Specific Activity Of Enzyme Research Articles

A navitoclax-loaded nanodevice targeting matrix metalloproteinase-3 for the selective elimination of senescent cells

Cellular senescence is implicated in the occurrence and progression of multiple age-related disorders. In this context, the selective elimination of senescent cells, senolysis, has emerged as an effective therapeutic strategy. However, the heterogeneous senescent phenotype hinders the discovery of a universal and robust senescence biomarker that limits the effective of senolytic with off-target toxic effects. Therefore, the development of more selective strategies represents a promising approach to increase the specificity of senolytic therapy. In this study, we have developed an innovative nanodevice for the selective elimination of senescent cells (SCs) based on the specific enzymatic activity of the senescent secretome. The results revealed that when senescence is induced in proliferating WI-38 by ionizing radiation (IR), the cells secrete high levels of matrix metalloproteinase-3 (MMP-3). Based on this result, mesoporous silica nanoparticles (MSNs) were loaded with the senolytic navitoclax (Nav) and coated with a specific peptide which is substrate of MMP-3 (NPs(Nav)@MMP-3). Studies in cells confirmed the preferential release of cargo in IR-induced senescent cells compared to proliferating cells, depending on MMP-3 levels. Moreover, treatment with NPs(Nav)@MMP-3 induced a selective decrease in the viability of SCs as well as a protective effect on non-proliferating cells. These results demonstrate the potential use of NPs to develop enhanced senolytic therapies based on specific enzymatic activity in the senescent microenvironment, with potential clinical relevance. Statement of significanceThe common β-galactosidase activity has been exploited to develop nanoparticles for the selective elimination of senescent cells. However, the identification of new senescent biomarkers is a key factor for the development of improved strategies. In this scenario, we report for the first time the development of NPs targeting senescent cells based on specific enzymatic activity of the senescent secretome. We report a navitoclax-loaded nanodevice responsive to the matrix metalloproteinase-3 (MMP-3) associated with the senescent phenotype. Our nanosystem achieves the selective release of navitoclax in an MMP-3-dependent manner while limiting off-target effects on non-senescent cells. This opens the possibility of using nanoparticles able to detect an altered senescent environment and selectively release its content, thus enhancing the efficacy of senolytic therapies.

Lactic acid production enhancement using metagenome-derived bifunctional enzyme immobilized on chitosan-alginate/nanocellulose hydrogel

Lactic acid (LA) production from renewable and abundant lignocellulosic resources has received great interest due to its various applications in food, cosmetic, pharmaceutical, and textile industries. Herein, the efficient immobilized bi-functional enzyme on an innovative hydrogel (nano)carrier with boosted stability/activity were prepared and utilized for hydrolyzing lignocellulosic biomass to enhance the fermentative production of LA. First, different samples of sodium alginate (SA)-chitosan (CH) hydrogel grafted to acrylic acid and N-isopropylacrylamide and reinforced with nanocellulose (NCs) were synthesized. Selected hydrogels of pristine SA-CH hydrogel and reinforced SA-CH/NCs hydrogel were utilized for the immobilization of a cellulase/xylanase enzyme (PersiCelXyn1). While free PersiCelXyn1 had the optimum temperature of 50 °C, the immobilized enzyme on SA-CH and SA-CH/NCs hydrogels showed optimum temperatures at 70, and 80 °C, respectively. Furthermore, the immobilization of PersiCelXyn1 onto both hydrogels shifted the optimum pH to alkaline pHs, and increased the enzyme specific activity. The enzyme immobilized on nanocarrier (PersiCelXyn1@SA-CH/NCs) showed higher reusability than PersiCelXyn1@SA-CH, after 14 cycles (70 % activity vs. 52 % activity, respectively), and less enzyme leaching. PersiCelXyn1@SA-CH/NCs showed higher efficiency in the hydrolysis of sugar beet pulp to obtain the hydrolysate containing fermentable sugar as a substrate for lactic acid (LA) production, than PersiCelXyn1@SA-CH hydrogel. After 96 h, the concentration of LA produced with the PersiCelXyn1@SA-CH/NCs was 33.1 g/L, while it was 26 g/L and 21.6 g/L, in the fermentation of hydrolysate with PersiCelXyn1@SA-CH hydrogel, and free PersiCelXyn1, respectively.

A radical containing micellar probe for assessing esterase enzymatic activity with ultra-low field Overhauser-enhanced magnetic resonance imaging.

The ability to track altered enzyme activity using a non-invasive imaging protocol is crucial for the early diagnosis of many diseases but is often challenging. Herein, we show that Overhauser magnetic resonance imaging (OMRI) can be used to monitor enzymatic conversion at an ultra-low field (206 μT) using a highly sensitive "off/on" probe with a nitroxide stable radical containing ester, named T2C12-T80. This TEMPO derivative containing probe forms stable electron paramagnetic resonance (EPR) silent micelles in water that are hydrolysed by esterases, thus yielding narrow EPR signals whose intensities correlate directly with specific enzymatic activity. The responsiveness of the probe to tumours, facilitated by increased esterase activity, was initially determined by comparing EPR signals measured upon incubation with 3T3 (healthy fibroblasts used as control), HepG2 (human hepatoma) and Hs766T (human pancreatic cancer cells) cell lysates and then with Hs766T and 3T3 living cells. Next, Overhauser MR images were detected on a phantom containing the probe and the esterases to show that the approach is well suited for being translated to the in vivo detection at the earth's magnetic field. Regarding detection sensitivity, ultra-low field OMRI (ULF-OMRI) is beneficial over OMRI at higher fields (e.g. 0.2 T) since Overhauser enhancements are significantly higher and the technique is safe in terms of the specific absorption rate.

Proteolysis Assays With Conserved or Aminofluorescein-Labeled Red Blood Cells.

Backgrounds: Various physiological functions and reaction cascades, as well as disease progression in the living systems, are controlled by the activity of specific proteolytic enzymes. We conducted the study to evaluate protease activity by assessing peptide fragments from either conserved or labeled red blood cells (RBCs) with aminofluorescein (AF) in the reaction media. Methods: RBCs were incubated in media containing trypsin. Subsequently, the concentration of peptide fragments in the reaction media, resulted by the digestion with trypsin from conserved cells, was estimated by 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) as an amine-reactive fluorogenic reagent. In a second approach, we conjugated AF to the conserved RBCs and then exposed AF-labeled RBCs to trypsin. This was followed by directly measuring the fluorescence intensity (FI) in the reaction media to estimate the concentration of AF-labeled peptide fragments resulting from the enzyme's activity. Results: Show a concentration- and time-dependent increase in FIs, reflecting the activity of trypsin as a proteolytic enzyme. The FIs increased significantly by 4 to 5 folds in samples treated with different enzyme concentrations, and by over 11 folds after 2 h incubation in media containing a 50 μL trypsin, as evidenced by CBQCA assays. Conclusion: These fast and affordable approaches could be applied with high reliability for the general estimation of protease activity in samples and customized for diagnostic purposes and prognostic evaluation in various diseases.

New Highly Sensitive Methods for Electroanalysis of the Catalytic Activity of Enzymes of Medical Significance

The review is devoted to new highly effective methods for analyzing the catalytic activity of enzymes of medical significance, such as cytochromes P450, trypsin, asparaginase, beta-lactamase, and nucleases. The methods are based on registration the specific activity of enzymes using electroanalytical methods. The review analyzes the experimental data obtained by the authors. Two platforms have been developed that allow quantitative measurement of catalytic activity based on the electrochemical properties of the enzyme (cytochrome P450, bactosomes, asparaginase) or substrate (trypsin, nucleases, restriction enzymes, beta-lactamase).

Development of optical probes with excellent intracellular retention

Small-molecule based activatable fluorescence probes for detecting specific enzyme activity with high sensitivity can visualize the expression site of marker genes and cancers where the enzyme is highly expressed. However, the enzyme-catalyzed fluorescent hydrolysis product easily leaks out and diffuses from the reaction site, making it difficult to perform long-term tracking and immunohistochemical analysis which needs washing/fixation procedure. Our group have focused on quinone methide chemistry and developed series of activatable fluorescence probes with excellent intracellular retention that are converted to quinone-methide or aza-quinone-methide intermediates upon reaction with enzymes, which are then react with intracellular nucleophiles such as proteins and glutathione to be retained in cells and to exhibit significant increase in fluorescence. Based on this molecular design, we have developed fluorescence probes targeting β-galactosidase and γ-glutamyltranspeptidase with different colors. We also developed photo-functional probes such as activatable photosensitizers and caged fluorophores. These probes can visualize or kill target enzyme-expressing cells with high selectivity by suppressing the leakage of hydrolysis products from target cells, and fluorescence imaging in combination with immunostaining was possible due to the high tolerance of the obtained fluorescence signal even after washing and fixation.

Site-directed mutagenesis of recombinant penicillin-G acylase from Escherichia coli and effect on the hydrolytic activity

Penicillin-derived antibiotics such as amoxicillin are one of the antibiotics that are widely used. Amoxicillin is synthesized through the availability of raw materials, 6-aminopenicillanic acid (6-APA), with penicillin-G acylase (PGA) as the catalyst. Previous studies revealed a recombinant PGA through the utilization of expression hosts. However, the low activity of the expressed enzyme is still an obstacle to the optimum utilization of its enzyme. In this study, we applied site-directed mutagenesis computationally for initial prediction by creating 36 PGA mutants through homology modeling and predicted their binding affinity as well as bounding interaction mode to penicillin-G through molecular docking. Our research found that four mutants had better potential activity than wild-type PGA. In the first experiment, only the βThr68Tyr PGA mutant was selected to be explored further. The mutant PGA gene inserted into pET-22b plasmid was transformed to Escherichia coli BL21(DE3) by heat shock method. For enzyme expression, IPTG and arabinose were used as inducers. As a result, the specific enzyme activity of mutant PGA showed insignificantly lower values in comparison to wild-type PGA. This activity appeared to be influenced by the formation of inclusion bodies during post-translational protein maturation, as described in this paper.

Screening and characterization of thermostable protease-producing bacteria isolated from slaughter site soil samples from Southern India

The current study was isolated and identified Bacillus cereus from an animal slaughter site in the South Indian state of Kerala, as a potent additive to detergents for enhancing stain removal efficacy. Identifying the isolates as B. cereus was confirmed through microbial biomass production, biochemical tests, protease-specific activity, and sequencing of the 16S rRNA gene. Following the growth condition optimization, the protease enzyme was extracted from the biomass, exhibiting a protease activity of 72.212 ± 0.235 U/mL. Calcium, magnesium, and sodium positively influenced protease activity under the tested conditions and were negatively impacted by zinc, copper, and iron. The polar solvent dimethyl sulfoxide (DMSO) supported enzyme activity, whereas the non-polar solvents hexane and chloroform completely inhibited it. The highest protease-specific activity of the crude extract following dialysis was 62.997 U/mg. The purified fractions of Sephadex G-100 and DEAE-Sephadex A50 anion exchange columns showed the highest enzyme-specific activity of 193.652 U/mg and 324.004 U/mg, respectively. The addition of crude protease to a generic detergent significantly enhanced its efficacy in removing fabric stains from cotton, polyester, and silk materials. Therefore, the isolated serine protease demonstrates significant potential for industrial applications.

Oxidoreductase activity of soybeen seedlings at the early stage of ontogenesis

The results of analysis of specific activity and multiple forms of oxidoreductase class enzymes are presented: antioxidant complex (catalase and peroxidase) and dehydrogenases (alcohol dehydrogenase, NAD+-malate dehydrogenase, glucose-6-phosphate dehydrogenase). Seven varieties of soybean (Glycine max (L.) Merrill) served as the object of the study. Dormant seeds and 3- and 7-day-old soybean seedlings were used for the analysis. Protein content was determined by the Lowry method, peroxidase activity was determined by colorimetric, catalase and the studied dehydrogenases – by spectrophotometric methods, electrophoretic spectra of enzymes – by electrophoresis on 7.5% polyacrylamide gel columns. Identification of the zones with enzymatic activity on the gel was performed by appropriate histochemical methods. Analysis of specific activity of antioxidant enzymes in dormant soybean seeds revealed increased activity of peroxidase and low activity of catalase. During seed germination, the inverse relationship of the specific activity of these enzymes is observed. On the 7th day the specific activity of catalase increases, that of peroxidase decreases to a minimum. In soybean germination, 5 forms of catalase were detected, indicating low polymorphism and stability of the enzyme, and 18 forms of peroxidase, which confirm high polymorphism and the possibility of using this enzyme as a marker of biochemical processes. Alcohol dehydrogenase and glucose-6-phosphate dehydrogenase were found to be of low heterogeneity, with the specific activity of these enzymes decreasing during germination compared to the dormant period of soybean seeds. The specific activity of NAD+-malate dehydrogenase increases slightly during germination. Eight forms of this enzyme were detected in the soybean varieties studied indicating increased polymorphism. In dormant soybean seeds, the electrophoretic spectra of NAD+-malate dehydrogenase exhibited varietal diversity allowing the enzyme to be used as a marker for further studies.

Isolation, Molecular Characterization, Optimization and Purification of Amylase Enzyme from Locally Isolated Bacillus Species in Different Regions of Munshiganj, Bangladesh

Amylase is a kind of enzyme that facilitates the breakdown of carbohydrates in the body. This enzyme has many applications in different industries. Generally, in pharmaceutical sector it is used for the treatment of pancreatic disorder, pancreatic enzyme replacement therapy (PERT) and as a digestive aid. In this study, the bacterial strain was isolated from soil samples collected from potato dumpsites in different areas of Munshiganj, Bangladesh. After subculturing on the nutrient agar plate, 31 colonies were obtained, of which 9 isolates were found as amylase producer on starch agar medium. Of these, 2 isolates (MC-04 and MC-15) were selected based on the starch hydrolysis clear zone ratio. The isolate MC-04 showed crude enzyme activity of 2.82 IU/ml and specific enzyme activity of 3.42 IU/mg, and isolate MC-15 showed crude enzyme activity of 3.16 IU/ml and specific enzyme activity of 3.77 IU/mg. The best isolate, MC-15, was then identified by morphology, biochemical and molecular characterization and confirmed by 16S-rRNA gene sequencing. After 16S-rRNA sequencing, the isolate (MC-15) was identified as Bacillus subtilis. This amylase production of this strain had also been optimized under certain conditions such as different incubation periods, pH, temperatures and different carbon sources. We found that the best incubation period was 48 h, the optimum pH-7.0, the optimum temperature at 40°C and 2% starch was considered as the best source of carbon. Finally, the crude amylase enzyme was purified by precipitation with ammonium sulfate, dialysis, and single-step gel filtration chromatography. The enzymatic activity of the purified amylase was found to be 8.91 IU/ml, that was 2.82-fold greater enzymatic activity than the raw enzyme. The experiments confirmed that Bacillus subtilis may be a good source of amylase enzyme for industrial application in Bangladesh. Dhaka Univ. J. Pharm. Sci. 22(2): 147-154, 2023 (December)

Newborn Screening of 6 Lysosomal Storage Disorders by Tandem Mass Spectrometry.

This study was designed to screen 6 lysosomal storage diseases (LSDs) in neonates using tandem mass spectrometry (MS/MS), and establish cutoff values for these LSDs with 3000 dried blood spots (DBS) samples. Cutoff values for α-L-iduronidase (IDUA), α-galactosidase (GLA), acid beta glucosidase (ABG), β-galactocerebrosidase (GALC), acid sphingomyelinase (ASM), and acid alpha glucosidase (GAA) were as follows: GLA, > 2.06 μmol/L·h; ABG, > 1.78 μmol/L·h; ASM, > 0.99 μmol/L·h; IDUA, > 1.33 μmol/L·h; GALC, > 0.84 μmol/L·h; and GAA, > 2.06 μmol/L·h. There were 30 positives in initial MS/MS screening test, and 15 samples were still positive with repeat testing. Their parents/guardians were recontacted and DBS samples were collected again for test. Only 1 child showed abnormal GAA enzyme activity after recontacting process, and was diagnosed with Pompe disease after genetic screening. Eventually, cutoff values of 6 specific enzyme activities were established and MS/MS is effective for early LSDs screening.

CYP-catalysed cycling of clozapine and clozapine-N-oxide promotes the generation of reactive oxygen species in vitro

Clozapine is an effective atypical antipsychotic indicated for treatment-resistant schizophrenia, but is under-prescribed due to the risk of severe adverse drug reactions such as myocarditis. A mechanistic understanding of clozapine cardiotoxicity remains elusive. This study aimed to investigate the contribution of selected CYP isoforms to cycling between clozapine and its major circulating metabolites, N-desmethylclozapine and clozapine-N-oxide, with the potential for reactive species production. CYP supersome™-based in vitro techniques were utilised to quantify specific enzyme activity associated with clozapine, clozapine-N-oxide and N-desmethylclozapine metabolism. The formation of reactive species within each incubation were quantified, and known intermediates detected. CYP3A4 predominately catalysed clozapine-N-oxide formation from clozapine and was associated with concentration-dependent reactive species production, whereas isoforms favouring the N-desmethylclozapine pathway (CYP2C19 and CYP1A2) did not produce reactive species. Extrahepatic isoforms CYP2J2 and CYP1B1 were also associated with the formation of clozapine-N-oxide and N-desmethylclozapine but did not favour one metabolic pathway over another. Unique to this investigation is that various CYP isoforms catalyse clozapine-N-oxide reduction to clozapine. This process was associated with the concentration-dependent formation of reactive species with CYP3A4, CYP1B1 and CYP1A1 that did not correlate with known reactive intermediates, implicating metabolite cycling and reactive oxygen species in the mechanism of clozapine-induced toxicity.

Gene cloning, expression, and characterization of two endo-xylanases from Bacillus velezensis and Streptomyces rochei, and their application in xylooligosaccharide production.

Endo-xylanase hydrolyzing xylan in cellulosic residues releasing xylobiose as the major product at neutral pH are desirable in the substitute sweeteners industry. In this study, two endo-xylanases were obtained from Streptomyces rochei and Bacillus velezensis. SrocXyn10 showed the highest identity of 77.22%, with a reported endo-xylanase. The optimum reaction temperature and pH of rSrocXyn10-Ec were pH 7.0 and 60°C, with remarkable stability at 45°C or pHs ranging from 4.5 to 11.0. rBvelXyn11-Ec was most active at pH 6.0 and 50°C, and was stable at 35°C or pH 3.5 to 10.5. Both rSrocXyn10-Ec and rBvelXyn11-Ec showed specific enzyme activities on wheat arabinoxylan (685.83 ± 13.82 and 2809.89 ± 21.26 U/mg, respectively), with no enzyme activity on non-xylan substrates. The Vmax of rSrocXyn10-Ec and rBvelXyn11-Ec were 467.86 U mg-1 and 3067.68 U mg-1, respectively. The determined Km values of rSrocXyn10-Ec and rBvelXyn11-Ec were 3.08 g L-1 and 1.45 g L-1, respectively. The predominant product of the hydrolysis of alkaline extracts from bagasse, corncob, and bamboo by rSrocXyn10-Ec and rBvelXyn11-Ec were xylooligosaccharides. Interestingly, the xylobiose content in hydrolysates by rSrocXyn10-Ec was approximately 80%, which is higher than most reported endo-xylanases. rSrocXyn10-Ec and rBvelXyn11-Ec could be excellent candidates to produce xylooligosaccharides at neutral/near-neutral pHs. rSrocXyn10-Ec also has potential value in the production of xylobiose as a substitute sweetener.

Altered rainfall greatly affects enzyme activity but has limited effect on microbial biomass in Australian dryland soils

Drylands support a substantial proportion of the worlds biodiversity and are important to food production but are sensitive to changes in rainfall regimes. Altered rainfall has been shown to impact plant growth and soil microbial activity in drylands but the longer-term effect on belowground communities and biogeochemical cycles remains uncertain. We explored how four years of reduced and increased rainfall influenced soil total and available carbon (C), nitrogen (N) and phosphorus (P) content, microbial biomass and potential extracellular enzyme activity under field conditions at six dryland sites in eastern Australia. The study coincided with a severe 3-year drought that resulted in low standing plant biomass and soil C content at all sites. Microbial biomass attributes varied considerably across sites, with rainfall treatment effects limited to decreased fungal biomass and lower fungal:bacterial ratios in semi-arid Nyngan and reduced fungal:bacterial ratios and microbial biomass C in semi-arid Quilpie in reduced treatments compared with increased rainfall plots. Similarly, available soil C, N and P varied considerably among sites, with more available N and P at four and all sites, respectively, in reduced rainfall treatments particularly when compared with increased rainfall treatments. Rainfall treatments consistently influenced enzyme activity across all sites, with higher rates in increased rainfall plots indicative of greater microbial activity and enhanced nutrient cycling. Enzymatic activity associated with N cycling showed a negative relationship with available N while enzymes associated with P cycling related positively to available C and negatively to available P. This indicates that microbes invested more in production of enzymes associated with less available nutrients. Enzyme activity was not related to microbial biomass suggesting a disconnect between biomass and enzyme production and that rainfall treatments altered the ecosystem's specific enzyme activity (activity per unit of microbial biomass). Our results suggest that altered rainfall consistently impacted dryland ecosystem function, but that microbial biomass is a poor proxy for rainfall-induced changes in soil processes.

Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold

Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.

Yeast diversity isolated from nypa palm leaf litter in the macrohabitat of the nypa palm worm (Namalycastis rhodochorde)

Yeasts are known to have broad and specific enzymatic activity, including cellulolytic activity. The diversity of yeast in the nypa palm mangrove area is thought to have a role in the organic degradation process of nypa leaf litter which supports the fermentation of nypa palm worm feed. This study aims to determine the composition and genera of yeast present in the habitat of nypa palm worms. The sampling process was determined using a purposive sampling method in Sungai Kakap Village, Kubu Raya Regency, West Kalimantan. Isolation was carried out using the pour plate method with serial dilution on the Saboraud Dextrose Agar Medium (SDA). The process of characterizing yeast genera was determined by observing the characteristics of microcolonies, type of budding cells, presence of pseudohyphae, type and number of spores, ascospore, and capsule presence. The results showed that the composition of the successfully isolated yeast was 11% of the total colonies growing on the SDA medium. Isolates NrYS1 and NrYS4 are similar to Torulaspora, while isolates NrYS2 and NrYS3 are similar to Schizosaccharomyces. Further research is needed on the potential of the four enzymatic yeasts to be used as probiotic feed for the nypa palm worm’s culture.

Zootechnical Parameters and Enzyme Activity in the Species Brycon moorei (Steindachner 1878)

Brycon moorei is an opportunistic omnivorous species. It is not known what its nutritional requirements are at any stage of its development; this study determined the influence of diet on the zootechnical parameters and enzyme activity of these fish. In a completely randomized quintuplicate design, an ad libitum dietary protocol was applied, which included Prochilodus magdalenae larvae, Artemia salina and balanced feed with increasing inclusion levels (35 to 55% crude protein (CP)). Larvae 22 h post-hatching (HPH), with an average initial weight and length of 3.07 ± 0.69 mg and 6.069 ± 0.22 mm, respectively, were seeded at a density of 17 larvae L−1 in tanks with an effective volume of 60 L. The main zootechnical parameters, water quality and enzyme activity were analyzed with respect to the experimental diets. Significantly, the best diet was that of 45% CP when compared with the other CP levels used. Specific serum enzyme activity was found from hatching, with fluctuating values, the specific pepsin-like activity started at 111 HPH. B. moorei larvae can receive a dry diet from 124 HPH and their requirement corresponds to 48.5% CP.

Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase

BackgroundIn whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E. coli for reaction flux optimization in whole cell conversions producing 3′-sialyllactose (3SL).ResultsDifference in enzyme specific activity (CSS from Neisseria meningitidis: 36 U/mg; α2,3-SiaT from Pasteurella dagmatis: 5.7 U/mg) was compensated by differential protein co-expression from tailored plasmid constructs, giving balance between the individual activities at a high level of both (α2,3-SiaT: 9.4 × 102 U/g cell dry mass; CSS: 3.4 × 102 U/g cell dry mass). Finally, plasmid selection was guided by kinetic modeling of the coupled CSS-SiaT reactions in combination with comprehensive analytical tracking of the multistep conversion (lactose, N-acetyl neuraminic acid (Neu5Ac), cytidine 5′-triphosphate; each up to 100 mM). The half-life of SiaT in permeabilized cells (≤ 4 h) determined the efficiency of 3SL production at 37 °C. Reaction at 25 °C gave 3SL (40 ± 4 g/L) in ∼ 70% yield within 3 h, reaching a cell dry mass-specific productivity of ∼ 3 g/(g h) and avoiding intermediary CMP-Neu5Ac accumulation.ConclusionsCollectively, balanced co-expression of CSS and SiaT yields an efficient (high-flux) sialylation module to support flexible development of E. coli whole-cell catalysts for sialo-oligosaccharide production.

Screening, cloning, immobilization and application prospects of a novel β-glucosidase from the soil metagenome

The soil environment for straw return is a rich and valuable library containing many microorganisms and proteins. In this study, we aimed to screen a high-quality β-glucosidase (BGL) from the soil metagenomic library and to overcome the limitation of the low extraction rate of resveratrol in Polygonum cuspidatum. This includes the construction of a soil metagenomic library, screening of BGL, bioinformatics analysis, cloning, expression, immobilization, enzymatic property analysis, and application for the transformation of polydatin. The results showed that the soil metagenomic library of straw return was successfully constructed, and a novel BGL was screened. The identified 1356 bp long BGL belonged to the glycoside hydrolase 1 (GH1) family and was named Bgl1356. After successful cloning and expression of Bgl1356, it was immobilized using chitosan. The optimum temperature of immobilized Bgl1356 was 50 °C, and the pH was 5. It exhibited good tolerance for various metal ions (CO2+, Ni2+, Cu2+, Mn2+, Na2+, Ca2+, and Ag+) and organic solvents (DMSO, Triton-X-10, and ethanol). Enzymatic kinetics assays showed that Bgl1356 had good affinity for the substrate, and the specific enzyme activity was 234.03 U/mg. The conversion rate of polydatin by immobilized Bgl1356 was 95.70 ± 1.08%, facilitating the production of high amounts of resveratrol. Thus, this paper reports a novel temperature-, organic solvent-, and metal ion-tolerant BGL that has good application prospects in the pharmaceutical industry.

Development of an Algorithm for Identification of N-terminal Acetyltransferases and Verification of Their Functional Activity

N-terminal acetyltransferases (NATs) of bacteria acetylate the alpha-amino group in amino acids and proteins, participate in the biosynthesis of lantibiotics, and inactivate a number of antibiotics. NATs are used in biotechnology for targeted acetylation of recombinant proteins and peptides. In this regard, the search for NATs that differ in terms of substrate specificity and are also capable of functioning in the reaction at elevated temperatures, a wide pH range, etc., seems relevant. In this work, we develop specific characteristics and a search algorithm for the identification of N-terminal acetyltransferases using the Thermus thermophilus thermophilic bacterium as an example. Out of 14 Abs annotated in the genome, we selected six «putative» NATs. Some of the genes encoding the selected NATs were successfully cloned, generated, and purified from E. coli cells. The specific enzymatic activity of a number of enzymes was confirmed.