scholarly journals Improving decolorization of dyes by laccase from Bacillus licheniformis by random and site-directed mutagenesis

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10267
Author(s):  
Tongliang Bu ◽  
Rui Yang ◽  
YanJun Zhang ◽  
Yuntao Cai ◽  
Zizhong Tang ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Methyl Orange ◽  
Bacillus Licheniformis ◽  
Ph Stability ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Dye Wastewater ◽  
Wild Type ◽  
Decolorization Rate ◽  
Acid Violet

Background Dye wastewater increases cancer risk in humans. For the treatment of dyestuffs, biodegradation has the advantages of economy, high efficiency, and environmental protection compared with traditional physical and chemical methods. Laccase is the best candidate for dye degradation because of its multiple substrates and pollution-free products. Methods Here, we modified the laccase gene of Bacillus licheniformis by error-prone PCR and site-directed mutagenesis and expressed in E. coli. The protein was purified by His-tagged protein purification kit. We tested the enzymatic properties of wild type and mutant laccase by single factor test, and further evaluated the decolorization ability of laccase to acid violet, alphazurine A, and methyl orange by spectrophotometry. Results Mutant laccase Lacep69and D500G were superior to wild type laccase in enzyme activity, stability, and decolorization ability. Moreover, the laccase D500G obtained by site-directed mutagenesis had higher enzyme activity in both, and the specific activity of the purified enzyme was as high as 426.13 U/mg. Also, D500G has a higher optimum temperature of 70 °C and temperature stability, while it has a more neutral pH 4.5 and pH stability. D500G had the maximum enzyme activity at a copper ion concentration of 12 mM. The results of decolorization experiments showed that D500G had a strong overall decolorization ability, with a lower decolorization rate of 18% for methyl orange and a higher decolorization rate of 78% for acid violet. Conclusion Compared with the wild type laccase, the enzyme activity of D500G was significantly increased. At the same time, it has obvious advantages in the decolorization effect of different dyes. Also, the advantages of temperature and pH stability increase its tolerance to the environment of dye wastewater.

scholarly journals Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human γ-glutamyl hydrolase

1999 ◽  
Vol 343 (3) ◽  
pp. 551-555 ◽  
Author(s):  
Karen J. CHAVE ◽  
John GALIVAN ◽  
Thomas J. RYAN
Keyword(s):  
Enzyme Activity ◽  
Structural Changes ◽  
Catalytic Mechanism ◽  
Iodoacetic Acid ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Mutant Proteins ◽  
Key Enzyme

γ-Glutamyl hydrolase (GH), which hydrolyses the γ-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target.

scholarly journals Improvement in the Thermostability of d-Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

2011 ◽  
Vol 77 (20) ◽  
pp. 7316-7320 ◽  
Author(s):  
Jin-Geun Choi ◽  
Yo-Han Ju ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Enzyme Activity ◽  
Agrobacterium Tumefaciens ◽  
Operation Time ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Conversion Yield ◽  
Content Type ◽  
Aromatic Stacking

ABSTRACTThe S213C, I33L, and I33L S213C variants ofd-psicose 3-epimerase fromAgrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer ofd-psicose.

scholarly journals Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans

1994 ◽  
Vol 302 (1) ◽  
pp. 291-295 ◽  
Author(s):  
A Moreau ◽  
M Roberge ◽  
C Manin ◽  
F Shareck ◽  
D Kluepfel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Enzyme Activity ◽  
Streptomyces Lividans ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Protein Mutation ◽  
Acidic Residues

On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.

scholarly journals Rat liver guanidinoacetate methyltransferase. Proximity of cysteine residues at positions 15, 90 and 219 as revealed by site-directed mutagenesis and chemical modification

1991 ◽  
Vol 277 (2) ◽  
pp. 399-406 ◽  
Author(s):  
Y Takata ◽  
T Date ◽  
M Fujioka
Keyword(s):  
Enzyme Activity ◽  
Chemical Modification ◽  
Rat Liver ◽  
Large Change ◽  
Spectroscopic Properties ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Nitrobenzoic Acid

Cys-90 of rat liver guanidinoacetate methyltransferase is a very reactive residue, and chemical modification of this residue results in a large decrease in activity [Fujioka, Konishi & Takata (1988) Biochemistry 27, 7658-7664]. To understand better the role of Cys-90 in catalysis, this residue was replaced with alanine by oligonucleotide-directed mutagenesis. The mutant is active and has kinetic constants similar to those of wild-type, indicating that Cys-90 is not involved in catalysis and substrate binding. The u.v.-absorption, fluorescence and c.d. spectra are also unchanged. Reaction of the mutant with an equimolar amount of 5,5′-dithiobis-(2-nitrobenzoic acid) or 2-nitro-5-thiocyanobenzoic acid results in an almost quantitative disulphide cross-linking between Cys-15 and Cys-21). The same treatment effects disulphide bond formation between Cys-15 and Cys-90 in wild type [Fujioka, Konishi & Takata (1988) Biochemistry 27, 7658-7664]. Since the mutant and wild-type enzymes appear to have similar secondary and tertiary structures, these results suggest that Cys-15, Cys-90 and Cys-219 of the methyltransferase occur spatially close together. The mutant cross-linked between Cys-15 and Cys-219 and the wild-type cross-linked between Cys-15 and Cys-90 show very similar spectroscopic properties. Although treatment of the mutant and wild-type enzymes with equimolar concentrations of 5,5′dithiobis-(2-nitrobenzoic acid) causes a large loss of enzyme activity in each case, kinetic analyses with the modified enzymes suggest that cross-linking of Cys-15 with Cys-90 or Cys-219 does not abolish activity and does not result in a large change in the Michaelis constants. Incubation of the mutant enzyme with excess 2-nitro-5-thiocyanobenzoic acid leads to modification of Cys-207 in addition to Cys-15 and Cys-219. Retention of considerable enzyme activity in the modified enzyme indicates that Cys-207 is also not an essential residue.

Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

RSC Advances ◽  
2015 ◽  
Vol 5 (93) ◽  
pp. 76040-76047 ◽  
Author(s):  
Zhenya Chen ◽  
Ye Li ◽  
Yue Feng ◽  
Liang Chen ◽  
Qipeng Yuan
Keyword(s):  
Active Site ◽  
Simulation Analysis ◽  
Docking Simulation ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Proteus Vulgaris ◽  
Molecular Docking Simulation ◽  
Activity Enhancement ◽  
First Time

Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

scholarly journals Mutant Firefly Luciferase Enzymes Resistant to the Inhibition by Sodium Chloride

2021 ◽  
Author(s):  
Satoshi Yawata ◽  
Kenichi Noda ◽  
Ai Shimomura ◽  
Akio Kuroda
Keyword(s):  
Sodium Chloride ◽  
Double Mutant ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Activity Level ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Original Activity ◽  
Double Mutation

Abstract ObjectivesFirefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study aims to expand the applications of firefly luciferase in the presence of sodium chloride.ResultsWe first obtained two mutant luciferase enzymes whose inhibition were alleviated and identified these mutations as Val288Ile and Glu488Val. Under dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into the wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride.ConclusionsThe mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The inhibition of luciferase CR under dialysis condition was more alleviated than either Val288Ile or Glu488Val alone, suggesting that the effect of the double mutation was cumulative. We discussed the effect of mutations on the alleviation of the inhibition by sodium chloride.

scholarly journals Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis

1991 ◽  
Vol 279 (1) ◽  
pp. 35-41 ◽  
Author(s):  
R Chambert ◽  
M F Petit-Glatron
Keyword(s):  
Bacillus Subtilis ◽  
Intermediate Formation ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Chain Elongation ◽  
Directed Mutagenesis ◽  
Water Mixture ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Sucrose Hydrolysis

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331→Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331→Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
G A Vehar ◽  
K M Tate ◽  
D L Higgins ◽  
W E Holmes ◽  
H L Heyneker
Keyword(s):  
Cho Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Single Chain ◽  
Serine Protease Family ◽  
The One ◽  
Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

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