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.     

Glucuronoyl esterases: diversity,properties and biotechnological potential. A review

Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 (CE15) are involved in microbial degradation of lignocellulosic plant materials. GEs are capable of degrading complex polymers of lignin and hemicellulose cleaving ester bonds between glucuronic acid residues in xylan and lignin alcohols. GEs promote separation of lignin, hemicellulose and cellulose which is crucial for efficient utilization of biomass as an energy source and feedstock for further processing into products or chemicals. Genes encoding GEs are found in both fungi and bacteria, but, so far, bacterial GEs are essentially unexplored, and despite being discovered >10?years ago, only a limited number of GEs have been characterized. The first laboratory scale example of improved xylose and glucuronic acid release by the synergistic action of GE with cellulolytic enzymes was only reported recently (improved C5 sugar and glucuronic acid yields) and, until now, not much is known about their biotechnology potential. In this review, we discuss the diversity, structure and properties of microbial GEs and consider the status of their action on natural substrates and in biological systems in relation to their future industrial use.     

Phylogeny,classification and metagenomic bioprospecting of microbial acetyl xylan esterases

Acetyl xylan esterases (AcXEs), also termed xylan deacetylases, are broad specificity Carbohydrate-Active Enzymes (CAZymes) that hydrolyse ester bonds to liberate acetic acid from acetylated hemicellulose (typically polymeric xylan and xylooligosaccharides). They belong to eight families within the Carbohydrate Esterase (CE) class of the CAZy database. AcXE classification is largely based on sequence-dependent phylogenetic relationships, supported in some instances with substrate specificity data. However, some sequence-based predictions of AcXE-encoding gene identity have proved to be functionally incorrect. Such ambiguities can lead to mis-assignment of genes and enzymes during sequence data-mining, reinforcing the necessity for the experimental confirmation of the functional properties of putative AcXE-encoding gene products.Although one-third of all characterized CEs within CAZy families 1⿿7 and 16 are AcXEs, there is a need to expand the sequence database in order to strengthen the link between AcXE gene sequence and specificity. Currently, most AcXEs are derived from a limited range of (mostly microbial) sources and have been identified via culture-based bioprospecting methods, restricting current knowledge of AcXEs to data from relatively few microbial species. More recently, the successful identification of AcXEs via genome and metagenome mining has emphasised the huge potential of culture-independent bioprospecting strategies. We note, however, that the functional metagenomics approach is still hampered by screening bottlenecks.The most relevant recent reviews of AcXEs have focused primarily on the biochemical and functional properties of these enzymes. In this review, we focus on AcXE phylogeny, classification and the future of metagenomic bioprospecting for novel AcXEs.     

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.     

Genome-wide identification,classification, and expression profiling of serine esterases and other esterase-related proteins in the tobacco hornworm,Manduca sexta

Serine esterases (SEs) are hydrolases that catalyze the conversion of carboxylic esters into acids and alcohols. Lipases and carboxylesterases constitute two major groups of SEs. Although over a hundred of insect genomes are known, systematic identification and classification of SEs are rarely performed, likely due to large size and complex composition of the gene family in each species. Considering their key roles in lipid metabolism and other physiological processes, we have categorized 144 M. sexta SEs and SE homologs (SEHs), 114 of which contain a motif of GXSXG. Multiple sequence alignment and phylogenetic tree analysis have revealed 39 neutral lipases (NLs), 3 neutral lipase homologs (NLHs), 11 acidic lipases (ALs), 3 acidic lipase homologs (ALHs), a lipase-3, a triglyceride lipase, a monoglyceride lipase, a hormone-sensitive lipase, and a GDSL lipase. Eighty-three carboxylesterase genes encode 29 α-esterases (AEs), 12 AEHs (e.g., SEH4-1–3), 20 feruloyl esterases (FEs), 2 FEHs, 2 β-esterases (BEs), 2 integument esterases (IEs), 1 IEH, 4 juvenile hormone esterases, 2 acetylcholinesterases, gliotactin, 6 neuroligins, neurotactin, and an uncharacteristic esterase homolog. In addition to these GXSXG proteins, we have identified 26 phospholipases and 13 thioesterases. Expression profiling of these genes in specific tissues and stages has provided insights into their functions including digestion, detoxification, hormone processing, neurotransmission, reproduction, and developmental regulation. In summary, we have established a framework of information on SEs and related proteins in M. sexta to stimulate their research in the model species and comparative investigations in agricultural pests or disease vectors.     

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 molecular diversity: Past and present Thom Award lecture

Journal of Industrial Microbiology &; Biotechnology -     

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.     

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。     

Systems for in vivo hypermutation: a quest for scale and depth in directed evolution

Traditional approaches to the directed evolution of genes of interest (GOIs) place constraints on the scale of experimentation and depth of evolutionary search reasonably achieved. Engineered genetic systems that dramatically elevate the mutation of target GOIs in vivo relieve these constraints by enabling continuous evolution, affording new strategies in the exploration of sequence space and fitness landscapes for GOIs. We describe various in vivo hypermutation systems for continuous evolution, discuss how different architectures for in vivo hypermutation facilitate evolutionary search scale and depth in their application to problems in protein evolution and engineering, and outline future opportunities for the field.     

Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions

Propionyl-CoA carboxylase (PCC) is a promising enzyme in the fields of biological CO₂ utilization, synthesis of natrual products, and so on. The activity and substrate specificity of PCC are dependent on its key subunit carboxyltransferase (CT). To obtain PCC with high enzyme activity, seven pccB genes encoding CT subunit from diverse microorganisms were expressed in recombinant E. coli, and PccB from Bacillus subtilis showed the highest activity in vitro. To further optimize this protein using directed evolution, a genetic screening system based on oxaloacetate availability was designed to enrich the active variants of PccB_Bs. Four amino acid substitutions (D46G, L97Q, N220I and I391T) proved of great assistance in PccB_Bs activity improvement, and a double mutant of PccB_Bs (N220I/I391T) showed a 94-fold increase of overall catalytic efficiency indicated by k_cat/K_m. Moreover, this PccB_Bs double mutant was applied in construction of new succinate biosynthetic pathway. This new pathway produces succinate from acetyl-CoA with fixation of two CO₂ molecules, which was confirmed by isotope labeling experiment with NaH¹³CO₃. Compared with previous succinate production based on carboxylation of phosphoenolpyruvate or pyruvate, this new pathway showed some advantages including higher CO₂ fixation potentiality and availability under aerobic conditions. In summary, this study developed a PCC with high enzyme activity which can be widely used in biotechnology field, and also demonstrated the feasibility of new succinate biosynthetic pathway with two CO₂ fixation reactions.     

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α-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α-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。     

Burkholderia sp. IDO3中靛蓝合成基因的克隆表达及其合成特性

【目的】克隆和表达靛蓝合成基因,并将其用于靛蓝合成研究。【方法】对菌株Burkholderia sp.IDO3中靛蓝合成基因进行克隆和大肠杆菌异源表达,构建能合成蓝色色素的基因工程菌。利用液相色谱和质谱对产物进行分析,采用单因素法对培养温度、转速、培养基成分等进行优化,并考察优化条件下的靛蓝合成曲线。【结果】构建了一株重组大肠杆菌E.coli IND_AB,该菌株能够在LB培养基生长的过程中合成蓝色色素,产物分析表明该色素为靛蓝;菌株IND_AB在30°C和150 r/min条件下能在LB培养基中合成22.9 mg/L靛蓝,优化培养条件后产量达到25.4 mg/L;优化LB培养基各组分浓度后产量可提高到35.1 mg/L;外加50.0 mg/L吲哚或0.1 g/L色氨酸后靛蓝产量可分别提高到57.7 mg/L和64.4 mg/L,相比初始产量提高了152.0%和181.2%;靛蓝合成曲线表明在添加吲哚或色氨酸的培养基中,菌株IND_AB前6 h没有靛蓝生成,6-15 h为靛蓝合成加速期,18 h达到产量平衡。【结论】重组大肠杆菌IND_AB可用于生物合成高纯度靛蓝,为靛蓝的微生物合成提供了有效的基因资源。     

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.     

Kidney esterases of Mus musculus: Further polymorphism of esterase-6, esterase-9, and a new esterase,esterase-20

The comparison of results obtained by different separation and staining techniques permits the definition of esterase-6 in comparison with esterase-9 and a new esterase, esterase-20. Alleles of Es-6 affect the product's ability to aggregate. Esterase-20 may be an aggregated product of Es-9. The close linkage of Es-6 and Es-9 is confirmed. Homology of esterase-6 with esterases from other mammalian species is also suggested.HRN was supported by the Medical Research Council. This is communication No. 32 of a research program devoted to the cellular distribution, genetics, and regulation of nonspecific esterases.