Linker-assisted engineering of chimeric xylanase-phytase for improved thermal tolerance of feed enzymes

Abstract

Biological enzymes are multifunctional macromolecules that can perform hundreds of reactions simultaneously. An enzyme must possess specific characteristics to meet industrial needs, such as stability over a wide pH and temperature range and high specific activity. A phytase and xylanase mixture is generally added to poultry feed to improve the bird’s health and productivity. Despite this, animal farmers have noticed no difference in productivity, and a leading cause is the high temperature at which feed is pulverized, which inactivates enzymes. A thermo-stable enzyme system can overcome these hitches. Commonly, coatings and immobilization reduce losses caused by physical-chemical factors in feed processing and digestion. To this end, we engineered the multifunctional xylanase-phytase domains on a single polypeptide fused by a helical linker. First, the ideal linker sequence was chosen by computing each selected linker’s root mean square deviation (RMSD). The selected helical linker provides sufficient structural flexibility for substrate binding and product release evaluated by molecular docking and molecular dynamic simulation studies. Furthermore, a domain-domain interaction has stabilized the bridging partners, attaining the thermal optima for xylanase and phytase at 90 °C. Even at the above-optimal temperature (100 °C), the recombinant PLX was relatively stable and retained 64.2% and 59.2% activity for xylanase and phytase, respectively, when surveyed for ten hours. So far, to this date, this is the highest degree of thermostability achieved by any recombinant phytase or xylanase.

Communicated by Ramaswamy H. Sarma

Keywords:

• Chimeric enzymes
• linker
• phytase
• xylanase
• thermostable

Acknowledgments

We thank the DST for INSPIRE FELLOWSHIP.

Author contribution

Dharti Patel: Methodology, Validation, Investigation, Writing original draft.

Gayatri Dave: Conceptualization, Supervision, Critical review.

Kruti Shah: Formal analysis, Investigation.

Ravi Rawat: Insilco analysis, Writing Insilco draft.

Shilpa Sharma: Computing Resources, Computational analysis.

Nayan Borsadiya: Formal analysis.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was financially supported by Department of Science and Technology (DST).