A main goal of green biotechnology is to reduce our dependence on fossil reserves and to increase the use of renewable materials. For this, lignocellulose, which is composed of cellulose, hemicellulose and lignin, represents the most promising feedstock. The latter is a complex aromatic heteropolymer formed by radical polymerization of guaiacyl, syringyl, and p-hydroxyphenyl units linked by b-aryl ether linkages, biphenyl bonds and heterocyclic linkages. Accordingly, lignin appears to be a potentially valuable renewable aromatic chemical, thus representing a main pillar in future biorefinery. The resistance of lignin to breakdown is the main bottleneck in this process, although a variety of white-rot fungi, as well as bacteria, have been reported to degrade lignin by employing different enzymes and catabolic pathways. Here, recent investigations have expanded the range of natural biocatalysts involved in lignin degradation/modification and significant progress related to enzyme engineering and recombinant expression has been made. The present review is focused primarily on recent trends in ligninolytic green biotechnology to suggest the potential (industrial) application of ligninolytic enzymes. Future perspectives could include synergy between natural enzymes from different sources (as well as those obtained by protein engineering) and other pretreatment methods that may be required for optimal results in enzyme-based, environmentally friendly, technologies.

D: -Amino acid oxidase (DAAO) is a biotechnologically relevant enzyme that is used in a variety of applications. DAAO is a flavine adenine dinucleotide-containing flavoenzyme that catalyzes the oxidative deamination of D-isomer of uncharged aliphatic, aromatic, and polar amino acids yielding the corresponding imino acid (which hydrolyzes spontaneously to the alpha-keto acid and ammonia) and hydrogen peroxide. This enzymatic activity is produced by few bacteria and by most eukaryotic organisms. In the past few years, DAAO from mammals has been the subject of a large number of investigations, becoming a model for the dehydrogenase-oxidase class of flavoproteins. However, DAAO from microorganisms show properties that render them more suitable for the biotechnological applications, such as a high level of protein expression (as native and recombinant protein), a high turnover number, and a tight binding of the coenzyme. Some important DAAO-producing microorganisms include Trigonopsis variabilis, Rhodotorula gracilis, and Fusarium solani. The aim of this paper is to provide an overview of the main biotechnological applications of DAAO (ranging from biocatalysis to convert cephalosporin C into 7-amino cephalosporanic acid to gene therapy for tumor treatment) and to illustrate the advantages of using the microbial DAAOs, employing both the native and the improved DAAO variants obtained by enzyme engineering.

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ,90% conversion of cephalosporin C to 7-aminocephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.Keywords: cephalosporin C; 7-amino cephalosporanic acid; protein engineering; directed evolution; site-saturation mutagenesis; enzymes; active sites; structure/function studies; protein sequencing; modification; mass spectrometry; protein structure prediction; kinetics Semisynthetic cephalosporins are the most widely used antibiotics and are primarily synthesized from 7-amino cephalosporanic acid (7-ACA), which is usually obtained by chemical deacylation of the natural antibiotic cephalosporin C (CephC). The chemical route includes, however, several expensive steps and requires treatment of toxic wastes. A two-step enzymatic route can also be used that, in two separate reactors, uses D-amino acid oxidase and glutaryl-7-amino cephalosporanic acid (gl-7ACA) acylase activity (see Scheme 1, below): Although this route solves the environmental safety problems, it is also expensive and not entirely satisfactory for industrial production. Therefore, enzymatic conversion of CephC to 7-ACA is of great interest to cephalosporin antibiotics manufacturers (an annual worldwide market of ,400 million US dollars has been estimated) (Pilone and Pollegioni 2002). The greatest hindrance to enzymatic industrial production is that acylase takes glutaryl-7-ACA (gl-7ACA) as a primary substrate, and its specificity (and activity) is too low for CephC. Glutaryl-7-ACA acylases are members of the N-terminal hydrolases (Ntn) class of hydrolytic enzymes. The gene structure of the open reading frame (ORF) of the members of this class consists of a signal peptide, followed by an a-subunit, a spacer sequence (which is not present in the acylase under investigation), and a b-subunit...

Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and D-isomer of amino acids to yield the corresponding ␣-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the k cat /K m ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the ␣2-␣3 loop (comprising residues 50 -60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg 54 could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide.

Over the years, accumulating evidence has indicated that D-serine represents the endogenous ligand for the glycine modulatory binding site on the NR1 subunit of N-methyl-D-aspartate receptors in various brain areas. Cellular concentrations of D-serine are regulated by synthesis due to the enzyme serine racemase (isomerization reaction) and by degradation due to the same enzyme(elimination reaction) as well as by the FAD-containing flavoenzyme D-amino acid oxidase (DAAO, oxidative deamination reaction).Several findings have linked low levels of D-serine to schizophrenia: D-serine concentrations in serum and cerebrospinal fluid have been reported to be decreased in schizophrenia patients while human DAAO activity and expression are increased; oral administration of D-serine improved positive, negative, and cognitive symptoms of schizophrenia as add-on therapy to typical and atypical antipsychotics.This evidence indicates that increasing NMDA receptor function, perhaps by inhibiting DAAO-induced degradation of D-serine may alleviate symptoms in schizophrenic patients. Furthermore, it has been suggested that co-administration of D-serine with a human DAAO inhibitor may be a more effective means of increasing D-serine levels in the brain. Here, we present an overview of the current knowledge of the structure-function relationships in human DAAO and of the compounds recently developed to inhibit its activity (specifically the ones recently exploited for schizophrenia treatment).

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In recent years, by investigating bacterial degradation of lignin, interesting enzymatic activities aimed at\ud breaking specific linkages were identified. In this work we focused on the tetrahydrofolate (THF)-\ud dependent O-demethylase LigM from the bacterium Sphingobium sp. strain SYK-6 which converts vanillic\ud acid to protocatechuic acid (PCA). The recombinant LigM was overexpressed in E. coli up to 80 mg L−1 fermentation\ud broth, and the main kinetic parameters on the best substrate vanillic acid (kcat = 0.19 s−1 and Km\ud = 54 μM) and the substrate specificity were identified. The substrate preference was rationalized using a\ud three-dimensional model of LigM structure in complex with THF, which also allowed us to propose a reaction\ud mechanism. LigM efficiently converts vanillic acid into PCA but the reaction requires a 10-fold molar\ud excess of the THF cofactor. In order to limit the cofactor consumption, the plant methionine synthase\ud MetE enzyme was also overexpressed in E. coli and used in combination with LigM. Under optimized conditions,\ud the dual enzyme system produced 5 mM PCA using 0.1 mM THF only, a 500-fold decrease in the\ud cofactor : substrate molar ratio compared to the mono-enzyme process. This represents the first regeneration\ud method for THF in a biocatalytic process