scholarly journals Engineering of Cyclodextrin Glycosyltransferase Reveals pH-Regulated Mechanism of Enhanced Long-Chain Glycosylated Sophoricoside Specificity

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Ruizhi Han ◽  
Jie Ni ◽  
Jieyu Zhou ◽  
Jinjun Dong ◽  
Guochao Xu ◽  
...  
Keyword(s):  
Water Solubility ◽  
Ph Regulation ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Cyclodextrin Glycosyltransferase ◽  
Sugar Chain ◽  
Content Type ◽  
Product Specificity ◽  
Pharmaceutical Industries ◽  
Long Chain

ABSTRACT Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among α-, β-, and γ-CGTases with different product specificities. Variants L174P, A156V/L174P, and A156V/L174P/A166Y greatly improved the product specificity for LCGS. pH significantly affected the extent of glycosylation catalyzed by the variants. Further investigations revealed that the pH-regulated mechanism for LCGS synthesis mainly depends on a disproportionation route at a lower pH (pH 4) and a cyclization-coupling route at a higher pH (pH 8) and equivalent effects of cyclization-coupling and disproportionation routes at pH 5. Whereas short-chain glycosylated sophoricosides (SCGS) are primarily produced via disproportionation of maltodextrin at pH 4 and secondary disproportionation of LCGS at pH 8. At pH 5, SCGS synthesis mainly depends on a hydrolysis route by the wild type (WT) and a secondary disproportionation route by variant A156V/L174P/A166Y. Kinetics analysis showed a decreased Km value of variant A156V/L174P/A166Y. Dynamics simulation results demonstrated that the improved LCGS specificity of the variant is possibly attributed to the enhanced affinity to long-chain substrates, which may be caused by the changes of hydrogen bond interactions at the –5, –6, and –7 subsites. Our results reveal a pH-regulated mechanism for product specificity of CGTase and provide guidance for engineering CGTase toward products with different sugar chain lengths. IMPORTANCE The low water solubility of sophoricoside seriously limits its applications in the food and pharmaceutical industries. Long-chain glycosylated sophoricosides show greatly improved water solubility. Here, the product specificity of cyclodextrin glycosyltransferase (CGTase) for long-chain glycosylated sophoricosides was significantly affected by pH. Our results reveal the pH-regulated mechanism of the glycosylated product specificity of CGTase. This work adds to our understanding of the synthesis of long-chain glycosylated sophoricosides and provides guidance for exploring related product specificity of CGTase based on pH regulation.

scholarly journals Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

2012 ◽  
Vol 78 (18) ◽  
pp. 6647-6655 ◽  
Author(s):  
Yu Zhang ◽  
Jiao An ◽  
Wei Ye ◽  
Guangyu Yang ◽  
Zhi-Gang Qian ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Divergent Evolution ◽  
Laboratory Evolution ◽  
Content Type ◽  
Hydrolytic Activities ◽  
Geobacillus Kaustophilus ◽  
Amidohydrolase Superfamily

ABSTRACTThe phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates forin vitroevolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL fromGeobacillus kaustophilusHTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (kcat/Km) for the OP pesticideethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 105-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

scholarly journals High-level production of γ-cyclodextrin glycosyltransferase in recombinant Escherichia coli BL21 (DE3): culture medium optimization, enzymatic properties characterization, and product specificity analysis

2020 ◽  
Vol 70 (1) ◽  
Author(s):  
Menglu Duan ◽  
Yan Wang ◽  
Guowu Yang ◽  
Jiao Li ◽  
Yi Wan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Performance ◽  
Low Cost ◽  
Cassava Starch ◽  
Transformation Process ◽  
Enzymatic Properties ◽  
Cyclodextrin Glycosyltransferase ◽  
High Productivity ◽  
Product Specificity ◽  
Pharmaceutical Industries

Abstract Purpose γ-Cyclodextrin glycosyltransferase (γ-CGTase) catalyzes the biotransformation of low-cost starch into valuable γ-cyclodextrin (γ-CD), which is widely applied in biotechnology, food, and pharmaceutical industries. However, the low specificity and activity of soluble γ-CGTase increase the production cost of γ-CD, thereby limiting its applications. Therefore, the present study aimed at optimizing an economical medium for high production of γ-CGTase by the recombinant Escherichia coli (E. coli) BL21 (DE3) and evaluating its enzymatic properties and product specificity. Methods The γ-CGTase production was optimized using the combination of Plackett-Burman experimental design (PBD) and Box-Behnken design-response surface methodology (BBD-RSM). The hydrolysis and cyclization properties of γ-CGTase were detected under the standard assay conditions with buffers of various pHs and different reaction temperatures. The product specificity of γ-CGTase was investigated by high-performance liquid chromatography (HPLC) analysis of three CDs (α-, β-, γ-CD) in the biotransformation product of cassava starch. Results The γ-CGTase activity achieved 53992.10 U mL−1 under the optimum conditions with the significant factors (yeast extract 38.51 g L−1, MgSO4 4.19 mmol L−1, NiSO4 0.90 mmol L−1) optimized by the combination of PBD and BBD-RSM. The recombinant γ-CGTase exhibited favorable stability in a wide pH and temperature range and maintained both the hydrolysis and cyclization activity under the pH 9.0 and 50 °C. Further analysis of the products from cassava starch catalyzed by the γ-CGTase reported that the majority (90.44%) of product CDs was the γ form, which was nearly 11% higher than the wild enzyme. Cyclododecanone added to the transformation system could enhance the γ-CD purity to 98.72%, which is the highest purity value during the transformation process reported so far. Conclusion The yield of γ-CGTase activity obtained from the optimized medium was 2.83-fold greater than the unoptimized medium, and the recombinant γ-CGTase exhibited a favorable thermal and pH stability, and higher γ-cyclization specificity. These results will provide a fundamental basis for the high productivity and purity of γ-CD in the industrial scale.

scholarly journals Methanogenic Paraffin Biodegradation: Alkylsuccinate Synthase Gene Quantification and Dicarboxylic Acid Production

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Lisa K. Oberding ◽  
Lisa M. Gieg
Keyword(s):  
Oil Recovery ◽  
Water Solubility ◽  
Enrichment Culture ◽  
Dicarboxylic Acids ◽  
Contaminated Site ◽  
Rrna Gene ◽  
Α Subunit ◽  
Content Type ◽  
Anoxic Environments ◽  
Long Chain

ABSTRACTParaffinicn-alkanes (>C17) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffinn-octacosane (C28H58). Compared with that in controls, the consumption ofn-octacosane was coupled to methane production, demonstrating its biodegradation under these conditions.Smithellawas postulated to be an important C28H58degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identifiedassAgene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from otherSmithellaorganisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays forassAdemonstrated significant increases in the abundance and expression of this gene in C28H58-degrading cultures compared with that in controls, suggestingn-octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C28H58-degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery.IMPORTANCEUnderstanding the methanogenic biodegradation of different classes of hydrocarbons has important applications for effective fuel-contaminated site remediation and for improved recovery from oil reservoirs. Previous studies have clearly demonstrated that short-chain alkanes (<C17) can be biodegraded anaerobically, but less is understood regarding the biodegradation of longer chain waxy alkanes (>C17) that comprise many fuel mixtures. Using an enrichment culture derived from a freshwater fuel-contaminated site, we demonstrate that the model waxy alkanen-octacosane can be biodegraded under methanogenic conditions by a presumedSmithellaphylotype. Compared with that of controls, we show an increased abundance and expression of theassAgene, which is known to be important for anaerobicn-alkane metabolism. Metabolite analyses revealed the presence of a range of α,ω-dicarboxylic acids found only inn-octacosane-degrading cultures, a novel finding that lends insight as to how anaerobic communities may access waxes as growth substrates in anoxic environments.

scholarly journals Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis

2013 ◽  
Vol 79 (24) ◽  
pp. 7562-7568 ◽  
Author(s):  
Ruizhi Han ◽  
Long Liu ◽  
Hyun-dong Shin ◽  
Rachel R. Chen ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Substrate Binding ◽  
Acid Synthesis ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Cyclodextrin Glycosyltransferase ◽  
Content Type ◽  
Paenibacillus Macerans ◽  
Ascorbic Acid Synthesis ◽  
Iterative Saturation Mutagenesis

ABSTRACT2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stablel-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase fromPaenibacillus maceransto improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.

scholarly journals Rational thermostabilisation of four-helix bundle dimeric de novo proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shin Irumagawa ◽  
Kaito Kobayashi ◽  
Yutaka Saito ◽  
Takeshi Miyata ◽  
Mitsuo Umetsu ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
Protein Complexes ◽  
Salt Bridge ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Helix Bundle ◽  
Stable Mutant ◽  
Four Helix Bundle ◽  
The Stability

AbstractThe stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

Determining the structure of pharmaceutical industry in Iran

2019 ◽  
Vol 13 (1) ◽  
pp. 101-115
Author(s):  
Sara Emamgholipour ◽  
Lotfali Agheli
Keyword(s):  
Pharmaceutical Industry ◽  
Economies Of Scale ◽  
Monopolistic Competition ◽  
Pharmaceutical Market ◽  
Data Availability ◽  
Market Demand ◽  
Firm Level ◽  
Content Type ◽  
Measure Concentration ◽  
Pharmaceutical Industries

Purpose As the pharmaceutical industry is one of the key sectors of the health-care system, the identification of its structure is of particular importance. This paper aims to determine the structure of the pharmaceutical industry in Iran to provide appropriate solutions for pricing and regulation by policymakers. Iran is a growing pharmaceutical market with over $4bn in sales, so the supply side needs to be examined to meet the domestic consumption. Design/methodology/approach This research is a descriptive and retrospective analytical study which examines the Iranian pharmaceutical industry through library studies and using pharmaceutical data of the country’s Food and Drug Administration during 1992-2016. Due to data availability in firm level, the concentration ratio of N leading firms and the Herfindahl–Hirschman index are used to measure the concentration of the pharmaceutical market in 2014 and 2016. Findings The results show that pharmaceutical manufacturing, importing companies and distributing companies play roles in monopolistic competition market, loose oligopoly market and oligopoly market, respectively. For all companies, the magnitudes of Herfindahl–Hirschman indices indicate non-competitive settings. As a result, these companies set their own prices, and market demand affects their sales. In addition, demand for medicines is shaped in the form of supply-induced demand. Research limitations/implications This research was accomplished with no computational limitation. However, it was confined to only one country, one industry and the mentioned period of study. Practical implications The pharmaceutical manufacturers have no influence on medicine prices, and government pricing regulations lessen the market power of such market agents. However, the easy entry to and exit from market stimulate producers to participate in manufacturing activities. The pharmaceutical importers may expand their imports in response to entry new actors; however, the new entrants weaken the coordination on pricing decisions. Social implications As pharmaceutical distributers act in an oligopoly market, they can collude, reduce competition and lower the welfare of pharmaceutical consumers. In such conditions, high investment requirements and economies of scale may discourage the entry of new firms. Originality/value Although there are various studies on market structure in non-pharmaceutical industries, this study is a new effort to measure concentration in the Iranian pharmaceutical market and to determine its structure.

scholarly journals The Seaweed Diet in Prevention and Treatment of the Neurodegenerative Diseases

Marine Drugs ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. 128
Author(s):  
Leonel Pereira ◽  
Ana Valado
Keyword(s):  
Multiple Sclerosis ◽  
Fatty Acids ◽  
Neurodegenerative Diseases ◽  
Marine Algae ◽  
Gelling Agents ◽  
Prevention And Treatment ◽  
Pharmaceutical Industries ◽  
Therapeutic Properties ◽  
Main Components ◽  
Long Chain

Edible marine algae are rich in bioactive compounds and are, therefore, a source of bioavailable proteins, long chain polysaccharides that behave as low-calorie soluble fibers, metabolically necessary minerals, vitamins, polyunsaturated fatty acids, and antioxidants. Marine algae were used primarily as gelling agents and thickeners (phycocolloids) in food and pharmaceutical industries in the last century, but recent research has revealed their potential as a source of useful compounds for the pharmaceutical, medical, and cosmetic industries. The green, red, and brown algae have been shown to have useful therapeutic properties in the prevention and treatment of neurodegenerative diseases: Parkinson, Alzheimer’s, and Multiple Sclerosis, and other chronic diseases. In this review are listed and described the main components of a suitable diet for patients with these diseases. In addition, compounds derived from macroalgae and their neurophysiological activities are described.

scholarly journals Novel Tandem Biotransformation Process for the Biosynthesis of a Novel Compound, 4-(2,3,5,6-Tetramethylpyrazine-1)-4′-Demethylepipodophyllotoxin

2011 ◽  
Vol 77 (9) ◽  
pp. 3023-3034 ◽  
Author(s):  
Ya-Jie Tang ◽  
Wei Zhao ◽  
Hong-Mei Li
Keyword(s):  
Cell Line ◽  
Alternaria Alternata ◽  
Water Solubility ◽  
Tumor Cell Line ◽  
Gibberella Fujikuroi ◽  
Hydroxyl Group ◽  
Epithelial Cell Line ◽  
The Novel ◽  
Content Type ◽  
Biotransformation Process

ABSTRACTAccording to the structure of podophyllotoxin and its structure-function relationship, a novel tandem biotransformation process was developed for the directional modification of the podophyllotoxin structure to directionally synthesize a novel compound, 4-(2,3,5,6-tetramethylpyrazine-1)-4′-demethylepipodophyllotoxin (4-TMP-DMEP). In this novel tandem biotransformation process, the starting substrate of podophyllotoxin was biotransformed into 4′-demethylepipodophyllotoxin (product 1) with the demethylation of the methoxyl group at the 4′ position byGibberella fujikuroiSH-f13, which was screened out from Shennongjia prime forest humus soil (Hubei, China). 4′-Demethylepipodophyllotoxin (product 1) was then biotransformed into 4′-demethylpodophyllotoxone (product 2) with the oxidation of the hydroxyl group at the 4 position byAlternaria alternataS-f6, which was screened out from the gatheredDysosma versipellisplants in the Wuhan Botanical Garden, Chinese Academy of Sciences. Finally, 4′-demethylpodophyllotoxone (product 2) and ligustrazine were linked with a transamination reaction to synthesize the target product 4-TMP-DMEP (product 3) byAlternaria alternataS-f6. Compared with podophyllotoxin (i.e., a 50% effective concentration [EC50] of 529 μM), the EC50of 4-TMP-DMEP against the tumor cell line BGC-823 (i.e., 0.11 μM) was significantly reduced by 5,199 times. Simultaneously, the EC50of 4-TMP-DMEP against the normal human proximal tubular epithelial cell line HK-2 (i.e., 0.40 μM) was 66 times higher than that of podophyllotoxin (i.e., 0.006 μM). Furthermore, compared with podophyllotoxin (i.e., logP= 0.34), the water solubility of 4-TMP-DMEP (i.e., logP= 0.66) was significantly enhanced by 94%. For the first time, the novel compound 4-TMP-DMEP with superior antitumor activity was directionally synthesized from podophyllotoxin by the novel tandem biotransformation process developed in this work.

Long-chain n-3 polyunsaturated fatty acids contained in liver and muscle tissues of Okinawan long-spine porcupinefish (Diodon holocanthus Linnaeus 1758)

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Yutaka Tashiro
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Polyunsaturated Fatty Acids ◽  
Ryukyu Islands ◽  
Okinawa Island ◽  
Content Type ◽  
Muscle Tissues ◽  
The Ryukyu Islands ◽  
Total Fatty Acids ◽  
Long Chain

Purpose This study aimed to analyze the lipid content and fatty acid composition in the liver and muscle of a porcupinefish species inhabiting waters around the Ryukyu Islands to investigate their potential as a source of long-chain n-3 polyunsaturated fatty acids (LC-PUFAs). Design/methodology/approach Porcupinefish were collected along the Okinawa Island coast. The composition of fatty acids and cholesterol in both liver and muscle were analyzed using a gas chromatograph mass spectrometer. Findings The liver of Okinawan long-spine porcupinefish was rich in lipids whose content correlated to the proportion of liver/body weight. Fatty acid compositions in their liver and muscles were similar to each other. LC-PUFAs occupied 44% of total fatty acids, with docosahexaenoic acid (DHA) being the dominant (42%), whereas eicosapentaenoic acid occupied 2.4%. The liver contained 1,690 mg of cholesterol and 14.8 g of DHA per 100 g, whose proportion decreased in summer compared to other seasons (p = 0.036). Originality/value The liver of Okinawan long-spine porcupinefish, which has not yet been commercially used although its non-toxicity is claimed, can be an excellent source of LC-PUFAs, especially DHA, accentuating its potential in food supplements’ production.

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