An up-scaled biotechnological approach for phosphorus-depleted rye bran as animal feed.

Side streams from the milling industry offer excellent nutritional properties for animal feed; yet their use is constrained by the elevated phosphorus (P) content, mainly in the form of phytate. Biotechnological P recovery fosters sustainable P management, transforming these streams into P-depleted animal feed through enzymatic hydrolysis. The enzymatic P mobilization not only enables P recovery from milling by-products but also supports the valorization of these streams into P-depleted animal feeds. Our study presents the scalability and applicability of the process and characterizes the resulting P-depleted rye bran as animal feed component. Batch mode investigations were conducted to mobilize P from 100 g to 37.1 kg of rye bran using bioreactors up to 400 L. P reductions of 89% to 92% (reducing from 12.7 gP/kg to 1.41-1.28 gP/kg) were achieved. In addition, High Performance Ion Chromatography (HPIC) analysis showed complete depletion of phytate. The successful recovery of the enzymatically mobilized P from the process wastewater by precipitation as struvite and calcium hydrogen phosphate is presented as well, achieving up to 99% removal efficiency. Our study demonstrates a versatile process that is easily adaptable, allowing for a seamless implementation on a larger scale.

A Screening Method for P450 BM3 Mutant Libraries Using Multiplexed Capillary Electrophoresis for Detection of Enzymatically Converted Compounds.

Capillary electrophoresis (CE) is an analytical method in which charged species are separated by attraction or repulsion performed in submillimeter diameter capillaries or micro- and nanofluidic channels through the application of a high voltage electric field. When capillary electrophoresis is assembled in a multicapillary instrument such as 96-well format (multiplexed), it becomes a powerful high-throughput system with the ability to simultaneously screen several types of samples like genetic mutations, metabolomes, kinase inhibitors, or enzymatic activities to name a few. The usage of a 96-multiplexed capillary electrophoresis system (96-MP-CE) represents a new platform for product-specific high-throughput screening of enzyme mutant libraries from directed evolution campaigns providing a comprehensive view on enzyme activity through the detection of all products formed. We describe the application of 96-MP-CE to screen mutant libraries of P450 BM3. MP-CE was used in directed evolution campaigns toward benzo-1,4-dioxane and α-isophorone.

Phytase blends for enhanced phosphorous mobilization of deoiled seeds.

Phytases are hydrolytic enzymes capable of a stepwise phosphate release from phytate which is the main phosphorous storage in seeds, cereals and legumes. Limitations such as low enzyme activity or incomplete phytate hydrolysis to inositol are a great challenge in phytase applications in food and feed. Herein we report a phytase blend of two enzymes with additive effects on phytate (InsP6) hydrolysis and its application in the enzymatic phosphorous recovery process. Blending the fast 6-phytase rPhyXT52 with the 3-phytase from Debaryomyces castellii, which is capable of fully hydrolyzing InsP6, we achieved rapid phosphate release with higher yields compared to the individual enzymes and a rapid disappearance of InsP6-3 intermediates, monitored by HPLC. NMR data suggest a nearly complete phytate hydrolysis to inositol and phosphate. The blend was applied for phosphate mobilization from phytate-rich biomass, such as deoiled seeds. For this emerging application, an up to 43% increased phosphate mobilization yield was achieved when using 1000 U of the blend per kg biomass compared to using only the E. coli phytase. Even so, the time of enzyme treatment was decreased by more than half (6 h instead of 16 h) when using 4000 U of blend, we reached a 78-90% reduction of the total phosphorous content in the explored deoiled seeds. In summary, the phytase blend of Dc phyt/rPhyXT52 was proven very efficient to obtain inositol phosphate depleted meal which has its potential application in animal feeding and is concomitant with the production of green phosphate from renewable resources.

Generation of phytase chimeras with low sequence identities and improved thermal stability.

Being able to recombine more than two genes with four or more crossover points in a sequence independent manner is still a challenge in protein engineering and limits our capabilities in tailoring enzymes for industrial applications. By computational analysis employing multiple sequence alignments and homology modeling, five fragments of six phytase genes (sequence identities 31-64 %) were identified and efficiently recombined through phosphorothioate-based cloning using the PTRec method. By combinatorial recombination, functional phytase chimeras containing fragments of up to four phytases were obtained. Two variants (PTRec 74 and PTRec 77) with up to 32 % improved residual activity (90 °C, 60 min) and retained specific activities of > 1100 U/mg were identified. Both variants are composed of fragments from the phytases of Citrobacter braakii, Hafnia alvei and Yersinia mollaretii. They exhibit sequence identities of ≤ 80 % to their parental enzymes, highlighting the great potential of DNA recombination strategies to generate new enzymes with low sequences identities that offer opportunities for property right claims.

Understanding substrate binding and the role of gatekeeping residues in PigC access tunnels.

Semi-rational redesign of the substrate binding pocket and access tunnels of prodigiosin ligase PigC enhanced the catalytic efficiency in the synthesis of pyrrolic anti-cancer agents more than 45 times. A molecular understanding was gained on residues V333 and T334 relevant to substrate binding and translocation of small pyrroles through PigC access tunnels.

Engineered P450 BM3 and cpADH5 coupled cascade reaction for ß-oxo fatty acid methyl ester production in whole cells.

Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product.

Insights on intermolecular FMN-heme domain interaction and the role of linker length in cytochrome P450cin fusion proteins.

Cytochrome P450s are an important group of enzymes catalyzing hydroxylation, and epoxidations reactions. In this work we describe the characterization of the CinA-CinC fusion enzyme system of a previously reported P450 using genetically fused heme (CinA) and FMN (CinC) enzyme domains from Citrobacter braaki. We observed that mixing individually inactivated heme (-) with FMN (-) domain in the CinA-10aa linker - CinC fusion constructs results in recovered activity and the formation of (2S)-2ß-hydroxy,1,8-cineole (174 µM), a similar amount when compared to the fully functional fusion protein (176 µM). We also studied the effect of the fusion linker length in the activity complementation assay. Our results suggests an intermolecular interaction between heme and FMN parts from different CinA-CinC fusion protein similar to proposed mechanisms for P450 BM3 on the other hand, linker length plays a crucial influence on the activity of the fusion constructs. However, complementation assays show that inactive constructs with shorter linker lengths have functional subunits, and that the lack of activity might be due to incorrect interaction between fused enzymes.

A colourimetric high-throughput screening system for directed evolution of prodigiosin ligase PigC.

A colourimetric high-throughput screening system was established for directed evolution of prodigiosin ligase PigC. The two-step system consists of a colony prescreening test and a subsequent photometric 96-well plate assay. Screening PigC epPCR libraries in Pseudomonas putida revealed a PigC variant that achieved a 2.9× increased yield of prodiginine derivatives.

Reversal of Regioselectivity in Zinc-Dependent Medium-Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives.

The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4-phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4-phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3.

The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450-1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.

Rapid and Robust Coating Method to Render Polydimethylsiloxane Surfaces Cell-Adhesive.

Polydimethylsiloxane (PDMS) is a synthetic material with excellent properties for biomedical applications because of its easy fabrication method, high flexibility, permeability to oxygen, transparency, and potential to produce high-resolution structures in the case of lithography. However, PDMS needs to be modified to support homogeneous cell attachments and spreading. Even though many physical and chemical methods, like plasma treatment or extracellular matrix coatings, have been developed over the last decades to increase cell-surface interactions, these methods are still very time-consuming, often not efficient enough, complex, and can require several treatment steps. To overcome these issues, we present a novel, robust, and fast one-step PDMS coating method using engineered anchor peptides fused to the cell-adhesive peptide sequence (glycine-arginine-glycine-aspartate-serine, GRGDS). The anchor peptide attaches to the PDMS surface predominantly by hydrophobic interactions by simply dipping PDMS in a solution containing the anchor peptide, presenting the GRGDS sequence on the surface available for cell adhesion. The binding performance and kinetics of the anchor peptide to PDMS are characterized, and the coatings are optimized for efficient cell attachment of fibroblasts and endothelial cells. Additionally, the applicability is proven using PDMS-based directional nanotopographic gradients, showing a lower threshold of 5 µm wrinkles for fibroblast alignment.

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles.

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min-1; R255L: 1168 ± 163 min-1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation.

Engineered phytases for emerging biotechnological applications beyond animal feeding.

Phytases are important industrial enzymes able to catalyze the release of up to six phosphates from phytate in a stepwise hydrolysis reaction. Phytases are almost exclusively used as a feed supplement. However, phytases are also used in human nutrition, food processing, non-food industrial products, and emerging applications like enzymatic phosphate recovery from renewable resources. Phytate, the main phosphorus storage form in seeds, and its hydrolysis products act as a chelator and reduce protein and mineral bioavailability in intestinal absorption. Full phosphate hydrolysis from the common storage compound phytate remains a challenge. Phytate hydrolysis patterns of tailored phytases and their protein engineering campaigns are discussed. The aim of our review is to give an overview on developed and emerging application areas (animal nutrition, food processing, and environmental resource management) and thereby generate an awareness for the importance of phosphorus stewardship in a circular bioeconomy. Emphasis will be given to processes using organic-bound phosphorus and related recycling strategy of this valuable resource. In detail, the main challenge in designing phytases to completely hydrolyze phosphate from phytate to inositol and the need for engineering campaigns to broaden their industrial use are described.

A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry.

The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNACUA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNACUA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity.

A hydroquinone-specific screening system for directed P450 evolution.

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 µM) and a broad linear detection range (5 to 250 µM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M).

A Comparative Reengineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis.

Positions identified in directed evolution campaigns or by (semi)rational design can be recombined iteratively or simultaneously. Iterative recombination has yielded many success stories and is beneficially used if screening capabilities are limited (four iterative SSMs generate 20×4=80 different enzyme variants). Simultaneous site saturation mutagenesis offers significantly higher diversity (204 =160 000 variants) and enables greater improvements to be found, especially if the selected positions are in close proximity to each other (cooperative effects). Here we report a first comprehensive comparison of iterative and simultaneous saturation of four residues in Candida parapsilosis alcohol dehydrogenase 5 (cpADH5) with methyl 3-hydroxyhexanoate as substrate. Screening of 7200 clones from 33 site saturation mutagenesis libraries (exploring 17 recombination paths) yielded the cpADH5 W286A variant, with a 82-fold improved initial activity toward methyl 3-hydroxyhexanoate. Screening 3500 clones from a single OmniChange library with four positions (C57, W116, L119, and W286; 1.8 % of the generated sequence space) yielded the cpADH5 C57V/W286S variant, with a 108-fold improvement in initial activity toward methyl 3-hydroxyhexanoate. A 1.8 % coverage of the sequence space of the simultaneous multisite saturation library was, in comparison to the investigated 17 recombination paths, sufficient to identify a cpADH5 variant with improved activity.

Improved microscale cultivation of Pichia pastoris for clonal screening.

BACKGROUND: Expanding the application of technical enzymes, e.g., in industry and agriculture, commands the acceleration and cost-reduction of bioprocess development. Microplates and shake flasks are massively employed during screenings and early phases of bioprocess development, although major drawbacks such as low oxygen transfer rates are well documented. In recent years, miniaturization and parallelization of stirred and shaken bioreactor concepts have led to the development of novel microbioreactor concepts. They combine high cultivation throughput with reproducibility and scalability, and represent promising tools for bioprocess development. RESULTS: Parallelized microplate cultivation of the eukaryotic protein production host Pichia pastoris was applied effectively to support miniaturized phenotyping of clonal libraries in batch as well as fed-batch mode. By tailoring a chemically defined growth medium, we show that growth conditions are scalable from microliter to 0.8 L lab-scale bioreactor batch cultivation with different carbon sources. Thus, the set-up allows for a rapid physiological comparison and preselection of promising clones based on online data and simple offline analytics. This is exemplified by screening a clonal library of P. pastoris constitutively expressing AppA phytase from Escherichia coli. The protocol was further modified to establish carbon-limited conditions by employing enzymatic substrate-release to achieve screening conditions relevant for later protein production processes in fed-batch mode. CONCLUSION: The comparison of clonal rankings under batch and fed-batch-like conditions emphasizes the necessity to perform screenings under process-relevant conditions. Increased biomass and product concentrations achieved after fed-batch microscale cultivation facilitates the selection of top producers. By reducing the demand to conduct laborious and cost-intensive lab-scale bioreactor cultivations during process development, this study will contribute to an accelerated development of protein production processes.

An Enzymatic Route to α-Tocopherol Synthons: Aromatic Hydroxylation of Pseudocumene and Mesitylene with P450 BM3.

Aromatic hydroxylation of pseudocumene (1 a) and mesitylene (1 b) with P450 BM3 yields key phenolic building blocks for α-tocopherol synthesis. The P450 BM3 wild-type (WT) catalyzed selective aromatic hydroxylation of 1 b (94 %), whereas 1 a was hydroxylated to a large extent on benzylic positions (46-64 %). Site-saturation mutagenesis generated a new P450 BM3 mutant, herein named "variant M3" (R47S, Y51W, A330F, I401M), with significantly increased coupling efficiency (3- to 8-fold) and activity (75- to 230-fold) for the conversion of 1 a and 1 b. Additional π-π interactions introduced by mutation A330F improved not only productivity and coupling efficiency, but also selectivity toward aromatic hydroxylation of 1 a (61 to 75 %). Under continuous nicotinamide adenine dinucleotide phosphate recycling, the novel P450 BM3 variant M3 was able to produce the key tocopherol precursor trimethylhydroquinone (3 a; 35 % selectivity; 0.18 mg mL-1 ) directly from 1 a. In the case of 1 b, overoxidation leads to dearomatization and the formation of a valuable p-quinol synthon that can directly serve as an educt for the synthesis of 3 a. Detailed product pattern analysis, substrate docking, and mechanistic considerations support the hypothesis that 1 a binds in an inverted orientation in the active site of P450 BM3 WT, relative to P450 BM3 variant M3, to allow this change in chemoselectivity. This study provides an enzymatic route to key phenolic synthons for α-tocopherols and the first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O2 .

Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3-Hydroxyalkanoates.

Expanding the substrate scope of enzymes opens up new routes for synthesis of valuable chemicals. Ketone-functionalized fatty acid derivatives and corresponding chiral alcohols are valuable building blocks for the synthesis of a variety of chemicals including pharmaceuticals. The alcohol dehydrogenase from Candida parapsilosis (cpADH5) catalyzes the reversible oxidations of chiral alcohols and has a broad substrate range; a challenge for cpADH5 is to convert alcohols with small substituents (methyl or ethyl) next to the oxidized alcohol moiety. Molecular docking studies revealed that W286 is located in the small binding pocket and limits the access to substrates that contain aliphatic chains longer than ethyl substituent. In the current manuscript, we report that positions L119 and W286 are key residues to boost oxidation of medium chain methyl 3-hydroxy fatty acids; interestingly the enantiopreference toward methyl 3-hydroxybutyrate was inverted. Kinetic characterization of W286A showed a 5.5 fold increase of Vmax and a 9.6 fold decrease of Km values toward methyl 3-hydroxyhexanoate (Vmax : 2.48 U mg- and Km : 4.76 mm). Simultaneous saturation at positions 119 and 286 library yielded a double mutant (L119M/W286S) with more than 30-fold improved activity toward methyl 3-hydroxyoctanoate (WT: no conversion; L119M/W286S: 30 %) and inverted enantiopreference (S-enantiomer ≥99 % activity decrease and R-enantiomer >20-fold activity improvement) toward methyl 3-hydroxybutyrate.

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.