Hydrolases, nucleic acids encoding them and methods for making and using them

Inventors

Barton, Nelson R. • Bueno, Analia • Cuenca, Joslin G. • Hitchman, Tim S. • Kline, Katie A. • Lyon, Jonathan D. • Miller, Mark L. • Wall, Mark A. • Dayton, Christopher L. G.

Assignees

DSM IP Assets BV • DSM Food Specialties BV

Abstract

Provided are hydrolases, including lipases, saturases, palmitases and/or stearatases, and polynucleotides encoding them, and methods of making and using these polynucleotides and polypeptides. Further provided are polypeptides, e.g., enzymes, having a hydrolase activity, e.g., lipases, saturases, palmitases and/or stearatases and methods for preparing low saturate or low trans fat oils, such as low saturate or low trans fat animal or vegetable oils, e.g., soy or canola oils.

Core Innovation

The invention provides isolated, synthetic, or recombinant nucleic acids and polypeptides having palmitase activity, including nucleic acids comprising sequences having sequence identity to SEQ ID NO:1 or encoding polypeptides having sequence identity to SEQ ID NO:2. The nucleic acids and encoded polypeptides include an amino acid residue modification of V163R, with sequence-identity thresholds used to define variant ranges.

The disclosure describes Tailored Multi-Site Combinatorial Assembly to build thermotolerant palmitase mutation libraries, generate defined mutation sets, and apply screened thermal tolerance selection. The resulting libraries are evaluated for thermal tolerance and oil-assay performance across temperature conditions, with thermal tolerance hits, top silent mutations, and constructed variants assessed for palmitate levels and improved performance profiles.

The disclosure also reports compiled fatty-acid profiling data, soy oil assay evaluation of evolved palmitase variants versus a parent SEQ ID NO:2, and thermostability characterization by residual activity after incubation at elevated temperatures. Related embodiments include expression constructs, promoters, expression cassettes, expression vectors, transformed cells, transgenic plants, and codon optimization for Pseudomonas fluorescens.

Claims Coverage

Two independent claims are identified. Across the independent claims, the core inventive coverage centers on isolated, synthetic, or recombinant nucleic acids encoding palmitase activity polypeptides with a V163R amino-acid residue modification and substantial sequence identity to SEQ ID NO:1 or SEQ ID NO:2.

The independent claims collectively define isolated nucleic acids encoding palmitase activity polypeptides that include the V163R amino-acid residue modification while maintaining substantial sequence identity to SEQ ID NO:1 and/or SEQ ID NO:2.

Stated Advantages

Documented Applications