Synthesis of propyl-β-d-galactoside with free and immobilized β-galactosidase from Aspergillus oryzae

Synthesis of propyl-β-galactoside catalyzed by Aspergillus oryzae β-galactosidase in soluble form was optimized using response surface methodology (RSM). Temperature and 1-propanol concentration were selected as explanatory variables; yield and productivity were chosen as response variables. Optimal reaction conditions were determined by weighing the responses through a desirability function. Then, synthesis of propyl-β-galactoside was evaluated at the optimal condition previously determined, with immobilized β-galactosidase in glyoxyl-agarose and amino-glyoxyl-agarose, and with cross-linked aggregates (CLAGs). Yields of propyl-β-galactoside obtained with CLAGs, amino-glyoxyl-agarose and glyoxyl-agarose enzyme derivatives were 0.75, 0.81 and 0.87 mol/mol and volumetric productivities were 5.2, 5.6 and 5.9 mM/h, respectively, being significantly higher than the corresponding values obtained with the soluble enzyme: 0.47 mol/mol and 4.4 mM/h. As reaction yield was increased twofold with the glyoxyl-agarose derivative, this catalyst was chosen for evaluating the synthesis of propyl-β-galactoside in repeated batch operations. Then, after ten sequential batches, the efficiency of catalyst use was 115% higher than obtained with the free enzyme. Enzyme immobilization also favored product recovery, allowing catalyst reuse, and avoiding browning reactions. Propyl-β-galactoside was recovery by extraction in 90%v/v acetone with a purity higher than 99% and its synthesis was confirmed by mass spectrometry.     

Production of a chiral polyester by Pseudomonas oleovorans grown with 5-phenyl-2,4-pentadienoic acid

Pseudomonas oleovorans has been previously shown to produce a polyester containing a phenyl pendant group when grown with 5-phenylpentanoic acid under nutrient-limiting conditions. The same polyester was produced when 5-phenyl-2,4-pentadienoic acid was the only carbon source, and a mixture of two different polymers was produced when this bacterium was grown on a mixture of 5-phenyl-2,4-pentadienoic acid and nonanoic acid. The polymer blend obtained was separated by fractional crystallization to yield poly(3-hydroxy-5-phenylpentanoate) and the copolymer which is normally produced with nonanoic acid alone.     

Poly(l-lactide) degradation by Kibdelosporangium aridum

A new poly(L-lactide) (PLA)-degrading actinomycete, Kibdelosporangium aridum, degraded more than 97 mg out of 100 mg added high molecular weight PLA film (Mn: 3.4 x 10(5)) within 14 d in liquid culture. L-Lactic acid, the monomeric degradation product of PLA, was totally assimilated by the strain. In solid culture, many distinct grooves formed by the morphology of filamentous microorganisms on the surface of a PLA film were observed by scanning electron microscopy.     

An untargeted liquid chromatography–mass spectrometry‐based workflow for the structural characterization of plant polyesters

Cell wall localized heterogeneous polyesters are widespread in land plants. The composition of these polyesters, such as cutin, suberin, or more plant‐specific forms such as the flax seed coat lignan macromolecule, can be determined after total hydrolysis of the ester linkages. The main bottleneck in the structural characterization of these macromolecules, however, resides in the determination of the higher order monomer sequences. Partial hydrolysates of the polyesters release a complex mixture of fragments of different lengths, each present in low abundance and therefore are challenging to structurally characterize. Here, a method is presented by which liquid chromatography–mass spectrometry (LC‐MS) profiles of such partial hydrolysates are searched for pairs of related fragments. LC‐MS peaks that show a mass difference corresponding to the addition of one or more macromolecule monomers were connected in a network. Starting from the lowest molecular weight peaks in the network, the annotation of the connections as the addition of one or more polyester monomers allows the prediction of consecutive and increasingly complex adjacent peaks. Multi‐stage MS (MSn) experiments further helped to reject, corroborate, and sometimes refine the structures predicted by the network. As a proof of concept, this procedure was applied to partial hydrolysates of the flax seed coat lignan macromolecule, and allowed to characterize 120 distinct oligo‐esters, consisting of up to six monomers, and containing monomers and linkages for which incorporation in the lignan macromolecule had not been described before. These results showed the capacity of the approach to advance the structural elucidation of complex plant polyesters.     

The biosynthesis of cutin and suberin as an alternative source of enzymes for the production of bio-based chemicals and materials

Oxygenated fatty acids such as ricinoleic acid and vernolic acid can serve in the industry as synthons for the synthesis of a wide range of chemicals and polymers traditionally produced by chemical conversion of petroleum derivatives. Oxygenated fatty acids can also be useful to synthesize specialty chemicals such as cosmetics and aromas. There is thus a strong interest in producing these fatty acids in seed oils (triacylglycerols) of crop species. In the last 15 years or so, much effort has been devoted to isolate key genes encoding proteins involved in the synthesis of oxygenated fatty acids and to express them in the seeds of the model plant Arabidopsis thaliana or crop species. An often overlooked but rich source of enzymes catalyzing the synthesis of oxygenated fatty acids and their esterification to glycerol is the biosynthetic pathways of the plant lipid polyesters cutin and suberin. These protective polymers found in specific tissues of all higher plants are composed of a wide variety of oxygenated fatty acids, many of which have not been reported in seed oils (e.g. saturated ω-hydroxy fatty acids and α,ω-diacids). The purpose of this mini-review is to give an overview of the recent advances in the biosynthesis of cutin and suberin and discuss their potential utility in producing specific oxygenated fatty acids for specialty chemicals. Special emphasis is given to the role played by specific acyltransferases and P450 fatty acid oxidases. The use of plant surfaces as possible sinks for the accumulation of high value-added lipids is also highlighted.     

A Novel PHB Depolymerase from a Thermophilic Streptomyces Sp.

A novel PHB depolymerase from a thermophilic Streptomyces sp. MG was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The molecular mass of the purified enzyme was 43 kDa as determined by size exclusion chromatography and 41 kDa by SDS-PAGE. The optimum pH and temperature were 8.5 and 60 °C respectively. The enzyme was stable at 50 °C and from pH 6.5–8.5. The enzyme hydrolyzed not only bacterial polyesters, i.e. poly(3-hydroxybutyric acid and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), but also synthetic, aliphatic polyesters such as polypropiolactone, poly(ethylene adipate) and poly(ethylene succinate). Revisions requested 9 November 2005; Revisions received 12 December 2005     

Review Degradation of microbial polyesters

Microbial polyhydroxyalkanoates (PHAs), one of the largest groups of thermoplastic polyesters are receiving much attention as biodegradable substitutes for non-degradable plastics. Poly(D-3-hydroxybutyrate) (PHB) is the most ubiquitous and most intensively studied PHA. Microorganisms degrading these polyesters are widely distributed in various environments. Although various PHB-degrading microorganisms and PHB depolymerases have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. Distributions of PHB-degrading microorganisms, factors affecting the biodegradability of PHB, and microbial and enzymatic degradation of PHB are discussed in this review. We also propose an application of a new isolated, thermophilic PHB-degrading microorganism, Streptomyces strain MG, for producing pure monomers of PHA and useful chemicals, including D-3-hydroxycarboxylic acids such as D-3-hydroxybutyric acid, by enzymatic degradation of PHB.     

Hydrolysis of polyesters by serine proteases

The substrate specificity of -chymotrypsin and other serine proteases, trypsin, elastase, proteinase K and subtilisin, towards hydrolysis of various polyesters was examined using poly(L-lactide) (PLA), poly(-hydroxybutyrate) (PHB), poly(ethylene succinate) (PES), poly(ethylene adipate) (PEA), poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBS/A), poly[oligo(tetramethylene succinate)-co-(tetramethylane carbonate)] (PBS/C), and poly(-caprolactone) (PCL). -Chymotrypsin could degrade PLA and PEA with a lower activity on PBS/A. Proteinase K and subtilisin degraded almost all substrates other than PHB. Trypsin and elastase had similar substrate specificities to -chymotrypsin.     

Biosynthesis of copolyesters consisting of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids from 1,3-butanediol or from 3-hydroxybutyrate by Pseudomonas sp. A33

Pseudomonas sp. A33 and other isolates of aerobic bacteria accumulated a complex copolyester containing 3-hydroxybutyric acid (3HB) and various medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from 3-hydroxybutyric acid or from 1,3-butanediol under nitrogen-limitated culture conditions. 3HB contributed to 15.1 mol/100 mol of the constituents of the polyester depending on the strain and on the cultivation conditions. The accumulated polymer was a copolyester of 3HB and 3HA_MCL rather than a blend of poly(3HB) and poly(3HA_MCL) on the basis of multiple evidence. 3-Hydroxyhexadecenoic acid and 3-hydroxyhexadecanoic acid were detected as constituents of polyhydroxyalkanoates, which have hitherto not been described, by¹³C nuclear magnetic resonance or by gas chromatography/mass spectrometric analysis. In total, ten different constituents were detected in the polymer synthesized from 1,3-butanediol by Pseudomonas sp. A33:besides seven saturated (3HB, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydrohexadecanoate) three unsaturated (3-hydroxydodecenoate, 3-hydroxytetradecenoate and 3-hydrohexadecanoate) hydroxyalkanoic acid constituents occured. The polyhydroxyalkanoate synthase of Pseudomonas sp. A33 was cloned, and its substrate specificity was evaluated by heterologous expression in various strains of P. putida, P. oleovorans and Alcaligenes eutrophus.