源于红树林土壤宏基因组文库的新型磷脂酶A1基因的筛选、克隆表达及酶学性质

【目的】利用宏基因组学的方法从红树林土壤中筛选新型酯水解酶类。【方法】构建红树林土壤宏基因组文库,采用以三丁酸甘油酯为底物的功能筛选方法,对筛选出的阳性克隆进行系统发育树分析,实现新型磷脂酶A1基因的原核表达,研究重组酶的酶学性质。【结果】筛选到一个新的磷脂酶A1编码基因phop1413 (GenBank登录号KF767097),测序表明其全长1 413 bp,可编码470个氨基酸残基,表达蛋白约51.7 kD,表达量高达220 mg/L,NCBI中Blast比对及系统进化树分析显示该蛋白属于磷脂酶类的fAMILY Ⅵ家族;酶学性质分析表明,该重组酶的最适反应底物为对硝基苯酚己酸酯,比酶活为124 U/mg;最适反应温度为54 °C,最适pH 7.8;50 °C热处理1.5 h剩余相对酶活为44%,表现出很好的热稳定性。【结论】通过构建宏基因组文库利用功能筛选方法获得一个新型磷脂酶A1基因;研究中获得的新型磷脂酶A1性质较好,可用于植物油酶法脱胶。     

纤维素酶水解啤酒糟的研究

研究了纤维素酶水解啤酒糟的适宜条件以及底物预处理方法对纤维素转化率和多糖水解率的影响。在适宜条件下,100g干啤酒糟可水解得10．8g还原糖。酶解液用于培养酵母菌提取麦角固醇,残渣是生产含菌体蛋白饲料的原料。     

降解木质纤维素放线菌的功能组学分析及工业应用前景

放线菌是一种高GC含量的革兰氏阳性细菌,在陆生、高温的木质纤维素降解生境中占据十分重要的地位.降解木质纤维素菌株的功能基因组分析发现降解纤维素的酶种类和数目相对较多,而降解半纤维素以及果胶成分的酶相对真菌较少.其中,降解纤维素的酶类主要以GH6家族外切酶为主,部分含有GH9和GH48家族的纤维素酶,基因组中还含有AA10家族的多糖裂解氧化酶,因此放线菌可通过持续性水解与氧化双重机制高效降解结晶纤维素.放线菌可通过双精氨酸转运系统快速将已正确折叠的降解酶类分泌至胞外,这些酶分子常具有多个功能结构域,具有耐高温、耐碱性以及高活力等特征.放线菌在木质纤维素降解及次级代谢产物等方面的特点与优势使得其具有巨大的工业应用前景.     

天麻发育过程中蛋白质、脂类及酶的组织化学定位初步观察

天麻胚内含有丰富的脂肪及总蛋白质,少量的碱性蛋白质,胚体萌动时还显示出明显的多酚氧化酶,酯酶及ATP酶的活性。在营养生长期,顶端分生组织,叶原基,维管束薄壁细胞,外皮层细胞以及大型维管柱细胞内含有丰富的总蛋白质,其分布与贮存多糖的分布相反。脂肪及碱性蛋白含量不明显。顶端分生组织,表皮层及维管束薄壁细胞内呈现出酸性磷酸酶,酯酶,ATP酶,多酚氧化酶及过氧化物酶较高的活性。开花期,球茎内这几种酶的活性均较高,在花序轴内ATP酶及过氧化物酶的活性比水解酶类的活性稍高些。球茎中央薄壁组织细胞由于贮存的多糖和蛋白质被逐渐消耗而形成空腔。果实成熟后,球茎内贮存的营养物质耗尽,酶类的活性显著下降。     

海藻工具酶——褐藻胶裂解酶研究进展

从海洋生物中筛选提取有价值的酶类,开发海洋多糖降解产物,已成为海洋生物资源开发的一个重要方面。因此,近年来对于海藻工具酶之一的褐藻胶裂解酶及其降解产物——褐藻寡糖的研究日益受到人们的普遍关注。从褐藻胶裂解酶的来源、分类、底物专一性、作用方式及结构与机理研究、酶活力测定和酶学性质等方面,结合本课题组的研究工作综述近十年来有关褐藻胶裂解酶的研究进展。     

氮沉降增加对森林凋落物分解酶活性的影响

氮沉降增加对森林凋落物分解酶产生的影响在世界范围受到关注。综述了凋落物分解酶的种类、影响酶的因素、酶的生态学意义和土壤酶研究技术的研究发展趋势。根据森林凋落物底物性质的不同,将凋落物分解酶分为纤维素分解酶类、木质素分解酶类、蛋白水解酶类和磷酸酶类。目前普遍认为,氮沉降增加,磷酸酶类活性随之增加,其它三类酶活性未呈现规律性变化。此外,还对氮沉降增加与土壤酶之间关系的研究前景进行了探讨。     

脂肪酶的表面展示及其应用

脂肪酶是一种广泛应用的水解酶类。脂肪酶的表面展示技术不仅是脂肪酶蛋白质工程中一种有效的高通量筛选方法,而且展示的脂肪酶与自由酶相比具备更高的温度稳定性、有机溶剂稳定性等优点,其作为全细胞催化剂与传统的固定化脂肪酶相比也具备诸多优点。脂肪酶表面展示的宿主包括噬菌体、细菌以及酵母等,本文将分别介绍这三种宿主中脂肪酶表面展示的概况以及其作为高通量筛选和全细胞等方面的应用。     

环状芽孢杆菌几丁质酶基因序列分析、表达和生物活性测定

真菌病害一直是影响作物的主要病害之一 ,每年造成巨大经济损失。几丁质酶可水解许多病原真菌细胞壁所含有的主要成分—几丁质 ,是研究得最多的抗真菌蛋白质。许多几丁质酶基因已从微生物中克隆到 ,芽孢杆菌是一类重要的几丁质酶产生菌。环状芽孢杆菌可产生并分泌多种多糖降解酶类 ,包括几丁质酶、β 1 ,3 葡聚糖酶、β 1 ,6葡聚糖酶和半纤维素酶[1] 。Ｗａｔａｎａｂｅ克隆了环状芽孢杆菌ＷＬ 1 2菌株的几丁质酶基因ｃｈｉＡ和ｃｈｉＤ ,对该几丁质酶基因的结构和功能进行了深入研究[2～ 4 ] 。我国的陈三凤克隆了黄杆菌的几丁质酶基因 ,…     

脂肪酶表面展示技术

脂肪酶是一种广泛应用的水解酶类.脂肪酶的表面展示技术不仅是脂肪酶蛋白质工程中一种有效的高通量筛选方法,而且展示的脂肪酶与自由酶相比具备更高的温度稳定性、有机溶剂稳定性等优点,其作为全细胞催化剂与传统的固定化脂肪酶相比也具备诸多优点.脂肪酶表面展示的宿主包括噬菌体、细菌以及酵母等,将分别介绍这三种宿主中脂肪酶表面展示的概况及其作为高通量筛选和全细胞等方面的应用.     

果胶分解菌的简便筛选方法

果胶醇是分解果胶的一类酶的总称,被广泛应用于食品工业中,也可用于麻类脱胶及木材防腐等。能产生果胶酶的微生物种类很多,果胶分解菌的筛选方法已有一些报道,如透明带法、十六烷基三甲基澳化铁（多糖沉淀剂）的透明圈法、钉红染色的透明圈法。这些方法或者效果不明显,或者果胶用量较大,或者成本较高,在实际工作中受到限制。刚果红染料可与多糖水解物形成有浓郁色泽的复合物［7］,因此Hen－driCkS等人及叶姜瑜分别利用刚果红对纤维素分解菌进行筛选和计数阳。我们经过试验,发现刚果红也可用于果胶分解菌的筛选和计数。1果胶琼脂…     

Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates

A plate assay based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of different dye-labelled polysaccharides as substrates, provides a specific, reliable and rapid simultaneous detection of corresponding polysaccharide-degrading microorganisms. It has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, in large numbers of natural samples. Diversely colored insoluble forms of amylose, xylan and hydroxyethyl-cellulose (HE-cellulose) were prepared as chromogenic substrates by using the cross-linking reagent 1,4-butanediol diglycidyl ether and the dyes Brilliant Red 3B-A, Cibacron Blue 3GA and Reactive Orange 14. Using the method, the bacteria with amylase or xylanase or cellulase or a combination of these activities were screened from soil and sludge samples, selected and identified according to 16S rDNA sequencing.     

β-Glucuronidase-coupled assays of glucuronoyl esterases

Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries.     

Plate screening methods for the detection of polysaccharase-producing microorganisms

Polysaccharide-degrading enzymes (polysaccharases) are widely applied in industry. One of the sources of these enzymes are polysaccharide-degrading microorganisms. To obtain such microorganisms from enrichment cultures, strain collections or gene libraries, efficient plate screening methods are required that discriminate between intact and degraded polysaccharide. This can be achieved by making use of specific physicochemical properties of the polysaccharide, such as complex formation with dyes and gelling capacity, or by the application of dye-labelled polysaccharides. This review presents a survey of plate methods based on these principles. Both theoretical and practical aspects of the methods are discussed.     

A medium-throughput screening assay to determine catalytic activities of oxygen-consuming enzymes: a new tool for functional characterization of cytochrome P450 and other oxygenases

A challenge of the post-genomic era is to determine the functions of a plethora of orphan genes. This is a more acute problem when dealing with large gene families, such as the superfamily encoding cytochrome P450 enzymes in higher plants. We propose here a new, simple, medium-throughput methodology to screen for potential substrates of orphan P450 mono-oxygenases. The same technique can also be applied to screening for inhibitors of the oxygenases involved in the biosynthesis of compounds essential for plant development, such as growth regulators. The method is based on a commercially available microplate system, which detects the oxygen consumed by the catalytic reaction via an oxygen-sensing fluorophore. It is optimized using as a model CYP73A1, the cinnamic acid hydroxylase from Helianthus tuberosus, expressed in yeast. We show that the procedure is suitable not only for the detection and real-time monitoring, but also for the quantitative evaluation of enzyme activity. This new method has broad application for the identification of candidate substrates and inhibitors in chemical libraries, to support determination of physiological substrates, development of plant growth regulators, investigations on herbicide and pollutant metabolism, synthesis of valuable compounds and drug design. It also provides a fast-assay platform for determination of catalytic and inhibition parameters. The method applies to plant P450 enzymes, but also to cytochromes P450 from other organisms, and all types of oxygenases. The critical steps, calculation of oxygen consumption from fluorescence signal, and limits of the methods are discussed.     

Microbial beta-glucanases different from cellulases

The beta-glucans different from cellulose are the most abundant class of polysaccharides. They are found in microorganisms and higher plants as structural entities of cell wall, as cytoplasmic and vacuolar reserve materials, and as extracellular substances. Enzyme systems capable to hydrolyze beta-glucans are produced by different microorganisms. The occurrence and nature of beta-glucanases and their substrates are reviewed. The regulation of biosynthesis of these enzymes, their properties, substrate and product specificities, mode of action and molecular cloning are described. The participation of beta-glucanases in the morphogenetic events of yeast cell is presented. The role and synergism of different types of 1,3-beta-glucanases in microbial cell wall lysis and the potential application for isolation of intracellular materials like proteins, carbohydrates, enzymes and as an analytical tool are discussed in the light of current knowledge.     

伊红、台盼蓝检测河蟹精子存活率的比较

对台盼蓝和伊红染色法检测河蟹(Eriocheir sinensis)精子存活率的方法进行了评价研究。结果表明,两种染色法死、活精子分别呈现出明显不同的染色特征:活精子无色透明,顶体中央凸起呈圆锥状,光镜下辐射臂及细胞边界清晰;死亡精子顶体着色,且中央有一染色较深的圆斑,核杯染色不明显,细胞体积变大,边界模糊。通过不同染色时间和不同染料浓度的比较发现,两种染色法最适染液浓度分别是0·25%的伊红和0·5%的台盼蓝,染色时间均以15min为佳。在此基础上,将新鲜精子和60℃水浴处理致死精子以不同的体积比混合,配成含致死精子比例为10%～90%的9个梯度样品,用伊红和台盼蓝分别测定各样品精子死亡率,并进行相关性分析。结果发现,各样品实测精子死亡率均略高于样品的理论死亡率,同时两种染色法实测值与样品理论值呈显著正相关(P<0·05),两种染色法之间亦呈显著正相关(P<0·05)。上述结果表明,伊红和台盼蓝可用于河蟹精子的活体染色,且两种染色法在对河蟹精子染色中具有一定的稳定性和可比性。     

A systematic approach for yielding a potential pool of enzymes: practical case for chiral resolution of (R,S)-ketoprofen ethyl ester

A systematic approach for the selection of potential biocatalysts from a natural source was developed and then a practical application was addressed. The approach that involves systematically combined conventional screening methods and current tools comprises the following consecutive steps: strain enrichment for activity screening, identification of positive strains, choosing whole genome-sequenced strains as candidates, gathering information about responsible enzymes, bioinformatic analyses and gene mining, probing genetic molecules and then functional expression. The target compound (R,S)-ketoprofen ethyl ester was to be resolved into an enantiomer, and a potential esterase from Pseudomonas fluorescens KCTC 1767 was prepared by the proposed procedure. The enzyme had a high activity and also strict selectivity for the enantiomer (S)-ketoprofen and was suitable therefore as a biocatalyst for practical use. The result achieved by the combined approach could not easily be obtained using other approaches with typical procedures. Hence the approach proposed here should be of considerable use for the screening of potential enzymes, particularly for enzymes with desired activity to unnatural substrates, from conditionally expressed and/or repressed proteins that are distributed widely in natural pools under normal conditions.     

Strains degrading polysaccharides produced by bacteria from paper machines

Biofilm-degrading enzymes are potential agents for slime control in paper machines. In this work, extracellular polysaccharides were produced by bacteria isolated from paper machines and the isolated polysaccharides were used as substrates for the screening of polysaccharide-degrading microbes. Polysaccharide yields of 1.5-3.5 g/l were obtained by ethanol precipitation from cultures of strains of Klebsiella pneumoniae, Bacillus licheniformis and Pseudomonas fluorescens on sucrose medium. Two K. pneumoniae strains apparently produced an identical heteropolysaccharide containing galacturonic acid. Fructose-containing polysaccharides were the main products of B. licheniformis and P. fluorescens. Bacteria capable of hydrolyzing the fructose-containing polymers (levans) appeared to be relatively common among the strains selected for screening. None of the bacteria or mixed cultures screened were able to utilize the Klebsiella heteropolysaccharides.     

The Tetracycline Fermentation and Its Regulation

Abstract

The β-glucans different from cellulose are the most abundant class of polysaccharides. They are found in microorganisms and higher plants as structural entities of cell wall, as cytoplasmic and vacuolar reserve materials, and as extracellular substances. Enzyme systems capable to hydrolyze β-glucans are produced by different microorganisms. The occurrence and nature of β-glucanases and their substrates are reviewed. The regulation of biosynthesis of these enzymes, their properties, substrate and product specificities, mode of action and molecular cloning are described. The participation of β-glucanases in the morphogenetic events of yeast cell is presented. The role and synergism of different types of 1,3-β-glucanases in microbial cell wall lysis and the potential application for isolation of intracellular materials like proteins, carbohydrates, enzymes and as an analytical tool are discussed in the light of current knowledge.     

Screening and characterization of microorganisms with glutaryl-7 ADCA acylase activity

A screening of microorganisms producing glutaryl-7 ADCA acylase, an enzyme able to hydrolyse glutaric acid selectively from glutaryl-3-deacetoxy-7-aminocephalosporanic acid (glutaryl-7 ADCA), has been carried out in soil samples. Five microorganisms expressing acylase activity were isolated and classified as Bacillus cereus, Achromobacter xylosooxidans, Bacillus sp., Pseudomonas sp. and Pseudomonas paucimobilis. The screening was carried out by preparing enrichment cultures containing glutaryl-7-ADCA or cephalosporin C as the selective carbon source. Four model compounds (adipoyl-, glutamyl- and glutaryl-p-nitroanilide and glutarylcoumarin), mimicking the glutaryl-7 ADCA -lactam moiety, were synthesized as substrates suitable for the rapid screening of the microorganisms (2500) isolated from the enrichment cultures. A total of 300 strains were active on the model substrates and only 5 displayed acylase activity on glutaryl-7 ADCA. The fermentation parameters, such as pH and inducer concentration, for the optimal acylase expression and acylase specificity towards the model substrates were different for each strain.