scholarly journals Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1229
Author(s):  
Yana Y. Berlina ◽  
Lada E. Petrovskaya ◽  
Elena A. Kryukova ◽  
Lyudmila N. Shingarova ◽  
Sultan Sh. Gapizov ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Half Life ◽  
Amino Acid Content ◽  
Typical Feature ◽  
Maximum Activity ◽  
Site Directed Mutagenesis ◽  
Permafrost Soil ◽  
Activity Profile ◽  
Cold Active

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops—a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to “the proline rule”. Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.

scholarly journals Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

2002 ◽  
Vol 68 (6) ◽  
pp. 2676-2682 ◽  
Author(s):  
Daohai Zhang ◽  
Xianzhen Li ◽  
Lian-Hui Zhang
Keyword(s):  
Amino Acid ◽  
Sequence Alignment ◽  
Half Life ◽  
Bacterial Isolate ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Maximum Activity ◽  
Amino Acid Residues ◽  
Sequence Domain ◽  
Gene Cloning And Characterization

ABSTRACT The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)8-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp241, Glu295, Asp369, His145, and His368) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a Km of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe3+ and Hg2+ and was enhanced by Mn2+ and Mg2+. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu498 and Arg310 with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.

scholarly journals A Novel Highly Thermostable Multifunctional Beta-Glycosidase from CrenarchaeonAcidilobus saccharovorans

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Vadim M. Gumerov ◽  
Andrey L. Rakitin ◽  
Andrey V. Mardanov ◽  
Nikolai V. Ravin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Half Life ◽  
Sequence Similarity ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Glycoside Hydrolases ◽  
Amino Acid Residues ◽  
High Tolerance ◽  
High Sequence Similarity

We expressed a putativeβ-galactosidase Asac_1390 from hyperthermophilic crenarchaeonAcidilobus saccharovoransinEscherichia coliand purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity withp-nitrophenyl (pNP) substrates followed the order pNP-β-D-galactopyranoside (328 U mg−1), pNP-β-D-glucopyranoside (246 U mg−1), pNP-β-D-xylopyranoside (72 U mg−1), and pNP-β-D-mannopyranoside (28 U mg−1). Thus the enzyme was actually a multifunctionalβ-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.

scholarly journals Isolation and Characterization of a Novel Cold-Active, Halotolerant Endoxylanase from Echinicola rosea Sp. Nov. JL3085T

Marine Drugs ◽  
2020 ◽  
Vol 18 (5) ◽  
pp. 245
Author(s):  
Jianlong He ◽  
Le Liu ◽  
Xiaoyan Liu ◽  
Kai Tang
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Maximum Velocity ◽  
Maximum Activity ◽  
Glycoside Hydrolases ◽  
Amino Acid Residues ◽  
Xylanase Gene ◽  
Isolation And Characterization ◽  
Cold Active

We cloned a xylanase gene (xynT) from marine bacterium Echinicola rosea sp. nov. JL3085T and recombinantly expressed it in Escherichia coli BL21. This gene encoded a polypeptide with 379 amino acid residues and a molecular weight of ~43 kDa. Its amino acid sequence shared 45.3% similarity with an endoxylanase from Cellvibrio mixtus that belongs to glycoside hydrolases family 10 (GH10). The XynT showed maximum activity at 40 °C and pH 7.0, and a maximum velocity of 62 μmoL min−1 mg−1. The XynT retained its maximum activity by more than 69%, 51%, and 26% at 10 °C, 5 °C, and 0 °C, respectively. It also exhibited the highest activity of 135% in the presence of 4 M NaCl and retained 76% of its activity after 24 h incubation with 4 M NaCl. This novel xylanase, XynT, is a cold-active and halotolerant enzyme that may have promising applications in drug, food, feed, and bioremediation industries.

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

Screening of amino acid constituents from date palm fruits

2016 ◽  
Vol 5 (10) ◽  
pp. 4972
Author(s):  
Lata Birlangi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
United Arab Emirates ◽  
Nutritional Value ◽  
Date Palm ◽  
Amino Acid Content ◽  
Essential Amino Acids ◽  
Cultivated Plants ◽  
Palm Fruits ◽  
Social Part

The date palm (Phoenix dactylifera L.) is one of mankind’s oldest cultivated plants. The fruit of the date palm is an important crop of the hot arid and semi-arid regions of the world. It has always played a genuine economic and social part in the lives of the people of these areas. The present objective in examining the amino acid content of different varieties of date palm fruits from Middle-East region; is to determine whether its protein could effectively supplement the nutritional value and it is also aimed in finding which variety is rich in number of amino acids. The phytochemical screening revealed the presence of eight essential amino acids and five non-essential amino acids in the date fruits. Among all the date fruit varieties taken as samples for the study, Dabbas cultivar of United Arab Emirates found to exhibit eight types of amino acids which includes five as non-essential ones. Total of thirteen amino acids were detected in the seven date cultivars. Determination of amino acid can serve as a guide to the possible nutritional value.

scholarly journals The effects of insulin and adrenaline on the amino-acid content of blood

1933 ◽  
Vol 27 (5) ◽  
pp. 1648-1654 ◽  
Author(s):  
James Murray Luck ◽  
Stanley Wallace Morse
Keyword(s):  
Amino Acid ◽  
Acid Content ◽  
Amino Acid Content
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