Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells.

Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.

Synthesis of α-l-Araf and ß-d-Galf series furanobiosides using mutants of a GH51 α-l-arabinofuranosidase.

The GH-51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus (TxAbf) possesses versatile catalytic properties, displaying not only the ability to hydrolyze glycosidic linkages but also to synthesize furanobiosides in α-l-Araf and ß-d-Galf series. Herein, mutants are investigated to evaluate their ability to perform self-condensation, assessing both yield improvements and changes in regioselectivity. Overall yields of oligo-α-l-arabino- and oligo-ß-d-galactofuranosides were increased up to 4.8-fold compared to the wild-type enzyme. In depth characterization revealed that the mutants exhibit increased transfer rates and thus a hydrolysis/self-condensation ratio in favor of synthesis. The consequence of the substitution N216W is the creation of an additional binding subsite that provides the basis for an alternative acceptor substrate binding mode. As a result, mutants bearing N216W synthesize not only (1,2)-linked furanobiosides, but also (1,3)- and even (1,5)-linked furanobiosides. Since the self-condensation is under kinetic control, the yield of homo-disaccharides was maximized using higher substrate concentrations. In this way, the mutant R69H-N216W produced oligo-ß-d-galactofuranosides in > 70% yield. Overall, this study further demonstrates the potential usefulness of TxAbf mutants for glycosynthesis and shows how these might be used to synthesize biologically-relevant glycoconjugates.

Feather protein lysate optimization and feather meal formation using YNDH protease with keratinolytic activity afterward enzyme partial purification and characterization.

Incubation parameters used for the creation of a protein lysate from enzymatically degraded waste feathers using crude keratinase produced by the Laceyella sacchari strain YNDH were optimized using the Response Surface Methodology (RSM); amino acids quantification was also estimated. The optimization elevated the total protein to 2089.5 µg/ml through the application of the following optimal conditions: a time of 20.2 h, a feather concentration (conc.) of 3 g%, a keratinase activity of 24.5 U/100 ml, a pH of 10, and a cultivation temperature of 50 °C. The produced Feather Protein Lysate (FPL) was found to be enriched with essential and rare amino acids. Additionally, this YNDH enzyme group was partially purified, and some of its characteristics were studied. Crude enzymes were first concentrated with an Amicon Ultra 10-k centrifugal filter, and then concentrated proteins were applied to a "Q FF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranging from 6 to 10 kDa. The maximum enzyme activity was observed at 70 °C and for a pH of 10.4. Most characteristics of this protease/keratinase group were found to be nearly the same when the activity was measured with both casein and keratin-azure as substrates, suggesting that these three protein bands work together in order to degrade the keratin macromolecule. Interestingly, the keratinolytic activity of this group was not inhibited by ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), or iron-caused activation, indicating the presence of a mixed serine-metallo enzyme type.

Thermophilic iron containing type superoxide dismutase from Cohnella sp. A01.

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH⁎, ΔS⁎, and ΔG⁎ were 187 min at 60 °C, 7.3 kJ.mol-1, -76.8 kJ.mol-1.°K-1, and 84.1 kJ.mol-1, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0-10.0) and temperatures (35-75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.

Cohnella 1759 cysteine protease shows significant long term half-life and impressive increased activity in presence of some chemical reagents.

Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme Km and Vmax values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme's co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.

Biosynthetic L-tert-leucine using Escherichia coli co-expressing a novel NADH-dependent leucine dehydrogenase and a formate dehydrogenase

BACKGROUND: L-tert-Leucine has been widely used in pharmaceutical, chemical, and other industries as a vital chiral intermediate. Compared with chemical methods, enzymatic methods to produce L-tert-leucine have unparalleled advantages. Previously, we found a novel leucine dehydrogenase from the halophilic thermophile Laceyella sacchari (LsLeuDH) that showed good thermostability and great potential for the synthesis of L-tertleucine in the preliminary study. Hence, we manage to use the LsLeuDH coupling with a formate dehydrogenase from Candida boidinii (CbFDH) in the biosynthesis of L-tert-leucine through reductive amination in the present study. RESULT: The double-plasmid recombinant strain exhibited higher conversion than the single-plasmid recombinant strain when resting cells cultivated in shake flask for 22 h were used. Under the optimized conditions, the double-plasmid recombinant E. coli BL21 (pETDute-FDH-LDH, pACYCDute-FDH) transformed 1 mol·L-1 trimethylpyruvate (TMP) completely into L-tert-leucine with greater than 99.9% ee within 8 h. CONCLUSIONS: The LsLeuDH showed great ability to biosynthesize L-tert-leucine. In addition, it provided a new option for the biosynthesis of L-tert-leucine.

Transformation of xylan into value-added biocommodities using Thermobacillus composti GH10 xylanase.

Enzymatic transformation of xylans into renewable fuels and value-added products is mediated by xylanases. Here we describe the biochemical and X-ray structural characterization of Thermobacillus composti GH10 xylanase (TcXyn10A) at 2.1 Å resolution aiming to unravel details of its recognition of glucurono- and arabinoxylan at a molecular level. TcXyn10A improves the efficiency of pretreated lignocellulosic biomass hydrolysis by a commercial enzyme cocktail causing a 15.35 % increase in xylose release and 4.38 % glucose release after 24 h of reaction. The enzyme releases predominantly xylobiose and xylotriose, as well as MeGlcA3 × 3 (from beechwood glucuronoxylan) and a range of decorated xylooligosaccharides (XOS) from rye arabinoxylan, with Ara2 × 2 being the major product. The enzyme liberates XOS with the yields of 29.09 % for beechwood glucuronoxylan and 16.98 % for rye arabinoxylan. Finally, TcXyn10A has a high thermal stability, halotolerance, and resistance to ethanol, biochemical properties that can be desirable for a number of industrial applications.

Kinetics, detergent compatibility and feather-degrading capability of alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 produced with fermentation of organic municipal solid wastes.

Alkaline proteases having activity and stability at alkaline pH possess a large variety of applications in many industries. Growing renewed interest urges the need to find a single alkaline protease with promising properties to be used in different industrial processes. Herein, alkaline proteases produced through fermentation of cheap and easily available organic municipal solid wastes by Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 were purified to investigate their kinetic and thermodynamic parameters, detergent compatibility, dehairing and feather-degrading capability. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the purified protease from B. subtilis and E. indicum had molecular mass of ∼45 and 75 kDa, respectively. The protease from B. subtilis and E. indicum showed highest activity at 55 and 50 °C having low K m 1.17 and 0.567 mg/mL and high V max 416.67 and 333.33 µmole/min, respectively. The activation energy and temperature quotient of protease from B. subtilis and E. indicum were 26.52 and 65.75 kJ/mole, and 1.0004 and 1.0003 at 20-55 and 20-50 °C, respectively. Thermodynamics analysis revealed the formation of more ordered enzyme-substrate complexes along with spontenity of enzyme reaction. The protease from E. indicum exhibited better compatibility at higher concentration of detergents compared to that from B. subtilis. However, both proteases could retain more than 80% of the activity in the presence of 0.1% commercial laundry detergents. The purified protease from the both sources could degrade almost 90% of barbs and 40% of dry weight of the native feather and that from E. indicum could dehair cow skin. Results reported herein suggest that the alkaline protease from B. subtilis AKAL7 and E. indicum AKAL11 has biotechnological implications in detergent, leather and poultry feather processing industries.

Structural and biochemical characterization of a novel thermophilic Coh01147 protease.

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/ß sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

Utilization of methyltrioctylammonium chloride as new ionic liquid in pretreatment of sugarcane bagasse for production of cellulase by novel thermophilic bacteria.

Fermentation of carbohydrates present in lignocellulosic (LC) biomass is facilitated by lignin removal, which is usually achieved by adopting various pretreatment methods to provide the enzymes proper access to their respective substrates. Pretreatment using ionic liquid (IL) is relatively recent advancement and considered as mild and green process. ILs can dissolve extensive quantities of biomass and depolymerize the cellulose. In this context, an abundantly available LC biomass, sugarcane bagasse (SB), was pretreated using alkali or with an IL, methyltrioctylammonium chloride, and was used for cellulase production from thermophilic bacteria. In all, 26 indigenously isolated thermophilic bacterial strains were quantitatively screened for cellulase production. 16S rDNA sequences of the promising isolates UE10 and UE27 revealed relatedness with Brevibacillus borstelensis, while the strain UE1 belonged to Aneurinibacillus thermoaerophilus. Cellulase production was compared by utilizing alkali pretreated and IL pretreated SB and the later was found more appropriate. UE1, UE10 and UE27 yielded 22.2, 22.18 and 33.3 IU mL-1 of endoglucanase, respectively, by fermenting IL pretreated SB. The changes in SB structure after pretreatment were evaluated by scanning electron microscopy. This study demonstrated the potential of novel thermophilic bacterial strains to utilize IL pretreated SB for production of industrially important enzyme, cellulase.

Tuning amino acid dehydrogenases with featured sequences for L-phosphinothricin synthesis by reductive amination.

Biosynthesizing unnatural chiral amino acids is challenging due to the limited reductive amination activity of amino acid dehydrogenase (AADH). Here, for the asymmetric synthesis of l-phosphinothricin from 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), a glutamate dehydrogenase gene (named GluDH3) from Pseudomonas monteilii was selected, cloned and expressed in Escherichia coli (E. coli). To boost its activity, a "two-step"-based computational approach was developed and applied to select the potential beneficial amino acid positions on GluDH3. l-phosphinothricin was synthesized by GluDH-catalyzed asymmetric amination using the d-glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH) for NADPH regeneration. Using lyophilized E. coli cells that co-expressed GluDH3_V375S and EsGDH, up to 89.04 g L-1 PPO loading was completely converted to l-phosphinothricin within 30 min at 35 °C with a space-time yield of up to 4.752 kg·L-1·d-1. The beneficial substitution V375S with increased polar interactions between K90, T193, and substrate PPO exhibited 168.2-fold improved catalytic efficiency (kcat/KM) and 344.8-fold enhanced specific activity. After the introduction of serine residues into other GluDHs at specific positions, forty engineered GluDHs exhibited the catalytic functions of "glufosinate dehydrogenase" towards PPO.

Production of l-ribose from l-arabinose by co-expression of l-arabinose isomerase and d-lyxose isomerase in Escherichia coli.

l-Ribose is an important pharmaceutical intermediate that is used in the synthesis of numerous antiviral and anticancer drugs. However, it is a non-natural and expensive rare sugar. Recently, the enzymatic synthesis of l-ribose has attracted considerable attention owing to its considerable advantages over chemical approaches. In this work, a new strategy was developed for the production of l-ribose from the inexpensive starting material l-arabinose. The l-arabinose isomerase (l-AIase) gene from Alicyclobacillus hesperidum and the d-lyxose isomerase (d-LIase) gene from Thermoflavimicrobium dichotomicum were cloned and co-expressed in Escherichia coli, resulting in recombinant cells harboring the vector pCDFDuet-Alhe-LAI/Thdi-DLI. The co-expression system exhibited optimal activity at a temperature of 70 °C and pH 6.0, and the addition of Co2+ enhanced the catalytic activity by 27.8-fold. The system containing 50 g L-1 of recombinant cells were relatively stable up to 55 °C. The co-expression system (50 g L-1 of recombinant cells) afforded 20.9, 39.7, and 50.3 g L-1 of l-ribose from initial l-arabinose concentrations of 100, 300, and 500 g L-1, corresponding to conversion rate of 20.9%, 13.2%, and 10.0%, respectively. Overall, this study provides a viable approach for producing l-ribose from l-arabinose under slightly acidic conditions using a co-expression system harboring l-AIase and d-LIase genes.

Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization.

L-glutaminase importance to use in the food industry and medicine has attracted much attention. Enzymes stability has always been a challenge while working with them. We heterologously expressed and characterized a novel stable L-glutaminase from an extremophile bacterium (Cohnella sp. A01, PTCC No: 1921). Km, Vmax, catalytic efficiency and specific activity of rSAM were respectively 1.8 mM, 49 µmol/min, 1851 1/(S.mM) and 9.2 IU/mg. Activation energy for substrate to product conversion and irreversible thermo-inactivation were respectively 4 kJ/mol and 105 kJ/mol from the linear Arrhenius plot. rSAM had the highest activity at temperature 50 °C, pH 8 and was resistant to a wide range of temperature and pH. In compare to the other characterized glutaminases, rSAM was the most resistant to NaCl. Mg2+, glycerol, DTT, and BME enhanced the enzyme activity and iodoacetate and iodoacetamide inhibited it. rSAM had only been partially digested by some proteases. According to the Fluorimetry and Circular dichroism analysis, rSAM in pH range from 4 to 11 and temperatures up to 60 °C had structural stability. A cysteine residue in the enzyme active site and a thiol bond were predicted upon the modeled tertiary structure of rSAM. Present structural studies also confirmed the presence of a thiol bond in its structure.

Improving the catalytic efficiency of aldo-keto reductase KmAKR towards t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via semi-rational design.

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The Km values of M3 for NADPH and (5R)-1 were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate vmax was 55.56 µmol/min/mg. Compared with M1, the catalytic efficiency kcat/Km of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5R)-1 at up to 100.0 g/L, producing 237.4 mmol/L (3R,5R)-2 in d.e.P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8 g/L/d.

In vitro digestibility of rice starch granules modified by ß-amylase, transglucosidase and pullulanase.

It is highly desirable to lower the glycemic index of rice starch-based foods. Herein, rice starch granules were treated sequentially with ß-amylase (BA), transglucosidase (TG) and pullulanase (PUL). The results indicated that compared with native rice starch and PUL-modified starch, degree of crystallinity, gelatinization temperature and enthalpy of BA/TG/PUL-modified starches increased remarkably. Moreover, the functionality of BA/TG/PUL-treated starch depended enormously on TG treatment time. BA/TG/PUL-modified starch possessed the highest relative crystallinity, gelatinization temperatures and gelatinization enthalpy and the content of resistant starch at TG treatment time of 20 h and the resistant crystals were formed largely from linear chains with DP 9-11. Whether before cooking or after cooking, BA/TG/PUL-modified starches had strong resistance to enzyme hydrolysis and had lower glycemic index. This outcome may offer a novel sight for the exploitation of starch-based functional foods with low glycemic index.

Heterologous expression, purification, immobilization and characterization of recombinant α-amylase AmyLa from Laceyella sp. DS3.

AmyLa α-amylase gene from Laceyella sp. DS3 was heterologously expressed in E. coli BL21. E. coli BL21 maximally expressed AmyLa after 4 h of adding 0.02 mM IPTG at 37 °C. The recombinant AmyLa α-amylase was purified 2.19-fold through gel filtration and ion exchange chromatography. We immobilized the purified recombinant AmyLa α-amylase on four carriers; chitosan had the best efficiency. The recombinant free and the immobilized AmyLa α-amylase showed optimum activity in the pH ranges of 6.0-7.0 and 4.0-7.0, respectively and possessed an optimum temperature of 55 °C. The free enzyme had activation energy, Km, and Vmax of 291.5 kJ, 1.5 mg/ml, and 6.06 mg/min, respectively. The immobilized enzyme had activation energy, Km, and Vmax of 309.74 kJ, 6.67 mg/ml, and 50 mg/min, respectively. The immobilized enzyme was calcium-independent and insensitive (relative to the free enzyme) to metals. It could also be reused for seven cycles.

Efficient Synthesis of Purine Nucleoside Analogs by a New Trimeric Purine Nucleoside Phosphorylase from Aneurinibacillus migulanus AM007.

Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) from Aneurinibacillus migulanus AM007 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The AmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0-9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived from Brevibacillus borstelensis LK01 and AmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP: AmPNP (2:20 µg/mL). Thus, the new trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.

Relative catalytic efficiencies and transcript levels of three d- and two l-lactate dehydrogenases for optically pure d-lactate production in Sporolactobacillus inulinus.

As the optical purity of the lactate monomer is pivotal for polymerization, the production of optically pure d-lactate is of significant importance. Sporolactobacillus inulinus YBS1-5 is a superior optically pure d-lactate-producing bacterium. However, little is known about the relationship between lactate dehydrogenases in S. inulinus YBS1-5 and the optical purity of d-lactate. Three potential d-lactate dehydrogenase (D-LDH1-3)- and two putative l-lactate dehydrogenase (L-LDH1-2)-encoding genes were cloned from the YBS1-5 strain and expressed in Escherichia coli D-LDH1 exhibited the highest catalytic efficiency toward pyruvate, whereas two L-LDHs showed low catalytic efficiency. Different neutralizers significantly affected the optical purity of d-lactate produced by strain YBS1-5 as well as the transcription levels of ldhDs and ldhLs. The high catalytic efficiency of D-LDH1 and elevated ldhD1 mRNA levels suggest that this enzyme is essential for d-lactate synthesis in S. inulinus YBS1-5. The correlation between the optical purity of d-lactate and transcription levels of ldhL1 in the case of different neutralizers indicate that ldhL1 is a key factor affecting the optical purity of d-lactate in S. inulinus YBS1-5.

Enantioselective synthesis of amines via reductive amination with a dehydrogenase mutant from Exigobacterium sibiricum: Substrate scope, co-solvent tolerance and biocatalyst immobilization.

In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance. Kinetics as well as inhibition effects were also studied and the suitability of this method for synthetic purpose was demonstrated with acetophenone as a model substrate. Even at an elevated substrate concentration of 50 mM, excellent conversion was achieved. In addition, the impact of water-miscible co-solvents was examined, and good activities were found when using DMSO of up to 30% (v/v). Furthermore, a successful immobilization of the EsLeuDH-DM was demonstrated utilizing a hydrophobic support and a support for covalent binding, respectively, as a carrier.

Exploring the aglycone subsite of a GH11 xylanase for the synthesis of xylosides by transglycosylation reactions.

Xylanases Tx-xyn10 and Tx-xyn11 were compared for their transxylosylation abilities in the presence of various acceptors. Tx-xyn10 exhibited a broad specificity for various acceptors, whereas xylanase Tx-xyn11 catalysed transxylosylation reactions only in presence of polyphenolic acceptors. A modelling approach was developed to study the molecular bottlenecks into the active site of the enzyme that could be responsible for this restricted specificity. The glycosyl-enzyme intermediate of Tx-xyn11 was modelled, and a rotamer of the Y78 residue was integrated. In silico mutations of some residues from the (+1) and (+2) subsites were tested for the deglycosylation step in the presence of non-polyphenolic acceptors. The results indicated that the mutant W126A was able to use aliphatic alcohols and benzyl alcohol as acceptors for transxylosylation. Experimental validation was tested by mutating the xylanase Tx-xyn11 at position W126 into alanine. The specific activity and catalytic efficiency of the W126A mutant during the hydrolysis of xylans decreased by 2-fold and 4-fold, respectively, compared to wild-type xylanase. Among tested acceptors, transxylosylation catalysed by mutant W126A was improved with benzyl alcohol leading to a 2-fold higher concentration of benzyl xylobioside, as predicted by in silico mutation. This improved transxylosylation in the presence of benzyl alcohol leading to higher synthesis of benzyl xylobioside could likely be explained by lowest steric hindrance in the aglycone subsite of the mutated xylanase. No secondary hydrolysis of benzyl xylobioside occurred for both wild-type and mutant xylanases. Finally, our results demonstrated that the modelling approach was limited and that accounting for protein dynamics can lead to improved models.