Enzyme Screening Kit

Our enzymes are not produced to a specific purity standard. While efforts are made to separate them from cell debris and fermentation components, some endogenous proteins and buffer salts may be present. These additional components often enhance stability.

We do not set expiration dates on research enzymes that have not yet been produced at a large scale. However, based on our experience, if stored dry at –20°C, most of our enzymes remain stable for 2 years.

The enzymes in the kit exhibit high selectivity, typically splitting about equally between (R) and (S) selectivity. We advise screening all enzymes rather than relying solely on historical notation (see the selectivity table below), as substrates may adopt unusual conformations that affect selectivity. It's important to note that the presence of non-carbon atoms in the substrate (e.g., halogens or sulfur) may lead to opposite selectivity due to Cahn-Ingold-Prelog isomer naming conventions. Please refer to the selectivity table in the Protocols section.

The stability and activity of Transaminases can vary due to several interdependent factors, including different substrates and products. Below is a Typical Parameter table providing performance parameters that may assist in further optimization after screening.

Pyruvic acid serves as a general amine acceptor for most enzymes in this kit and is ideal for testing your reaction system.

Typical reaction conditions include 25 mM amine acceptor, 25 mM 2-(4-nitrophenyl)ethan-1-amine (amine acceptor), 100 mM potassium phosphate buffer (pH 7.4), and 1 mM pyridoxal 5-phosphate (PLP).

We recommend screening in potassium phosphate buffer at pH 7.4 initially. Once hit(s) are identified, buffer type and pH can be optimized. Selectivity is typically not pH dependent, though activity may vary. Borate buffer is suitable for evaluating performance at high pH.

Most of the enzymes in the kit remain stable with up to 10% v/v DMSO as a co-solvent.

Enzymes in the kit operate effectively within a pH range of 7 to 8.5, with a small subset tolerant to pH 9 to10. It's not recommended to decrease the pH below 7.0.

The variants exhibit differing stability but generally operate between 20°C and 60°C. Refer to the Typical Parameters table for more details.

If the substrate is soluble in DMSO and can be added equally to each vial, partial insolubility in the reaction mixture is usually not an issue.

Try allowing the reaction to run for a longer time, increasing the temperature (to 40°C), increasing substrate concentration, and/or increasing the enzyme concentration. If issues persist, consider consulting the Twist®-bitBiome® TA screening kit or contacting Twist for further options. You can also run a control reaction using pyruvic acid as an amine acceptor to verify your reaction solution.

Consider stopping the reaction earlier or repeating the screen using a lower enzyme loading. If repeating, maintain DMSO concentration at <10% v/v.

Please see our Transaminase Screening FAQs for suggestions on optimizing the reaction conditions.

Typical amine donors include alanine, ethylamine, 1- and 2-propylamine, 1- and 2-butylamine, and others. The ketone/aldehyde form of these donors also act as good amine acceptors during chiral resolution.

Refer to our Transaminase Screening FAQs for answers to other questions you may have, or feel free to contact us.

We recommend preparing solutions fresh on the day of the experiment. Over time, both the substrate and cofactor may degrade, potentially reducing the overall performance of the reaction. If storing and reusing the solution is necessary, it should be kept cold and shielded from light. Additionally, we advise checking its performance against fresh solutions whenever possible.

For many enzymes, incorporating 10% v/v DMSO into the reaction mixture and increasing the temperature to 40–45°C can enhance substrate solubility. Refer to the Typical Parameters table for the typical conditions tolerated by each enzyme. Additionally, increasing the pH might aid solubility, although this varies depending on the substrate. We advise against decreasing pH below 7.0. Cloudiness in the reaction mixture, indicating incomplete substrate solubility, is typically acceptable and does not usually impede the reaction. It's best to add the substrate neat or from a DMSO (or similar solvent) solution to ensure even distribution across all reactions.

It's generally optimal to add the enzyme after all other reagents and solvents have been incorporated, and pH adjustment has been completed. If feasible, dissolve the enzyme in buffer before adding it to the reaction mixture. Occasionally, altering the order of reagent addition can significantly impact reaction performance, either positively or negatively.

It's crucial to comprehend the inherent nature of both the substrate and product, as these are properties the enzyme cannot alter (e.g., solubility, pH, and temperature stability). Additionally, it's important to avoid conditions that may promote side reactions, lead to downstream issues, or are incompatible with the substrate or product. Evaluate the reaction performance at different pH levels (recommended: pH 7.4, 9, and 10 using potassium phosphate buffer at pH 7.4 and sodium borate at pH 9 and 10). Once an optimal pH is determined, investigate the effect of temperature on reaction performance (recommended temperature range: 30–55°C). Furthermore, consider evaluating the reaction at higher substrate loadings to optimize performance. If the substrate is not soluble in the reaction mixture, it may lead to substrate mass transfer limitation. In such cases, consider investigating the addition of a solubilizing co-solvent. Refer to the Typical Parameters table for solvent concentration tolerance; besides DMSO, other solvents such as short-chain alcohols, DMF, THF, and acetonitrile can also be explored.

We check the activity of each enzyme in every batch using a standard colorimetric method. This ensures that each enzyme meets our activity standards and performs reliably in your applications.

We measure the powder lysate of specific enzymes in the kit for each batch. This measurement ensures that there is an adequate amount of lysate, guaranteeing the effectiveness of our kits.

To ensure environmental safety, we confirm that there are no living expression hosts (such as E. coli) in the lysate powder. This step is crucial for maintaining the safety and integrity of our product.

We conduct rigorous testing to confirm the shelf life of each batch. This ensures that our enzyme kits remain effective and stable for the duration of their intended use.

The absence of living expression hosts, like E. coli, in the lysate powder is important to prevent any potential contamination and to ensure the safety of users and the environment.

A colorimetric method is a technique used to determine the concentration of an enzyme based on its color change when reacting with a specific substrate. It is used because it provides a reliable and quantifiable measure of enzyme activity.

If a batch does not meet our stringent quality standards, it is not released for sale. Instead, it undergoes further analysis and corrective actions to address any issues before it can be reconsidered for distribution.

Our quality control processes are conducted using validated methods and equipment, operated by trained professionals. Regular audits and reviews are also conducted to ensure ongoing compliance with industry standards and best practices.

Yes, customers can request detailed quality control reports for their specific batch. These reports provide transparency and assurance regarding the quality and integrity of the enzyme kit they receive.