Three New Lipases from Actinomycetes and Their Use in Organic Reactions

Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates.     

ENANTIOSELECTIVE ACYLATION OF β-PHENYLALANINE ACID AND ITS DERIVATIVES CATALYZED BY PENICILLIN G ACYLASE FROM Alcaligenes faecalis

This study developed a simple, efficient method for producing racemic β-phenylalanine acid (BPA) and its derivatives via the enantioselective acylation catalyzed by the penicillin G acylase from Alcaligenes faecalis (Af-PGA). When the reaction was run at 25°C and pH 10 in an aqueous medium containing phenylacetamide and BPA in a molar ratio of 2:1, 8 U/mL enzyme and 0.1 M BPA, the maximum BPA conversion efficiency at 40 min only reached 36.1%, which, however, increased to 42.9% as the pH value and the molar ratio of phenylacetamide to BPA were elevated to 11 and 3:1, respectively. Under the relatively optimum reaction conditions, the maximum conversion efficiencies of BPA derivatives all reached about 50% in a relatively short reaction time (45–90 min). The enantiomeric excess value of product (ee_p ) and enantiomeric excess value of substrate (ee_s ) were all above 98% and 95%, respectively. These results suggest that the method established in this study is practical, effective, and environmentally benign and may be applied to industrial production of enantiomerically pure BPA and its derivatives.     

Novozyme 435-Catalyzed Asymmetric Acylation of (R,S)-3-n-Butylphthalide in Hexane

Abstract

The asymmetric acylation of (R, S)-3-n-butylphthalide could be efficiently catalyzed by Novozyme 435. The effect of various reaction parameters such as water activity, temperature, molar ratio of acetic anhydride to (R, S)-3-n-butylphthalide, and reaction time on the asymmetric acylation were studied. The optimums of the reaction parameters were water activity 0.62, temperature 30°C, molar ratio of acetic anhydride to (R, S)-3-n-butylphthalide 8:1, and reaction time 48 h, respectively. Under the optimum conditions, enantiopure 3-n-butylphthalide with an optical purity of 95.7% enantiomeric excess and 49.1% yield could be obtained. Furthermore, the enantiomeric excess of product was over 98%.     

Perspectives and industrial potential of PGA selectivity and promiscuity

Penicillin G acylases (PGAs) are robust industrial catalysts used for biotransformation of β-lactams into key intermediates for chemical production of semi-synthetic β-lactam antibiotics by hydrolysis of natural penicillins. They are used also in reverse, kinetically controlled synthetic reactions for large-scale productions of these antibiotics from corresponding beta-lactam nuclei and activated acyl donors. Further biocatalytic applications of PGAs have recently been described: catalysis of peptide syntheses and the resolutions of racemic mixtures for the production of enantiopure active pharmaceutical ingredients that are based on enantioselective acylation or chiral hydrolysis. Moreover, PGAs rank among promiscuous enzymes because they also catalyze reactions such as trans-esterification, Markovnikov addition or Henry reaction. This particular biocatalytic versatility represents a driving force for the discovery of novel members of this enzyme family and further research into the catalytic potential of PGAs. This review deals with biocatalytic applications exploiting enantioselectivity and promiscuity of prokaryotic PGAs that have been recently reported. Biocatalytic applications are discussed and presented with reaction substrates converted into active compounds useful for the pharmaceutical industry.     

Stereoselective Synthesis of (3R)‐3‐Alkyl‐4,1‐Benzoxazepine‐2,5‐Diones

Novel 3‐alkyl‐4,1‐benzoxazepine‐2,5‐diones were synthesized in good ee exploiting the chiral pool methodology, an economical way of asymmetric synthesis. Various anthranilic acids are coupled with different α‐haloacids to afford N‐acylated anthranilic acid intermediates which undergo cyclization to (3R)‐3‐alkyl‐4,1‐benzoxazepines‐2,5‐diones. Chirality 25:865–870, 2013. © 2013 Wiley Periodicals, Inc.     

Deciphering sulfoglycolipids of Mycobacterium tuberculosis

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways.     

Insights into metalloregulation by M-box riboswitch RNAs via structural analysis of manganese-bound complexes

The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses on other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base-stacking, electrostatic, and hydrogen-bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal binding sites, we resolved a manganese-chelated M-box RNA complex to 1.86 Å by X-ray crystallography. These data support the presence of at least eight well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal-binding cores within the M-box RNA that facilitate a network of long-range interactions within the metal-bound, compacted conformation.     

A quantum mechanics/molecular mechanics study of the reaction mechanism of the hepatitis C virus NS3 protease with the NS5A/5B substrate

Combined quantum mechanics and molecular mechanics (QM/MM) calculations were carried out to characterize the reaction mechanism of the NS3 protease with its preferred substrate (NS5A/5B). The main purpose of this study was to locate the barrier states and intermediates along the distinguished coordinate path (DCP) involved in this process. These structures, and in particular the one corresponding to the first barrier state and intermediate (B1 and I1), could be a starting point for the synthesis of inhibitors of this protease, which could be used to treat hepatitis C. The two first steps of the reaction mechanism were studied, i.e., the acylation step and the breaking of the peptide bond. The first step takes place through a tetracoordinated intermediate, as suggested from previous works on other Serine proteases. The importance of the different amino acid residues was also considered (perturbation study where the MM charges of each residue were set to zero independently). The residues of the oxyanion hole were confirmed as the most important for the electrostatic stabilization of the tetracoordinate intermediate. Moreover, the role of other residues, e.g., Arg-155 and Asp-79, was also explained.     

Synthesis of L‐Lys‐Aminoxy‐Goralatide

Natural tetrapeptide Goralatide inhibits primitive hematopoietic cell proliferation but reported to be rather unstable in solution (half‐life 4.5 min). In this work, we report the synthesis of an aminoxy analog of Goralatide. Aminoxy moiety is expected to provide increased stability and bioavailability of the Goralatide analog. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Bottlenecks in erucic acid accumulation in genetically engineered ultrahigh erucic acid Crambe abyssinica

Erucic acid is a valuable industrial fatty acid with many applications. The main producers of this acid are today high erucic rapeseed (Brassica napus) and mustard (Brassica juncea), which have 45%–50% of erucic acid in their seed oils. Crambe abyssinica is an alternative promising producer of this acid as it has 55%–60% of erucic acid in its oil. Through genetic modification (GM) of three genes, we have previously increased the level of erucic acid to 71% (68 mol%) in Crambe seed oil. In this study, we further investigated different aspects of oil biosynthesis in the developing GM Crambe seeds in comparison with wild‐type (Wt) Crambe, rapeseed and safflower (Carthamus tinctorius). We show that Crambe seeds have very low phosphatidylcholine‐diacylglycerol interconversion, suggesting it to be the main reason why erucic acid is limited in the membrane lipids during oil biosynthesis. We further show that GM Crambe seeds have slower seed development than Wt, accompanied by slower oil accumulation during the first 20 days after flowering (DAF). Despite low accumulation of erucic acid during early stages of GM seed development, nearly 86 mol% of all fatty acids accumulated between 27 and 50 DAF was erucic acid, when 40% of the total oil is laid down. Likely bottlenecks in the accumulation of erucic acid during early stages of GM Crambe seed development are discussed.