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Novel Use for the Osmolyte Trimethylamine N-oxide: Retaining the Psychrophilic Characters of Cold-Adapted Protease Deseasin MCP-01 and Simultaneously Improving its Thermostability

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Abstract

The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k cat /K m) by 290% at 0°C. In the presence of 1 M TMAO, the thermostability (t 1/2) of MCP-01 increased by two- to fivefold at 60∼40°C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40°C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T m) of MCP-01 by 10.5°C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.

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Abbreviations

TMAO:

trimethylamine N-oxide

CD:

circular dichroism

UV:

ultraviolet

FAAF:

N-succinyl-Phe-Ala-Ala-Phe-p-nitroanilide

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Acknowledgment

The work was supported by Hi-Tech Research and Development program of China (2006AA09Z414, 2007AA091903), National Natural Science Foundation of China (30770040, 40706001), Program for New Century Excellent Talents in University (NCET-06-0578), COMRA Program (DYXM-115-02-2-6), Specialized Research Fund for the Doctoral Program of Higher Education, Foundation for Young Scientists in Shandong Province (4020867).

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Authors and Affiliations

  1. The State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan, 250100, China

    Hai-Lun He, Xiu-Lan Chen, Xi-Ying Zhang, Cai-Yun Sun, Bai-Chen Zou & Yu-Zhong Zhang

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  1. Hai-Lun He
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  2. Xiu-Lan Chen
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  3. Xi-Ying Zhang
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  5. Bai-Chen Zou
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  6. Yu-Zhong Zhang
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Corresponding author

Correspondence to Yu-Zhong Zhang.

Additional information

Xiu-Lan Chen and Hai-Lun He have made equal contribution to this work.

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Fig. S1

Lineweaver–Burk plots for the hydrolysis of FAAF by MCP-01 at 0°C in the absence (a) and presence (b) of 1 M TMAO. Lineweaver–Burk plots were made by linear regression with initial rates determined between 0 and 1 mg/mL of FAAF. K m and V m values of MCP-01 with FAAF as substrate at 0°C were determined by these plots. k cat was calculated by the formula k cat = V m/[E], where [E] is the concentration of MCP-01 in the reaction. The data shown are the mean of three repeats with standard errors ≤5%. (DOC 294 kb)

Fig. S2

Arrhennius plots for the hydrolysis of FAAF by MCP-01 at 0∼30°C in the absence and presence of 1 M TMAO. Arrhennius plots were made based on the k cat values which were calculated based on the reaction rates measured at given temperatures. The activation energy E a values of the reaction catalyzed by MCP-01 and with FAAF as substrate were determined from the corresponding Arrhenius plot with the equation of ln k cat = ln A  − E a/RT. The data shown are the mean of three repeats with standard errors ≤5%. (DOC 336 kb)

Fig. S3

Irreversible thermal inactivation plots of MCP-01 in the absence (a) and presence (b) of 1 M TMAO and subtilisin Carlsberg (c). Irreversible thermal inactivation of subtilisin Carlsberg and MCP-01 in the absence and presence of 1 M TMAO was determined by incubating the enzymes in sealed Eppendorf tubes at temperatures ranging from 40°C to 60°C for varying periods of time. At various time intervals, aliquots were taken and cooled immediately in ice water. The residual activity was assayed with casein as substrate at 40°C using the method described previously (Chen et al. 2003). filled squares 40°C, filled circles 45°C, filled triangles 50°C, filled diamonds 55°C, filled stars 60°C. The thermal inactivation constant k i were directly determined from the slopes of the first-order plots of LN (residual activity) versus time. (DOC 580 kb)

Fig. S4

The Arrhenius plots (ln k i versus 1/T) for MCP-01 in the absence (filled squares) and presence of 1 M TMAO (filled triangles) and subtilisin Carlsberg (filled circles). Activation energy (E ai) of enzyme inactivation was determined from these slopes. (DOC 188 kb)

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He, HL., Chen, XL., Zhang, XY. et al. Novel Use for the Osmolyte Trimethylamine N-oxide: Retaining the Psychrophilic Characters of Cold-Adapted Protease Deseasin MCP-01 and Simultaneously Improving its Thermostability. Mar Biotechnol 11, 710–716 (2009). https://doi.org/10.1007/s10126-009-9185-2

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  • DOI: https://doi.org/10.1007/s10126-009-9185-2

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