Enzymology has a long and illustrious history, dating back to the seminal work by Sumner on crystallization of urease and demonstration that enzymes were proteins (as reviewed in 1) .

Isolating and purifying individual enzymes, followed by determining the enzyme’s properties, has been a mainstay of enzymology for the past 90 years. This type of work is still valuable and remains the backbone of enzymology.

I purified my first enzyme as a college freshman in 1973. At that time, there was no choice but to use native sources of the enzyme. A limited number of chromatography media types were available, and all suffered from slow flow and poor resolution. Complete protein sequences were practically unknown, and temperature and pH optima, and K_m and V_max values on defined small-molecule substrates were the major defining properties that defined the enzyme. Over the past 45 years, enzymology has seen quantum leaps in technology:

Edman degradation made determining protein sequences a tedious, but routine practice, opening up enzymology to asking questions about sequence versus function 2.

Determining the sequence of enzymes by DNA sequencing 3.

Cloning of genes to eliminate the need for native sources 4.

Improved promoters for high level expression combined with enzyme tags and affinity chromatography 5.

Complete chemical synthesis of genes coding for enzymes

High throughput enzyme crystal structure determination 6.

The result of these technology leaps is a generally faster, easier route to pure, single enzymes. What is needed, as we approach the start of the second century of enzymology, is a new set of challenges for enzymology. Some of these challenges include:

Natural product production by assembling biosynthetic pathways in vitro.

Degradation of complex natural substrates by single enzymes, mixtures of enzymes, and enzyme complexes such as cellulosomes 7.

Non-traditional enzymes including single-turnover enzymes such as Cas9 8.

Enzymology of microbial immune systems 9.

Role of glycosylation 10 and other post-translational modifications on enzyme activity.

Functional characterization of “hypothetical proteins” identified in genomic and metagenomic sequencing 11.

There are many routes forward for enzymology in this dawning new century. Enzymologists should boldly explore new techniques and new collaborations to continue advancing the field.