scholarly journals Elucidating the degradation pattern of a new cold-tolerant pectate lyase used for efficient preparation of pectin oligosaccharides

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ling Zheng ◽  
Zilong Guo ◽  
Shengsheng Cao ◽  
Benwei Zhu
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
High Specific Activity ◽  
Pectate Lyase ◽  
Catalytic Efficiency ◽  
High Catalytic Activity ◽  
Ionization Mass ◽  
Biotechnological Applications ◽  
Product Analysis ◽  
Cold Tolerant

AbstractThe cold-active pectate lyases have drawn increasing attention in food and biotechnological applications due to their ability to retain high catalytic efficiency under lower temperatures, which could be helpful for energy saving, cost reduction and flavor preservation. Herein, a new cold-tolerant pectate lyase (ErPelPL1) gene from Echinicola rosea was cloned and heterologously expressed in Escherichia coli. Interestingly, ErPelPL1 retained high catalytic activity even at a low temperature (4 °C). ErPelPL1 exhibited optimal activity at 35 ℃, pH 8.0 with 1 mM of Ca2+. It showed high specific activity towards polygalacturonic acid (34.7 U/mg) and sodium polygalacturonate (59.3 U/mg). The combined thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC) and electrospray ionization mass spectrometry (ESI-MS) results indicated that ErPelPL1 endolytically degraded pectic substances into the oligosaccharides with degrees of depolymerization (Dps) of 1–6. In conclusion, this study mainly conducted biochemical characterization and product analysis of a cold-tolerant pectate lyase. Therefore, it provides a promising enzyme candidate for food and biotechnological applications. Graphical Abstract

scholarly journals Structure-Based Engineering of Amidase from Pantoea sp. for Efficient 2-Chloronicotinic Acid Biosynthesis

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Xiao-Ling Tang ◽  
Jian-Qiang Jin ◽  
Zhe-Ming Wu ◽  
Li-Qun Jin ◽  
Ren-Chao Zheng ◽  
...  
Keyword(s):  
Structure Function ◽  
Pyridine Ring ◽  
Double Mutant ◽  
Specific Activity ◽  
Function Analysis ◽  
Catalytic Efficiency ◽  
Saturation Mutagenesis ◽  
Nucleophilic Attack ◽  
High Catalytic Activity ◽  
Chlorine Substitution

ABSTRACT 2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for 2-chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electronic effect caused by 2-position chlorine substituent of the pyridine ring. In this study, an amidase from a Pantoea sp. (Pa-Ami) was designed and engineered to have improved catalytic properties. Single mutant G175A and double mutant G175A/A305T strains exhibited 3.2- and 3.7-fold improvements in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased, with kcat/Km values 3.1 and 10.0 times higher than that of the wild type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide was shortened in the G175A mutant, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the G175A/A305T double mutant as a biocatalyst, a maximum of 1,220 mM 2-chloronicotinic acid was produced with a 94% conversion, and the space-time yield reached as high as 575 gproduct liter−1 day−1. These results provide not only a novel robust biocatalyst for the production of 2-chloronicotinic acid but also new insights into amidase structure-function relationships. IMPORTANCE In recent years, the demand for 2-chloronicotinic acid has been greatly increased. To date, several chemical methods have been used for the synthesis of 2-chloronicotinic acid, but all include tedious steps and/or drastic reaction conditions, resulting in both economic and environmental issues. It is requisite to develop an efficient and green synthesis route. We recently screened Pa-Ami and demonstrated its potential for synthesis of 2-chloronicotinic acid from 2-chloronicotinamide. However, chlorine substitution on the pyridine ring of nicotinamide significantly affected the activity of Pa-Ami. Especially for 2-chloronicotinamide, the enzyme activity and catalytic efficiency were relatively low. In this study, based on structure-function analysis, we succeeded in engineering the amidase by structure-guided saturation mutagenesis. The engineered Pa-Ami exhibited quite high catalytic activity toward 2-chloronicotinamide and could serve as a promising biocatalyst for the biosynthesis of 2-chloronicotinic acid.

scholarly journals A Novel Digestive α-Amylase from Blue Crab (Portunus segnis) Viscera: Purification, Biochemical Characterization and Application for the Improvement of Antioxidant Potential of Oat Flour

2021 ◽  
Vol 22 (3) ◽  
pp. 1070
Author(s):  
Hana Maalej ◽  
Amina Maalej ◽  
Sawsan Affes ◽  
Noomen Hmidet ◽  
Moncef Nasri
Keyword(s):  
Blue Crab ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Potato Starch ◽  
High Specific Activity ◽  
Biochemical Properties ◽  
Antioxidant Potential ◽  
Tween 20 ◽  
Industrial Sectors ◽  
Oat Flour

This study reports on the purification and characterization of a digestive α-amylase from blue crab (Portunussegnis) viscera designated Blue Crab Amylase (BCA). The enzyme was purified to homogeneity by ultrafiltration, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with the final purification fold of 424.02, specific activity of 1390.8 U mg−1 and 27.8% recovery. BCA, showing a molecular weight of approximately 45 kDa, possesses desirable biotechnological features, such as optimal temperature of 50 °C, interesting thermal stability which is enhanced in the presence of starch, high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), high specific activity, quite high storage and broad pH range stability. The enzyme displayed Km and Vmax values, of 7.5 ± 0.25 mg mL−1 and 2000 ± 23 μmol min−1 mg−1 for potato starch, respectively. It hydrolyzed various carbohydrates and produced maltose, maltotriose and maltotetraose as the major end products of starch hydrolysis. In addition, the purified enzyme was successfully utilized for the improvement of the antioxidant potential of oat flour, which could be extended to other cereals. Interestingly, besides its suitability for application in different industrial sectors, especially food industries, the biochemical properties of BCA from the blue crab viscera provide novel features with other marine-derived enzymes and better understanding of the biodegradability of carbohydrates in marine environments, particularly in invasive alien crustaceans.

scholarly journals Peroxidase from Coleus Forskohlii: Purification and Biochemical Characterization

2020 ◽  
Vol 5 (1) ◽  
pp. 9-20
Author(s):  
Yaaser Q. Almulaiky ◽  
Yaaser Q. Almulaiky
Keyword(s):  
Ion Exchange ◽  
Peroxidase Activity ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Coleus Forskohlii ◽  
Sds Page ◽  
Optimum Ph

In this study, a peroxidase from new source was purified using ion exchange and gel filtration techniques. The recovery for peroxidase activity was 19% with 11-fold purification and specific activity of 749 unit/mg protein. Purified peroxidase demonstrated a molecular mass of 39 kDa using gel filtration and was confirmed as a single band on SDS-PAGE. The purified peroxidase revealed a broad optimum pH activity at 6.0-6.5 and 50°C temperature. The kinetic parameters for purified peroxidase toward H2O2 and guaiacol as substrates were found to be Km = 3.355, 5.395 mM, Kcat = 99.52, 79.56 s-1 and Vmax =1.531, 1.242 µmole ml-1 min-1, respectively. The catalytic efficiency (kcat/Km) of the purified peroxidase was 14.75 and 29.66 s−1 mM−1 for guaiacol and H2O2, respectively. Peroxidase activity was observed to be enhanced by Cu2+, Co2+, Ni2+ and inhibited in the presence of Sn2+, Al3+, Hg2+, NaN3, EDTA and urea. Characterization showed that peroxidase purified from C. forskohlii has the ability to be used for food industrial applications.

scholarly journals Comparative Catalytic Evaluation of Nano-ZrOx Promoted Manganese Catalysts: Kinetic Study and the Effect of Dopant on the Aerobic Oxidation of Secondary Alcohols

2017 ◽  
Vol 2017 ◽  
pp. 1-14 ◽  
Author(s):  
Mohamed E. Assal ◽  
Mufsir Kuniyil ◽  
Mujeeb Khan ◽  
Mohammed Rafi Shaik ◽  
Abdulrahman Al-Warthan ◽  
...  
Keyword(s):  
Reaction Time ◽  
Specific Activity ◽  
Aerobic Oxidation ◽  
Chemical Properties ◽  
High Specific Activity ◽  
Catalytic Efficiency ◽  
Manganese Carbonate ◽  
Spectroscopic Techniques ◽  
Oxidation Of Alcohols ◽  
Physical And Chemical

This work reports the zirconia (ZrOx) nanoparticles doped MnCO3 catalysts prepared by facile and simple coprecipitation technique and the synthesis of zirconia-manganese carbonate [X% ZrOx–MnCO3] (where X% = 0–7%) catalyst which upon calcination at 400°C is converted to zirconia-manganese dioxide [1% ZrOx–MnO2] and when calcined at 500°C is converted to zirconia-manganic trioxide [1% ZrOx–Mn2O3]. A comparative catalytic study was performed to investigate the catalytic efficiency between carbonate and oxides for the selective oxidation of 1-phenylethanol by using molecular O2 as a clean oxidant. The influence of several parameters such as w/w% of ZrOx, reaction time, calcination temperature, catalyst amount, and reaction temperature has been thoroughly examined using oxidation of 1-phenylethanol as a model substrate. The 1% ZrOx–MnCO3 precalcined at 300°C exhibited the best catalytic efficiency. It was found that ZrOx nanoparticles also play an essential role in enhancing the effectiveness of the catalytic system for the aerobic oxidation of alcohols. Furthermore, the physical and chemical properties of synthesized catalysts were evaluated by microscopic and spectroscopic techniques. An extremely high specific activity of 40 mmol·g−1·h−1 with a 100% conversion of oxidation product and selectivity of >99% was achieved within extremely short reaction time (6 min).

scholarly journals Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Junji Hayashi ◽  
Tomonari Seto ◽  
Hironaga Akita ◽  
Masahiro Watanabe ◽  
Tamotsu Hoshino ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Specific Activity ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
High Catalytic Activity ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase

ABSTRACT A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP+ and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P)+-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.

Biochemical characterization of a thermophilic β-mannanase from Talaromyces leycettanus JCM12802 with high specific activity

2014 ◽  
Vol 99 (3) ◽  
pp. 1217-1228 ◽  
Author(s):  
Caihong Wang ◽  
Huiying Luo ◽  
Canfang Niu ◽  
Pengjun Shi ◽  
Huoqing Huang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
High Specific Activity

scholarly journals Biochemical Characterization of a Novel l-Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

2013 ◽  
Vol 80 (5) ◽  
pp. 1561-1569 ◽  
Author(s):  
Linhua Huang ◽  
Yu Liu ◽  
Yan Sun ◽  
Qiaojuan Yan ◽  
Zhengqiang Jiang
Keyword(s):  
Amino Acid ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
High Specific Activity ◽  
Rhizomucor Miehei ◽  
Drug Dosage ◽  
Amino Acid Sequences ◽  
Content Type ◽  
Leukemia Treatment ◽  
Glutaminase Activity

ABSTRACTA novel fungal gene encoding theRhizomucor mieheil-asparaginase (RmAsnase) was cloned and expressed inEscherichia coli. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reportedl-asparaginases. The purifiedl-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, thisl-asparaginase was found to potentiate a lectin's induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia.

scholarly journals Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells

2003 ◽  
Vol 373 (2) ◽  
pp. 423-435 ◽  
Author(s):  
Edward McKENZIE ◽  
Kathryn YOUNG ◽  
Margaret HIRCOCK ◽  
James BENNETT ◽  
Maina BHAMAN ◽  
...  
Keyword(s):  
Insect Cells ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Expression System ◽  
Heparan Sulphate ◽  
High Specific Activity ◽  
Vitro Assay ◽  
Tumour Metastasis

The mammalian endoglycosidase heparanase (Hpa1) is primarily responsible for cleaving heparan sulphate proteoglycans (HSPGs) present on the basement membrane of cells and its potential for remodelling the extracellular matrix (ECM) could be important in embryonic development and tumour metastasis. Elevated expression of this enzyme has been implicated in various pathological processes including tumour cell proliferation, metastasis, inflammation and angiogenesis. The enzyme therefore represents a potential therapeutic target. Hpa1 protein is initially synthesized as an inactive 65 kDa proenzyme that is then believed to be subsequently activated by proteolytic cleavage to generate an active heterodimer of 8 and 50 kDa polypeptides. By analysis of a series of Hpa1 deletion proteins we confirm that the 8 kDa subunit is essential for enzyme activity. We present here for the first time an insect cell expression system used for the generation of large amounts of recombinant protein of high specific activity. Individual subunits were cloned into baculoviral secretory vectors and co-expressed in insect cells. Active secreted heterodimer protein was recovered from the medium and isolated by a one-step heparin–Sepharose chromatography procedure to give protein of >90% purity. The recombinant enzyme behaved similarly to the native protein with respect to the size of HS fragments liberated on digestion, substrate cleavage specificity and its preference for acidic pH. A significant amount of activity, however, was also detectable at physiological pH values, as measured both by an in vitro assay and by in vivo degradation of cell-bound heparan sulphate.

scholarly journals Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Samaneh Mosallatpour ◽  
Saeed Aminzadeh ◽  
Mehdi Shamsara ◽  
Reza Hajihosseini
Keyword(s):  
Food Industry ◽  
Tertiary Structure ◽  
Biochemical Characterization ◽  
Arrhenius Plot ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Cysteine Residue ◽  
Enzyme Active Site ◽  
Wide Range ◽  
Ph Range

AbstractL-glutaminase importance to use in the food industry and medicine has attracted much attention. Enzymes stability has always been a challenge while working with them. We heterologously expressed and characterized a novel stable L-glutaminase from an extremophile bacterium (Cohnella sp. A01, PTCC No: 1921). Km, Vmax, catalytic efficiency and specific activity of rSAM were respectively 1.8 mM, 49 µmol/min, 1851 1/(S.mM) and 9.2 IU/mg. Activation energy for substrate to product conversion and irreversible thermo-inactivation were respectively 4 kJ/mol and 105 kJ/mol from the linear Arrhenius plot. rSAM had the highest activity at temperature 50 °C, pH 8 and was resistant to a wide range of temperature and pH. In compare to the other characterized glutaminases, rSAM was the most resistant to NaCl. Mg2+, glycerol, DTT, and BME enhanced the enzyme activity and iodoacetate and iodoacetamide inhibited it. rSAM had only been partially digested by some proteases. According to the Fluorimetry and Circular dichroism analysis, rSAM in pH range from 4 to 11 and temperatures up to 60 °C had structural stability. A cysteine residue in the enzyme active site and a thiol bond were predicted upon the modeled tertiary structure of rSAM. Present structural studies also confirmed the presence of a thiol bond in its structure.

scholarly journals Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming

2015 ◽  
Vol 81 (17) ◽  
pp. 5714-5723 ◽  
Author(s):  
Cheng Zhou ◽  
Jintong Ye ◽  
Yanfen Xue ◽  
Yanhe Ma
Keyword(s):  
Directed Evolution ◽  
Textile Industry ◽  
Specific Activity ◽  
High Specific Activity ◽  
Pectate Lyase ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Content Type ◽  
Alkaline Pectate Lyase

ABSTRACTThermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase fromBacillussp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca2+. However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in thet50(time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in thet50value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry.

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