# Dissecting enzyme function at scale using synergistic advances in microfluidics and genetic code expansion

> **NIH GM F32** · STANFORD UNIVERSITY · 2026 · $79,348

## Abstract

PROJECT SUMMARY.
 Noncanonical amino acids (ncAAs) have myriad valuable applications in the biochemical and biophysical
sciences. Their site-specific incorporation into proteins of interest can directly install systematically perturbed
residues, sensitive biophysical probes, bio-orthogonal handles, and post-translational modifications (PTMs) at
positions of interest. While promising, these applications have been greatly limited by costly materials and labor-
intensive, low-yielding preparations. To realize the full potential of ncAAs, I will leverage the recently developed
high-throughput microfluidic enzyme kinetics (HT-MEK) platform from the Fordyce and Herschlag laboratories
at Stanford University to enable the parallel expression, purification, and quantitative assay of >1,000 ncAA-
harboring protein variants on a single microfluidic device. With this approach, it will become feasible and routine
to collect >10,000 gold-standard biochemical measurements of ncAA-containing proteins while using less
material and effort than is typically required to collect a single such measurement.
 To illustrate the power and utility of this technique, I will first apply it towards understanding the catalytic
mechanisms governing proton transfer at carbon in the model system alanine racemase (AlaR), an important
pyridoxal 5’-phosphate (PLP)-dependent enzyme involved in cell-wall biosynthesis. PLP-dependent enzymes
account for 4% of all classified enzymatic activities and ~1.5% of prokaryotic reading frames, and they are
increasingly important in biotechnology. Although we have a reasonable understanding of how the small-
molecule cofactor itself can influence catalysis, the specific contributions of the protein scaffold remain
speculative, qualitative, or both. Previous studies that have used traditional site-directed mutagenesis—altering
many properties simultaneously—and only examined a handful of variants have failed to deliver a unified view
of how this enzyme achieves its c

## Key facts

- **NIH application ID:** 11249544
- **Project number:** 5F32GM156066-02
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Patrick James Almhjell
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** GM
- **Fiscal year:** 2026
- **Award amount:** $79,348
- **Award type:** 5
- **Project period:** 2024-12-01T00:00:00 → 2026-11-30T00:00:00

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11249544

## Citation

> US National Institutes of Health, RePORTER application 11249544, Dissecting enzyme function at scale using synergistic advances in microfluidics and genetic code expansion (5F32GM156066-02). Retrieved via AI Analytics 2026-05-18 from https://api.ai-analytics.org/grant/nih/11249544. Licensed CC0.

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