Gly or Ala substitutions for Pro210Thr211Asn212 at the β8–β9 turn of subtilisin Carlsberg increase the catalytic rate and decrease thermostability

Fuchita, N.; Arita, Saori; Ikuta, Junya; Miura, Masahiro; Shimomura, K.; Motoshima, Hiroyuki

doi:10.1016/j.bbapap.2012.01.015

Cited by 5 publications

(8 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…So these variants are different from other variants reported previously. 20,25) For example, substitutions of the β8-β9 turn in subtilisin Carlsberg increased the catalytic activity, but decreased the thermostability of the enzyme. 25) This phenomenon is commonly recognized as a "tradeoff" between activity and stability.…”

Section: Temperature-dependent Caseinolytic Activitymentioning

confidence: 98%

“…20,25) For example, substitutions of the β8-β9 turn in subtilisin Carlsberg increased the catalytic activity, but decreased the thermostability of the enzyme. 25) This phenomenon is commonly recognized as a "tradeoff" between activity and stability. 7) However, there are examples that integrate thermostability and activity into a single variant by protease engineering.…”

Section: Temperature-dependent Caseinolytic Activitymentioning

confidence: 98%

See 1 more Smart Citation

Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP)

Zhao

Liu

et al. 2016

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of k/K value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.

show abstract

Section: Temperature-dependent Caseinolytic Activitymentioning

confidence: 98%

Section: Temperature-dependent Caseinolytic Activitymentioning

confidence: 98%

Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP)

Zhao

Liu

et al. 2016

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

show abstract

“…Proline incorporation at position 39 or 290 increases the kinetic stability of Lip5. Previous report showed that the subtilis in mutants P210G and P210A at the β8–β9 turn could result in decreased stability of the enzyme . Thus, proline sites in the loop or turn may play an essential role in influencing the stability of enzymes.…”

Section: Resultsmentioning

confidence: 97%

“…Formula t 1/2 = ln2/ к was used for calculating the half‐lives of Lip5 and mutants. The differences in activation free energy of thermal inactivation between wild‐type and mutants (ΔΔG) were calculated as described before .…”

Section: Methodsmentioning

confidence: 99%

“…Common strategies toward improving protein stability include introducing new interactions, such as salt bridge , hydrogen bond , hydrophobic contact , and disulfide bond , as well as increasing conformational rigidity . By reducing the configurational entropy of unfolding and thus increasing rigidity, proline incorporation has been repeatedly reported to successfully improve the stability of enzymes . Proline incorporation mostly focuses on flexible loop region or β‐turn in protein, based on 3D structure.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Sequence‐based proline incorporation improves the thermostability of Candida albicans lipase Lip5

Yuan

Zhao

Wang

et al. 2015

Euro J Lipid Sci & Tech

View full text Add to dashboard Cite

Increasing kinetic stability of industrial enzymes is a key objective of protein engineering. In this study, a cold-active Candida albicans lipase Lip5 was selected as a model to improve its stability. With no 3D structure available, multiple-sequence alignment of homologous lipases with different kinetic stabilities in the same superfamily was used to identify the mostly likely positions relevant to protein stability; these positions (V39, I52, and I290) were selected for proline incorporation in Lip5 as a strategy to increase the protein stability. Of the mutants generated, two of them (V39P and I290P) displayed a twofold increase in kinetic stability and improved thermodynamic stability as well. None of the mutants exhibited secondary or tertiary structure changes as shown by circular dichroism and fluorescence spectroscopy analyses. These results demonstrate that in the absence of 3D structure, multiple-sequence alignment could be a useful tool to direct proline incorporation for lipase stability engineering.Practical applications: In the absence of a 3D structure, multiple-sequence alignment-based proline incorporation is a useful tool to engineer lipase towards improved stability.

show abstract

Improved thermostability of D-allulose 3-epimerase from Clostridium bolteae ATCC BAA-613 by proline residue substitution

Wang

Chen

Zhao

et al. 2022

Protein Expression and Purification

View full text Add to dashboard Cite

Gly or Ala substitutions for Pro210Thr211Asn212 at the β8–β9 turn of subtilisin Carlsberg increase the catalytic rate and decrease thermostability

Cited by 5 publications

References 29 publications

Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP)

Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP)

Sequence‐based proline incorporation improves the thermostability of Candida albicans lipase Lip5

Improved thermostability of D-allulose 3-epimerase from Clostridium bolteae ATCC BAA-613 by proline residue substitution

Contact Info

Product

Resources

About