产纤维素酶黏细菌在生物转化的作用

目的:预处理对木质纤维素降解的影响.方法:从土壤中分离筛选到高纤维素酶活的黏细菌菌株So ce sh1008.该菌具有CMC酶活(CMCase)及微晶纤维素酶活性.研究NaOH联合黏细菌降解盐蒿、稻草、棉花秸秆和甘蔗渣四种木质纤维素的情况.结果:碱(2％ NaOH) -黏细菌处理的方法优于黏细菌-碱的方法,其中降解棉花秸秆降解效果最明显,以5.0g木质纤维素为原料,其最终干重损失达2.1g,溶液中总糖含量和还原糖含量均值分别为12.8 mg/mL和0.93 mg/mL.酵母菌发酵产乙醇的研究结果表明,最佳发酵时间为47h,碱-黏细菌甘蔗渣降解液发酵效果最好,乙醇产出达6.0％.结论:黏细菌联合2％ NaOH能有效降解甘蔗渣,提高乙醇产量.     

解淀粉芽孢杆菌MN-8对玉米秸秆木质纤维素的降解

微生物降解木质纤维素既是生物质资源化利用中的关键问题,也是亟需解决的难点问题.本文在前期获得木质素降解菌——解淀粉芽孢杆菌MN-8菌株的基础上,进一步研究该菌株对玉米秸秆木质纤维素的降解作用.研究利用玉米秸秆粉-MSM培养基对MN-8菌株进行固态发酵,监测发酵过程中木质纤维素酶活力和木质纤维素含量变化情况,并通过傅立叶红外光谱(FTIR)和气质联用色谱(GC/MS)对木质纤维素的降解情况及产物进行分析.结果表明:解淀粉芽孢杆菌MN-8菌株可产生木质素过氧化物酶、锰过氧化物酶、纤维素酶和半纤维素酶等木质纤维素降解酶,在发酵10～16 d陆续达到酶活力峰值,最高酶活力分别为55.0、16.7、45.4和60.5 U·g-1.发酵24 d后,玉米秸秆中木质素、纤维素和半纤维素的降解率可分别达到42.9％、40.6％和27.1％.FTIR光谱数据表明,玉米秸秆发酵后木质素、纤维素和半纤维素的特征吸收峰强度均有一定程度的下降,表明木质纤维素被部分降解.GC/MS分析结果也证实,解淀粉芽孢杆菌MN-8能有效降解秸秆木质纤维素.MN-8菌株可断裂玉米秸秆木质素单体之间的连接键β-O-4,将秸秆木质素解聚为苯丙胺、苯丙酮和苯丙酸等保留木质素苯丙烷结构的单体化合物,并将部分单体化合物进一步氧化为Cα羰基化合物,如2-氨基-1-苯丙酮和紫丁香基苯乙酮等.在对纤维素和半纤维素降解产物的GC/MS分析中发现,降解产物包含葡萄糖、甘露糖和半乳糖等多种单糖化合物以及甲酸、乙酸、丙酸、1,1-乙二醇和3-羟基丁酸等代谢产物.表明解淀粉芽孢杆菌MN-8对秸秆木质纤维素表现出强降解作用,且该作用依赖于菌株产木质纤维素降解酶的能力.     

三株高效秸秆纤维素降解真菌的筛选及其降解效果

【目的】利用多种筛选方法,获得高效秸秆纤维素降解真菌,并研究其秸秆纤维素的降解能力。【方法】采用滤纸片孔洞法、滤纸条降解法、羧甲基纤维素钠(CMC-Na)水解圈测定法、秸秆失重法、纤维素分解率测定法、胞外酶活测定法等常规秸秆纤维素降解菌的筛选方法。【结果】筛选到3株具有较强纤维素降解能力的真菌菌株,经初步鉴定菌株98MJ为草酸青霉(Penicillium oxalicum)、菌株W3为木霉(Trichoderma sp.)、菌株W4为扩张青霉(Penicillium expansum)。菌株W4具有非常强的秸秆纤维素降解能力,10d内对秸秆的降解率可达56.3%,对纤维素、半纤维素和木质素的分解率分别为59.06%、78.75%和33.79%。菌株W4的胞外纤维素酶活力在14.25-49.75U/mL之间。【结论】筛选获得3株高效秸秆纤维素降解真菌菌株,其中菌株W4的纤维素酶活高于已报道的菌株,是一株十分具有研究开发潜力的纤维素酶生产菌株。     

一株木质纤维素降解菌的筛选及其全基因组分析北大核心CSCD

【目的】筛选和鉴定有木质纤维素降解能力的1株细菌,测定其相关酶活力并进行全基因组分析,为构建木质纤维素降解工程菌提供依据。【方法】采用3种木质素类似物(天青-B;酚红;愈创木酚)的脱色/染色法,从腐木和被枝叶覆盖的土壤中分离和筛选出1株具有较强木质纤维素降解能力的细菌。通过16S r RNA基因和全基因组序列分析对该菌进行种属鉴定。使用紫外分光光度法测定其锰过氧化物酶(Mn P)、漆酶(Lac)、羧甲基纤维素酶(CMCase)以及滤纸酶(FPA)活力,了解该菌相关酶活力大小在一定时间内的变化趋势。使用Illumina Miseq和454 GS Junior测序平台获取该菌的全基因组序列,将其全基因组序列经过注释的基因蛋白质序列提交COG和KEGG数据库进行BLASTp比对分析,确定该菌潜在的重要酶类和代谢途径,并对部分注释基因进行定量RT-PCR验证。【结果】筛选得到1株优势菌株S12,该菌经鉴定后命名为解鸟氨酸拉乌尔菌(Raoultella ornithinolytica)。在液体CMC-Na培养基中发酵28 h,菌体生长达到稳定期,纤维素降解相关酶活力也在此时达到峰值。生物信息学分析结果表明,菌株S12具有木质素降解通路中重要酶类的编码基因,如过氧化物酶、Fe-Mn型超氧化物歧化酶、邻苯二酚1,2-双加氧酶和原儿茶酸-3,4-双加氧酶等,这些基因在以碱性木质素为碳源的培养条件下表达量不同程度地高于以葡萄糖为碳源的培养条件。另外,菌株S12具备完整的纤维素降解和乙醇生成通路。【结论】本研究首次揭示了Raoultella ornithinolytica S12具备有效的木质纤维素降解性能,这对于推动木质纤维素应用产业的发展具有重要意义。     

平板混合培养木质纤维素降解菌研究进展

微生物的混合培养已广泛应用于木质纤维素类物质的转化与降解领域.不同木质纤维素降解菌在混合培养时的相互关系在很大程度上影响混合培养的效果.目前对这种相互关系的研究主要依托平板混合培养展开,所用到的平板主要有基础培养基平板和改进培养基平板两种.其中基础培养基平板法主要根据菌落形态、菌丝体颜色、胞外挥发性有机化合物成分和典型胞外酶活性等进行研究,而改进培养基平板则是将基础培养基平板中的碳源更换为天然木质纤维素类物质进行对比研究.本文综述了采用平板混合培养不同木质纤维素降解菌菌株的研究现状和进展,并对该领域研究应重点关注的问题进行了展望.     

居瘤胃解纤维素菌细胞密度与纤维素酶的合成

居瘤胃解纤维素菌(Cellulosilyticum ruminicola)H1是本实验室分离自青海牦牛瘤胃的一株新的纤维素降解细菌。前期研究发现,菌株H1在滤纸纤维素上连续传代数次后无法生长,只有在纤维素降解产物纤维二糖中培养后方能继续在纤维素中生长,并恢复其纤维素降解活性。这与纤维素酶合成受"代谢产物抑制"的传统认识相悖。【目的】证明菌株H1的纤维素酶合成受细胞密度调控。【方法】检测菌株H1的纤维素酶活和转录水平在高和低密度细胞培养物中差异,并检测高密度细胞培养物中的寡肽对低密度细胞纤维素酶活和转录水平的促进作用。【结果】菌株H1的高密度细胞培养物的纤维素酶活和转录水平比低密度细胞的高3-10倍;并且高密度细胞培养液能显著提高低密度细胞纤维素酶活和转录水平。【结论】居瘤胃解纤维素菌(Cellulosilyticum ruminicola)H1纤维素酶的合成受细胞密度调控。     

一株多糖降解菌的分离、鉴定与琼脂糖降解能力

【目的】筛选海洋来源的多糖降解菌,分析其多糖降解能力并初探机制。【方法】碘液染色法从海泥中初筛琼脂糖降解菌,唯一碳源生长法分析菌株的多糖利用能力,克隆16S rRNA基因以分析系统分类地位。用硫酸铵沉淀法制备胞外粗酶制剂,DNS-还原糖法测定琼胶酶活性,活性染色法分析胞外琼胶酶系的组成特征。分离、纯化琼脂糖的酶解产物,通过TLC测定寡糖Rf值、阳离子质谱测定分子量。【结果】分离到1株能液化琼脂糖的海洋细菌JZB09,鉴定至桃色杆菌属(Persicobacter)。JZB09能利用11种不同的多糖为唯一碳源生长,在利用琼脂糖、纤维素和木聚糖时生长较好。胞外粗酶制剂的琼胶酶活力约77.2U/mg,含有至少2条琼胶酶,大小约45kDa、70kDa。酶制剂降解琼脂糖后的产物是系列新琼寡糖,四糖是主产物,表明β-琼胶酶在胞外琼胶酶系降解琼脂糖时起关键作用。【结论】海洋细菌Persicobacter sp.JZB09是1株多能型多糖降解菌,可分泌β-琼胶酶降解琼脂糖且活性显著,具有潜在开发价值。     

不同碳源条件下广叶绣球菌转录组分析

【背景】广叶绣球菌(Sparassis latifolia)是一种名贵的食用菌,其木质纤维素降解的分子机制尚不明确。【目的】了解广叶绣球菌在不同碳源条件下木质纤维素降解相关基因表达动态。【方法】通过转录组测序技术对分别以葡萄糖、纤维素+木质素、纤维素及松木屑为碳源的广叶绣球菌基因表达谱进行分析。以葡萄糖为碳源的样本为对照,分别对不同碳源下广叶绣球菌显著差异表达的基因进行功能分析。【结果】Geneontology(Go)富集分析表明,以葡萄糖为碳源的样本为对照,差异表达基因主要富集在碳水化合物利用的过程,如多糖催化过程、碳水化合物催化过程、碳水化合物代谢过程及多糖代谢过程等。碳水化合物活性酶(Carbohydrate-activeenzymes,CAZymes)功能注释表明,碳源种类主要影响了半纤维素和纤维素降解相关糖苷水解酶家族基因的表达,其中涉及半纤维素降解的相关酶基因上调幅度最大。同时,在纤维素+木质素、松木屑为碳源的处理组中一些转录因子基因上调表达显著。【结论】不同碳源显著影响了广叶绣球菌基因表达谱,这种对碳源的适应也可能反映了广叶绣球菌攻击植物细胞壁的机制,研究结果为深入了解广叶绣球菌木质纤维素降解的分子机理和相关功能基因提供了一些参考。     

基于转录组和加权基因共表达网络的广叶绣球菌木质纤维素降解特性分析

【目的】分析广叶绣球菌（Sparassis latifolia）在不同木质纤维素诱导条件下基因表达差异，为广叶绣球菌木质纤维素降解关键基因和分子机制研究提供参考。【方法】以松木、杉木、甘蔗渣和天然堆积发酵后的杉木和发酵后的甘蔗渣为碳源，在液体培养条件下培养诱导广叶绣球菌，对其转录组进行测序研究，并对不同木质纤维素诱导样本进行加权基因共表达网络分析（weighted gene co-expression network analysis，WGCNA）。【结果】杉木培养与松木培养比较组差异表达基因最少（20个），蔗渣培养与松木培养比较组差异表达基因最多（486个）。基因本体（gene ontology，GO）富集分析结果表明，差异表达基因主要涉及氧化还原酶活性、单加氧酶活性和铁离子结合活性等，京都基因和基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析结果表明，差异表达基因主要涉及戊糖和葡萄糖醛酸转换、甲烷代谢和乙醛酸盐和二羧酸盐代谢等通路。发酵甘蔗渣为碳源培养时，纤维素和半纤维素降解相关的糖苷水解酶基因表达量总体上较高，而未发酵的松木、杉木和甘蔗渣为碳源培养时木质素降解或修饰相关的碳水化合物辅助酶基因表达量总体上较高。利用WGCNA共鉴定出10个共表达模块，其中green模块与未发酵蔗渣诱导显著正相关，blue模块与发酵甘蔗渣诱导显著正相关，magenta和turquoise模块与发酵杉木诱导显著正相关。GO富集分析结果表明，turquoise模块内基因显著富集到尿素跨膜转运子活性、甲基转移酶活性和单加酶活性等，blue模块基因显著富集到水解酶活性和β-甘露糖苷酶活性。KEGG通路富集分析结果表明，blue模块内基因显著富集的通路有半乳糖代谢、果糖和甘露糖代谢、苯丙氨酸代谢、精氨酸和脯氨酸代谢等。通过构建互作网络图挖掘到12个核心基因，其可能参与了基质降解及相关基因的表达调控。【结论】不同木质纤维素类型显著影响了广叶绣球菌木质纤维素降解基因的差异表达轮廓，这种差异反映了广叶绣球菌对不同木质纤维素特异的降解策略。     

大熊猫肠道纤维素分解菌的分离鉴定及产酶性质

【目的】从健康大熊猫新鲜粪便中分离具有纤维素酶活性的菌株,并对其进行菌种鉴定及产酶性质研究。【方法】利用羧甲基纤维素钠培养基分离纯化具有较高纤维素酶活性的菌株,根据形态学特征、生理生化特性以及16S rDNA分析对其进行分类鉴定,研究影响该菌株纤维素酶的产酶条件,以及对不同纤维素底物的降解情况。【结果】分离得到一株纤维素酶产生菌株P2,该菌株为好氧的革兰氏阳性细菌,生长温度范围20-50℃(最适温度37℃),pH范围6.0-9.0(最适pH7.0),NaCl浓度范围0%-15%(最适2%NaCl),培养24h达到产酶高峰。16S rDNA基因序列分析显示,菌株P2与解淀粉芽胞杆菌(Bacillusamyloliquefaciens)NBRC15535相似性为99.66%。该菌株对四种纤维素底物(滤纸、脱脂棉、秸秆、竹纤维)均有不同程度的降解,内切葡聚糖酶、外切葡聚糖酶、β-葡萄糖苷酶和总酶活具有不同的酶活变化。【结论】本研究首次从大熊猫粪便中分离出了好氧纤维素分解菌,并鉴定为解淀粉芽胞杆菌,对上述四种纤维结构均有一定的破坏和分解作用,为进一步研究大熊猫竹纤维消化机制提供了菌源。     

Determination of the roles of active sites in F1-ATPase by controlled affinity labeling

The affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl) [alpha-32P]ATP (FDNP-[alpha-32P]ATP) and 3'-O-(5-fluoro-2,4-dinitrophenyl) [8-14C]ATP (FDNP-[14C]ATP) were synthesized and used to characterize the structure and function of the three active sites in F1-ATPase. FDNP-[alpha-32P]ATP was found to bind covalently to F1 up to two DNP-[alpha-32P]ATP labels per F1 in the absence of Mg2+ without decreasing the ATPase activity. However, when MgCl2 was subsequently added to the reaction mixture, the enzyme could be further labeled with concomitant decrease in ATPase activity that is consistent with the complete inactivation of one enzyme molecule by an affinity label at the third ATP-binding site. Partial hydrolysis of the FDNP-[14C]ATP-labeled enzyme and sequencing of the isolated peptide indicated that the affinity label was attached to Lys-beta 301 at all three active sites. Samples of F1 with covalent affinity label on Lys-beta 301 were also used to reconstitute F1-deficient submitochondrial particles. The reconstituted particles were assayed for ATPase and oxidative phosphorylation activities. These results show that the catalytic hydrolysis of ATP either by F1 in solution or by F0F1 complex attached to inner mitochondrial membrane takes place essentially at only one active site, but is promoted by the binding of ATP at the other two active sites, and that ATP synthesis during oxidative phosphorylation takes place at all three active sites [corrected].     

一株产右旋糖酐酶青霉的分离及酶的纯化和性质

[目的]从土壤中筛选到一株新的产右旋糖酐酶的真菌F1001,为酶法制备药用级右旋糖酐提供新的右旋糖酐酶产生菌株.[方法]通过形态特征和ITS rDNA序列分析方法鉴定菌株.利用硫酸铵盐析、Sepharose 6B凝胶柱纯化,得到纯度较高的酶蛋白.以右旋糖酐70 kDa为底物,对右旋糖酐酶酶学性质及催化机理进行研究.[结...     

A sensitive method for the assay of guanylate cyclase activity.

A method for the assay of guanylate cyclase is described utilizing alpha-[32P]-GTP as substrate for the enzyme reaction. 100-150 microgram of enzyme protein is incubated in a 15.6 mM Tris-HCl buffer incubation mixture, pH 7.6. The reaction is stopped by the addition of EDTA. The [32P]-cyclic GMP formed is separated by a two-step column chromatography on Dowex 50W-X4 ion-exchange resin and neutral alumina. The recovery for cyclic GMP was about 70%. The blank values ranged from 0.001-0.003% of the added alpha-[32P]-GTP which had been purified by Dowex 50W-X4 column chromatography. This method was employed for the assay of guanylate cyclase activities in different tissues.     

Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway

Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters. It was the first example of propionic fermentation from ethanol. Morphologic and physiologic characterizations of the strain are presented here. This strain is described as type strain of a new species, Clostridium neopropionicum sp. nov. Whole cells of strain X4 ferment [1-¹³C]ethanol and CO₂ to [2-¹³C]propionate, [1-¹³C]acetate and [2-¹³C]propanol, suggesting the absence of a randomizing pathway during the propionate formation. Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation. The same pathway may be used for the degradation of lactate or acrylate to acetate.     

Human red cell acetyltransferase

Acetyltransferase was isolated by histone-Sepharose affinity chromatography from human cord blood red cells. The enzyme was detected only in very young red cells. The semipurified enzyme and [14C]acetyl-CoA were used to acetylate isolated Hb F tetramer and alpha and gamma subunits. The in vitro acetylated products were characterized by globin chain separation by CM-cellulose chromatography and tryptic peptide analysis by reverse-phase HPLC. Acetylation of both the gamma-chains and the alpha-chains could occur within the Hb F tetramer. Acetylation also could take place on intact subunits. It appears that some Hb FIC could be formed in the cells by utilizing Hb F or free gamma-chains as acetylation substrate.     

红灯食烷菌(Alcanivorax hongdengensis)黄素结合单加氧酶(AlmA)的基因克隆及其烷烃诱导表达

【目的】研究海洋烷烃降解菌新种模式菌株Alcanivorax hongdengensis A-11-3降解长链烷烃的分子机制。【方法】PCR克隆编码黄素结合单加氧酶的基因序列,利用生物信息学软件对序列进行分析,运用RT-PCR和实时荧光定量PCR技术分析基因在不同烷烃诱导下的表达水平。【结果】从菌株A-11-3中克隆获得了两个黄素结合单加氧酶基因片段(almA1和almA2)。它们编码的氨基酸序列与菌株Acinetobacter sp.DSM17874的AlmA同源性分别为58.6%和53.2%。实时荧光定量PCR分析表明,almA1基因只在长链烷烃(C28-C32)的诱导下上调表达,而almA2基因中能在更宽范围的长链烷烃(C24-C34)和支链烷烃诱导下上调表达。两者均在C9-C22的烷烃诱导下没有上调表达。【结论】黄素结合单加氧酶可能是A-11-3降解长链烷烃和支链烷烃的关键酶。     

腐皮镰孢菌壳聚糖酶的酶学性质研究及其在酿酒酵母工业菌株中的表达

摘要：【目的】本研究旨在了解腐皮镰孢菌（Fusarium solani）壳聚糖酶的基本酶学性质及其在壳寡糖生产中的应用,构建能高效分泌表达壳聚糖酶的酿酒酵母工业菌株。【方法】采用RT-PCR扩增腐皮镰孢菌壳聚糖酶的cDNA序列;通过组氨酸标签,纯化得到E. coli表达的重组壳聚糖酶,并进行基本酶学性质研究;以薄层层析、高效液相色谱等技术对该酶的酶解产物进行分析;通过马克斯克鲁维酵母（Kluyveromyces marxianus）菊粉酶信号肽（INU1A）实现壳聚糖酶在酿酒酵母工业菌株N-27中的分泌表     

Comparison of the hydrolysis of polyethylene terephthalate fibers by a hydrolase from Fusarium oxysporum LCH I and Fusarium solani f. sp. pisi

The hydrolysis of polyethylene terephthalate (PET) fibers by two fungal hydrolases was investigated. The hydrolase from a newly isolated Fusarium oxysporum strain (LCH 1) was more efficient in releasing terephthalic acid from PET fibers compared to the enzyme from F. solani f. sp. pisi DSM 62420 when equal amounts of p-nitrophenyl butyrate-hydrolyzing activity were employed. PET fabrics treated under the same conditions with the enzyme from F. oxysporum LCH 1 also showed a considerably higher increase in hydrophilicity compared to fabrics treated with the enzyme from F. solani f. sp. pisi DSM 62420.     

Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A.?saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A.?saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50?°C, and at 60?°C it had a half-life of approximately 6?h.     

Identification of the lysine residue to which the 4-nitrobenzofurazan group migrates after the bovine mitochondrial F1-ATPase is inactivated with 7-chloro-4-nitro[14C]benzofurazan

When bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, was inactivated by greater than 90% with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.4, 1.15 mol of 4-nitrobenzofurazan [14C]Nbf were incorporated per mol of enzyme. Reactivation of a sample of the modified enzyme with dithiothreitol removed 0.82 mol of [14C]Nbf/mol of the F1-ATPase indicating that, of the 1.15 mol of [14C]Nbf incorporated, 0.82 mol were present on tyrosine residues and 0.33 mol on lysine residues. Incubation of the modified enzyme at pH 9.0 for 18 h at 23 degrees C led to an increase of 0.64 mol of [14C]Nbf-N'-Lys/mol of the F1-ATPase which occurred as a consequence of an O----N migration. About 15% enzyme reactivation occurred simultaneously with the migration indicating that the fraction of the [14C]Nbf group originally present on tyrosine which did not migrate was lost by hydrolysis. Examination of a tryptic digest of the labeled enzyme after the O----N migration by reversed-phase high-pressure liquid chromatography revealed a single major radioactive peptide. The labeled tryptic fragment was purified and subjected to automatic Edman degradation. This analysis revealed that Lys-beta-162 was specifically labeled during the O----N migration of the [14C]Nbf group.