Stable Enzyme Research Articles

Genomics of Alkaliphiles.

Alkalinicity presents a challenge for life due to a "reversed" proton gradient that is unfavourable to many bioenergetic processes across the membranes of microorganisms. Despite this, many bacteria, archaea, and eukaryotes, collectively termed alkaliphiles, are adapted to life in alkaline ecosystems and are of great scientific and biotechnological interest due to their niche specialization and ability to produce highly stable enzymes. Advances in next-generation sequencing technologies have propelled not only the genomic characterization of many alkaliphilic microorganisms that have been isolated from nature alkaline sources but also our understanding of the functional relationships between different taxa in microbial communities living in these ecosystems. In this review, we discuss the genetics and molecular biology of alkaliphiles from an "omics" point of view, focusing on how metagenomics and transcriptomics have contributed to our understanding of these extremophiles. Graphical Abstract.

Isolation, screening and identification of haloarchaea with chitinolytic activity from hypersaline lakes of Iran

Halophiles produce stable enzymes under extreme conditions. The scant information about chitinolytic haloarchaea led us to conduct the present study in order to isolate and screen native halophilic archaea with chitinolytic activity and to optimize the enzyme production conditions. Among 500 haloarchaeal strains isolated from water samples from different hypersaline lakes of Iran, five strains showed chitinolytic activity. Based on biochemical, morphological and molecular analyses, we established that all five potent strains belonged to the genus Natrinema. Besides, observing chitinase function in culture media, through an additional molecular test the presence of the chitinase gene in chitinase-producing strains was also confirmed by PCR amplification. Compared with other potent strains, Natrinema sp. strain BS5 showed significant chitinase production. The production of chitinase in strain BS5 accompanied growth, started at the logarithmic phase and increased to its maximum level at the beginning of the stationary phase. Maximum chitinase production was obtained at 37?C, pH 7.5, 3 M NaCl and 1% colloidal chitin. The strain BS5 showed 38%, 30%, 24% and 28% decreases in enzyme production at 40?C, pH 8, 3.5 M NaCl and 0.5% substrate, respectively. This strain was able to produce the enzyme in NaCl 4 M and in the absence of MgCl2 and MgSO4. This study revealed the strong potential of the genus Natrinema to produce chitinase at high salt concentrations without Mg2+ requirement.

Purification and Partial Characterization of α-Amylase Produced by a Thermo-Halophilic Bacterium Isolate PLS 75

Bio-based industries require stable enzymes in a broad range of environmental conditions. Extremophiles have attracted more interests as the source of such enzymes, one of which is α-amylase. This study aimed to purify and characterize α-amylase produced by a thermo-halophilic bacterium PLS 75 isolated from underwater fumaroles. Ammonium sulfate precipitation results showed that the highest specific α-amylase activity (21.7 U/mg) obtained at 40-60% saturation level, with a purity of 7.7-fold of the crude extract with 16.2% yield. Further purification using DEAE Sepharose column chromatography increased the enzyme purity 11.1-fold of the crude extract with 7.1% yield. Specific activity after column chromatography purification was 31.3 U/mg. The pure enzyme had a low molecular weight of 14 kDa. The enzyme showed the highest activity at 80 °C and pH 5. The activity increased to 126% when in methanol, while decreased when in ethyl acetate and chloroform. The characteristics of α-amylase with low molecular weight, which was active in acidic condition, stable in polar and non-polar solvents, may be used for for specific industrial needs.

Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles.

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0 ± 0.1μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe3O4@Chitosan@Xylanase in the harsh temperature condition of relevant industries.

Repetitive Batch Mode Facilitates Enzymatic Synthesis of the Nucleotide Sugars UDP-Gal, UDP-GlcNAc, and UDP-GalNAc on a Multi-Gram Scale.

The availability of nucleotide sugars is considered as bottleneck for Leloir-glycosyltransferases mediated glycan synthesis. A breakthrough for the synthesis of nucleotide sugars is the development of salvage pathway like enzyme cascades with high product yields from affordable monosaccharide substrates. In this regard, the authors aim at high enzyme productivities of these cascades by a repetitive batch approach. The authors report here for the first time that the exceptional high enzyme cascade stability facilitates the synthesis of Uridine-5'-diphospho-α-d-galactose (UDP-Gal), Uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), and Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in a multi-gram scale by repetitive batch mode. The authors obtained 12.8 g UDP-Gal through a high mass based total turnover number (TTNmass ) of 494 [gproduct /genzyme ] and space-time-yield (STY) of 10.7 [g/L*h]. Synthesis of UDP-GlcNAc in repetitive batch mode gave 11.9 g product with a TTNmass of 522 [gproduct /genzyme ] and a STY of 9.9 [g/L*h]. Furthermore, the scale-up to a 200 mL scale using a pressure operated concentrator was demonstrated for a UDP-GalNAc producing enzyme cascade resulting in an exceptional high STY of 19.4 [g/L*h] and 23.3 g product. In conclusion, the authors demonstrate that repetitive batch mode is a versatile strategy for the multi-gram scale synthesis of nucleotide sugars by stable enzyme cascades.

Biomimetic oxidase sensor based on functionalized surface of carbon nanotubes and iron prophyrins for catechol detection.

A novel and highly stable biomimetic oxidase sensor system was designed for catehol detection. FePP used as biomimetic horseradish peroxidase (HRP) was immobilized onto modified multi-walled carbon nanotubes (MWCNTs). Functional groups such as -OH, -NH2 and -COOH were introduced onto the surface of MWCNTs to provide biomimetic microenvironment for iron porphyrins (FePP). Stable biomimetic enzyme electrode has been developed to detect catechol as a simple, economical and efficient method. At optimal condition, the detection limit of OH-MWCNTs/FePP/Nafion was 3.754 × 10- 6 M. After stored at -4°C for 35 days, the oxidation current value still maintained 98.3% of initial activity. In repetitive nature test, relative standard deviation (RSD) of oxidation current remained within 1.0% after ten consecutive measurements in the same concentration of catechol solution, while most of reported oxidase sensor was within 2.0% under the same condition.

RNA-Mediated Dimerization of the Human Deoxycytidine Deaminase APOBEC3H Influences Enzyme Activity and Interaction with Nucleic Acids

Human APOBEC3H is a single-stranded (ss)DNA deoxycytidine deaminase that inhibits replication of retroelements and HIV-1 in CD4+ T cells. When aberrantly expressed in lung or breast tissue, APOBEC3H can contribute to cancer mutagenesis. These different activities are carried out by different haplotypes of APOBEC3H. Here we studied APOBEC3H haplotype II, which is able to restrict HIV-1 replication and retroelements. We determined how the dimerization mechanism, which is mediated by a double-stranded RNA molecule, influenced interactions with and activity on ssDNA. The data demonstrate that the cellular RNA bound by APOBEC3H does not completely inhibit enzyme activity, in contrast to other APOBEC family members. Despite degradation of the cellular RNA, an approximately 12-nt RNA remains bound to the enzyme, even in the presence of ssDNA. The RNA-mediated dimer is disrupted by mutating W115 on loop 7 or R175 and R176 on helix 6, but this also disrupts protein stability. In contrast, mutation of Y112 and Y113 on loop 7 also destabilizes RNA-mediated dimerization but results in a stable enzyme. Mutants unable to bind cellular RNA are unable to bind RNA oligonucleotides, oligomerize, and deaminate ssDNA in vitro, but ssDNA binding is retained. Comparison of A3H wild type and Y112A/Y113A by fluorescence polarization, single-molecule optical tweezer, and atomic force microscopy experiments demonstrates that RNA-mediated dimerization alters the interactions of A3H with ssDNA and other RNA molecules. Altogether, the biochemical analysis demonstrates that RNA binding is integral to APOBEC3H function.

Stable amylaze-produsing micromycetes isolated from soils of South Caucasus Primary tabs

Production of stable enzymes is one of the actual problems in bio- and enzyme technology. Conducting of the fermentation processes at pasteurization temperature is of great importance (2800 strains) because of making possible to minimize pollution of the reaction medium. Collection of micellar fungi isolated from different ecological niches of the Caucasus has been created in Durmishidze Institute of Biochemistry and Biotechnology. 39 strains - producers of amylase were revealed in the collection as a result of screening. Most of these strains belong to the genus Aspergillus. The temperature optimum of thermophilic strains was studied. In the cultural liquids obtained after the submerged cultivation of selected strains a-amylase producers were tested in the temperature range 30-45 oC, at 5 oC intervals. The temperature optimum of the strains was established to be within the range 65-70 oC, making possible to use them in bio- and enzymatic technologies to diminish the pollution of the reaction medium while conducting the fermentation process at pasteurization temperature (65 oC). Consequently, the selection of stable, operable at pasteurization and higher temperatures amylase preparation is of great importance. Proposed technology is cost-effective, ecologically safe, competitive and provides deep hydrolysis of starch to fermentable sugar at the elevated temperature (65-70 oC) in one step, by means of only one, stable amylase preparation.

Superactive β-galactosidase inclusion bodies.

Bacterial inclusion bodies (IBs) were historically considered one of the major obstacles in protein production through recombinant DNA techniques and conceived as amorphous deposits formed by passive and rather unspecific structures of unfolded proteins aggregates. Subsequent studies demonstrated that IBs contained an important quantity of active protein. In this work, we proved that recombinant β-galactosidase inclusion bodies (IBβ-Gal) are functional aggregates. Moreover, they exhibit particular features distinct to the soluble version of the enzyme. The particulate enzyme was highly active against lactose in physiological and in acid pH and also retained its activity upon a pre-incubation at high temperature. IBβ-Gal washing or dilution induced the spontaneous release of active enzymes from the supramolecular aggregates. Along this process, we observed a continuous change in the values of several kinetic parameters, including specific activity and Michaelis-Menten constant, measured in the IBβ-Gal suspensions. Simultaneously, IBβ-Gal turned into a more heterogeneous population where smaller particles appeared. The released protein exhibited secondary structure features more similar to those of the soluble species than to the aggregated enzyme. Concluding, IBβ-Gal represents a reservoir and packed source of highly active and stable enzyme.

Biochemical characterization of a stable azoreductase enzyme from Chromobacterium violaceum: Application in industrial effluent dye degradation

The presence of dye, including azo functional group (NN) containing dyes, in industrial waste water is one of the major causes of water pollution. This report showcases the functional role of azoreductase from Chromobacterium violaceum (MTCC No: 2656) as a valuable enzyme for degradation of azo dyes. The enzyme was cloned, expressed, purified and biochemically characterized and further tested for degradation efficiency of azo group containing dyes like methyl red, amaranth and methyl orange. The degraded azo dye products (metabolites) resulted by the action of azoreductase enzyme had reduced toxicity on fibroblast cell lines (L929) as compared to raw and intact dye. Further, good stability of the enzyme makes it more suitable for various applications related to the degradation and decolourisation of effluent dyes.

An Unusual Two‐Step Hydrolysis of Nerve Agents by a Nanozyme

AbstractOrganophosphate‐based nerve agents irreversibly inhibit acetylcholinesterase enzyme, leading to respiratory failure, paralysis and death. Several organophosphorus hydrolases are capable of degrading nerve agents including pesticides and insecticides. Development of stable artificial enzymes capable of hydrolysing nerve agents is important for the degradation of environmentally toxic organophosphates. Herein, we describe a Zr‐incorporated CeO2 nanocatalyst that can be used for an efficient capture and hydrolysis of nerve agents such as methyl paraoxon to less toxic monoesters. This unusual sequential degradation pathway involves a covalently linked nanocatalyst‐phosphodiester intermediate.

Use of Candida rugosa lipase as a biocatalyst for L-lactide ring-opening polymerization and polylactic acid production

Abstract Candida rugosa lipase (CRL, EC Number: 232–619-9) is a stable enzyme, and its use is relatively widespread in the field of biotechnology, especially in hydrolysis reactions, esterification, transesterification and enantioselective biotransformation. This study examined the role of CRL as a biocatalyst for L-lactide ring-opening polymerization in the production of metal-free polylactic acid (PLA). The ring-opening polymerization reaction was conducted at various temperatures (70, 90, 110 and 130 °C) and CRL concentrations (1, 2, 3 (%w/w)). The results revealed a strong relationship between PLA formation and CRL activity. The highest CRL activity was obtained in ring-opening polymerization of L-lactide at a temperature of 90 °C and concentration of 2% (w/w). Under these conditions, the weight-average molecular weight (Mw) and number-average molecular weight (Mn) of PLA analyzed by gel permeation chromatography was highest at 5428 and 2854 g/mol respectively, with a yield of 93% and the enzyme activity 0.39 U. The polymerization of lactide at 90 °C occurred only in the presence of the catalyst. The crystallinity and melting point of PLA were 31% and 120 °C, respectively. Scanning electron microscopy (SEM) analysis of the morphology of PLA revealed smooth and uniform pores in each region, with mass percentage of the elements carbon (C) and oxygen (O) of 52% and 48%, respectively.

A L-proline/O2 biofuel cell using L-proline dehydrogenase (LPDH) from Aeropyrum pernix.

To utilize amino acids from food waste as an energy source, L-proline/O2 biofuel cell was constructed using a stable enzyme from hyperthermophilic archaeon for long-term operation. On the anode, the electrocatalytic oxidation of L-proline by L-proline dehydrogenase from Aeropyrum pernix was observed in the presence of ferrocenecarboxylic acid as mediator. On the cathode, electrocatalytic oxygen reduction was detected. Ketjenblack modification of carbon cloth substrate increased the current density due to increased laccase loading and enhanced electron transfer reaction. The biofuel cell using these electrodes achieved a current density of 6.00µA/cm2. We successfully constructed the first biofuel cell that generates power from L-proline.

Effect of disulfide bridge on hydrolytic characteristics of xylanase from Penicillium janthinellum

Highly efficient and stable enzymes are required for application in biotechnology, to meet the technical, environmental, and economic industrial demands. Xylanases are hemicellulolytic enzymes that degrade the heteroxylan constituent of the lignocellulosic plant cell wall. In this study, an acidic xylanase designated Pjxyn (pH 4.0) from Penicillium janthinellum was engineered by the introduction of a disulfide bridge. This strategy exploited the influence of the bridge on hydrolysis characteristics and enhanced hydrolysis was achieved. Three mutants [PjxynS(27)S(39), PjxynS(27)S(186), and PjxynS(39)S(186)] produced more xylose and xylobiose as hydrolysis products compared with the wild-type Pjxyn, when commercial xylans and lab-prepared water-insoluble corncob-xylan were used as the substrates, especial for the PjxynS(27)S(39) mutant, the content of xylose and xylobiose was 87.62% (using beechwood xylan) and 69.91% (using oat-spelt xylan) higher than that in the hydrolysis products of Pjxyn. Moreover, each mutant combined with the xylanase mutant T-XynFM effectively decreased the production of xylose with an optimum xylobiose yield. The findings demonstrate the potential industrial value of engineering xylanase to improve its hydrolytic properties and thermostability.

Active biocatalyst for biodiesel production from spent coffee ground

Physical adsorption preserving activity and support reusability was used to directly bond lipase from Thermomyces lanuginosus on citric acid (CA) modified Fe3O4/Au magnetic nanoparticles. A new faster approach has been used for CA ligand exchange, which ensures an high payload of stable enzyme. The immobilized lipase was tested for the biodiesel production from spent coffee ground in a solvent free system. It retains, after 60 days, more than 90% of its initial activity. Biodiesel yield of 51.7%, after 3 h of synthesis, which increases up to ∼100% after 24 h indicating an enzymatic fast kinetic, was measured. No significant decrease, during the first three cycles of use, of the lipase activity occurs. The biodiesel presents an ester content of 98.4 ± 0.23 in agreement with the EN14214. The iodine value of 76.67 (g iodine/100 g) is in agreement with the European standard.

Preparation of Expoxy-Functionalized Magnetic Nanoparticles for Immobilization of Glycerol Dehydrogenase.

Immobilization of glycerol dehydrogenase (GDH) from Serratia marcescens H30 onto epoxy functional magnetic nanoparticles by covalent attachment was carried out. The optimal immobilization conditions were obtained as follows: enzyme/support 6.08 mg/g, temperature 25 °C, pH 7.0 and time 8 h. Under these conditions, a high immobilization yield above 90% was obtained. The characterization of the immobilized GDH indicated that enhanced pH and thermal stability were achieved. Kinetic parameters Km of free and immobilized GDH were determined as 10.35 mM and 15.76 mM, respectively. The immobilized GDH retained about 85% initial activity after ten cycles. These results suggested that GDH immobilized onto magnetic nanoparticles is a simple and efficient way for preparation of stable enzyme. And the immobilized GDH has potential applications in the production of DHA.

Partial purification and characterization of the quality deteriorating enzymes from Indian pink guava (Psidium guajava L.), var. Lalit.

Peroxidase (POD), polyphenol oxidase (PPO), ascorbate oxidase (AO) and pectin methylesterase (PME) from 'Lalit' guava variety have been purified using BioGel P-100 and UNOsphere Q resins. POD, PPO, AO, and PME were partially purified to 28, 30, 36 and 30 fold; single band on SDS-PAGE represented a molecular weight of 21, 70, 75, 50kDa, respectively. Optimum pH for POD and AO was 6.5 whereas, PPO and PME showed optimum pH in the alkaline range of pH 8.0 and 8.5, respectively. AO showed optimum activity at 25°C, POD and PPO showed optimum activity at 35°C; however, PME showed maximum activity at 55°C. AO was the most heat labile whereas, PME was the most heat stable enzyme studied and may carry an important role in the thermal processing of guava pulp as an indicator of the efficacy of treatment.

Engineering of Saccharomyces cerevisiae for enhanced production of L-lactic acid by co-expression of acid-stable glycolytic enzymes from Picrophilus torridus

L-lactic acid, as a monomer of polylactic acid, has attracted much attention because of the growing market for biodegradable bioplastics to reduce landfill waste. As an industrial L-lactic acid producer, Saccharomyces cerevisiae is generally used because it survives in low pH. However, in S. cerevisiae, production of L-lactic acid causes a decrease in intracellular pH, which leads to slow glycolytic flux, and consequently results in a lower productivity of L-lactic acid. For this reason, yeast strains that maintain their growth and the activities of metabolic enzymes during lactic acid production need to be developed for industrial applications. Herein, acid stable enzymes from acidophilic archaea Picrophilus torridus were expressed in L-lactic acid producing S. cerevisiae to increase glycolytic flux at low intracellular pH conditions for a higher L-lactic acid titer. Enzymes of lower glycolysis including phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase from P. torridus were introduced to develop a novel L-lactic acid producing strain. It was clearly shown that the production of lactic acid in the developed strain increased by 20% compared to the parental strain. To the best of our knowledge, this is the first report of P. torridus enzymes used in metabolic engineering to enhance the metabolic flux at a lower intracellular pH. Moreover, it is expected that the new strain will have an enhanced glycolytic flux at a low pH expressing acid stable enzymes that could be used to produce other valuable organic acids with increased titers.

Analysis of Cell Viability by the Lactate Dehydrogenase Assay.

A common method for determining cytotoxicity is based on measuring the activity of cytoplasmic enzymes released by damaged cells. Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is found in all cells. LDH is rapidly released into the cell culture supernatant when the plasma membrane is damaged, a key feature of cells undergoing apoptosis, necrosis, and other forms of cellular damage. LDH activity can be easily quantified by using the NADH produced during the conversion of lactate to pyruvate to reduce a second compound in a coupled reaction into a product with properties that are easily quantitated. This protocol measures the reduction of a yellow tetrazolium salt, INT, by NADH into a red, water-soluble formazan-class dye by absorbance at 492 nm. The amount of formazan is directly proportional to the amount of LDH in the culture, which is, in turn, directly proportional to the number of dead or damaged cells.

AR Signaling in Prostate Cancer Regulates a Feed-Forward Mechanism of Androgen Synthesis by Way of HSD3B1 Upregulation.

3βHSD1 enzymatic activity is essential for synthesis of potent androgens from adrenal precursor steroids in prostate cancer. A germline variant in HSD3B1, the gene that encodes 3βHSD1, encodes for a stable enzyme, regulates adrenal androgen dependence, and is a predictive biomarker of poor clinical outcomes after gonadal testosterone deprivation therapy. However, little is known about HSD3B1 transcriptional regulation. Generally, it is thought that intratumoral androgen synthesis is upregulated after gonadal testosterone deprivation, enabling development of castration-resistant prostate cancer. Given its critical role in extragonadal androgen synthesis, we sought to directly interrogate the transcriptional regulation of HSD3B1 in multiple metastatic prostate cancer cell models. Surprisingly, we found that VCaP, CWR22Rv1, LNCaP, and LAPC4 models demonstrate induction of HSD3B1 upon androgen stimulation for approximately 72 hours, followed by attenuation around 120 hours. 3βHSD1 protein levels mirrored transcriptional changes in models harboring variant (LNCaP) and wild-type (LAPC4) HSD3B1, and in these models androgen induction of HSD3B1 is abrogated via enzalutamide treatment. Androgen treatment increased flux from [3H]-dehydroepiandrosterone to androstenedione and other downstream metabolites. HSD3B1 expression was reduced 72 hours after castration in the VCaP xenograft mouse model, suggesting androgen receptor (AR) regulation of HSD3B1 also occurs in vivo. Overall, these data suggest that HSD3B1 is unexpectedly positively regulated by androgens and ARs. These data may have implications for the development of treatment strategies tailored to HSD3B1 genotype status.