Presence Of Various Organic Solvents Research Articles

Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance.

Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased KM value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.

Crystallographic and Spectroscopic Snapshots of Multi‐Stimuli‐Responsive Dinuclear Cobalt Metal−Organic Frameworks

AbstractInvestigating the reversible structural transformation of stimuli‐responsive metal–organic frameworks (MOFs) through single‐crystal diffraction and establishing a structure–property relationship pose a significant challenge. Herein, the controllable synthesis of Co‐MOF‐1 and Co‐MOF‐2 by incorporating water into the solvothermal synthesis process is presented. In comparison to the trinuclear [Co3(COO)8]2− clusters found in Co‐MOF‐1, Co‐MOF‐2 presents a variable dinuclear [Co2(COO)4(H2O)5] cluster. Single crystal diffraction studies, utilizing synchrotron radiation, confirm the reversible two‐step transformation. The presence of various organic solvents induces the single‐crystal‐to‐single‐crystal (SC‐to‐SC) transformation of Co‐MOF‐2. This transformation is initiated by the cleavage of three coordinated water molecules and subsequent coordination of carboxylates in the ligand, resulting in solvatochromic behavior. Further, the reversible stimuli‐responsive behavior can also be rapidly induced under the stimuli of heat, vacuum, and X‐rays. This study successfully demonstrates the crystallographic and spectroscopic snapshots of dynamic coordination bonds in MOFs and contributes to a deeper understanding of the structure–property relationship.

Evaluation of Lipase from an Indigenous Isolated Bacillus Strain for Biodiesel Production

Lipases are utilized in biodiesel production utilizing various types of substrates. The use of lipase in bioenergy production aims to reduce energy crises and environmental pollution. Lipase-producing indigenous bacteria Bacillus licheniformis (Accession no. OP56979) and Bacillus rugosus (Accession no. OP56980) were isolated from various oil-contaminated sites. The isolated potential lipolytic bacteria were screened for maximum lipase production. Then, the bacteria showing the highest lipolytic activity were subjected to identification using the 16s rRNA technique while other isolated were identified biochemically. Lipase [LipBL-WII(c)] from Bacillus licheniformis having the highest lipolytic activity expressed various characteristics. Characterization of crude LipBL-WII(c) expressed that it showed stability in a wide range of pH (4 to 10) with optimum lipolytic activity observed at pH 8. It was then found to be active at a temperature range from 20°C to 80°C with optimal at 50°C. Lipase activity was also stimulated in metal ions such as Ca+1, Mg2+, and Zn2+ the most. Furthermore, LipBL-WII(c) retained lipolytic activity in the presence of various organic solvents and surfactants. The kinetic parameters (Km and Vmax) for LipBL-WII(c) were ascertained using Lineweaver- Burk plot. LipBL-WII(c) showed a potential for biodiesel production using olive oil as a source. Lipase gave 84% yield of biodiesel production from olive oil. Thus, it could be employed as a potential candidate for green biodiesel production using oil sources.

A GH42 β-galactosidase from Bifidobacterium thermophilum: Biochemical characterization and synthesis of galactosyl glycerol by reverse hydrolysis.

In this study, a β-galactosidase gene (btgal42) was first cloned from Bifidobacterium thermophilum and successfully expressed in Escherichia coli. The molecular weight of the purified BtGal42 was estimated to be 78kDa by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 225kDa by the size-exclusion chromatography, indicating that the native BtGal42 was a homotrimer. BtGal42 belonged to the glycosidase hydrolase family 42, exhibiting the maximum activity at pH of 7 and at temperature of 50°C. The enzyme displayed a strictly specific activity toward substrates with β-galactosyl linkages at the nonreducing ends, of which the activity on 4-nitrophenyl-β-d-galactopyranoside was the highest, followed by 2-nitrophenyl-β-d-galactopyranoside and lactose. Among the tested metals and reagents, BtGal42 showed tolerance in the presence of various organic solvents. Importantly, BtGal42 exhibited a high reverse hydrolysis activity when using galactose as the donor and the di-alcohol ethylene glycol and the trialcohol glycerol as the acceptors. Under unoptimized reaction conditions, the galactosyl glycerol yield reached 62.2g/L (galactose conversion rate, 41.2%). This study might provide a feasible method for the biosynthesis of galactosyl glycerol from low-cost glycerol and galactose, which was associated with high conversion efficiency and few byproducts.

Identification and Characterization of a Thermostable GH36 α-Galactosidase from Anoxybacillusvitaminiphilus WMF1 and Its Application in Synthesizing Isofloridoside by Reverse Hydrolysis.

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0–9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as Km (0.12 mM), Vmax (1.10 × 10−3 mM s−1), and Kcat/Km (763.92 mM−1 s−1). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.

Characterization of a novel lipase from Bacillus licheniformis NCU CS-5 for applications in detergent industry and biodegradation of 2,4-D butyl ester

Enzymatic degradation has become the most promising approach to degrading organic ester compounds. In this study, Bacillus licheniformis NCU CS-5 was isolated from the spoilage of Cinnamomum camphora seed kernel, and its extracellular lipase was purified, with a specific activity of 192.98 U/mg. The lipase was found to be a trimeric protein as it showed a single band of 27 kDa in SDS-PAGE and 81 kDa in Native-PAGE. It was active in a wide range of temperatures (5–55 °C) and pH values (6.0–9.0), and the optimal temperature and pH value were 40 °C and 8.0, respectively. The enzyme was active in the presence of various organic solvents, metal ions, inhibitors and surfactants. Both crude and purified lipase retained more than 80% activity after 5 h in the presence of commercial detergents, suggesting its great application potential in detergent industry. The highest activity was found to be towards medium- and long-chain fatty acids (C6-C18). Peptide mass spectrometric analysis of the purified lipase showed similarity to the lipase family of B. licheniformis. Furthermore, it degraded more than 90% 2,4-D butyl ester to its hydrolysate 2,4-D within 24 h, indicating that the novel lipase may be applied to degrade organic ester pesticides.

Zinc(II)-Organic Framework Films with Thermochromic and Solvatochromic Applications.

Multiple-stimuli-responsive photoluminescence films based on a ZnII -organic framework, {[Zn2 (Htpim)(3,4-pydc)2 ]⋅4 DMF⋅4 H2 O}n (1, Htpim=2,4,5-tri(4-pyridyl)imidazole, 3,4-H2 pydc=3,4-pyridinedicarboxylic acid), were fabricated. This compound consisted of a 2D corrugated layer, {Zn(3,4-pydc)}n , which was further pillared using a Y-shaped pillar N-donor ligand (Htpim) to form a 3D-pillared-layer framework with 1D open channels. The rectangular channels in the as-synthesized compound are fully occupied by guest DMF and H2 O molecules. The framework exhibits instant and reversible thermochromic properties corresponding to the removal of different H2 O and DMF guest molecules as temperature increases. The pale-yellow crystal undergoes significant redshifting to a greenish emission centered at 530 nm. Compound 1 also showed remarkable solvatochromic effects in the presence of various organic solvents without affecting its structural integrity. In addition, polycrystalline MOF films were grown on an α-Al2 O3 support for switchable and fast-response thermochromic and solvatochromic sensors.

Investigation of Organic Solvents Effects on Kenaf (Hibiscus cannabinus L.) Biomass Conversion in Subcritical Water

Kenaf biomass material was hydrolyzed in subcritical water condition in presence of various organic solvents. The solvents tested were tetrahydrofurane (THF), acetone, xylene and methanol. The organic compounds released into hydrolysates, total organic contents, water soluble total phenol and the molecular weight distributions of the polysaccharides in the hydrolysates, solid residues leftover after hydrolysis and gaseous products formed during the solubilization process were determined. The results showed that organic solvents significantly enhanced the dissolution of kenaf biomass (methanol < xylene ≤ acetone ~ tetrahydrofurane). The hydrolysis percentage was found to be between 75-82% depending on the type of the solvent. Hydrolysis yield and total organic carbons released into hydrolysates highly differed when the solubilization process was performed under carbondioxide pressure and this effect considerably varied based on the type of solvent used in hydrolysis process. The main gas product formed during hydrolysis process was carbon dioxide with ~80% composition. Morphological measurements of the solid biomass residues left after hydrolysis showed substantial degradations with increasing number of pores on the biomass surfaces.

Biochemical and structural characterization of 3‐ketosteroid‐Δ1‐dehydrogenase, a candidate enzyme for efficient bioconversion of steroids

AbstractBackground3‐ketosteroid‐Δ1‐dehydrogenase, a key enzyme involved in microbial catabolism of sterols, catalyzes the 1, 2‐desaturation of steroidal substrates using FAD as a cofactor. Recombinant 3‐ketosteroid‐Δ1‐dehydrogenase from Arthrobacter simplex (KsdD4) was expressed in Escherichia coli BL21 (DE3).ResultsKsdD4 exhibited optimal activity at pH 7.0 and 35°C. KsdD4 had the highest activity toward 4‐androstene‐3,17‐dione (AD) and a moderate activity toward cortisone acetate and hydrocortisone. Structure‐based homology modeling revealed that Y118, Y355, Y528 and G532 are located in the active site, thus the most likely catalytic residues. The substrate‐binding cavity was composed of hydrophobic residues with small side chains, including A49, V328, A390 and A531, which facilitate the recognition of steroidal substrates with large C17 side‐chains. E332 forms a unique hydrogen bond with substrates. Recombinant E. coli resting cells expressing KsdD4 showed high catalytic activity in the presence of various organic solvents. A fed‐batch bioconversion using resting cells resulted in efficient production of 2.66 g L−1 androst‐1,4‐diene‐3,17‐dione (ADD).ConclusionBiochemical data and structural analysis greatly advanced our understanding of the KsdD family enzymes, and the fed‐batch strategy improved the bioconversion of steroids. © 2018 Society of Chemical Industry

Biochemical Characterization of Lipases Obtained from <i>Acinetobacter psychrotolerans</i> Strains

In this study, extracellular lipases obtained from Acinetobacter psychrotolerans strains (Xg1 and Xg2) were characterized. The effects of varying pH values (3.0-10.0) and various temperatures (10-90 °C) on lipase activities were examined. Also the effects of different metal ions, organic solvents and detergents on lipases were studied. The extracellular crude lipases were concentrated using ultrafiltration. Zymogram analysis of these lipases was performed. Lipases exhibited maximum activity at pH 8 and 30 °C. While lipase obtained from the Xg1 strain exhibited the highest stability in the presence of various organic solvents, including hexane, ethyl acetate, chloroform and N,N dietil formamide, lipase obtained from the Xg2 strain was sensitive in the presence of isopropanol, acetonitrile, and butan-1-ol. The lipases of the Xg1 and Xg2 strains were inhibited in the presence of Cu 2+ and Zn 2+ . Also, the lipase of the Xg1 strain was inhibited in the presence of Fe 3+ . In the presence of EDTA, the lipase activities of the Xg1 and Xg2 strains were partially inhibited. In presence of SDS, they were exactly inhibited. According to the zymogram results, the molecular weights of the lipases obtained from the Acinetobacter psychrotolerans Xg1 and Xg2 strains have been found approximately 37 and 30 kDa, respectively.

Characterization of pH-tolerant and thermostable GH 134 β-1,4-mannanase SsGH134 possessing carbohydrate binding module 10 from Streptomyces sp. NRRL B-24484

A GH 134 β-1,4-mannanase SsGH134 from Streptomyces sp. NRRL B-24484 possesses a carbohydrate binding module (CBM) 10 and a glycoside hydrolase 134 domain at the N- and C-terminal regions, respectively. Recombinant SsGH134 expressed in Escherichiacoli. SsGH134 was maximally active within a pH range of 4.0-6.5 and retained >80% of this maximum after 90min at 30°C within a pH range of 3.0-10.0. The β-1,4-mannanase activity of SsGH134 towards glucomannan was 30% of the maximal activity after an incubation at 100°C for 120min, indicating that SsGH134 is pH-tolerant and thermostable β-1,4-mannanase. SsGH134, SsGH134-ΔCBM10 (CBM10-linker-truncated SsGH134) and SsGH134-G34W (substitution of Gly34 to Trp) bound to microcrystalline cellulose, β-mannan and chitin, regardless of the presence or absence of CBM10. These indicate that GH 134 domain strongly bind to the polysaccharides. Although deleting CBM10 increased the catalytic efficiency of the β-1,4-mannanase, its disruption decreased the pH, solvent and detergent stability of SsGH134. These findings indicate that CBM10 inhibits the β-1,4-mannanase activity of SsGH134, but it is involved in stabilizing its enzymatic activity within a neutral-to-alkaline pH range, and in the presence of various organic solvents and detergents. We believe that SsGH134 could be useful to a diverse range of industries.

Detergent‐Compatible Robust Alkaline Protease from Newly Isolated Halotolerant Salinicoccus sp. UN‐12

AbstractCurrently, enzyme‐containing blended detergent preparations are favored in the detergent industry. The detergent compatibility of an enzyme depends on its robustness to withstand harsh operating conditions and maintain efficient functioning in the presence of various surfactants and detergents. Alkaline proteases from halotolerant microbes having the capability to work in demanding environments are desirable for their application in the detergent industry. The protease secreted by a halotolerant soil isolate Salinicoccus sp. UN‐12 was evaluated for its suitability in detergent formulations. The studied enzyme is an alkaline serine protease (63 kDa) with an optimum temperature of 55 °C and optimum pH of 8.5. The protease was purified 17‐fold with a 4% yield. The protease was active in NaCl (up to 4 mol) and showed enhanced activity in the presence of CaCl2, KCl, and MnCl2. In addition, the proteolytic activity was sustained in the presence of various organic solvents, cyclodextrin, disodium cocoamphodiacetate, ionic liquids, hydrotropes, and ultrasound. This report is the first to document the multifarious robustness and novel attributes of a protease secreted by the newly isolated Salinicoccus sp. UN‐12. The protease‐detergent formulation efficiently removes blood, egg, grass, and tomato sauce stains. This protease is now available for efficient and eco‐friendly applications in the detergent industry.

The use of various organic solvents to tailor the properties of ibuprofen–glucosamine HCl solid dispersions

Ibuprofen is a Biopharmaceutical classification system class II drug that exhibits poor dissolution rate in the gastrointestinal tract. The aim of the present study is to enhance the dissolution of ibuprofen in presence of glucosamine. To this end, different ratios of ibuprofen:glucosamine were dissolved in various organic solvents to obtain the solid dispersions of ibuprofen–glucosamine mixtures. The solid state analysis of the samples (differential scanning calorimetry, Fourier infrared spectroscopy and X-ray powder diffraction) and the morphology (scanning electron microscope) were investigated. Particle size analysis and in vitro dissolution studies showed that the type of solvent has a significant influence on dissolution rate. Ibuprofen–glucosamine solid dispersions obtained from acetone produced better dissolution compared to that of other organic solvents. The effect of water in binary mixtures of either acetone or ethanol was also explored and the results showed that when the ratio of acetone to water was 75:25, the highest dissolution was obtained. Solid state analysis ruled out any chemical interaction between the drug and carrier even in the presence of various organic solvents which indicates a good stability of the solid dispersions to enhance the dissolution rate of ibuprofen. It was also investigated via XRPD analysis that the ibuprofen retained its crystallinity without any adverse effect on the dissolution rates. In conclusion, the present study showed that a manipulation of the organic solvent:water ratio and a change in the type of organic solvent impacted on ibuprofen–glucosamine achieving very high dissolution rate without compromising its crystallinity and physical stability.

Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India.

In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg−1 and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0–14.0 pH and 30–80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of β-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel β-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.

Polyurethane Synthesis from Sugarcane Bagasse Organosolv and Kraft Lignin

Sugarcane bagasse (SCB) was subjected to a single-step fractionation and hydrolysis reaction in the presence of various organic solvents. The reaction was performed at 170 °C for 3 h when sodium hydroxide was used as catalyst. The results showed that suitable solvents significantly enhanced the yield of lignin fractionation and simultaneous hydrolysis reaction took place indicated by an increase of hydroxyl groups in lignin molecules. Lignin-based polyurethane (LPU) from SCB organosolv lignin polyols had relatively better mechanical and thermal resistant properties compared to LPU from liquefied Kraft lignin from pulp and paper industry.

Hide Dehairing and Laundry Detergent Compatibility Testing of Thermostable and Solvents Tolerant Alkaline Protease from Hot Spring Isolate <i>Bacillus cohnii</i>U3

In the present study, alkaline protease producing thermophilic Bacillus cohniiU3 strain was isolation from Unnai hot spring, India. The maximum production of protease (344 U/mL) was reported after 72 h of inoculation at 50°C in shake flask culture using the gelatin casein medium. The protease was found to remain active up to 96 h and production was growth dependent. The activity of parital purified protease was reported in abroad range of pH ranging from 4.0 to 11.0 with an optimum of 9.0 pH; Enzymes was indicated the highest activity at 50°C temperature. Salt independent catalysis and activity with a broad range of substrate concentration is the key feature of the protease. Thermos table nature, the stability in alkaline pH and stability in high salt concentration for 45 min were outstanding features of protease. The enzyme was stable in the presence of various organic solvents like Dimethyl Sulphoxide (DMSO), Methanol, Butanol, n-Hexane, Benzene at 25% (v/v) concentration. A good compatibility of the enzyme with most commercial detergents indicated its application in detergent industry. The remarkable dehairing in goat hide and destaining of blood spot in 2 h using 10 U/mL of protease assure that it could be a potential candidate for leather and detergent industries.

Isolation of an organic solvent‐tolerant bacterium Bacillus licheniformis PAL05 that is able to secrete solvent‐stable lipase

In this study, seven lipase-producing bacterial strains were isolated from salt-enriched and cattle farm soil samples after incubation in toluene- and benzene-enriched media. One strain (PAL05) showed significantly greater lipase activity on spirit blue agar medium and stability in organic solvents. The positive strain (PAL05) was identified as Bacillus licheniformis by 16S rRNA gene sequencing. Lipase production was optimized in a medium containing glycerol as the carbon source and Tween 80 as an inducer (0.5% glycerol+0.5% Tween 80) at pH 8.0 and a temperature of 30 °C. In addition, the enzyme was moderately halotolerant as it exhibited increased activity in the presence of 2.5% NaCl. Optimized conditions increased the lipase production threefold. Crude lipase retained its activity for 14 days of incubation in the presence of various organic solvents at a level of 25% and 50%. The enzyme was stable at 25% in most solvents; some of the solvents such as hexane, benzene, and ethanol actually stimulated enzyme activity. The organic solvent stability of the lipase produced by the strain PAL05 enables the enzyme to be used as a potential biocatalyst for ester synthesis and other applications in nonaqueous conditions.

Isolation, Characterization and investing the Industrial Applications of Thermostable and Solvent Tolerant Serine Protease from Hot Spring Isolated Thermophililic Bacillus licheniformis U1

Protease is the largest selling enzyme in the world due to its various applications in the making of detergent, food and leather, meat tenderisation and pharmaceutical industries. The aim of the study is to isolate and identify thermophilic Bacillus licheniformis U1 strains for thermostable protease production. The partial purified enzyme was characterized under different conditions using Anson-Hagihara’s method. Casein as a substrate in the concentration of 0.6 % w/v optimum for enzyme activity and tolerant up to 2.0% casein concentration. An optimum enzyme activity was reported at pH 7 and decreased with increasing in pH, while temperature optimum was found at 50 °C. The enzyme was stable at 40 °C to 50 °C for half an hour and nearly 50% residual activity was indicated at 60 °C. NaCl was not required for catalysis. Stability of enzymes in the presence of various organic solvents and different detergents was remarkable. The enzyme was stable up to 3 days into various solvents and slowly denatured with prolonged incubation. The result of the washing performance with detergent was clearly indicated. Moreover the removal of blood stains and dehairing in goat skin suggests the crucial application in the commercial production at large scale.DOI: http://dx.doi.org/10.3126/ijasbt.v2i1.9519Int J Appl Sci Biotechnol, Vol. 2(1): 75-82

Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool

Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger. All enzymes were active toward p-nitrophenyl-β-d-galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose β-1,4 linked to either N-acetylglucosamine or fucose, such as N-glycans derived from bovine fibrin and Caenorhabditis elegans. In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae, all enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-013-5192-3) contains supplementary material, which is available to authorized users.

Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

Enzymes of the isoprenoid biosynthetic pathway in halophilic archaea remain poorly characterized, and parts of the pathway remain cryptic. This situation may be explained, in part, by the difficulty of expressing active, functional recombinant forms of these enzymes. The use of newly available expression plasmids and hosts has allowed the expression and isolation of catalytically active Haloferax volcanii 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (EC 2.3.310). This accomplishment has permitted studies that represent, to the best of our knowledge, the first characterization of an archaeal hydroxymethylglutaryl CoA synthase. Kinetic characterization indicates that, under optimal assay conditions, which include 4 M KCl, the enzyme exhibits catalytic efficiency and substrate saturation at metabolite levels comparable to those reported for the enzyme from nonhalophilic organisms. This enzyme is unique in that it is the first hydroxymethylglutaryl CoA synthase that is insensitive to feedback substrate inhibition by acetoacetyl-CoA. The enzyme supports reaction catalysis in the presence of various organic solvents. Haloferax 3-hydroxy-3-methylglutaryl CoA synthase is sensitive to inactivation by hymeglusin, a specific inhibitor known to affect prokaryotic and eukaryotic forms of the enzyme, with experimentally determined Ki and kinact values of 570 ± 120 nM and 17 ± 3 min(-1), respectively. In in vivo experiments, hymeglusin blocks the propagation of H. volcanii cells, indicating the critical role that the mevalonate pathway plays in isoprenoid biosynthesis by these archaea.