scholarly journals Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability

2019 ◽  
Vol 20 (19) ◽  
pp. 4701 ◽  
Author(s):  
Shahar Yoav ◽  
Johanna Stern ◽  
Orly Salama-Alber ◽  
Felix Frolow ◽  
Michael Anbar ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Random Mutagenesis ◽  
Cellulose Hydrolysis ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Glucose Yield ◽  
Multiple Alignments ◽  
Subsequent Hydrolysis ◽  
Hydrolysis Of

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.

scholarly journals Mutational Replacement of Leu-293 in the Class C Enterobacter cloacae P99 β-Lactamase Confers Increased MIC of Cefepime

2002 ◽  
Vol 46 (6) ◽  
pp. 1966-1970 ◽  
Author(s):  
Sergei B. Vakulenko ◽  
Dasantila Golemi ◽  
Bruce Geryk ◽  
Maxim Suvorov ◽  
James R. Knox ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Broad Spectrum ◽  
Random Mutagenesis ◽  
Enterobacter Cloacae ◽  
The Other ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Wide Range ◽  
Class C

ABSTRACT The class C β-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum β-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the E. cloacae P99 ampC gene, we obtained a Leu-293-Pro mutant of the P99 β-lactamase conferring a higher MIC of cefepime (MIC, 8 μg/ml, compared with 0.5 μg/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other β-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified E. cloacae P99 β-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (k cat/Km ) of the Leu-293-Pro mutant β-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 β-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant β-lactamase.

scholarly journals The role of the pseudo-disaccharide neamine as an intermediate in the biosynthesis of neomycin

1976 ◽  
Vol 159 (3) ◽  
pp. 601-606 ◽  
Author(s):  
C J Pearce ◽  
J E G. Barnett ◽  
C Anthony ◽  
M Akhtar ◽  
S D Gero
Keyword(s):  
Incubation Medium ◽  
Streptomyces Rimosus ◽  
Wild Type ◽  
Streptomyces Fradiae ◽  
Subunit Assembly ◽  
Subsequent Hydrolysis ◽  
Hydrolysis Of

By using wild-type and deoxystreptamine-negative mutants of Streptomyces fradiae grown in media containing [6(-3)H]glucose or [U-14C]glucose, and by subsequent hydrolysis of the labelled neomycin produced, neamines labelled with 3H in both rings I and II, but with 14C in ring I only, were prepared. A mixture of these two forms of neamine was converted by deoxystreptamine-negative Streptomyces rimosus forma paromomycinus into neomycin (not paromomycin) with a 30% yield. The3H: 14C ratio in this neomycin was the same as the measured in neamine produced by hydrolysis of the neomycin, and in unused neamine reisolated from the incubation medium. The 3H:14C ratio in the neomycin was not affected by the presence of unlabelled deoxystreptamine during the incubation. The radioactivity in the neomycin was associated with rings I and II only. It is concluded that the added neamine is incorporated into antibiotic intact, without initial hydrolysis, and that the probable first step in the subunit assembly of neomycin is the formation of neamine.

scholarly journals Altering Catalytic Properties of 3-Chlorocatechol-Oxidizing Extradiol Dioxygenase fromSphingomonas xenophaga BN6 by Random Mutagenesis

2001 ◽  
Vol 183 (7) ◽  
pp. 2322-2330 ◽  
Author(s):  
Ulrich Riegert ◽  
Sibylle Bürger ◽  
Andreas Stolz
Keyword(s):  
Amino Acid ◽  
Random Mutagenesis ◽  
Ring Cleavage ◽  
Amino Acid Substitutions ◽  
The Novel ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Extradiol Dioxygenase ◽  
Mutant Forms ◽  
Sphingomonas Xenophaga

ABSTRACT The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.

scholarly journals Engineering Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Optimal Ring Expansion Activity toward Penicillin G

2003 ◽  
Vol 69 (4) ◽  
pp. 2306-2312 ◽  
Author(s):  
Chia-Li Wei ◽  
Yunn-Bor Yang ◽  
Wen-Ching Wang ◽  
Wen-Chi Liu ◽  
Jyh-Shing Hsu ◽  
...  
Keyword(s):  
Random Mutagenesis ◽  
Fold Increase ◽  
Streptomyces Clavuligerus ◽  
Ring Expansion ◽  
Relative Activity ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Triple Mutant

ABSTRACT The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k cat/Km ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k cat/Km values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k cat/Km ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.

Application of directed evolution in the development of enantioselective enzymes

2000 ◽  
Vol 72 (9) ◽  
pp. 1615-1622 ◽  
Author(s):  
Manfred T. Reetz
Keyword(s):  
Organic Chemistry ◽  
Gene Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinetic Resolution ◽  
Random Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Hydrolytic Kinetic Resolution ◽  
Novel Approach

A novel approach to developing enantioselective enzymes for use in organic chemistry has been devised which is independent of structural or mechanistic aspects. The underlying idea is to combine appropriate methods of random mutagenesis, gene expression, and high-throughput screening for enantioselectivity. If these actions are performed in repetitive cycles, an evolutionary pressure is created that leads to sequential improvements of the enantioselectivity of a given enzyme-catalyzed reaction. The concept is illustrated by an example involving the lipase-catalyzed hydrolytic kinetic resolution of an α-chiral ester, the enantio-selectivity increasing from ee = 2% (E =1.1) for a wild-type enzyme to ee = 90-93% (E = 25) for the best mutants.

scholarly journals Rapid burst kinetics in the hydrolysis of 4-nitrophenyl acetate by penicillin G acylase from Kluyvera citrophila. Effects of mutation F360V on rate constants for acylation and de-acylation

1996 ◽  
Vol 316 (2) ◽  
pp. 409-412 ◽  
Author(s):  
A Roa ◽  
M L Goble ◽  
J L García ◽  
C Acebal ◽  
R Virden
Keyword(s):  
Rate Constants ◽  
Acetyl Derivative ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Side Chain ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Burst Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1·s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1·s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

scholarly journals Development of Improved Cellulase Variants for the Conversion of Spent Mushroom Substrate Supplemented with Wheat Straw by Directed Evolution

2021 ◽  
Author(s):  
Valentina Mauriello ◽  
Anna Pennacchio ◽  
Irantzu Alegria Dallo ◽  
Laura Garcia Saez ◽  
Petri Ihalainen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Wheat Straw ◽  
Recombinant Expression ◽  
Scale Up ◽  
Wild Type ◽  
Spent Mushroom Substrate ◽  
Glucose Yield ◽  
Hydrolysis Of ◽  
Selection Of

Abstract To improve the Spent mushroom substrate (SMS) saccharification, cloning, recombinant expression in Escherichia coli and characterization of two new GH5 family cellulases (Cel1 and Cel2) were performed. Based on enzymes properties, Cel2 was selected for the generation of 30,000 random mutants by directed evolution in order to develop improved biocatalysts. Error-prone Polymerase Chain Reaction was used for diversity generation in cel2 gene and the screening for activity of mutants allowed selection of 63 improved variants that were subjected to a scale up production. Among these, 13 clones exhibited two-fold higher activity than Cel2 and a higher thermoresistance after 72h. The performances of these mutants in the hydrolysis of pretreated SMS/ wheat straw (40/60) were compared to the wild type Cel2 in conjunction with a commercial enzymatic mixture (MetZyme® SUNO™ BOOSTER 144). All the mutants exhibited a glucose yield two-fold or four fold higher than wild-type Cel2 after 72h of incubation.

scholarly journals Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase

2005 ◽  
Vol 71 (9) ◽  
pp. 5433-5439 ◽  
Author(s):  
Michiyo Yanase ◽  
Hiroki Takata ◽  
Kazutoshi Fujii ◽  
Takeshi Takaha ◽  
Takashi Kuriki
Keyword(s):  
Heat Treatment ◽  
Random Mutagenesis ◽  
Cumulative Effect ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Triple Mutant ◽  
Original Activity ◽  
Glucan Phosphorylase

ABSTRACT The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.

scholarly journals Effects of Specific Amino Acid Substitutions on Activities of Dinitrogenase Reductase-Activating Glycohydrolase fromRhodospirillum rubrum

2001 ◽  
Vol 183 (19) ◽  
pp. 5743-5746 ◽  
Author(s):  
Babu S. Antharavally ◽  
Russell R. Poyner ◽  
Yaoping Zhang ◽  
Gary P. Roberts ◽  
Paul W. Ludden
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Paramagnetic Resonance ◽  
Specific Amino Acid ◽  
Dinitrogenase Reductase ◽  
Electron Paramagnetic ◽  
Hydrolysis Of

ABSTRACT Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

scholarly journals Site-directed mutagenesis and substrate-induced inactivation of β-lactamase I

1992 ◽  
Vol 288 (3) ◽  
pp. 1045-1051 ◽  
Author(s):  
S J Thornewell ◽  
S G Waley
Keyword(s):  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Extracellular Medium ◽  
The Stability ◽  
Hydrolysis Of ◽  
Reduced Activity

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.

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