MELDB: a database for microbial esterases and lipases

MELDB is a comprehensive protein database of microbial esterases and lipases which are hydrolytic enzymes important in the modern industry. Proteins in MELDB are clustered into groups according to their sequence similarities based on a local pairwise alignment algorithm and a graph clustering algorithm (TribeMCL). This differs from traditional approaches that use global pairwise alignment and joining methods. Our procedure was able to reduce the noise caused by dubious alignment in the distantly related or unrelated regions in the sequences. In the database, 883 esterase and lipase sequences derived from microbial sources are deposited and conserved parts of each protein are identified. HMM profiles of each cluster were generated to classify unknown sequences. Contents of the database can be keyword-searched and query sequences can be aligned to sequence profiles and sequences themselves.     

Dioxygenase- and monooxygenase-catalysed synthesis of <Emphasis Type="Italic">cis</Emphasis>-dihydrodiols,catechols, epoxides and other oxygenated products

Oxidoreductases are an emerging class of biotechnologically relevant enzymes due to their regio- and stereo-specificity. The selective oxygenation of aromatic compounds by oxidoreductases has received much attention and a wide range of reactions have been documented using these enzymes from various microbial sources. This review gives an overview of various dioxygenase, monooxygenase and oxidase enzymes that have been manipulated for the synthesis of products such as cis-dihydrodiols, catechols, epoxides and other oxygenated products. The use of protein engineering and its advancement in the synthesis of recombinant enzymes is also discussed.     

Improvement of thermostable aldehyde dehydrogenase by directed evolution for application in Synthetic Cascade Biomanufacturing

The aldehyde dehydrogenase from Thermoplasma acidophilum, which was previously implemented as a key enzyme in a synthetic cell-free reaction cascade for the production of alcohols, was optimized by directed evolution. Improvements have been made to enhance reaction velocity and solubility. Using a random approach followed by site-directed and saturation mutagenesis, three beneficial amino acid mutations were found after screening of ca. 20,000 variants. Mutation Y399C enhanced the protein solubility after recombinant expression in Escherichia coli 6-fold. Two further mutations, F34M and S405N, enhanced enzyme activity with the cofactor NAD⁺ by a factor of eight. Impacts on enzyme stability and substrate specificity were negligible.     

Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches

Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are ubiquitous enzymes that catalyze the breakdown of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoglycerols and glycerol. Besides this, they are also efficient in various reactions such as esterification, transesterification and aminolysis in organic solvents. Therefore, those enzymes are nowadays extensively studied for their potential industrial applications. Examples in the literature are numerous concerning their use in different fields such as resolution of racemic mixtures, synthesis of new surfactants and pharmaceuticals, oils and fats bioconversion and detergency applications. However, the drawbacks of the extensive use of lipases (and biocatalysts in general) compared to classical chemical catalysts can be found in the relatively low stability of enzyme in their native state as well as their prohibitive cost. Consequently, there is a great interest in methods trying to develop competitive biocatalysts for industrial applications by improvement of their catalytic properties such as activity, stability (pH or temperature range) or recycling capacity. Such improvement can be carried out by chemical, physical or genetical modifications of the native enzyme. The present review will survey the different procedures that have been developed to enhance the properties of lipases. It will first focus on the physical modifications of the biocatalysts by adsorption on a carrier material, entrapment or microencapsulation. Chemical modifications and methods such as modification of amino acids residues, covalent coupling to a water-insoluble material, or formation of cross-linked lipase matrix, will also be reviewed. Finally, new and promising methods of lipases modifications by genetic engineering will be discussed.     

Enzymatic and whole cell catalysis: Finding new strategies for old processes

The use of enzymes and whole bacterial cells has allowed the production of a plethora of compounds that have been used for centuries in foods and beverages. However, only recently we have been able to master techniques that allow the design and development of new biocatalysts with high stability and productivity. Rational redesign and directed evolution have lead to engineered enzymes with new characteristics whilst the understanding of adaptation mechanisms in bacterial cells has allowed their use under new operational conditions. Bacteria able to thrive under the most extreme conditions have also provided new and extraordinary catalytic processes. In this review, the new tools available for the improvement of biocatalysts are presented and discussed.     

Biocatalysis in ionic liquids for lignin valorization: Opportunities and recent developments

Lignin holds tremendous potential as a renewable feedstock for upgrading to a number of high-value chemicals and products that are derived from the petroleum industry at present. Since lignin makes up a significant fraction of lignocellulosic biomass, co-utilization of lignin in addition to cellulose and hemicelluloses is vital to the economic viability of cellulosic biorefineries. The recalcitrant nature of lignin, originated from the molecule's compositional and structural heterogeneity, however, poses great challenges toward effective and selective lignin depolymerization and valorization. Ionic liquid (IL) is a powerful solvent that has demonstrated high efficiency in fractionating lignocellulosic biomass into sugar streams and a lignin stream of reduced molecular weight. Compared to thermochemical methods, biological lignin deconstruction takes place at mild temperature and pressure while product selectivity can be potentially improved via the specificity of biocatalysts (lignin degrading enzymes, LDEs). This review focuses on a lignin valorization strategy by harnessing the biomass fractionating capabilities of ILs and the substrate and product selectivity of LDEs. Recent advances in elucidating enzyme-IL interactions as well as strategies for improving enzyme activity in IL are discussed, with specific emphases on biocompatible ILs, thermostable and IL-tolerant enzymes, enzyme immobilization, and surface charge engineering. Also reviewed is the protein engineering toolsets (directed evolution and rational design) to improve the biocatalysts' activity, stability and product selectivity in IL systems. The alliance between IL and LDEs offers a great opportunity for developing a biocatalytic route for lignin valorization.     

Directed evolution of aldolases for exploitation in synthetic organic chemistry

This review focuses on the directed evolution of aldolases with synthetically useful properties. Directed evolution has been used to address a number of limitations associated with the use of wild-type aldolases as catalysts in synthetic organic chemistry. The generation of aldolase enzymes with a modified or expanded substrate repertoire is described. Particular emphasis is placed on the directed evolution of aldolases with modified stereochemical properties: such enzymes can be useful catalysts in the stereoselective synthesis of biologically active small molecules. The review also describes some of the fundamental insights into mechanistic enzymology that directed evolution can provide.     

微生物肥料及其生产应用中的问题

微生物肥料和微生物制剂是新世纪实行绿色农业的重要技术保障,关键是因其中含有大量的有效微生物的生命活动产生特定的肥效或其他生理功能,导致增产,微生物肥料包括根瘤菌肥,解磷菌肥、解钾菌肥、5406菌肥,植物根际促生菌,VA菌根等等,有机肥料堆制剂中含有多种降解农业有机废料的菌种,可缩短堆肥周期,提高养份利用率,由于微生物本身的特殊性,相关市场监督机制不健全,微生物肥料的生产,应用领域存在很多问题,甚至出现明显的伪科学,科研、生产、行政各有关部门应加强基础研究,提高全民科普水平,健全市场监督机制,依靠专家设计严格试验,优选可靠技术,保证良好的经济效益和社会效益。     

Microbial pectate lyases: characterization and enzymological properties

Pectate lyase plays an important role in plant pathogenesis. The enzyme is widely distributed in diverse families of microorganisms. The current knowledge including biochemical studies on microbial pectate lyases, such as isozymes, structure, reaction mechanism, purification and properties like molecular mass, pI, optimum pH and temperature, substrate specificity, metal ion requirement, inhibitors and activators, and kinetic parameters of the enzyme are reviewed.     

Microbial molecular diversity: Past and present Thom Award lecture

Journal of Industrial Microbiology &; Biotechnology -     

酶的定向进化研究及其在工业生物催化中的应用

综合概括了各种蛋白质改造与微生物代谢途径改造的非理性定向进化技术的原理、特点,介绍了这些技术在高性能工业生物催化剂改造中的应用。     

东亚飞蝗山西两地理种群酯酶特性的比较研究

对东亚飞蝗山西临猗和永济2个地理种群的酯酶特性进行了比较研究.非变性聚丙烯酰胺凝胶电泳图谱显示:以a-乙酸萘酯为底物染色,2个东亚飞蝗种群谱带差别不明显.但是,酯酶动力学研究结果表明:以a-乙酸萘酯和α-丁酸萘酯为底物时,永济种群的酯酶活性分别是临猗种群的1.81倍和1.20倍.永济种群酯酶活性的增高可能与非变性聚丙烯酰胺凝胶电泳图谱显示出较临猗种群多出的酶带有关.体外酯酶抑制动力学研究表明:永济和临猗2种群所含酯酶大都为B型酯酶,其含量分别为84.94%和91.47%.永济种群对对氧磷的耐受性要高于临猗种群,我们推测可能与2种群马拉硫磷使用背景不同有关.     

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method. Joaquin Sanchis, Layla Fernández, and J. Daniel Carballeira contributed equally.     

Microbial Biomass, N Mineralization and Nitrification, Enzyme Activities, and Microbial Community Diversity in Tea Orchard Soils

Understanding the chronological changes in soil microbial and biochemical properties of tea orchard ecosystems after wasteland has been reclaimed is important from ecological, environmental, and management perspectives. In this study, we determined microbial biomass, net N mineralization, and nitrification, enzyme (invertase, urease, proteinase, and acid phosphatase) activities, microbial community diversity assessed by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA polymerase chain reaction (PCR) products, and related ecological factors in three tea orchard systems (8-, 50-, and 90-year-old tea orchards), adjacent wasteland and 90-year-old forest. Soil microbial biomass C (C_mic) and activity, i.e., soil basal respiration (R_mic), microbial biomass C as a percent of soil organic C (C_mic/C_org), N mineralization, invertase, urease, proteinase, and acid phosphatase, significantly increased after wasteland was reclaimed; however, with the succeeding development of tea orchard ecosystems, a decreasing trend from the 50- to 90-year-old tea orchard became apparent. Soil net nitrification showed an increasing trend from the 8- to 50-year-old tea orchard and then a decreasing trend from the 50- to 90-year-old tea orchard, and was significantly higher in the tea orchards compared to the wasteland and forest. Urea application significantly stimulated soil net nitrification, indicating nitrogen fertilizer application may be an important factor leading to high-nitrification rates in tea orchard soils. The Shannon’s diversity index (H) and richness (S) based on DGGE profiles of 16S rRNA genes were obviously lower in all three tea orchards than those in the wasteland; nevertheless, they were significantly higher in all three tea orchards than those in the forest. As for the three tea orchard soils, comparatively higher community diversity was found in the 50-year-old tea orchard.     

Controls on Microbial Production of Methane and Carbon Dioxide in Three Sphagnum-Dominated Peatland Ecosystems as Revealed by a Reciprocal Field Peat Transplant Experiment

We examined controls on mineralization of carbon to methane (CH₄) and carbon dioxide (CO₂) in Sphagnum (moss)-dominated peatland ecosystems by transplanting surface (5 cm deep) and subsurface (40 cm deep) peat samples reciprocally among three sites for periods ranging from 4 to 25 months. The sites were Big Run Bog in West Virginia, USA, Bog Lake Bog in Minnesota, USA, and Bog 307 in Ontario, Canada. Immediately upon retrieval, we incubated the peat samples in the laboratory at 12 and 22°C under both anoxic and oxic conditions to estimate rates of carbon mineralization. Transplanting affected surface peat more than subsurface peat. Peat incubated within Bog Lake Bog in Minnesota had the highest rates of CH₄ production, regardless of origin, whereas transplanting did not affect rates of CO₂ production measured concomitantly. Peat that originated in Big Run Bog in West Virginia generally maintained higher rates of CH₄ production and CO₂ production than peat from the other two sites after incubation in the field. The temperature dependence (Q ₁₀) of CH₄ production and CO₂ production varied among transplant sites, but not among peat origins, suggesting physiological adaptations of microbial communities to local environmental conditions. Differences in organic matter quality of the peat, particularly lignin chemistry, helped explain the results: (a) CH₄ production correlated with fresher lignin derived from Carex sedges, and (b) CO₂ production correlated with woody lignin. We concluded that, although both site conditions (climate, nutrient status, and microbial communities) and organic matter quality influence carbon mineralization in peat, interactive effects occur and may differ depending on peat temperature. Moreover, CH₄ production and CO₂ production respond differently to environmental regulators.     

Recent advances in discovery,heterologous expression,and molecular engineering of cyclodextrin glycosyltransferase for versatile applications

Cyclodextrin glycosyltransferase (CGTase) is an important enzyme with multiple functions, in particular the production of cyclodextrins. It is also widely applied in baking and carbohydrate glycosylation because it participates in various types of catalytic reactions. New applications are being found with novel CGTases being isolated from various organisms. Heterologous expression is performed for the overproduction of CGTases to meet the requirements of these applications. In addition, various directed evolution techniques have been applied to modify the molecular structure of CGTase for improved performance in industrial applications. In recent years, substantial progress has been made in the heterologous expression and molecular engineering of CGTases. In this review, we systematically summarize the heterologous expression strategies used for enhancing the production of CGTases. We also outline and discuss the molecular engineering approaches used to improve the production, secretion, and properties (e.g., product and substrate specificity, catalytic efficiency, and thermal stability) of CGTase.     

Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase,an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate,is not involved in floral scent production in snapdragon flowers

Using a functional genomic approach we have isolated and characterized a cDNA that encodes a salicylic acid carboxyl methyltransferase (SAMT) from Antirrhinum majus. The sequence of the protein encoded by SAMT has higher amino acid identity to Clarkia breweri SAMT than to snapdragon benzoic acid carboxyl methyltransferase (BAMT) (55 and 40% amino acid identity, respectively). Escherichia coli-expressed SAMT protein catalyzes the formation of the volatile ester methyl salicylate from salicylic acid with a K(m) value of 83 microM. It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM. Snapdragon flowers do not emit methyl salicylate. The potential involvement of SAMT in production and emission of methyl benzoate in snapdragon flowers was analyzed by RNA gel blot analysis. SAMT mRNA was not detected in floral tissues by RNA blot hybridization, but low levels of SAMT gene expression were detected after real-time RT-PCR in the presence of SAMT-specific primers, indicating that this gene does not contribute significantly, if at all, in methyl benzoate production and emission in snapdragon flowers. Expression of SAMT in petal tissue was found to be induced by salicylic and jasmonic acid treatments.     

Immobilization of Candida cylindracea lipase on poly lactic acid,polyvinyl alcohol and chitosan based ternary blend film: Characterization,activity, stability and its application for N-acylation reactions

The ecofriendly ternary blend polymer film was prepared from the chitosan (CH), polylactic acid (PLA) and polyvinyl alcohol (PVA). Immobilization of Candida cylindracea lipase (CCL) was carried out on ternary blend polymer via entrapment methodology. The ternary blend polymer and immobilized biocatalyst were characterized by using N₂ adsorption–desorption isotherm, SEM, FTIR, DSC, and (%) water content analysis through Karl Fischer technique. Biocatalyst was then subjected for the determination of practical immobilization yield, protein loading and specific activity. Immobilized biocatalyst was further applied for the determination of biocatalytic activity for N-acylation reactions. Various reaction parameters were studied such as effect of immobilization support (ratio of PLA:PVA:CH), molar ratio (dibutylamine:vinyl acetate), solvent, biocatalyst loading, time, temperature, and orbital speed rotation. The developed protocol was then applied for the N-acylation reactions to synthesize several industrially important acetamides with excellent yields. Interestingly, immobilized lipase showed fivefold higher catalytic activity and better thermal stability than the crude extract lipase CCL. Furthermore various kinetic and thermodynamic parameters were studied and the biocatalyst was efficiently recycled for four successive reuses. It is noteworthy to mention that immobilized biocatalyst was stable for period of 300 days.     

Distribution and probable physiological role of esterases in reproductive,digestive, and fat-body tissues of the adult cotton boll weevil,Anthonomus grandis Boh

Polyacrylamide gel electrophoresis was used to examine gut, Malpighian tube, fat-body, testes, and ovariole tissues of the adult cotton boll weevil, Anthonomus grandis Boh. Esterases for which the inheritance has been reported previously by Terranova using whole-body homogenates were detected in dissected tissues and the probable physiological function of each allozyme is suggested. EST-1 occurs most frequently in ovarioles and female fat bodies. EST-2 is most often found in fat bodies and may be important in lipid turnover. No sex difference was observed. EST-3^S is found in fat bodies and reproductive tissue, while EST-3^F is always located in gut tissues, indicating that EST-3 is not controlled by a single autosomal locus with two codominant alleles as previously reported. EST-4, the most abundant esterase, can be detected in gut tissue at any age and is probably involved in digestion. EST-5 contains four allozymes which appear most frequently in testes and may be important during reproduction.