A growth selection system for the directed evolution of amine forming or converting enzymes

Fast screening of enzyme variants is crucial for tailoring biocatalysts for the asymmetric synthesis of non natural chiral chemicals, such as amines. However, most existing screening methods either are limited by the throughput or require specialized equipment. Herein, we report a simple, high throughput, low equipment dependent, and generally applicable growth selection system for engineering amine forming or converting enzymes and apply it to improve biocatalysts belonging to three different enzyme classes. This results in i an amine transaminase variant with 110 fold increased specific activity for the asymmetric synthesis of the chiral amine intermediate of Linagliptin; ii a 270 fold improved monoamine oxidase to prepare the chiral amine intermediate of Cinacalcet by deracemization; and iii an ammonia lyase variant with a 26 fold increased activity in the asymmetric synthesis of a non natural amino acid. Our growth selection system is adaptable to different enzyme classes, varying levels of enzyme activities, and thus a flexible tool for various stages of an engineering campaig

Mechanism Based Design of Efficient PET Hydrolases

Polyethylene terephthalate PET is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal methods for a circular plastic economy, only a handful of benchmark enzymes have been thoroughly described and subjected to protein engineering for improved properties over the last 16 years. By analyzing the specific material properties of PET and the reaction mechanisms in the context of interfacial biocatalysis, this Perspective identifies several limitations in current enzymatic PET degradation approaches. Unbalanced enzyme substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temperatures, and inhibition caused by oligomeric degradation intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative experimental and computational approaches have been published extensively in recent years in this thriving research field and are summarized and discussed in detail here. The acquired knowledge and experience will be applied in the near future to address plastic waste contributed by other mass produced polymer types e.g., polyamides and polyurethanes that should also be properly disposed by biotechnological approache

Multiple Substrate Binding Mode Guided Engineering of a Thermophilic PET Hydrolase

[Image: see text] Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substrate-binding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 °C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 °C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic recycling processes

Solving the material and energy challenges of the future

This year has been proclaimed the International Year of Chemistry by the United Nations. This year long celebration allows chemists to highlight the rich history and successes of their scientific discipline and to explain how chemistry can help to solve the global challenges that mankind faces today and tomorrow

Screening of commercial hydrolases for the degradation of Ochratoxin A

Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin. The toxin is a common contaminant of various foods and feeds and poses a serious threat to the health of both humans and animals. A number of commercial hydrolases were screened for the ability to degrade OTA to nontoxic compounds. A crude lipase from Aspergillus niger (Amano A) proved to substantially hydrolyze OTA to the nontoxic OTα and phenylalanine, as confirmed by HPLC with fluorescence detection. The enzyme was purified by anion exchange chromatography to homogeneity. Activity staining of the purified enzyme with α-naphthyl acetate/Fast Red revealed only one band exhibiting hydrolytic activity. The specific activity of the purified enzyme toward OTA was 2.32 units/mg.Articl

Purification and Reconstitution of an Integral Membrane Protein, the Photoreaction Center of Rhodobacter sphaeroides, Using Synthetic Sugar Esters

Detergents are indispensable reagents for the extraction and solubilization of integral membrane proteins, but their removal from a reconstituted phospholipid-protein complex is usually desirable. In this paper, we describe a novel method in which the synthetic sugar esters 6-O-octanoyl-β-Dglucose (OG) or 6-O-octanoyl-β-D-mannose (OM) are used as detergents for both the isolation and the rapid reconstitution of the photosynthetic reaction center protein of Rhodobacter sphaeroides. Following solubilization of the reaction center with OG or OM and reconstitution of this protein in liposomes, a convenient removal of these detergents was achieved within less than two hours by hydrolytic cleavage of the sugar esters using immobilized lipases. Best results were achieved with lipase from Bacillus sp. immobilized on silica gel

Enzymatic removal of carboxyl protecting groups. III. Fast removal of allyl and chloroethyl esters by Bacillus subtilis esterase (BS2)

(Chemical Equation Presented) An esterase from Bacillus subtilis (BS2) allows the fast and selective removal of allyl, 2-chloroethyl, and 2,2,2-chloroethyl esters under mild conditions in high yields. In addition, BS2 easily hydrolyzes phenacyl esters, while the hydrolysis of sterically hindered diphenylmethyl esters is slow, requiring longer reaction time and higher enzyme/substrate ratio. © 2007 American Chemical Society

Conversion of a mono- and diacylglycerol lipase into a triacylglycerol lipase by protein engineering.

Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono- and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa. By protein engineering, and with only one mutation, we managed to convert this enzyme into a typical triacylglycerol lipase (the wild-type lipase does not accept triacylglycerides). The variant Q282L accepts a broad spectrum of triacylglycerides, although the catalytic behavior is altered to some extent. From in silico analysis it seems that specific hydrophobic interactions are key to the altered substrate specificity. A mono- and diglyceride lipase was engineered to a triacylglyceride lipase by introducing a single point mutation (Q282L). The variant has broad substrate specificity on triacylglycerides. The results indicate that the main reason that the wild-type enzyme does not accept triacylglycerides is not their bulkiness, but specific hydrophobic interactions