scholarly journals Saturation mutagenesis at Ser196 in BaCsn46A from Bacillus Amyloliquefaciens Enhances Enzyme Activity and Thermostability

2021 ◽  
Author(s):  
Wang Yi ◽  
Gao Wenjun ◽  
Wang Hailong ◽  
Xu Kepan ◽  
Luo Wen ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Secondary Structure ◽  
Industrial Production ◽  
Bacillus Amyloliquefaciens ◽  
Three Dimensional ◽  
Saturation Mutagenesis ◽  
Specific Enzyme ◽  
Mutation Site ◽  
Excellent Thermal Stability ◽  
Specific Enzyme Activity

Abstract The chitosanase (BaCsn46A) was extracted from Bacillus amyloliquefaciens (GenBank: QEK97559.1) and synthesized after codon optimization. The saturation mutation site was determined by analyzing the sequence and three-dimensional protein model. WT and mutant chitosanase genes were cloned and expressed in E. coli BL21 (DE3). The enzymatic properties of WT and mutants were compared, including the optimal reaction pH, temperature and thermostability. Three mutants S196F, S196Y and S196A with the highest specific enzyme activity were selected for further study. Compared with WT, the specific enzyme activity of S196Y increased by 144.76% (more than other two mutants), and the thermostability was not significantly improved. While the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. From the perspective of industrial production, S196A is more in line with the requirements of industrial production because of its excellent thermal stability at 60°C. From the results of circular dichroism spectrum, the mutation of chitosanase at Ser196 did not change the secondary protein structure. In addition, CD analysis showed that the secondary structure of WT and mutants did not change significantly, indicating that the improvement of thermostability of S196A was not related to the secondary structure.

Evolution of enzyme structure

1979 ◽  
Vol 205 (1161) ◽  
pp. 443-452 ◽  
Keyword(s):  
Enzyme Activity ◽  
Metabolic Pathways ◽  
Serine Proteases ◽  
Selective Pressure ◽  
Three Dimensional ◽  
Enzyme Structure ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
Evolution Of Metabolic Pathways ◽  
Molecular Morphology

Three-dimensional structures of enzymes offer evidence about their evolution. There are clear examples of divergent families (e. g. mammalian serine proteases) and convergence (e. g. chymotrypsin and subtilisin). Topological similarities in dehydrogenases may reflect an ancient divergence or merely chemical constraints on protein architectures. Further experimental evidence is desirable to back up arguments based on molecular morphology. By growing microorganisms on novel foodstuffs in a chemostat, one can focus selective pressure on a specific enzyme activity. Experiments will be described in which such pressure is focused on pentitol metabolism. Examination of the fine structure of the genes responsible for this pentitol metabolism has given clues about the evolution of metabolic pathways.

Specific Enzyme Activity

2019 ◽  
Author(s):  
Ján Labuda ◽  
Richard P. Bowater ◽  
Miroslav Fojta ◽  
Günter Gauglitz ◽  
Zdeněk Glatz ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Specific Enzyme ◽  
Specific Enzyme Activity

scholarly journals Preparation of gram quantities of a purified R-factor-mediated penicillinase from Escherichia coli strain W3310

1972 ◽  
Vol 130 (1) ◽  
pp. 55-62 ◽  
Author(s):  
J. Melling ◽  
G. K. Scott
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Coli Strain ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
E Coli ◽  
R Factor

Purified penicillinase, in gram quantities, has been prepared from Escherichia coli strain W3310 by using methods developed to handle large amounts of material. The final product had a specific enzyme activity of 3.08 units/μg of protein, which was over twice as high as that reported previously (Datta & Richmond, 1966). The purified enzyme was similar to that from E. coli strain TEM, but different in molecular weight and some other respects. The differences observed may be a result of the greater purity obtained.

Detection in HeLa cell extracts of a 7-methyl guanosine specific enzyme activity that cleaves m7GpppNm

Cell ◽  
1975 ◽  
Vol 6 (1) ◽  
pp. 21-27 ◽  
Author(s):  
D.L. Nuss ◽  
Y. Furuichi ◽  
G. Koch ◽  
A.J. Shatkin
Keyword(s):  
Enzyme Activity ◽  
Hela Cell ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
Cell Extracts

Further studies of L-arginine biosynthesis in germinating pea seeds. Evidence for the presence of argininosuccinate synthetase in cotyledon extracts

1969 ◽  
Vol 47 (4) ◽  
pp. 467-475 ◽  
Author(s):  
P. D. Shargool ◽  
E. A. Cossins
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Magnesium Ions ◽  
Argininosuccinate Synthetase ◽  
Argininosuccinate Lyase ◽  
Specific Enzyme ◽  
Arginine Biosynthesis ◽  
Specific Enzyme Activity ◽  
Pea Seeds

The synthesis and metabolism of arginine in germinating peas was examined by supplying micromolar quantities of L-citruiline-carbamyl-14C, DL-arginine-carbamyl-14C, and DL-arginine-5-14C to imbibing seeds. Citrulline was readily incorporated into arginine, but the labelled arginine solutions were not extensively metabolized.Extracts of 1-day-old cotyledons were found to catalyze the synthesis of arginine from citrulline in a reaction having absolute requirements for ATP, L-aspartate, and magnesium ions. The extracts were fractionated by (NH4)2SO4 precipitation followed by gel filtration on columns of Sephadex G-50 and G-200. These treatments increased the specific enzyme activity by approximately 36 times. After such treatments the preparations still contained appreciable amounts of argininosuccinate lyase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) activity. The rate of arginine synthesis was altered by increasing the concentrations of L-citrulline, L-aspartate, and ATP. The latter compounds were found to be inhibitory at concentrations of 1 μmole/ml and 4 μmoles/ml, respectively. Arginine synthesis was markedly affected by pH and by additions of arginine and argininosuccinate. It is concluded that germinating pea cotyledons contain appreciable levels of argirrinosuccmate synthetase (L-citrulline:L-aspartate ligase (AMP), EC 6.3.4.5), and furthermore, that this enzyme has importance in arginine biosynthesis during germination.

Persistent elevation of paraoxonase-1 specific enzyme activity after weight reduction in obese non-diabetic men with metabolic syndrome

2011 ◽  
Vol 412 (19-20) ◽  
pp. 1835-1841 ◽  
Author(s):  
Kae-Woei Liang ◽  
Wen-Jane Lee ◽  
I.-Te Lee ◽  
Wen-Lieng Lee ◽  
Shih-Yi Lin ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
Enzyme Activity ◽  
Weight Reduction ◽  
Paraoxonase 1 ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
Persistent Elevation

Quantitative assay system for specific enzyme activity using antibody: the case of protease, subtilisin BPN′

1998 ◽  
Vol 66 (2-3) ◽  
pp. 157-163 ◽  
Author(s):  
Yoshiaki Miyota ◽  
Shingo Komada ◽  
Haruo Momose ◽  
Seiichi Taguchi
Keyword(s):  
Enzyme Activity ◽  
Assay System ◽  
Quantitative Assay ◽  
Specific Enzyme ◽  
Specific Enzyme Activity

Combined Cytochemical Detection of Müller-Cell-Specific Enzyme Activity and Permeability Tracers with 1 color plate

1991 ◽  
Vol 202 (2) ◽  
pp. 94-99 ◽  
Author(s):  
Akira Hirata ◽  
Takashi Kitaoka ◽  
Hitoshi Ishigooka ◽  
Satoki Ueno
Keyword(s):  
Enzyme Activity ◽  
Müller Cell ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
Color Plate ◽  
Muller Cell

scholarly journals Effects of stimulation of muscarinic and of β-catecholamine receptors on the intracellular distribution of protein kinase C in guinea pig exocrine glands

1987 ◽  
Vol 242 (3) ◽  
pp. 749-754 ◽  
Author(s):  
E Machado-de Domenech ◽  
H D Söling
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Parotid Gland ◽  
Particulate Fraction ◽  
Amylase Secretion ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
Kinase C ◽  
Stimulation Of

Stimulation of exocrine cells via muscarinic receptors is associated with an activation of protein kinase C [Padel & Söling (1985) Eur. J. Biochem. 151, 1-10]. We show here that stimulation of isolated parotid gland lobules with 8 X 10(-6) M-carbamoylcholine leads to a translocation of protein kinase C from the cytosolic to the particulate compartment within 30 s (25% and 45% of total activity recovered in the particulate fraction of controls and stimulated samples respectively). The specific enzyme activity in the particulate fraction increased to 169% of the corresponding control value. After 10 min the changes started to reverse and, after 30 min, cytosolic protein kinase C was higher in stimulated than in unstimulated lobules. Isoproterenol (2 X 10(-5) M) stimulated the release of amylase more than did carbamoylcholine, but did not significantly affect intracellular distribution of protein kinase C during the observation time of 30 min. In isolated pancreatic lobules a significant carbamoylcholine-mediated translocation of protein kinase C into the particulate fraction could be observed after 5 and 20 min, but not after 1 min. After 5 min the specific enzyme activity in the particulate fraction had increased to 153% of the corresponding controls. The corresponding decrease (-38%) in the specific enzymic activity of cytosolic protein kinase C stayed constant up to 30 min. In isolated parotid gland lobules alpha-amylase secretion proceeded at a linear rate already during the first 1 min of stimulation, whereas in pancreatic lobules a measurable rate of alpha-amylase secretion did not occur before 5 min. These differences in time course paralleled the differences in the onset of translocation of protein kinase C. The results support a direct involvement of protein kinase C in carbamoylcholine-mediated but not in isoproterenol-mediated stimulation of exocytosis in exocrine cells.

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