Enzymes for biocatalysis and enzymes for cell culture - frequently asked questions

What are the benefits of enzymes for biocatalysis? What are the benefits of enzymes for cell culture? Get the answer to these and other questions about enzymes.

Enzymes for biocatalysis - frequently asked questions:

(or go to: Enzymes for cell culture – frequently asked questions)

What are enzymes?
Enzymes are biological catalysts in the form of proteins that catalyze chemical reactions in the cells of living organisms. In biocatalysis, natural catalysts such as enzymes are used in place of chemical catalysts in synthetic processes. 

What are the sources of enzymes? How are enzymes generally produced?
At Novozymes, industrial enzymes are produced using a process called submerged fermentation. This involves growing carefully selected microorganisms (bacteria and fungi) in closed vessels containing a rich broth of nutrients (the fermentation medium) and a high concentration of oxygen (aerobic conditions). As the microorganisms break down the nutrients, they produce the desired enzymes. Most often the enzymes are secreted into the fermentation medium. 

What are the other areas and applications in which Novozymes enzymes are used?
Novozymes enzymes are used in number of applications in household care, food and nutrition, brewing and bioenergy. 

Where are Novozymes enzymes for biocatalysis manufactured?
Most of the enzymes for biocatalysis are manufactured in Denmark. Other key manufacturing facilities are based in USA and China. 

What do you mean by GMO and non-GMO?
GMO stands for Genetically Modified Organism. Novozymes produces enzymes by fermenting microorganisms which can be genetically modified to improve the production process. Enzymes are proteins, not living organisms, and contain no genetically modified organisms. Information on the production microorganism is provided in the Product Data Sheet for the enzyme products. The fermentation takes place in contained steel tanks. Novozymes uses well tested and safe microorganisms only. By the end of the production process the microorganisms are separated from the enzymes and inactivated. Novozymes also uses gene technology to develop new enzymes. 

How can I get a sample of an enzyme?
You can request an enzyme sample from your account manager or by clicking the contact us link at the top of the page. 

What is the standard timeframe in which enzyme samples can be provided?
Standard timeframe for delivery of enzyme samples is generally 2-3 weeks from the request date. 

Handling, usage and formulation
How should I store enzymes? What are the correct storage conditions for enzymes?
Recommended storage conditions for particular enzymes are provided in the respective Product Data Sheets (PDS). For long term storage it is recommended that the enzyme samples are stored at 5-10ºC but not placed in a freezer. 

Do enzymes only work in aqueous solutions?
Enzymes function optimally in aqueous solutions or water, however many Novozymes enzymes - especially lipases - work very well in organic solvents or in an organic solvent/buffer mixture. 

Can enzymes act on substrates other than those on which they act in nature?
In addition to the substrates on which they act in nature, many Novozymes enzymes also work well on other substrates. 

What are the typical conditions under which enzymes can be used for biocatalysis?
Typically for enzymes, Novozymes enzyme products have optimum pH and temperature conditions under which they work. The optimum pH and temperature curves which show the best working conditions for Novozymes enzyme products are available in the  Novozymes customer center or can be obtained by clicking the contact us link at the top of the page.  

What enzyme formulations are available?
Enzymes are available both in liquid and solid formulations. Liquid formulations are a solution of the enzyme protein in water with several additives including stabilizers and salts. Solid enzymes can be either granulate enzymes or immobilized enzymes. Granulate enzymes are enzymes which are granulated with salts. Immobilized enzymes are enzymes adsorbed onto a solid support, typically a polymeric resin.

What is the shelf life of the products?
Most Novozymes enzyme products have a shelf life of 1-2 years, provided they are stored under the recommended conditions. Stability data information for Novozymes enzyme products can be obtained from your account manager or by clicking the contact us link at the top of the page. 

What are the general precautions to be taken when handling enzymes?
Proteins are abundant in nature. Like many other proteins foreign to the human body, enzymes are potential allergens if inhaled. All enzymes have to be handled safely using the correct protective clothing and equipment.
Each Novozymes enzyme product has a Material Safety Data Sheet (MSDS) which gives directions for safe use of the enzyme. An MSDS is supplied with both samples and consignments of enzymes. 

What is the composition of an enzyme product? 
The composition of each Novozymes enzyme product is given in its Product Data Sheet. 

Which reactions can lipases be used in? Which reactions can proteases be used in?
Lipases can be used in the following reactions: 

  • Kinetic resolution of alcohols, esters and amines
  • Dynamic kinetic resolution of alcohols, esters and amines
  • Desymmetrization of di-esters
  • Trans esterification of alcohols
  • Regio-selective hydrolysis of acyl esters
  • Inter esterification of  carboxylic esters
  • Amide formation
Proteases can be used in the following reactions:
  • Kinetic resolution of amino esters and selected esters
  • Dynamic kinetic resolution of amino esters
What types of buffers can be used for enzymatic reactions?
Phosphate buffers are recommended for lipase and protease-based enzymatic reactions. Based on the pH required for the reaction, a specific phosphate buffer can be prepared or used. 

What do you mean by immobilized enzymes? What are the different immobilized enzyme products offered by Novozymes?
Immobilized enzymes are enzymes which are immobilized on a solid support, typically a polymeric resin. Immobilization can be through either adsorption of the enzyme on the solid support or by covalent linkage of the enzyme to the polymer support.
Novozymes mainly offers adsorption-based immobilized enzymes. The three immobilized enzymes offered by Novozymes are Novozym®435, Lipozyme®TL IM and Novozym®40085.

What is enzyme activity and how is this measured?
Enzyme activity is generally measured by measuring the rate at which a particular chemical reaction is catalyzed by a specific amount of enzyme. Activity of the enzyme is defined by a specific unit which is usually based on the method of analysis. Unit examples include LU (Lipase unit), PLU (Propyl Laurate Unit) and IUN (Interesterification Unit). The activity of a Novozymes enzyme can be measured using Novozymes method of analysis.

Is the method of analysis used to measure activity available to customers?
You can request the method of analysis used to measure enzyme activity from your account manager.

What is the difference between food grade and technical grade enzymes?
Food grade enzymes conform to food safety regulations and have additional certifications while technical grade enzymes do not. Technical grade enzymes are widely used in pharmaceutical applications as there is no requirement for food grade enzymes in these applications.

Which grades of enzymes are available from Novozymes?
Most of the enzyme products in the Novozymes biocatalysis portfolio are technical grade but some food grade enzymes are also offered.

Customer technical support
What level of customer support can Novozymes provide?
Technical information related to use of enzymes for biocatalysis and enzyme related information, including analytical methods, are available to Novozymes customers.

Regulatory affairs
Which regulatory documents are available for enzymes?
Novozymes offers regulatory documents including animal origin-free production certificates relevant to animal-derived contamination including Bovine Serum Encephalopathy (BSE) and Transmissible Spongiform Encephalopathy (TSE). Novozymes offers certifications required for pharmaceutical drug master file (DMF) filings. Novozymes also offers other documents for regulatory filings.

Where can I get regulatory documents for enzymes?
You can request regulatory documents for Novozymes enzymes from your account manager.

How will be regulatory queries be handled?
Novozymes has a dedicated regulatory department to handle regulatory queries. Please contact your account manager with any regulatory queries.

How do you estimate protein content from enzymes in the final Active Pharmaceutical Ingredient (API) or intermediate?
Novozymes does not have a unique method for estimating protein content in Active Pharmaceutical Ingredients. Standard protein estimation methods can be used for estimating protein content in APIs/intermediates.

What is the general manufacturing process flow for enzymes?
At Novozymes, industrial enzymes are produced using a process called submerged fermentation. This involves growing carefully selected microorganisms (bacteria and fungi) in closed vessels containing a rich broth of nutrients (the fermentation medium) and a high concentration of oxygen (aerobic conditions). As the microorganisms break down the nutrients, they produce the desired enzymes. The first step in harvesting enzymes from the fermentation medium is to remove insoluble products, primarily microbial cells. This is normally done by centrifugation or microfiltration steps. As most industrial enzymes are extracellular – secreted by cells into the external environment – they remain in the fermented broth after the biomass has been removed. The enzymes in the remaining broth are then concentrated by evaporation, membrane filtration or crystallization depending on their intended application.

Does Novozymes have Current Good Manufacturing Practice (cGMP) certification for enzyme products?
Most enzymes produced by Novozymes are technical grade enzymes and therefore do not require cGMP certification. Some Novozymes food grade enzymes are produced under food cGMP conditions.
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Enzymes for cell culture – frequently asked questions:

What is rTrysin?
Recombinant Trypsin (rTrypsin) is an animal origin-free alternative to animal-derived Trypsin. rTrypsin hydrolyzes peptide bonds where the carboxyl part of the peptide bond is contributed by either Lysine (Lys) or Arginine (Arg). 

Does Novozyme provide rTrypsin in a solution format?
No, rTrypsin is only available as a solid product (granulate). 

In what unit size does Novozymes sell rTrypsin?
We offer rTrypsin in pack sizes of 100g and 1 kg. 

How can I get a sample of rTrypsin?
You can obtain a sample of rTrypsin by clicking the contact us link at the top of this page.

Handling and formulation
How should I store rTrypsin?
Recommended storage is 0-25°C (32-77°F). Packaging must be kept intact, dry and away from sunlight. The product performs optimally when stored as recommended and used prior to the best before date. When you have formulated the rTrypsin granulate to a liquid form we recommend that you freeze the liquid at the required end concentration and divide your solution into small aliquots in order to avoid future freeze and thaw.

Which buffer is required for dissolving rTrypsin?
We recommend using a PBS buffer adjusted to pH 7. Depending on the cell line, the suggested rTrypsin concentration should be adjusted to 0.2 mg/ml (corresponding to 200 U/ml rTrypsin with an activity of 1000 USP/mg) and sterile filtered. For some cell lines EDTA needs to be added. The addition of EDTA usually accelerates the dissociation phase.
Incubating cells with too high a trypsin concentration for too long a time period will damage cell membranes and kill the cells. If unsure about the concentration of trypsin to use, use a low concentration (0.02% - 0.05%).

Is there EDTA in rTrypsin?
rTrypsin contains enzyme concentrate, sodium chloride and around 1% water. There is no EDTA in the product. The product is free from undesirable proteases such as carboxypeptidase and chymotrypsin.
Which concentration of EDTA I can add for good detachment?
It depends on the cell line but we recommend 0.5 – 1 mM EDTA. 

What is the activity unit of Novozymes recombinant trypsin?
The unit activity is USP/mg and the lower limit is 800 USP/mg.

I am not familiar with USP unit calculation; how does it correspond to the BAEE Unit?
The USP units are determined as follows:

USP Unit

Adheres to the Schwert & Takenaka method* but activities are 1/3 of the S & T-units (BAEE Units).
1 USP-unit catalyzes a change in adsorbance at 253 nm of 0.003 per minute at 25°, pH 7.6 using N-benzoyl-L-arginine ethyl ester (BAEE) as substrate.
*Schwert & Takenaka devised an assay method for tryptic activity using N-α-benzoyl-L-arginine ethyl ester (BAEE) as substrate. 1 S & T-unit catalyzes a change in absorbance at 253 nm of 0.001 per minute at 25 °, pH 7.6 in a reaction volume of 3.2 ml.
1 USP Unit is approximately equivalent to 3.0 BAEE Units.

Customer technical support
Gamma irradiation is typically performed on animal origin trypsin to ensure inactivation of any animal origin viruses. Is it necessary to perform gamma irradiation on rTrypsin?
rTrypsin is manufactured by an animal origin-free process so it is not necessary to perform gamma irradiation on rTrypsin.

What is the PI (Isoelectric point) of rTrypsin?
The theoretical PI is 8.52 but our laboratory has measured the PI at 9.5 (a slight difference from the theoretical PI).

What is the cleavage specificity compared to porcine trypsin?
Our scientists have compared the cleavage specificity of rTrypsin to porcine trypsin and found the cleavage specificity of rTrypsin to be very similar. Both trypsin products cleave after R (Arginine) and K (Lysine).

Regulatory affairs
According to which standards is rTrypsin produced?
rTrypsin is a technical product manufactured under ISO standards. It is not produced under GMP and is not pharmaceutical grade.