Pilot-Scale Production of Peroxygenase from Agrocybe aegerita.

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L-1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L-1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

Production of Hydroxynitrile Lyase from Davallia tyermannii (DtHNL) in Komagataella phaffii and Its Immobilization as a CLEA to Generate a Robust Biocatalyst.

Hydroxynitrile lyase from the white rabbit's foot fern Davallia tyermannii (DtHNL) catalyzes the enantioselective synthesis of α-cyanohydrins, which are key building blocks for pharmaceutical and agrochemical industries. An efficient and competitive process necessitates the availability and robustness of the biocatalyst. Herein, the recombinant production of DtHNL1 in Komagataella phaffii, yielding approximately 900 000 U L-1 , is described. DtHNL1 constitutes approximately 80 % of the total protein content. The crude enzyme was immobilized. Crosslinked enzyme aggregates (CLEAs) resulted in significant enhancement of the biocatalyst stability under acidic conditions (activity retained after 168 h at pH 2.4). The DtHNL1-CLEA was employed for (R)-mandelonitrile synthesis (99 % conversion, 98 % enantiomeric excess) in a biphasic system, and evaluated for the synthesis of (R)-hydroxypivaldehyde cyanohydrin under reaction conditions that immediately inactivated non-immobilized DtHNL1. The results show the DtHNL1-CLEA to be a stable biocatalyst for the synthesis of enantiomerically pure cyanohydrins under acidic conditions.

Mini-review: recent developments in hydroxynitrile lyases for industrial biotechnology.

Hydroxynitrile lyases (HNLs) catalyze the cleavage as well as the formation of cyanohydrins. The latter reaction is valuable for the stereoselective C-C bond formation by condensation of HCN with carbonyl compounds. The resulting cyanohydrins serve as versatile building blocks for a broad range of chemical and enzymatic follow-up reactions. A significant number of (R)- and (S)-selective HNLs are known today and the number is still increasing. HNLs not only exhibit varying substrate scope but also differ in sequence and structure. Tailor-made enzymes for large-scale manufacturing of cyanohydrins with improved yield and enantiomeric excess are very interesting targets, which is reflected in a solid number of patents. This review will complement and extend our recent review with a strong focus on applications of HNLs for the synthesis of highly functionalized, chiral compounds with newest literature, recent and current patent literature.

Enzyme immobilisation in biocatalysis: why, what and how.

In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).

Nitriliruptor alkaliphilus gen. nov., sp. nov., a deep-lineage haloalkaliphilic actinobacterium from soda lakes capable of growth on aliphatic nitriles, and proposal of Nitriliruptoraceae fam. nov. and Nitriliruptorales ord. nov.

A novel bacterial strain, designated ANL-iso2(T), was obtained from an enrichment culture inoculated with a mixture of soda lake sediments by using isobutyronitrile (iBN) as the carbon, energy and nitrogen source at pH 10. The enrichment resulted in a stable binary culture containing iBN-degrading Gram-positive rods and a satellite Gram-negative gammaproteobacterium Marinospirillum sp. strain (ANL-isoa) scavenging the products of nitrile hydrolysis. Cells of the iBN-degrading strain, ANL-iso2(T), were short, non-motile, non-spore-forming rods. Strain ANL-iso2(T) was capable of utilizing propionitrile (C(3)), butyronitrile (C(4)), isobutyronitrile (C(4)), valeronitrile (C(5)) and capronitrile (C(6)) as the only growth substrate. Growth on nitriles was biphasic with fast initial hydrolysis of nitriles to the corresponding amides, carboxylic acids and ammonia and slow further utilization of these products resulting in biomass growth. Cells of strain ANL-iso2(T) grown with iBN were capable of extremely active hydration of a wide range of nitriles into the corresponding amides and much slower hydrolysis of these amides to the corresponding carboxylic acids. This indicated the presence of the nitrile hydratase/amidase pathway of nitrile degradation in the novel bacterium. Strain ANL-iso2(T) showed obligately alkaliphilic growth on iBN within the pH range 8.4-10.6, with optimum growth at 9.0-9.5. It was moderately salt-tolerant, with a salt range for growth of 0.1-2.0 M Na(+) and an optimum salt concentration for growth of 0.2-0.3 M. The dominant fatty acids in the polar lipids were C(16 : 0), iso-C(14), C(14 : 0), iso-C(16) and C(16 : 1)omega7. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid. Phylogenetic analysis placed strain ANL-iso2(T) within the class Actinobacteria as an independent lineage with only uncultured bacteria from soda lakes as its nearest relatives. On the basis of its unique phenotype and distinct phylogeny, strain ANL-iso2(T) is considered to represent a novel species of a new genus, for which the name Nitriliruptor alkaliphilus gen. nov., sp. nov. is proposed. The type strain of the type species, Nitriliruptor alkaliphilus, is ANL-iso2(T) (=DSM 45188(T)=NCCB 100119(T)=UNIQEM U239(T)). Phylogenetic data suggest that the novel bacterium forms the basis of a new family Nitriliruptoraceae fam. nov. and a novel order Nitriliruptorales ord. nov. within the class Actinobacteria.

Utilization of aliphatic nitriles under haloalkaline conditions by Bacillus alkalinitrilicus sp. nov. isolated from soda solonchak soil.

Enrichment with isobutyronitrile as the sole carbon, energy and nitrogen source at pH 10, using soda solonchak soils as an inoculum, resulted in the selection of a binary culture consisting of two different spore-forming phenotypes. One of them, strain ANL-iso4, was capable of growth with isobutyronitrile as a single substrate, while the other phenotype only utilized products of isobutyronitrile hydrolysis, such as isobutyroamide and isobutyrate. Strain ANL-iso4 is an obligate alkaliphile and a moderately salt-tolerant bacterium. Apart from isobutyronitrile, it grew on other (C3-C6) aliphatic nitriles at pH 10. Resting cells of ANL-iso4 actively hydrolyzed a number of aliphatic and arylaliphatic nitriles and their corresponding amides. The latter, together with the intermediate formation of amides during nitrile hydrolysis, indicated the presence of a nitrile hydratase/amidase system in the novel bacterium. Although present in an alkaliphilic bacterium, both nitrile- and amide-hydrolyzing activities had a pH optimum within the neutral range, probably due to their intracellular localization. On the basis of phenotypic and phylogenetic analyses, strain ANL-iso4 is proposed as a new species Bacillus alkalinitrilicus sp. nov.

Microbial isobutyronitrile utilization under haloalkaline conditions.

The utilization of isobutyronitrile (iBN) as a C and N source under haloalkaline conditions by microbial communities from soda lake sediments and soda soils was studied. In both cases, a consortium consisting of two different bacterial species capable of the complete degradation and utilization of iBN at pH 10 was selected. The soda lake sediment consortium consisted of a new actinobacterium and a gammaproteobacterium from the genus Marinospirillum. The former was capable of fast hydrolysis of aliphatic nitriles to the corresponding amides and much-slower further hydrolysis of the amides to carboxylic acids. Its partner cannot hydrolyze nitriles but grew rapidly on amides and carboxylic acids, thus acting as a scavenger of products released by the actinobacterium. The soda soil consortium consisted of two Bacillus species (RNA group 1). One of them initiated nitrile hydrolysis, and the other utilized the hydrolysis products isobutyroamide (iBA) and isobutyrate (iB). In contrast to the actinobacterium, the nitrile-hydrolyzing soil Bacillus grew rapidly with hydrolysis products, but it was dependent on vitamins most probably supplied by its product-utilizing partner. All four bacterial strains isolated were moderately salt-tolerant alkaliphiles with a pH range for growth from pH 7.0 to 8.5 up to 10.3 to 10.5. However, both their nitrile hydratase and amidase activities had a near-neutral pH optimum, indicating an intracellular localization of these enzymes. Despite this fact, the study demonstrated a possibility of whole-cell biocatalytic hydrolysis of various nitriles at haloalkaline conditions.

Acetonitrile degradation under haloalkaline conditions by Natronocella acetinitrilica gen. nov., sp. nov.

Nitriles are important environmental compounds, both as natural products and industrial pollutants. Until now, there have been no data on the possibility of microbial nitrile degradation at high pH/salt conditions. Acetonitrile (CH(3)C(triple bond)N) is the simplest organic nitrile. Here, evidence is provided of microbial utilization of acetonitrile as a carbon, energy and nitrogen source at extremely high pH and moderate salinity. Positive enrichment cultures with acetonitrile at pH 10 and salt content equivalent to 0.6 M total Na(+) were obtained from mixed sediment samples from soda lakes, but not from soda soils. Purification of these cultures resulted in the isolation of two bacterial strains capable of growth with acetonitrile as sole carbon, energy and nitrogen source under haloalkaline conditions. Apart from acetonitrile, the bacteria also grew with propionitrile. Nitrile hydrolysis to acetamide was identified as the rate-limiting step of acetonitrile degradation via the nitrile hydratase/amidase pathway. The new bacteria belonged to moderately salt-tolerant obligate alkaliphiles with optimum growth at pH 10 and 0.5 M total Na(+). The cells were yellow-coloured due to a high concentration of carotenoids dominated by zeaxanthin. Phylogenetic analysis placed the isolates into a new lineage within the family Ectothiorhodospiraceae in the Gammaproteobacteria. On the basis of unique phenotypic properties and their separate phylogenetic position, the new bacteria are placed into a new genus and species for which the name Natronocella acetinitrilica gen. nov., sp. nov is proposed.