Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis: recent insights and promises

Nitrile-converting enzymes, including nitrilase and nitrile hydratase (NHase), have received increasing attention from researchers of industrial biocatalysis because of their critical role as a tool in organic synthesis of carboxylic acids and amides from nitriles. To date, these bioconversion approaches are considered as one of the most potential industrial processes using resting cells or purified enzymes as catalysts for production of food additives, pharmaceutical, and agrochemical precursors. This review focuses on the distribution and catalytic mechanism research of nitrile-converting enzymes in recent years. Molecular biology aspects to improve the biocatalytic performance of microbial nitrilase and NHase are demonstrated. The process developments of microbial nitrilase and NHase for organic synthesis are also discussed.     

Biosynthesis of nicotinic acid from 3-cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150

In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0–12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50–55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid.     

Selection and screening for enzymes of nitrile metabolism

This work critically reviews the assays of nitrile-converting and nitrile-forming enzymes (nitrilases, nitrile hydratases, amidases, aldoxime dehydratases). Most of the strains producing such enzymes were obtained by selection on media with nitriles, amides or aldoximes as nitrogen sources. Activity and enantioselectivity of the enzymes was usually assayed by time-consuming chromatographic analysis of substrates and the corresponding reaction products. Attempts at introducing faster assays resulted in several spectrophotometric methods for reaction product (ammonia, hydroxamate, methacrylamide, benzamide, etc.) determination. Recently, new methods for colorimetric and fluorimetric determination of ammonia have been developed, which appear promising for high-throughput assays. Alternatively, methods consisting in determination of NADH consumed in a coupled amination reaction or pH-responsive methods are promising for this purpose. All the above selection and screening methods establish fundamental conditions for the design of hierarchical screening projects. However, the potential of these principles, in particular spectrophotometric and fluorimetric methods, will be probably further exploited and adapted to multiwell plate and robotic systems.     

Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase

Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase.     

Nitrile biotransformation by Aspergillus niger

A nitrile-converting enzyme activity was induced in Aspergillus niger K10 by 3-cyanopyridine. The whole cell biocatalyst was active at pH 3–11 and hydrolyzed the cyano group into acid and/or amide functions in benzonitrile as well as in its meta- and para-substituted derivatives, cyanopyridines, 2-phenylacetonitrile and thiophen-2-acetonitrile. Amides constituted a significant part of the total biotransformation products of 2- and 4-cyanopyridine, 4-chlorobenzonitrile, 4-tolunitrile and 1,4-dicyanobenzene, while -substituted acrylonitriles gave amides as the sole products.     

Catalysing new reactions during evolution: economy of residues and mechanism

The diversity of function in some enzyme superfamilies shows that during evolution, enzymes have evolved to catalyse different reactions on the same structure scaffold. In this analysis, we examine in detail how enzymes can modify their chemistry, through a comparison of the catalytic residues and mechanisms in 27 pairs of homologous enzymes of totally different functions. We find that evolution is very economical. Enzymes retain structurally conserved residues to aid catalysis, including residues that bind catalytic metal ions and modulate cofactor chemistry. We examine the conservation of residue type and residue function in these structurally conserved residue pairs. Additionally, enzymes often retain common mechanistic steps catalyzed by structurally conserved residues. We have examined these steps in the context of their overall reactions.     

细菌群体感应淬灭酶的研究进展

细菌的群体感应系统（Quorum sensing,QS）参与许多生物学功能的调控,其中包括动植物病原细菌致病因子的生成以及人类某些病原细菌生物膜的形成。酰基高丝氨酸内酯（N—acylhomoserine laetone,AHL）是调控群体感应系统的关键信号分子。近年的研究表明,不同生物体包括细菌和真核生物中都存在类别不同的能够降解AHL的群体感应淬灭酶（Quorum—quenching enzyme）。在AHL依赖型致病菌和转基因植物中表达AHL降解酶能有效地抑制QS信号分子的积累,从而阻断了病原细菌的发病机制,提高了植物的抗病性。这些新颖的群体感应淬灭酶的发现,不仅为防治细菌侵染提供了可行的途径,也对研究它们在宿主中的功能和对生态系统的潜在影响提出挑战。     

Molecular evolution of carotenoid biosynthesis from bacteria to plants

β-Carotene and derivatives are important pigments in plant photosynthesis. They are found not only in green plants but also accumulate in archea, prokaryotes and fungi. For β -carotene biosynthesis, enzymes are necessary to catalyse the formation of phytoene, several desaturation steps and cyclization reactions. This review is focused on the molecular phylogeny of the enzymes, the genes involved and their diversity. It outlines how genes and enzymes from prokaryotes and archea were modified to give rise to the corresponding plant constituents. In the cases of phytoene synthase, a direct line of evolution can be drawn. For other carotenogenic enzymes, new genes and enzymes have been acquired at certain stages of evolution. In addition, phytoene desaturases and lycopene cyclases are examples of convergent evolution of different types of enzymes, which are structurally completely unrelated but functionally identical. Finally, several gene duplications led to homologous enzymes with different catalytic functions including those involved in the synthesis of α -carotene.     

Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes.

The three classes of enzymes which inactivate aminoglycosides and lead to bacterial resistance are reviewed. DNA hybridization studies have shown that different genes can encode aminoglycoside-modifying enzymes with identical resistance profiles. Comparisons of the amino acid sequences of 49 aminoglycoside-modifying enzymes have revealed new insights into the evolution and relatedness of these proteins. A preliminary assessment of the amino acids which may be important in binding aminoglycosides was obtained from these data and from the results of mutational analysis of several of the genes encoding aminoglycoside-modifying enzymes. Recent studies have demonstrated that aminoglycoside resistance can emerge as a result of alterations in the regulation of normally quiescent cellular genes or as a result of acquiring genes which may have originated from aminoglycoside-producing organisms or from other resistant organisms. Dissemination of these genes is aided by a variety of genetic elements including integrons, transposons, and broad-host-range plasmids. As knowledge of the molecular structure of these enzymes increases, progress can be made in our understanding of how resistance to new aminoglycosides emerges.     

RNA helicases--one fold for many functions

RNA helicases are a large group of enzymes that function in virtually all aspects of RNA metabolism. Although RNA helicases share a highly conserved structure, different enzymes display a wide array of biochemical activities, including RNA duplex unwinding, protein displacement from RNA and strand annealing. Recent structural and functional studies have started to illuminate the mechanisms by which this remarkable diversity of functions can be conducted by the conserved helicase fold.     

Structural aspects of recently discovered viral deubiquitinating activities

Protein ubiquitination has been identified as a regulatory mechanism in key cellular activities, and deubiquitination is recognized as an important step in processes governed by ubiquitin and ubiquitin-like modifiers. Viruses are known to target ubiquitin and ubiquitin-like modifier pathways using various strategies, including the recruitment of host deubiquitinating enzymes. Deubiquitinating activities have recently been described for proteins from three different virus families (adenovirus, coronavirus and herpesvirus), and predicted for others. This review centers on structural-functional aspects that characterize the confirmed viral deubiquitinating enzymes, and their relationships to established families of cellular deubiquitinating enzymes.     

Starch-hydrolyzing enzymes from thermophilic archaea and bacteria

Extremophlic microorganisms have developed a variety of molecular strategies in order to survive in harsh conditions. For the utilization of natural polymeric substrates such as starch, a number of extremophiles, belonging to different taxonomic groups, produce amylolytic enzymes. This class of enzyme is important not only for the study of biocatalysis and protein stability at extreme conditions but also for the many biotechnological opportunities they offer. In this review, we report on the different molecular properties of thermostable archaeal and bacterial enzymes including alpha-amylase, alpha-glucosidase, glucoamylase, pullulanase, and cyclodextrin glycosyltransferase. Comparison of the primary sequence of the pyrococcal pullulanase with other members of the glucosyl hydrolase family revealed that significant differences are responsible for the mode of action of these enzymes.     

Isolation of Moderately Halophilic <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> Producing Extracellular Hydrolytic Enzymes from Persian Gulf

Extracellular hydrolytic enzymes such as amylases, proteases, lipases and DNases have quite diverse potential usages in different areas such as food industry, biomedical sciences and chemical industries, also it would be of great importance to have available enzymes showing optimal activities at different values of salt concentrations and temperature. Halophiles are the most likely source of such enzymes, because not only their enzymes are salt-tolerant, but many are also thermotolerant. The purpose of this study was isolation of hydrolytic extracellular enzyme producing halophilic bacteria from water and sediment of the Persian Gulf. Isolated bacteria from water and sediment were inoculated in media with concentration of 0–20% NaCl to determine the optimum salt concentration for growth, isolates were also inoculated in 4 types of solid medium containing substrates of 3 extracellular hydrolytic enzymes including amylase, Protease and Lipase, to determine the quantitative detection of enzyme production, selected strains after more accurate physiological and biochemical studies were identified regarding phylogeny and molecular characteristics using 16S rRNA technique. Isolated enzyme producing bacteria belong to Pseudoalteromonas genera.     

Accessing DNA damage in chromatin: Preparing the chromatin landscape for base excision repair

DNA damage in chromatin comes in many forms, including single base lesions that induce base excision repair (BER). We and others have shown that the structural location of DNA lesions within nucleosomes greatly influences their accessibility to repair enzymes. Indeed, a difference in the location of uracil as small as one-half turn of the DNA backbone on the histone surface can result in a 10-fold difference in the time course of its removal in vitro. In addition, the cell has evolved several interdependent processes capable of enhancing the accessibility of excision repair enzymes to DNA lesions in nucleosomes, including post-translational modification of histones, ATP-dependent chromatin remodeling and interchange of histone variants in nucleosomes. In this review, we focus on different factors that affect accessibility of BER enzymes to nucleosomal DNA.     

DNA polymerases β and λ and their roles in DNA replication and repair

DNA-dependent DNA polymerases are the main enzymes that catalyze DNA replication. Higher eukaryotic cells have 19 DNA polymerases with strikingly different properties [1]. Mitochondrial DNA polymerase γ of the A family and most of the nuclear enzymes of the B family are high-fidelity DNA polymerases that are involved not only in genomic DNA replication but also in DNA repair. Among the other 15 proteins, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are also involved in repair, including base excision repair and nonhomologous end joining. Some of them play a specific role in replication of damaged DNA templates. This process is referred to as translesion synthesis (TLS). DNA polymerases β and λ, which belong to the X structural family, are polyfunctional enzymes; their properties and functions are discussed.     

刀孢轮枝菌胞外几丁质酶的基因克隆及系统发育分析

食线虫真菌是植物寄生线虫的重要天敌,它们所产生的胞外水解酶(蛋白酶、几丁质酶和胶原蛋白酶等)能够降解线虫体壁和卵壳中的蛋白质及几丁质等结构成分并在侵染过程中发挥着重要的作用。本文中,我们发现刀孢轮枝菌Lecanicillium psalliotae对南方根结线虫Meloidogyne incognita卵具有较强的侵染能力。为了进一步研究刀孢轮枝菌胞外几丁质酶的性质,我们通过简并引物设计和DNA walking方法从刀孢轮枝菌的基因组中成功地克隆得到一个内切几丁质酶基因Lpchi1,该几丁质酶编码基因含有3个内含子,编码423个氨基酸。同源性和系统发育分析表明,不同生防真菌来源的几丁质酶具有较高的同源性并根据分子量的大小形成三个不同的进化分枝。     

Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics

Application of polyester-degrading enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. Many hydrolases from several fungi and bacteria have been discovered and successfully evaluated for their activity towards different aliphatic polyesters (PHA, PBS, PBSA, PCL, PLA), aromatic polyesters (PET, PBT, PMT) as well as their co-polyesters (PBST, PBAT, PBSTIL). This revision gives an up-to-date overview on the main biochemical features and biotechnological applications of those reported enzymes which are able to degrade polyester-based plastics, including different microbial polyester depolymerases, esterases, cutinase-like enzymes and lipases. Summarized information includes available protein sequences with the corresponding accession numbers deposited in NCBI server, 3D resolved structures, and data about optimal conditions for enzymatic activity and stability of many of these microbial enzymes that would be helpful for researchers in this topic. Although screening and identification of new native polyester hydrolases from microbial sources is undeniable according to literature, we briefly highlight the importance of the design of improved enzymes towards recalcitrant aromatic polyesters through different approaches that include site-directed mutagenesis and surface protein engineering.     

自噬相关去泛素化酶及其小分子抑制剂的研究进展

自噬是进化上高度保守并受到多途径严密调控的细胞生物学过程,其向溶酶体递送多种细胞质组分以进行细胞内物质的降解以及再循环.这一过程涉及到细胞器的更新、错误折叠蛋白质和蛋白质聚集体以及细胞内病原体的清除.因此,自噬对于细胞稳态的维持至关重要,与许多人类疾病的发生发展密切相关.随着细胞自噬调节机制研究的不断深入,越来越多的去泛素化酶被证明在自噬相关的泛素信号调控系统中发挥了重要的作用.这些去泛素化酶作用于细胞自噬的不同阶段,靶向调节不同的泛素化自噬功能元件或自噬底物.去泛素化酶作为包括神经退行性疾病以及肿瘤在内的细胞自噬相关疾病的治疗靶点受到了广泛的关注,其中各类小分子抑制剂的发现为进一步研究去泛素化酶的自噬调节活性及相关疾病的治疗提供了可能.