Class Of Enzymes Research Articles

Deciphering the biotransformation mechanism of dialkylresorcinols by CYP4F11

Cytochrome P450 enzymes (CYPs) are one of the most important classes of oxidative enzymes in the human body, carrying out metabolism of various exogenous and endogenous substrates. In order to expand the knowledge of these enzymes’ specificity and to obtain new natural product derivatives, CYP4F11, a cytochrome P450 monooxygenase, was used in the biotransformation of dialkylresorcinols 1 and 2, a pair of antibiotic microbial natural products. This investigation resulted in four biotransformation products including two oxidative products: a hydroxylated derivative (3) and a carboxylic acid derivative (4). In addition, acetylated (5) and esterified products (6) were isolated, formed by further metabolism by endogenous yeast enzymes. Oxidative transformations were highly regioselective, and took place exclusively at the ω-position of the C-5 alkyl chain. Homology modeling studies revealed that optimal hydrogen bonding between 2 and the enzyme can only be established with the C-5 alkyl chain pointing towards the heme. The closely-related CYP4F12 was not capable of oxidizing the dialkylresorcinol 2. Modeling experiments rationalize these differences by the different shapes of the binding pockets with respect to the non-oxidized alkyl chain. Antimicrobial testing indicated that the presence of polar groups on the side-chains reduces the antibiotic activity of the dialkylresorcinols.

Complex pectin metabolism by Lactobacillus and Streptococcus suggests an effective control approach for Maillard harmful products in brown fermented milk

Harmful Maillard reaction products (HMRPs) derived from brown fermented milk pose a potential threat to human health, but the conversion mechanism during the manufacturing process remains elusive and urgently needs to be controlled. Acrylamide (FC 2.14, adjusted p-value = 0.041), 5-hydroxymethylfurfural (FC 2.61, adjusted p-value = 0.026) and methylglyoxal (FC 2.07, adjusted p-value = 0.019) were identified as the significantly increased HMRPs after browning in this study and the analysis of proteomics integrated with untargeted metabolomics demonstrated that the degradation of HMRPs was jointly accomplished by Streptococcus thermophilus and Lactobacillus bulgaricus. The galactose oligosaccharide metabolism in Streptococcus thermophilus was identified as a key biochemical reaction for HMRPs degradation, and the hydrolysates of pectin could be utilized as prebiotics for Streptococcus thermophilus. Eighteen classes of enzymes of L. bulgaricus and Streptococcus thermophilus related to energy metabolism were upregulated in the pectin-added group, indicating that the entry of acrylamide and methylglyoxal into the tricarboxylic acid cycle was accelerated. NAD-aldehyde dehydrogenase and alanine dehydrogenase are enzymes belonging to Streptococcus thermophilus, and their downregulation accelerated the efflux of acetate, which was beneficial for the proliferation of L. bulgaricus and prevented the conversion of pyruvate to l-alanine, thus facilitating the energy metabolism. The recoveries and relative standard deviations of the intraday and interday precision experiments were 89.1%-112.5%, 1.3%-8.4% and 2.1%-9.4%, respectively, indicating that the developed approach was credible. Sensory evaluation results revealed that the brown fermented milk added with pectin had a better flavor, which was due to the fact that the supplement of polysaccharide promoted the fatty acid metabolism of lactic acid bacteria and increased the aroma substances including octoic acid and valeric acid. This study provided an insight into the formation and degradation mechanism of HMRPs in brown fermented milk, aiming to reduce the intake of advanced glycation end products in the diet.

First Identification of a Large Set of Serine Hydrolases by Activity-Based Protein Profiling in Dibutyl Phthalate-Exposed Zebrafish Larvae.

Despite the involvement of several serine hydrolases (SHs) in the metabolism of xenobiotics such as dibutyl phthalate (DBP), no study has focused on mapping this enzyme class in zebrafish, a model organism frequently used in ecotoxicology. Here, we survey and identify active SHs in zebrafish larvae and search for biological markers of SH type after exposure to DBP. Zebrafish were exposed to 0, 5, and 100 µg/L DBP from 4 to 120 h post-fertilization. A significant decrease in vitellogenin expression level of about 2-fold compared to the control was found in larvae exposed to 100 µg/L DBP for 120 h. The first comprehensive profiling of active SHs in zebrafish proteome was achieved with an activity-based protein profiling (ABPP) approach. Among 49 SHs identified with high confidence, one was the carboxypeptidase ctsa overexpressed in larvae exposed to 100 µg/L DBP for 120 h. To the best of our knowledge, this is the first time that a carboxypeptidase has been identified as deregulated following exposure to DBP. The overall results indicate that targeted proteomics approaches, such as ABPP, can, therefore, be an asset for understanding the mechanism of action related to xenobiotics in ecotoxicology.

Comprehensive screening of marine metabolites against class B1 metallo-β-lactamases of Klebsiella pneumoniae using two-pronged in silico approach

The emergence of antibiotic resistance is one of the major global threats in healthcare. Metallo-β-Lactamases (MBL) are a class of enzymes in bacteria that cleave β-lactam antibiotics and confer resistance. MBLs are further divided into subclasses B1, B2 and B3. Of these, subclasses B1-MBLs (including NDM-1, VIM-2 and IMP-1) constitute the clinically prevalent lactamases conferring resistance. To date, no effective drugs are available clinically against MBLs. In this work, we aim to identify potent inhibitors for the B1 subclass of MBL from available marine metabolites in Comprehensive Marine Natural Product database through integrated in silico approaches. We have used two methods, namely, the high-throughput strategy and the pharmacophore-based strategy to identify potential inhibitors from marine metabolites. High-throughput virtual screening identified N-methyl mycosporine-Ser, which had the highest binding affinity to NDM-1. The pharmacophore-based approach based on co-crystallized ligands identified makaluvic acid and didymellamide with higher binding affinity across B1-MBLs. Taking into account of the advantage of a pharmacophore model-based approach with higher binding affinity, we conclude that both makaluvic acid and didymellamide show potential broad-spectrum effects by binding to all three B1-MBL receptors. The study also indicates the need to take multiple in silico approaches to screen and identify novel inhibitors. Together, our study reveals promising inhibitors that can be identified from marine systems. Communicated by Ramaswamy H. Sarma

β-Xylosidase SRBX1 Activity from Sporisorium reilianum and Its Synergism with Xylanase SRXL1 in Xylose Release from Corn Hemicellulose.

Sposisorium reilianum is the causal agent of corn ear smut disease. Eleven genes have been identified in its genome that code for enzymes that could constitute its hemicellulosic system, three of which have been associated with two Endo-β-1,4-xylanases and one with α-L-arabinofuranosidase activity. In this study, the native protein extracellular with β-xylosidase activity, called SRBX1, produced by this basidiomycete was analyzed by performing production kinetics and its subsequent purification by gel filtration. The enzyme was characterized biochemically and sequenced. Finally, its synergism with Xylanase SRXL1 was determined. Its activity was higher in a medium with corn hemicellulose and glucose as carbon sources. The purified protein was a monomer associated with the sr16700 gene, with a molecular weight of 117 kDa and optimal activity at 60 °C in a pH range of 4-7, which had the ability to hydrolyze the ρ-nitrophenyl β-D-xylanopyranoside and ρ-Nitrophenyl α-L-arabinofuranoside substrates. Its activity was strongly inhibited by silver ions and presented Km and Vmax values of 2.5 mM and 0.2 μmol/min/mg, respectively, using ρ-nitrophenyl β-D-xylanopyranoside as a substrate. The enzyme degrades corn hemicellulose and birch xylan in combination and in sequential synergism with the xylanase SRXL1.

Comprehensive analysis of the carboxylesterase gene reveals that NtCXE22 regulates axillary bud growth through strigolactone metabolism in tobacco.

Carboxylesterases (CXE) are a class of hydrolytic enzymes with α/β-folding domains that play a vital role in plant growth, development, stress response, and activation of herbicide-active substances. In this study, 49 Nicotiana tabacum L. CXE genes (NtCXEs) were identified using a sequence homology search. The basic characteristics, phylogenetic evolution, gene structure, subcellular location, promoter cis-elements, and gene expression patterns of the CXE family were systematically analyzed. RNA-seq data and quantitative real-time PCR showed that the expression level of CXEs was associated with various stressors and hormones; gene expression levels were significantly different among the eight tissues examined and at different developmental periods. As a new class of hormones, strigolactones (SLs) are released from the roots of plants and can control the germination of axillary buds.NtCXE7, NtCXE9, NtCXE22, and NtCXE24 were homologous to Arabidopsis SLs hydrolase AtCXE15, and changes in their expression levels were induced by topping and by GR24 (a synthetic analogue of strigolactone). Further examination revealed that NtCXE22-mutant (ntcxe22) plants generated by CRISPR-Cas9 technology had shorter bud outgrowth with lower SLs content. Validation of NtCXE22 was also performed in NtCCD8-OE plants (with fewer axillary buds) and in ntccd8 mutant plants (with more axillary buds). The results suggest that NtCXE22 may act as an efficient SLs hydrolase and affects axillary bud development, thereby providing a feasible method for manipulating endogenous SLs in crops and ornamental plants.

Contribution of UGT Enzymes to Human Drug Metabolism Stereoselectivity: A Case Study of Medetomidine, RO5263397, Propranolol, and Testosterone.

The enantiomeric forms of chiral compounds have identical physical properties but may vary greatly in their metabolism by individual enzymes. Enantioselectivity in UDP-glucuronosyl transferase (UGT) metabolism has been reported for a number of compounds and with different UGT isoforms involved. However, the impact of such individual enzyme results on overall clearance stereoselectivity is often not clear. The enantiomers of medetomidine, RO5263397, and propranolol and the epimers testosterone and epitestosterone exhibit more than a 10-fold difference in glucuronidation rates by individual UGT enzymes. In this study, we examined the translation of human UGT stereoselectivity to hepatic drug clearance considering the combination of multiple UGTs to overall glucuronidation, the contribution of other metabolic enzymes such as cytochrome P450s (P450s), and the potential for differences in protein binding and blood/plasma partitioning. For medetomidine and RO5263397, the high individual enzyme (UGT2B10) enantioselectivity translated into ∼3- to >10-fold differences in predicted human hepatic in vivo clearance. For propranolol, the UGT enantioselectivity was irrelevant in the context of high P450 metabolism. For testosterone, a complex picture emerged due to differential epimeric selectivity of various contributing enzymes and potential for extrahepatic metabolism. Quite different patterns of P450- and UGT-mediated metabolism were observed across species, as well as differences in stereoselectivity, indicating that extrapolation from human enzyme and tissue data are essential when predicting human clearance enantioselectivity. SIGNIFICANCE STATEMENT: Individual enzyme stereoselectivity illustrates the importance of three-dimensional drug-metabolizing enzyme-substrate interactions and is essential when considering the clearance of racemic drugs. However, translation from in vitro to in vivo can be challenging as contributions from multiple enzymes and enzyme classes must be combined with protein binding and blood/plasma partitioning data to estimate the net intrinsic clearance for each enantiomer. Preclinical species may be misleading as enzyme involvement and metabolism stereoselectivity can differ substantially.

A growth selection system for the directed evolution of amine-forming or converting enzymes

Fast screening of enzyme variants is crucial for tailoring biocatalysts for the asymmetric synthesis of non-natural chiral chemicals, such as amines. However, most existing screening methods either are limited by the throughput or require specialized equipment. Herein, we report a simple, high-throughput, low-equipment dependent, and generally applicable growth selection system for engineering amine-forming or converting enzymes and apply it to improve biocatalysts belonging to three different enzyme classes. This results in (i) an amine transaminase variant with 110-fold increased specific activity for the asymmetric synthesis of the chiral amine intermediate of Linagliptin; (ii) a 270-fold improved monoamine oxidase to prepare the chiral amine intermediate of Cinacalcet by deracemization; and (iii) an ammonia lyase variant with a 26-fold increased activity in the asymmetric synthesis of a non-natural amino acid. Our growth selection system is adaptable to different enzyme classes, varying levels of enzyme activities, and thus a flexible tool for various stages of an engineering campaign.

Mapping of the ocular surface enzymes in healthy subjects

AbstractPurpose: This study aimed to pool identified enzymes from numerous comprehensive open‐access proteomic studies to profile and classify detected enzymes on the ocular surface (OS).Methods: Data were taken from studies that analysed tear samples of healthy subjects either collected by capillary tubes (CT (tears), n = 5) or Schirmer strips (ScS, n = 5). Enzymes were mapped using UniProt ‘enzyme class’ (EC) and Panther software. Functional analysis was done using Metascape.Results: In total, 1010 enzymes were identified from all studies which are classified as oxidoreductases (EC1, 13.4%), transferases (EC2–30.3%), hydrolases (EC 3–42.3%), lyases (EC 4–4.3%), isomerases (EC5–4.1%), ligases (EC6–4.1%), translocases (EC 7–1.4%). More isomerases (5% vs. 3.4%) and ligases (4.7% vs. 4.1%) could be identified in ScS samples. Isomerases formed 5.6% of specific enzymes detected only in ScS while no specific isomerase was found in tears. Antioxidant and antimicrobial enzymes, EC1 class, were formed by dehydrogenases 36.2%, reductases 23.3%, oxidoreductases 18.1%, peroxidases 10.3%, oxygenases 7.8% and oxidases 4.3%. Alcohol oxidoreductases (EC1.1) were the most represented subgroup of EC1 with 46 enzymes (34.3%). Of all enzymes EC2.7 (phosphotransferases), EC3.1 (esterases) and EC3.4 (peptidases) were the most represented subclasses with 156, 151, 172 enzymes, respectively. The metabolic process of carbohydrate (14.7%), nucleobase‐containing small molecule (15.6%), cellular amide (14.7%) and carbohydrate derivative biosynthetic process (12%) were the most representative biological processes of all identified enzymes. Neutrophil degranulation, carbon and amino acid metabolism were the main pathways respectively that these enzymes are involved.Conclusions: Open‐access proteomic studies enable the pooling of all detected proteins and enzymes to have a great quantity of data. OS harbours hundreds of enzymes, which are involved in the homeostasis and defence mechanisms of OS. Enzymes could be attractive targets for novel treatments in OS pathologies due to their druggability property.

Efficient Activity Enhancement of a Lipase from Sporisorium reilianum for the Synthesis of a Moxifloxacin Chiral Intermediate via Rational Design

Lipase-catalyzed stereoselective resolution of cis -(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate ( cis -(±)-1) is an attractive route for the synthesis of ( S,S )-2,8-diazobicyclo[4.3.0]nonane, an important chiral intermediate of the fluoroquinolone antibiotic, moxifloxacin. In our previous study, a lipase from Sporisorium reilianum (SRL) was identified to possess excellent thermostability and pH stability. However, the low enzymatic activity of the SRL is a challenge that must be addressed. A rational design was initially employed for SRL tailoring according to the engineered Candida antarctica lipase B (CALB), resulting in a beneficial variant called SRL-I194N/V195L. Subsequently, two key amino acid residues in loop 6, L145 and L154, which might modulate the lid conformation between open and closed, were identified. A tetra-site variant, SRL-I194N/V195L/L145V/L154G (V13), with a significantly enhanced activity of 87.8 U∙mg −1 was obtained; this value was 2195-fold higher than that of wild-type SRL. Variant V13 was used to prepare optically pure (2 S ,3 R )-dimethyl 1-acetylpiperidine-2,3-dicarboxylate ((2 S ,3 R )-1), resolving 1 mol∙L −1 cis -(±)-1 with a conversion of 49.9% in 2 h and absolute stereoselectivity ( E > 200). Excellent stability with a half-life of 92.5 h was also observed at 50 °C. Overall, the study findings reveal a lipase with high activity toward cis -(±)-1 at an industrial level and may offer a general strategy for enhancing the enzyme activity of other lipases and other classes of enzymes with a lid moiety.

In vitro studies of the protein-interaction network of cell-wall lytic transglycosylase RlpA of Pseudomonas aeruginosa

The protein networks of cell-wall-biosynthesis assemblies are largely unknown. A key class of enzymes in these assemblies is the lytic transglycosylases (LTs), of which eleven exist in P. aeruginosa. We have undertaken a pulldown strategy in conjunction with mass-spectrometry-based proteomics to identify the putative binding partners for the eleven LTs of P. aeruginosa. A total of 71 putative binding partners were identified for the eleven LTs. A systematic assessment of the binding partners of the rare lipoprotein A (RlpA), one of the pseudomonal LTs, was made. This 37-kDa lipoprotein is involved in bacterial daughter-cell separation by an unknown process. RlpA participates in both the multi-protein and multi-enzyme divisome and elongasome assemblies. We reveal an extensive protein-interaction network for RlpA involving at least 19 proteins. Their kinetic parameters for interaction with RlpA were assessed by microscale thermophoresis, surface-plasmon resonance, and isothermal-titration calorimetry. Notable RlpA binding partners include PBP1b, PBP4, and SltB1. Elucidation of the protein-interaction networks for each of the LTs, and specifically for RlpA, opens opportunities for the study of their roles in the complex protein assemblies intimately involved with the cell wall as a structural edifice critical for bacterial survival.

Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

Cofactor and Process Engineering for Nicotinamide Recycling and Retention in Intensified Biocatalysis

There is currently considerable interest in the intensification of biocatalytic processes to reduce the cost of goods for biocatalytically produced chemicals, including pharmaceuticals and advanced pharmaceutical intermediates. Continuous-flow biocatalysis shows considerable promise as a method for process intensification; however, the reliance of some reactions on the use of diffusible cofactors (such as the nicotinamide cofactors) has proven to be a technical barrier for key enzyme classes. This minireview covers attempts to overcome this limitation, including the cofactor recapture and recycling retention of chemically modified cofactors. For the latter, we also consider the state of science for cofactor modification, a field reinvigorated by the current interest in continuous-flow biocatalysis.

Phase 1 Safety and Dose Escalation of Purinostat Mesylate, a Uniquely Potent and Selective Inhibitor of HDAC I and IIb in Relapsed or Refractory Lymphoma and Multiple Myeloma

Introduction Current treatment options for patients with relapsed or refractory (RR) lymphoma and multiple myeloma (MM) are limited, highlighting the unmet need for effective therapies in these disease settings. Purinostat Mesylate (PM) is a small molecule that is designed to selectively inhibit histone deacetylase (HDAC) enzymes class I and IIb, which are members of common oncogenic pathways in hematologic malignancies. This study examined PM monotherapy in patients with RR lymphoma and MM. Methods This open-label, non-randomized, first-in-human, phase 1 two-center trial enrolled adult patients with lymphoma or MM who were refractory to or relapsed after ≥1 prior regimens. PM was administered by 30-minute intravenous infusion in a standard 3+3 dose escalation design. The trial was conducted in three stages sequentially as follows: the first stage as a single dose (Day 1), the second stage as multiple doses (Day 8,11, and 15), and the third stage as extended doses (Day1, 4, 8, 11 in 21-day cycles). The decision to proceed to the third stage is based on patient tolerability in the previous two stages, disease status, and continued benefit determined by investigators. Initially dosing started at 1.2 mg/m2, escalated to 2.4 mg/m2, 4.0 mg/m2, 6.0 mg/m2, 8.4 mg/m2, 11.2 mg/m2 and finally 15.0 mg/m2. Patients continued to receive PM until disease progression or other treatment discontinuation criteria were met. The primary objective was to determine the maximum-tolerated dose and recommended phase 2 dose (RP2D); secondary objectives were to assess the safety and tolerability, pharmacokinetic profiles, and preliminary anti-tumor activity. This ongoing trial is registered at Chinese Clinical Trial Registry, number ChiCTR20191121. Results A total of 11 heavily pretreated MM patients(11/27,41%) and 16 lymphoma patients(16/27,59%) were enrolled to receive PM up to a maximum of 15.0 mg/m2 in the dose escalation. Most adverse events (AEs) were grade 1 to 2 and recovered with observation or symptomatic management. The most common Grade ≥3 AEs were thrombocytopenia (n=48.1%), neutropenia (n=48.1%), and anemia (11.1%), which occurred primarily during the extended dosing stages. AEs led to dose reductions in 4 patients. Dose limiting toxicities (DLTs) have not been observed. Twenty-seven patients were evaluable for responses. Eleven patients with RR MM had a disease control rate (DCR) of 72.7%. Eight patients had stable disease (SD) in cycle 1 and entered the third stage with a maximum of 6 extended cycles, of which 1 patient achieved a minimal remission (MR) in cycle 2. Nine out of 16 patients with RR lymphoma achieved objective responses rates (ORR) of 56.2%. Among them, six of 10 patients with diffuse large B-cell lymphoma (DLBCL) achieved objective responses (3 complete remissions [CR], 2 partial remissions [PR], and 1 MR). CRs occurred in 2 patients with DLBCL in cycle 3 (8.4mg/m2, 11.2 mg/m2) and 1 patient in cycle 2 (11.2 mg/m2). PRs occurred in 4 patient in cycle 1 (2 in 8.4 mg/m2, 2 in 11.2 mg/m2 ) and another patient who relapsed after 2 lines of prior therapy including autologous stem cell transplantation(ASCT) in cycle 3 (11.2 mg/m2). One MR and 1 SD have been observed in patients with double-expression DLBCL in cycle 1(15.0 mg/m2, 8.4 mg/m2) . Thus, the ORR in patients with RR DLBCL was 60.0% (6/10) and DCR was 70.0% (7/10). Two out of 4 follicular lymphoma patients relapsed after 2~4 lines of prior therapy also achieved objective responses (1 MR in cycle 1 and 1 PR in cycle 4). Of note, one patient with peripheral T-cell lymphoma who relapsed after four lines of treatment, including ASCT and Chidamide combination treatment with R-CHOP, obtained PR in cycle 1. Up to date, eight active response patients are still being treated. On the basis of the confirmed objective response and tolerability profile, the RP2D of PM was determined to be 8.4mg/m2 and 11.2mg/m2. Conclusions PM has demonstrated tolerability and good anti-tumor activity across all dosing schedules studied, especially multiple objective responses and disease control in patients with DLBCL. These results support continued development of PM, alone or in combination with other therapies, for the treatment of RR DLBCL. An open-label, non-randomized, phase 2, multicenter trial will be soon conducted in China to evaluate the efficacy of PM monotherapy in RR DLBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pectinase Production by Aspergillus niger and Its Applications in Fruit Juice Clarification

Pectinases (EC 3.2.1.15) are a class of enzymes that catalyze the depolymerization or de-esterification reactions that degrade pectic substances. In the present study, we have isolated the Aspergillus niger strain from soil samples, in India and evaluated pectinase production. The highest pectinase producing A. niger strain was further evaluated and optimized with various agricultural wastes. Plackett-Burman design (PBD) and Central composite design ‘(CCD)’ were used to determine the best parameters for maximum pectinase production. Pectinase activity was increased to 99.21 U/ml after optimizing the production medium using PBD and CCD statistical analysis. A positive correlation of pectinase activity between predicted (112.65 U/ml) and experimental (99.21 U/ml with SD=0.005) optimum was observed. Maximum pectinase was produced by A. niger under submerged fermentation, utilizing orange peel, which is a cost-effective, adaptable, and environmentally friendly approach. The partially purified pectinase showed significant application for apple juice clarification and showed the ability to degrade pectin and therefore the colour change was observed in apple juice within 120 min. Maximum pectinase was produced by A. niger using agricultural waste orange peel under submerged fermentation which is an economical, versatile and eco-friendly process and pectinase showed a significant application for apple juice clarification.

The role of noncoding RNAs in cancer lipid metabolism.

Research on noncoding ribonucleic acids (ncRNAs) is mostly and broadly focused on microRNAs (miRNAs), cyclic RNAs (circRNAs), and long ncRNAs (lncRNAs), which have been confirmed to play important roles in tumor cell proliferation, invasion, and migration. Specifically, recent studies have shown that ncRNAs contribute to tumorigenesis and tumor development by mediating changes in enzymes related to lipid metabolism. The purpose of this review is to discuss the characterized ncRNAs involved in the lipid metabolism of tumors to highlight ncRNA-mediated lipid metabolism-related enzyme expression in malignant tumors and its importance to tumor development. In this review, we describe the types of ncRNA and the mechanism of tumor lipid metabolism and analyze the important role of ncRNA in tumor lipid metabolism and its future prospects from the perspectives of ncRNA biological function and lipid metabolic enzyme classification. However, several critical issues still need to be resolved. Because ncRNAs can affect tumor processes by regulating lipid metabolism enzymes, in the future, we can study the unique role of ncRNAs from four aspects: disease prevention, detection, diagnosis, and treatment. Therefore, in the future, the development of ncRNA-targeted therapy will become a hot direction and shoulder a major task in the medical field.

High-Throughput MALDI-TOF Mass Spectrometry-Based Deubiquitylating Enzyme Assay for Drug Discovery.

Ubiquitylation (or ubiquitination) is the reversible conjugation of a 76-amino-acid polypeptide (ubiquitin) to a target protein to modulate various biological processes. Deubiquitylating enzymes (DUBs) are a class of enzymes that specifically remove ubiquitin from a substrate. In recent years DUBs have garnered significant attention as a new class of targets in multiple therapeutic areas. The recent development of high-throughput Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) has provided new tools to perform drug discovery screening. Here we present a facile and high-throughput step-by-step protocol of the MALDI-TOF MS-based DUB assay for screening the activity of DUBs in vitro. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains. The presented protocol takes advantage of nanoliter dispensing robotics and automated MALDI-TOF MS analysis to screen large and diverse compound libraries.

Identification of Deubiquitinase Substrates in Xenopus Egg Extract.

Deubiquitinases (DUBs) antagonize protein ubiquitination by removing ubiquitin from substrates. Identifying the physiological substrates of each DUB is critical for understanding DUB function and the principles that govern the specificity of this class of enzymes. Since multiple DUBs can act on the same substrate, it can be challenging to identify substrates using inactivating a single enzyme. Here, we outline a method that enables the identification of proteins whose stability depends on DUB activity and an approach to profile DUB specificity in Xenopus egg extract. By coupling broad DUB inhibition with quantitative proteomics, we circumvent DUB redundancy to identify DUB substrates. By adding back recombinant DUBs individually to the extract, we pinpoint DUBs sufficient to counteract proteasomal degradation of these newly identified substrates. We apply this method to Xenopus egg extract but suggest that it can also be adapted to other cell lysates.

Chemical Tools for the Study of DNA Repair.

ConspectusDNA repair enzymes continuously provide surveillance throughout our cells, protecting the enclosed DNA from the damage that is constantly arising from oxidation, alkylating species, and radiation. Members of this enzyme class are intimately linked to pathways controlling cancer and inflammation and are promising targets for diagnostics and future therapies. Their study is benefiting widely from the development of new tools and methods aimed at measuring their activities. Here, we provide an Account of our laboratory's work on developing chemical tools to study DNA repair processes in vitro, as well as in cells and tissues, and what we have learned by applying them.We first outline early work probing how DNA repair enzymes recognize specific forms of damage by use of chemical analogs of the damage with altered shapes and H-bonding abilities. One outcome of this was the development of an unnatural DNA base that is incorporated selectively by polymerase enzymes opposite sites of missing bases (abasic sites) in DNA, a very common form of damage.We then describe strategies for design of fluorescent probes targeted to base excision repair (BER) enzymes; these were built from small synthetic DNAs incorporating fluorescent moieties to engender light-up signals as the enzymatic reaction proceeds. Examples of targets for these DNA probes include UDG, SMUG1, Fpg, OGG1, MutYH, ALKBH2, ALKBH3, MTH1, and NTH1. Several such strategies were successful and were applied both in vitro and in cellular settings; moreover, some were used to discover small-molecule modulators of specific repair enzymes. One of these is the compound SU0268, a potent OGG1 inhibitor that is under investigation in animal models for inhibiting hyperinflammatory responses.To investigate cellular nucleotide sanitation pathways, we designed a series of "two-headed" nucleotides containing a damaged DNA nucleotide at one end and ATP at the other; these were applied to studying the three human sanitation enzymes MTH1, dUTPase, and dITPase, some of which are therapeutic targets. The MTH1 probe (ARGO) was used in collaboration with oncologists to measure the enzyme in tumors as a disease marker and also to develop the first small-molecule activators of the enzyme.We proceed to discuss the development of a "universal" probe of base excision repair processes (UBER), which reacts covalently with abasic site intermediates of base excision repair. UBER probes light up in real time as the reaction occurs, enabling the observation of base excision repair as it occurs in live cells and tissues. UBER probes can also be used in efficient and simple methods for fluorescent labeling of DNA. Finally, we suggest interesting directions for the future of this field in biomedicine and human health.

(Un)coupling the factors contributing to the interfacial activation of Streptomyces rimosus lipase: computational and spectrophotometric study

Streptomyces rimosus lipases (SrLip) present an enzyme class that catalyzes the hydrolysis of triacylglycerols producing fatty acids and glycerols. The abrupt increase in catalytic activity of SrLip when found at water-oil interface is assigned as interfacial activation (IA). Since the latter is a complex function of interface charge, chemical composition, as well as substrate orientation and concentration, the role of individual factors and their combinations that trigger IA remains rather elusive. The aim of this work is to unravel the contribution of particular factor in IA by examining SrLip activity toward p-nitrophenylpalmitate (p-NPP) as a substrate incorporated in three chemically different self-assembled aggregates: gum Arabic, sodium deoxycholate and liposomes made of 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) lipids, and at two different pH values (pH = 6.8 and pH = 10.0). Spectrophotometric (UV/Vis) study revealed that SrLip catalyzes p-NPP hydrolysis when the substrate is embedded in sodium deoxycholate at both pH values, suggesting that the coupling of favorable interface charge and substrate orientation drives the interfacial activation. Moreover, especially in alkaline conditions (pH = 10.0), hydrolysis of the ester bond of the substrate incorporated in DOPC bilayers occurs when appropriate orientation, concentration and arrangement are accomplished. All experiments were conducted in parallel on an inactive mutant SrLipS10/A as a reference. The molecular-level details of particular interface and accompanied substrates examined by all-atom molecular dynamics simulations revealed that the availability of p-NPP at these three interfaces decreases in the following order: sodium-deoxycholate > DOPC > model gum Arabic.