Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling

ABSTRACT The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.

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Faculty Opinions recommendation of DNA shuffling as a tool for protein crystallization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1026442.325143 ◽

2005 ◽

Author(s):

Michael Sundström

Keyword(s):

Protein Crystallization ◽

Dna Shuffling

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Association of Novel Domain in Active Site of Archaic Hyperthermophilic Maltogenic Amylase fromStaphylothermus marinus

Journal of Biological Chemistry ◽

10.1074/jbc.m111.304774 ◽

2012 ◽

Vol 287 (11) ◽

pp. 7979-7989 ◽

Cited By ~ 20

Author(s):

Tae-Yang Jung ◽

Dan Li ◽

Jong-Tae Park ◽

Se-Mi Yoon ◽

Phuong Lan Tran ◽

...

Keyword(s):

Active Site ◽

Maltogenic Amylase

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Mutations affecting the activity of the cyclodextrin glucanotransferase of Paenibacillus pabuli US132: insights into the low hydrolytic activity of cyclodextrin glucanotransferases

Biologia ◽

10.2478/s11756-012-0055-4 ◽

2012 ◽

Vol 67 (4) ◽

Cited By ~ 3

Author(s):

Sonia Jemli ◽

Mamdouh Ben-Ali ◽

Hajer Ben-Hlima ◽

Bassem Khemakhem ◽

Samir Bejar

Keyword(s):

Hydrolytic Activity ◽

Influential Factor ◽

Rational Approach ◽

Cyclodextrin Glucanotransferase ◽

Hydrolytic Activities ◽

Maltogenic Amylase ◽

Structural Barrier ◽

First Time

AbstractThe cyclodextrin glucanotransferase from Paenibacillus pabuli US132 (US132 CGTase) was engineered using a rational approach in an attempt to provide it with anti-staling properties comparable to those of the commercial maltogenic amylase (Novamyl). The study aimed to concurrently decrease the cyclization activity and increase the hydrolytic activity of US132 CGTase. A five-residue loop (PAGFS) was inserted, alone or with the substitution of essential residues for cyclization (G180, L194 and Y195), mimicking the case of Novamyl. The findings indicate that, unlike the case of the CGTase of Thermoanerobacterium thermosulfurigenes strain EM1 whose initial high hydrolytic activity was exceptional, these mutations completely abolished the cyclization and hydrolytic activities of the US132 CGTase. This suggests that those mutations are not able to convert conventional CGTases, whose hydrolytic activities are very weak, into hydrolases. Accordingly, and for the first time, a structural barrier at subsite −3 was advanced as an influential factor which might explain the low hydrolytic activity of conventional CGTases.

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Novaferon, a novel recombinant protein produced by DNA-shuffling of IFN-α, shows antitumor effect in vitro and in vivo

Cancer Cell International ◽

10.1186/1475-2867-14-8 ◽

2014 ◽

Vol 14 (1) ◽

pp. 8 ◽

Cited By ~ 6

Author(s):

Meng Li ◽

Chunming Rao ◽

Dening Pei ◽

Lan Wang ◽

Yonghong Li ◽

...

Keyword(s):

Recombinant Protein ◽

Antitumor Effect ◽

Dna Shuffling

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Development of Innovative Pediocin PA-1 by DNA Shuffling among Class IIa Bacteriocins

Applied and Environmental Microbiology ◽

10.1128/aem.00558-07 ◽

2007 ◽

Vol 73 (16) ◽

pp. 5292-5299 ◽

Cited By ~ 20

Author(s):

Tatsuya Tominaga ◽

Yoshinori Hatakeyama

Keyword(s):

Dairy Products ◽

Dairy Product ◽

Food Additives ◽

Dna Shuffling ◽

Activity Assay ◽

Effective Version ◽

Activity Screening ◽

Leuconostoc Lactis ◽

Enterocin A ◽

Increased Activity

ABSTRACT Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products.

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Engineering Plant Bioreactors For Biofuel Applications: Evolution of A. thaliana Glycosyl Hydrolases by Template Assisted DNA Shuffling and In Vitro Recombination

The FASEB Journal ◽

10.1096/fasebj.26.1_supplement.lb187 ◽

2012 ◽

Vol 26 (S1) ◽

Author(s):

Leroy Keith Davis

Keyword(s):

Dna Shuffling ◽

Glycosyl Hydrolases ◽

Engineering Plant ◽

In Vitro Recombination ◽

Plant Bioreactors

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Application of DNA Shuffling as a Tool for Hydrolase Activity Improvement of Pseudomonas Strain

Biointerface Research in Applied Chemistry ◽

10.33263/briac111.77357745 ◽

2020 ◽

Vol 11 (1) ◽

pp. 7735-7745

Keyword(s):

Protein Engineering ◽

Protoplast Fusion ◽

Concentration Gradient ◽

First Generation ◽

Human Life ◽

Genome Shuffling ◽

Dna Shuffling ◽

High Concentration ◽

Mutant Strains ◽

High Concentrations

Biotechnology is considered one of the most influential technologies in various areas of human life, including health, economics, and the environment. Protein engineering is one of the major biotechnology tools in the field of modification and advancement of biocatalysts capabilities. Among the most effective protein engineering methods, in particular, to improve the industrial strain capabilities, is the shuffling genome method. This study aimed to follow knowledge and biocatalysts engineering techniques based on DNA shuffling methods. In the first step, two procedures were followed (DES method and compatibility according to the concentration gradient of Diazinon) to obtain mutant strains. Acquired mutant strains from both methods were resistant to high concentrations of poison up to 3000 mg/L. The activity of these strains also demonstrated their elevated activity compared to parent samples. The highest activity was related to four strains IR1.G1, IR1.D8, IR1.D4, and IR1.D5, which were 0.234 U/ml, 0.1 U/ml, 0.098 U/ml, and 0.066 U/ml, respectively. The improved strain was obtained via the concentration gradient of the diazinon method (IRL1.G1 strain) in comparison with IRL1.D8 strain (owning highest activity through DES method) possesses excessive activity in 3000 mg/L concentration of Diazinon. The evaluated results of first-generation genome shuffling of strains (the first round of protoplast fusion) also indicated that those shuffled strains with the ability to grow in the vicinity of the toxin (3000 mg/L concentration of Diazinon) showed better activity than obtained mutated strains by both methods (concentration gradient of the toxin and the DES method). In the final stage, the best results were related to IRL1.F2, IRL1.F3, and IRL1.F1 shuffled strains with 0.541 mg/L, 0.523 mg/L, and 0.509 mg/L, respectively. The highest activity belonged to the IRL1.F2 genome shuffled strain (first round of protoplast fusion). This strain could grow in a high concentration of toxin, and also, the activity was increased 30, 3.6, and 2.3 times in comparison with the parent strain (IRL1), IRL.D8 mutant, and IRL1.G1, respectively.

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