Biotechnology Institute

About: Biotechnology Institute is a nonprofit organization based out in Washington D.C., District of Columbia, United States. It is known for research contribution in the topics: Gene & Catalysis. The organization has 93 authors who have published 230 publications receiving 13050 citations.

Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different

Abstract: Enzymes responsible for deamination reactions are widespread throughout intermediary metabolism and serve to incorporate and recycle nitrogen among key metabolites essential for DNA and protein synthesis. Some are members of an amidohydrolase protein superfamily, which catalyze at least 30% of the steps in four intermediary metabolic pathways (22). Recently, a new class of bacterial amidohydrolases has been identified; they catalyze the hydrolytic displacement of amino groups and chlorine substituents from s-triazine ring compounds (22, 32). The s-triazine compounds have numerous applications throughout industry and agriculture (10, 19, 25, 30). Those containing N-alkyl substituents, like atrazine (2-chloro-4-N-ethylamino-6-N-isopropylamino-1,3,5-triazine), have been applied successfully as herbicides (3). Atrazine and analogous chlorinated s-triazines were initially considered to be incompletely metabolized by microorganisms (10, 19). However, 40 years after the initial introduction of atrazine into the environment, bacteria with the ability to completely mineralize this herbicide have been isolated (12, 26, 31, 40). Subsequently, bacteria were shown to initiate atrazine metabolism via dechlorination to yield hydroxyatrazine (2, 8, 12, 26, 31, 40). In 1996, the dechlorinating enzyme atrazine chlorohydrolase (AtzA) was purified and shown, via [18O]water experiments, to catalyze a hydrolytic displacement reaction (Fig. ​(Fig.1)1) (13). FIG. 1 Comparison of the reactions catalyzed by melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 (A) and AtzA from Pseudomonas sp. strain ADP (B). The substrate specificity of AtzA from Pseudomonas sp. strain ADP was recently investigated (35). AtzA catalyzes the hydrolytic removal of a chlorine or fluorine substituent but does not remove cyano, azido, methoxy, thiomethyl, or amino substituents from compounds structurally analogous to atrazine. AtzA is also not active with any of the pyrimidine substrates tested (35). Melamine (2,4,6-triamino-1,3,5-triazine), a related s-triazine that predates the use of atrazine (29), is also metabolized by soil bacteria. Worldwide production of melamine in 1994 was estimated to be 900 million lb (21). Melamine is most commonly used in the production of melamine-formaldehyde resins, which are used in laminates, adhesives, fire retardants, molding compounds, coatings, and concrete plasticizers (29). Prior to the identification of atrazine-mineralizing bacteria, Cook and Hutter isolated melamine-metabolizing pseudomonads (11). One of these, Pseudomonas sp. strain NRRL B-12227, catalyzes consecutive hydrolysis of the three amino substituents of melamine, producing the intermediates ammeline, ammelide, and cyanuric acid (Fig. ​(Fig.1).1). Another bacterium, Pseudomonas sp. strain NRRL B-12228, was unreactive with melamine but catalyzed deamination of ammeline to ammelide and of ammelide to cyanuric acid (11). Genes for ammeline and ammelide deamination, trzB and trzC, respectively, have been cloned from Pseudomonas sp. strain NRRL B-12228 (17). Detailed restriction site pattern analysis revealed conservation of trzC but not trzB in Pseudomonas sp. strain NRRL B-12227 (17). The genes encoding the enzyme for melamine or ammeline deamination in Pseudomonas sp. strain NRRL B-12227, however, were not reported. Furthermore, Pseudomonas sp. strain NRRL B-12227 was shown not to metabolize atrazine (11). Given the similarity in structure between melamine and atrazine and their similar hydrolytic metabolism, it was hypothesized here that AtzA gene probes and antibodies might be used to identify the melamine deaminase gene and protein, respectively, in Pseudomonas sp. strain NRRL B-12227. Using this strategy, the melamine deaminase gene, designated triA, was identified, cloned, and sequenced. The melamine deaminase gene from Pseudomonas sp. strain NRRL B-12227 was 99% identical to the atzA gene from Pseudomonas sp. strain ADP. The cloned melamine deaminase was expressed in Escherichia coli DH5α and shown to catalyze the deamination of melamine and ammeline. Melamine deaminase had no activity with any of the chlorotriazine substrates tested. Taken together with the known substrate specificity of AtzA, these studies identified two nearly identical proteins that catalyze clearly distinct biochemical reactions.

Bacterial community structures are unique and resilient in full-scale bioenergy systems

Abstract: Anaerobic digestion is the most successful bioenergy technology worldwide with, at its core, undefined microbial communities that have poorly understood dynamics. Here, we investigated the relationships of bacterial community structure (>400,000 16S rRNA gene sequences for 112 samples) with function (i.e., bioreactor performance) and environment (i.e., operating conditions) in a yearlong monthly time series of nine full-scale bioreactor facilities treating brewery wastewater (>20,000 measurements). Each of the nine facilities had a unique community structure with an unprecedented level of stability. Using machine learning, we identified a small subset of operational taxonomic units (OTUs; 145 out of 4,962), which predicted the location of the facility of origin for almost every sample (96.4% accuracy). Of these 145 OTUs, syntrophic bacteria were systematically overrepresented, demonstrating that syntrophs rebounded following disturbances. This indicates that resilience, rather than dynamic competition, played an important role in maintaining the necessary syntrophic populations. In addition, we explained the observed phylogenetic differences between all samples on the basis of a subset of environmental gradients (using constrained ordination) and found stronger relationships between community structure and its function rather than its environment. These relationships were strongest for two performance variables—methanogenic activity and substrate removal efficiency—both of which were also affected by microbial ecology because these variables were correlated with community evenness (at any given time) and variability in phylogenetic structure (over time), respectively. Thus, we quantified relationships between community structure and function, which opens the door to engineer communities with superior functions.

Hydrolases in Organic Synthesis: Regio- and Stereoselective Biotransformations

Abstract: 1 Introduction. 2 Designing Enantioselective Reactions. 2.1 Kinetic Resolutions. 2.2 Asymmetric Syntheses. 3 Choosing Reaction Media: Water and Organic Solvents. 3.1 Hydrolysis in Water. 3.2 Transesterifications and Condensations in Organic Solvents. 3.3 Other Reaction Media. 3.4 Immobilization. 4 Protein Sources and Optimization of Biocatalyst Performance. 4.1 Accessing Biodiversity. 4.2 Creating Improved Biocatalysts. 4.3 Catalytic Promiscuity in Hydrolases. 5 Lipases and Esterases. 5.1 Availability, Structures and Properties. 5.2 Survey of Enantioselective Lipase-Catalyzed Reactions. 5.3 Chemo-and Regioselective Lipase-Catalyzed Reactions. 5.4 Reactions Catalyzed by Esterases. 6 Proteases and Amidases. 6.1 Occurrence and Availability of Proteases and Amidases. 6.2 General Features of Subtilisin, Chymotrypsin, and Other Proteases and Amidases. 6.3 Structures of Proteases and Amidases. 6.4 Survey of Enantioselective Protease-and Amidase-Catalyzed Reactions. 7 Phospholipases. 7.1 Phospholipase A1. 7.2 Phospholipase A2. 7.3 Phospholipase C. 7.4 Phospholipase D. 8 Epoxide Hydrolases. 8.1 Introduction. 8.2 Mammalian Epoxide Hydrolases. 8.3 Microbial Epoxide Hydrolases. 9 Hydrolysis of Nitriles. 9.1 Introduction. 9.2 Mild Conditions. 9.3 Regioselective Reactions of Dinitriles. 9.4 Enantioselective Reactions. 10 Other Hydrolases. 10.1 Glycosidases. 10.2 Haloalcohol Dehalogenases. 10.3 Phosphotriesterases. Abbreviations. References. Index.