产纤维素酶,从中分离到β-葡萄糖苷酶,分析其理化特性,确定其最佳活性条件。方法:灰绿曲霉Eu7-22发酵液离心后,上清液经硫酸铵沉淀、PHEnyl 6 fAST flOW(HIgHSub)疏水层析和SEPHACryl S-200凝胶层析,获得纯化的β-葡萄糖苷酶。结果:纯酶的比活性为5.1 Iu/Mg,得率为13.89%。SdS-PAgE凝胶电泳分析表明该酶是单亚基蛋白,其分子量为56.2 kdA。在PH4.0--6.0范围内,β-葡萄糖苷酶具有较高的稳定性,该酶的最适酶促反应PH为5.0。当β-葡萄糖苷酶在温度低于60℃的缓冲液中温育1 H后,酶活损失不大,表现了较好的稳定性;当该酶在温度高于60℃的缓冲液中温育1 H后,酶活迅速丧失。β-葡萄糖苷酶在70℃时具有最大催化活性。结论:灰绿曲霉Eu7-22发酵产生的β-葡萄糖苷酶具有较高活性,具有分子量较小、最佳催化温度较高的特点。Objective:β-glucosidase produced by Aspergillus glaucus EU7-22 was purified and characterized.Method:The β-glucosidase of Aspergillus glaucus EU722 was purified from ferment supernatant liquid by three steps of purification,ammonium sulfate precipitation(80%,W/V),Phenyl 6 Fast Flow(high sub) column chromatography and Sephacryl S-200 column chromatography,with a specific activity of 5.1 IU/mg and a yield of 13.89%.Result:The purified β-glucosidase was determined as a monomeric protein with a molecular weight of 56.2 kDa.The enzyme exhibited high stability when it was kept in the buffer at pH 4.0～6.0 and at the temperature below 60℃.β-glucosidase exhibited its optimal activity at 70℃.Conclusion: β-glucosidase produced by Aspergillus glaucus EU7-22 with small molecular showed high activity under the optimal conditions.国家“973”计划项目(2010CB732201);国际科技合作重点项目(2009DFA60930)资

木聚糖酶原位协同水解预处理大米草的研究

实验主要研究了氨水/双氧水结合球磨预处理对重组木聚糖酶协同水解大米草的增强效果。首先使用氨水与双氧水预处理大米草，预处理物与原料大米草随后用于球磨。然后将预处理底物分别用于结构表征及木聚糖酶的协同酶解。结果说明氨水结合双氧水预处理能有效脱除大米草中的木质素(63.81%)，球磨处理能对纤维素结晶结构有所破坏，两者有效提高木聚糖酶的水解率。而经预处理过的大米草的酶水解物主要成分为木糖(12.54%)，木二糖(40.38%)，及少量阿拉伯糖(5.50%)。以上结果说明碱预处理结合球磨预处理对后续木聚糖酶协同水解大米草产木寡糖具有明显的促进作用。厦门大学能源发展基金(2018NYFZ03

Thermal Degradation of Bamboo Lignin in Alkaline Ethanol Solvent and Product Analysis

本研究以nAOH-乙醇水溶液为溶剂体系对竹木质素进行热降解,主要考察了nAOH浓度、反应温度、反应时间、乙醇用量等条件对竹木质素降解转化为酚类化合物的影响。通过gC-MS及fT-Ir对降解产物进行分析检测,得出最佳反应条件为:竹木质素5 g,nAOH浓度(基于乙醇水溶液)20 g/l,乙醇10 Ml,反应时间2 H,反应温度240℃。在此条件下,降解产物中总的酚类化合物的相对峰面积为73.88%,残渣率为30.67%。竹木质素的降解主要产物是酚类化合物:苯酚(17.98%)、2-甲氧基苯酚(16.49%)及1,2-苯二酚(10.03%)。与现有文献相比,本文竹木质素在碱性乙醇溶剂体系中降解能够获得较高产量的酚类化合物,有望实现竹木质素的高值化利用。The thermal degradation of bamboo lignin in NaOH-ethanol solvent system was studied in this work.The effects of NaOH concentration, reaction temperature, reaction time, and dosage of ethanol on the phenolic products were investigated, respectively.The degradation products were analyzed by GC-MS and FT-IR.The optimum conditions were achieved as follows: mbamoo lignin = 5 g, CNaOH(in alcohol aqueous systems) = 20 g/L, Vethanol = 10 mL, reaction temperature is 240 oC and reaction time is 2 h.The total relative peak area of phenolic products in the degradation products was 73.88%, and the yield of residue was 30.67%.The main products of bamboo lignin degradation were phenols, such as phenol(17.98%), 2-methoxyphenol(16.49%) and 1,2-benzenediol(10.03%).The results indicated that higher yield of phenolic products was obtained under the optimized conditions compared with the reports.This work showed the potential to degrade bamboo lignin into high value products in NaOH-ethanol solvent system.农业部“引进国际先进农业科学技术”项目(2013-Z70); 福建省自然科学基金(2012J05029