Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase

Arjomand, Maryam Rezaei; Habibi-Rezaei, Mehran; Ahmadian, Gholamreza; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Asadifar, Mandana; Amanlou, Massoud

doi:10.1016/j.ijbiomac.2016.08.039

Cited by 18 publications

(20 citation statements)

References 31 publications

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“…Efficient production of inulinases on an industrial scale can significantly reduce the costs of biocatalyst, reduce energy consumption and improve the efficiency of fructose production [8]. Some exo-inulinase genes have been expressed in bacteria Escherichia coli and yeast Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris and fungi Penicillium canescens [14][15][16][17][18][19][20]. The goal of scientists is to obtain the recombinant inulinases that are more thermostable at different pH than the native strains.…”

Section: Introductionmentioning

confidence: 99%

“…There have been many publications in the literature on recombinant exo-inulinases from fungi A. niger [14,17,18,20], A. awamori [15] and from yeast K. cicerisporus [16] and K. marxianus [19]. However, processes involving this recombinant exo-inulinase for industrial purposes cannot be designed and optimized without knowing the kinetic parameters of the process and thus the effect of temperature on activity recombinant exo-inulinase [21].…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, it is necessary to determine the optimum temperature T opt , activation energy E a and activation energy of the deactivation process E d for recombinant exo-inulinases. The method of determining the parameters based on experimental data on the effect of temperature on the activity recombinant of exo-inulinases has been presented [14][15][16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

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The inulin hydrolysis by recombinant exo-inulinases: determination the optimum temperatures and activation energies

Miłek

2021

J Therm Anal Calorim

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The advantages of recombinant enzymes over native include the control in a production environment, product purity and also high yield. The paper presents the determination the optimum temperatures and the activation energies for various origin recombinant exo-inulinases, among others from Aspergillus niger, A. awamori, Kluyveromyces marxianus and K. cicerisporus. The parameters were estimated based on the literature of the activity curves versus temperature for hydrolysis of inulin. It was assumed that both the hydrolysis reaction process and the deactivation process of recombinant exo-inulinase were first-order reactions by the enzyme concentration. A mathematical model describing the effect of temperature on recombinant exo-inulinase activity was used. Based on the comparison analysis, values of the activation energies $${E_{\rm a }}$$ E a were in the range from $${32.01 \pm 7.80}$$ 32.01 ± 7.80 to $${43.83 \pm 4.87}$$ 43.83 ± 4.87 kJ mol$$^{-1}$$ - 1 , the deactivation energies $${E_{\rm d }}$$ E d were in the range from $${83.93 \pm 4.82}$$ 83.93 ± 4.82 to $${352.44 \pm 14.26}$$ 352.44 ± 14.26 kJ mol$$^{-1}$$ - 1 and the optimum temperature $${T_{\rm opt }}$$ T opt were obtained in the range from $${318.91 \pm }$$ 318.91 ± 1.19 to $${328.76 \pm 0.25}$$ 328.76 ± 0.25 K.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The inulin hydrolysis by recombinant exo-inulinases: determination the optimum temperatures and activation energies

Miłek

2021

J Therm Anal Calorim

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“…Mutagenesis reports on the thermal performance of exo-inulinases are scarce. Deletion of the Ω-loop fragment, 74 Tyr-Gly-Ser-Asp-Val-Thr 79 , from Aspergillus niger exo-inulinase resulted in thermostability loss at 60°C [ 19 ]. Substituting the histidine from 189 Ala-Glu-Leu-His 192 of the A. niger exo-inulinase with alanine resulted in thermostability loss at 60°C [ 20 ].…”

Section: Discussionmentioning

confidence: 99%

Removal of N-terminal tail changes the thermostability of the low-temperature-active exo-inulinase InuAGN25

Zhang

Shen

et al. 2020

Bioengineered

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Exo-inulinases are members of the glycoside hydrolase family 32 and function by hydrolyzing inulin into fructose with yields up to 90-95%. The N-terminal tail contributes to enzyme thermotolerance, which plays an important role in enzyme applications. However, the role of N-terminal amino acid residues in the thermal performance and structural properties of exo-inulinases remains to be elucidated. In this study, three and six residues of the N-terminus starting from Gln23 of the exo-inulinase InuAGN25 were deleted and expressed in Escherichia coli. After digestion with human rhinovirus 3 C protease to remove the N-terminal amino acid fusion sequence that may affect the thermolability of enzymes, wild-type RfsMInuAGN25 and its mutants RfsMutNGln23Δ3 and RfsMutNGln23Δ6 were produced. Compared with RfsMInuAGN25, thermostability of RfsMutNGln23Δ3 was enhanced while that of RfsMutNGln23Δ6 was slightly reduced. Compared with the N-terminal structures of RfsMInuAGN25 and RfsMutNGln23Δ6, RfsMutNGln23Δ3 had a higher content of (1) the helix structure, (2) salt bridges (three of which were organized in a network), (3) cation-π interactions (one of which anchored the N-terminal tail). These structural properties may account for the improved thermostability of RfsMutNGln23Δ3. The study provides a better understanding of the N-terminus-function relationships that are useful for rational design of thermostability of exo-inulinases.

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“…It is difficult to overcome the activity–stability trade-off of enzymes during mutagenesis [ 31 ]. For example, deletion of the Ω-loop fragment 74 YGSDVT 79 from Aspergillus niger exo-inulinase resulted in a 12°C decrease in optimum temperature and thermostability loss at 60°C [ 32 ]. Substituting the histidine residue from 189 AELH 192 of the A. niger exo-inulinase with alanine residue resulted in a 5°C decrease in optimum temperature and thermostability loss at 60°C [ 33 ].…”

Section: Discussionmentioning

confidence: 99%

Improving low-temperature activity and thermostability of exo-inulinase InuAGN25 on the basis of increasing rigidity of the terminus and flexibility of the catalytic domain

Zhang

Shen

et al. 2020

Bioengineered

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Enzymes displaying high activity at low temperatures and good thermostability are attracting attention in many studies. However, improving low-temperature activity along with the thermostability of enzymes remains challenging. In this study, the mutant Mut8S, including eight sites (N61E, K156R, P236E, T243K, D268E, T277D, Q390K, and R409D) mutated from the exo-inulinase InuAGN25, was designed on the basis of increasing the number of salt bridges through comparison between the low-temperature-active InuAGN25 and thermophilic exo-inulinases. The recombinant Mut8S, which was expressed in Escherichia coli , was digested by human rhinovirus 3 C protease to remove the amino acid fusion sequence at N-terminus, producing RfsMut8S. Compared with wild-type RfsMInuAGN25, the mutant RfsMut8S showed (1) lower root mean square deviation values, (2) lower root mean square fluctuation (RMSF) values of residues in six regions of the N and C termini but higher RMSF values in five regions of the catalytic pocket, (3) higher activity at 0–40°C, and (4) better thermostability at 50°C. This study proposes a way to increase low-temperature activity along with a thermostability improvement of exo-inulinase on the basis of increasing the rigidity of the terminus and the flexibility of the catalytic domain. These findings may prove useful in formulating rational designs for increasing the thermal performance of enzymes.

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Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase

Cited by 18 publications

References 31 publications

The inulin hydrolysis by recombinant exo-inulinases: determination the optimum temperatures and activation energies

The inulin hydrolysis by recombinant exo-inulinases: determination the optimum temperatures and activation energies

Removal of N-terminal tail changes the thermostability of the low-temperature-active exo-inulinase InuAGN25

Improving low-temperature activity and thermostability of exo-inulinase InuAGN25 on the basis of increasing rigidity of the terminus and flexibility of the catalytic domain

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