A look at our Enzyme Technology: Molecular Modeling

This post has been written by ZYMVOL's Senior Researchers Marina Cañellas and Lur Alonso.

 

Zymvol’s core technology is Molecular Modeling, which – along with bioinformatics – allows us to understand the mechanistic details under enzymatic function and use the generated knowledge to search and tailor biocatalysts towards improved properties.

In this way, by integrating molecular modeling-based strategies at distinct levels of theory, we can identify the best target enzymes and perform their custom design in less than one month!

So, what is Molecular Modeling?

Molecular Modeling refers to a collection of in silico methods that model or mimic the behavior of molecules, ions and/or particles (Nature Subjects). Their main goal is to provide knowledge on the chemistry, structure, dynamics and function of these systems. 

By definition, In silico refers to “conducted or produced by means of computer modelling or computer simulation” (Oxford Dictionary). Simply put, as these methods require huge amounts of calculations, they are performed by computers.

Molecular Modeling has a wide range of applications in many different areas:  computational chemistry, computational biology, drug design, material science… and for this reason it has become a rapidly growing field during the last decades. 

The large variety of systems that can be modeled range from small chemical systems (like the reaction mechanism between two substrates) to larger biological molecules (such as enzymes, antibodies or DNA) and material/molecular assemblies (like supramolecular polymers (Bochiccio, 2017; Frederix, 2018), protein-based assemblies (Soni, 2017), and molecular machines (Aprahamian, 2020).

Although many limitations still exist, they have shed light on some features like functions, processes, and catalytic pathways.

QM and MM

Because of the high complexity underneath molecular systems, such as enzyme-catalyzed reactions, there is not a single in silico technique that suffices for their full modeling. In this way, two main categories of computational methods have been developed at different levels of description: 

  • Those describing systems from an electronic point of view (Quantum Mechanics (QM) methods, this is the deepest level of accuracy to study a system!)
  • Those that describe systems at the atomic level (Molecular Mechanics (MM) methods).

QM methods provide an accurate representation of the system, enabling the description of reaction mechanisms at the electron level, but are computationally very expensive.

On the other hand, MM methods allow the study of larger molecules like proteins, enabling sampling the overall system flexibility, but cannot represent bond-breaking/bond-forming events. As a solution to this issue, the benefits of both strategies can synergize into hybrid schemes, such as combined Quantum Mechanics/Molecular Mechanics methods (QM/MM), enabling the accurate description of large biological phenomena and reactions. QM/MM was awarded with the Nobel Prize in 2013 to scientists Karplus, Levitt, and Warshel.

Adapted from Dr. Marina Cañellas PhD thesis “In silico molecular modelling and design
of heme-containing peroxidases for industrial applications” 2018.

 

The benefits of Molecular Modeling

Why do we think that relying only on bioinformatics is not enough?

Of course, there are some perks to it.

  • Allows to simulate an entire chemical system in the computer
  • It has unique potential to offer detailed atomic-level insights into the studied systems 
  • Allows to predict new mutant variants beyond currently available data.
  • Allows to perform millions of screening/mutations in one day, saving up to 90% of time (compared to experimental procedure). 
  • Saves resources since only a low number of enzymes are tested in the lab (maximum of 300 enzymes tested in the lab with Zymvol’s technology instead of up to millions using exclusively experimental approaches)

As pointed out before, Molecular Modeling techniques are always complemented with sequence-based strategies; the increasing field of bioinformatics complements the predictions and adds new hints hidden on the protein sequence! 

Bioinformatics evaluates the vast amount of data collected daily worldwide and extracts extremely valuable information by applying the proper tools and algorithms. 

Molecular modeling complements wet lab experiments. The way of maximizing success and development in our services Enzyme Search and In silico design is through the close collaboration between dry and wet lab. Considering these two areas as iterative approaches allows scientists to gather a deeper and faster understanding of, in this case, biocatalysis. While computational predictions need to be validated by experimental techniques, wet lab experiments can also benefit from computational approaches by reducing the research time and costs and giving valuable atomic-level insights. In this way, by analyzing huge datasets (sequences, mutant libraries, enzymatic properties, …), lab work is significantly reduced. This enables scientists to obtain results in shorter times and accelerates research/industrial projects, which is of imperious importance for the Industrial Sector (Truppo 2017).

Molecular Modeling in use

Due to the exponential increase that computational resources and strategies have undergone in the last decade, in silico simulations have gained a spot guiding the experimental work in a wide range of areas. (Check the following references for more information on these topics: Hollingsworth & Dror, 2018, Schwaigerlehner et al., 2018, Blog article Ebejer and Baron 2020, as well as more trend research like the one related to COVID19 (Talk by Strauch, 2021)). 

Some fields that are benefited by these in silico methodologies include:

  • Drug discovery (disease mechanisms)
  • Neuroscience (for example, protein-protein interactions)
  • Advanced therapies (for example, antibody engineering)
  • Biocatalysis (for example, directed evolution)

Some practical examples:

  • Elucidate enzymatic mechanisms, understanding enzymes’ catalytic power and enzyme design.
  • Simulation of binding-free energies of small molecules (e.g drugs to their targets).
  • Search for wild type enzymes to perform a particular non-natural reaction.

Getting the best out of Molecular Modeling

As it has been stated, Molecular Modeling is one of the most important approaches we use to provide our Enzyme Search and In Silico Design services. Thanks to our innovative approach, at ZYMVOL we are able to take into account the following features:

  • Solvent effects (by incorporating explicit waters in our simulations) 
  • Quantic effects 
  • Dynamic changes of the protein backbone (backbone flexibility)
  • Distant mutations
  • Side chain flexibility

 

 

Normally, it's hard to find companies who work with all these features, since techniques are very expensive, time consuming or require high specialization. 

The truth is that in this area, the customer moves in the trade off triangle of price-time-expertise: either techniques are very expensive (and/or time-consuming) or require scientists with outstanding degree of specialization to successfully carry out projects. 

What you usually find in the market is:

  • Companies relying only in bioinformatics and experimental work (↑time, ↑price, ↓specific expertise)
  • Companies offering only experimental approach (↑time, ↑price, ↓specific expertise)
  • Companies that offer excellent software, but it is very difficult to achieve quick, reliable results without the assistance of an expert.  (↑time, ↓price, ↓specific expertise).

However, taking all of these features into account is truly beneficial, as it means:

  • More realistic simulations
  • More accurate results and with the unique advantage 
  • The possibility of obtain results in significant short times

Molecular Modeling is a truly powerful tool and it has contributed significantly to our enzyme discovery and design projects. Thanks to it, we can help many industries transition to the use of biocatalysts and make the industry greener.

Do you still have questions related to molecular modeling? Drop us an email: info@zymvol.com

 


References:

Aprahamian, I. (March 3, 2020) The Future of Molecular Machines. ACS Cent. Sci., 6(3), 347-359

Bochicchio, D.; Pavan, G.M. (February 11, 2018) Molecular modelling of supramolecular polymers. Advances in Physics:X, 3(1)

Ebejer, J.P; Baron, B. (June 14, 2020) Dry- and wet-lab research: two sides of the same coin. Times of Malta. https://timesofmalta.com/articles/view/dry-and-wet-lab-research-two-sides-of-the-same-coin.798297

Frederix, P.W.J.M.; Patmanidis, I.; Marrink, S.J. (April 24, 2018). Molecular simulations of self-assembling bio-inspired supramolecular systems and their connection to experiments. Chem. Soc. Rev., 47, 3470-3489

Hollingsworth, S.A.; Dror, R.O. (Sept 19, 2018) Molecular dynamics simulation for all. Neuron, 99(6), 1129-1143

Nature. Molecular Modelling. https://www.nature.com/subjects/molecular-modelling

Soni, N.; Mashusudhan, M.S. (June, 2017) Computational modeling of protein assemblies. Current Opinion in Structural Biology, 44, 179-189

Schwaigerlehner, L; Pechlaner, M; Mayrhofer, P; Oostenbrink, C; Kunert, R. (May 11, 2018) Lessons learned from merging wet lab experiments with molecular simulation to improve mAb humanization. Peds, 31(7-8), 257-265

Truppo, M.D. (April 18, 2017) Biocatalysis in the Pharmaceutical Industry: The Need for Speed. ACS Med Chem Lett. 8(5): 476-480


Why is Green Chemistry important? Origins and Industry Impact

For the past few decades, the scientific community as well as society as a whole has raised its voice on the impact our actions have on the environment, pressing authorities and looking for solutions to address the problem. One of the main focuses has been set on Chemistry, as many “traditional” chemical processes are not sustainable in the long run, with devastating consequences for the environment and quality of life.

In this context, there’s a term that has been slowly, but steadily, gaining traction: Green Chemistry

As the International Union of Pure and Applied Chemistry (IUPAC) puts it, Green Chemistry (also known as Sustainable Chemistry) encompasses the “invention, design, and application of chemical products and processes to reduce or to eliminate the use and generation of hazardous substances”.

But what does this all mean? Why is green chemistry important and how does it contribute to the world’s sustainable development?

Let’s start by going back in history…

The Origins of Green Chemistry

According to the American Chemical Society (ACS), the term “Green Chemistry” was first coined by the US Environmental Protection Agency - Office of Pollution Prevention and Toxins around the 1990s.

The idea of a greater consciousness regarding chemistry had been gaining power since the 60s and 70s, however, it was mostly focused on banning dangerous toxins like DDT and “cleaning up” the aftermath of certain chemical activities. It was not until the 80s and 90s that scientists started thinking differently about their way of doing chemistry, shifting the focus on how to prevent pollution before it even took place.

Then, in 1998, two scientists named Paul Anastas and John C. Warner published what today is popularly known as the "Twelve Principles of Green Chemistry".

The Twelve Principles of Green Chemistry

First published in the book “Green Chemistry: Theory and Practice”, the Twelve Principles of Green Chemistry is a set of guidelines that other chemists can consult to work towards a more sustainable chemistry. The book marked a new era, by helping consolidate a movement that was destined to define how modern chemistry is made.

The principles highlighted in Anastas and Warner’s book are:

  1. Waste Prevention
  2. Atom Economy
  3. Less Hazardous Chemical Syntheses
  4. Designing Safer Chemicals
  5. Safer Solvents and Auxiliaries
  6. Design for Energy Efficiency
  7. Use of Renewable Feedstocks
  8. Reduce Derivatives
  9. Catalysis
  10. Design for Degradation
  11. Real-time analysis for Pollution Prevention
  12. Safer Chemistry for Accident Prevention

Green Chemistry in Industry

Now that sustainability is on everybody’s top-of-mind, Green Chemistry is more important than ever. Just think about the amount of industries that rely on chemistry and whose activity has a great impact on the environment: pharma, agriculture, colorants, materials, consumer products... to name a few. 

This in part is due to the clear increase in awareness about environmental pollution. Especially since the last decades, people are becoming aware that we, as humans, are stressing the planet’s finite resources, and acknowledging that our consumption and waste have to go somewhere. And the current COVID crisis has consolidated this feeling. Just to mention some facts, there is fair evidence about:

If you watch the news, you might have noticed that some institutions are working non-stop to keep this issue present:

And special laws and regulations are being developed to promote more environmentally friendly, sustainable production processes and industries: REACH normative in European Union, ISO 14001, to name some of them.

In parallel, consumers are concerned about the impact the products they buy have in the environment and in their own bodies. Multiple initiatives inviting consumers to choose more “consciously” are nowadays on the front line, and they are demanding products that are respectful in the whole production chain: from manufacturing to recyclability. 

Having said all this, perhaps you are not very sure yet about the difference between green chemistry and general chemistry. The best way to describe it is that Green Chemistry’s main goal is to achieve the same or equivalent chemical reactions with a decrease in environmental damage.

And how is this accomplished?

Some techniques that are used for this aim include:

  • Catalysis: as mentioned in our first blog post, catalysis is the process of increasing a chemical reaction’s rate by the addition of an element denominated catalyst (like enzymes!), that is not consumed during the reaction and therefore can act repeatedly.
  • Synthetic biology: by applying engineering principles, the goal is to redesign and create new biological systems with the aim of providing novel solutions. Examples of this could be the creation of lab-grown meat, synthetic insulin or biofuels produced by algae.
  • Chemical synthesis: as the Nature journal definition says, chemical synthesis is “the process by which one or more chemical reactions are performed with the aim of converting a reactant or starting material into a product or multiple products”.

Why are industrial enzymes an example of Green Chemistry?

Maybe you are still wondering how green chemistry can help decrease pollution.

Like we mentioned earlier, Green Chemistry focuses on sustainability by looking for ways of preventing pollution, hazardous activity and resource waste. That’s why the use of industrial enzymes is such a sought-after solution for many companies who want to shift to greener production processes.

As biocatalysts, enzymes have a set of properties more beneficial than their non-enzymatic counterparts:

  • They are highly selective and specific, which allows chemists to have more control over their desired results.
  • They are effective under mild temperatures, which means significant energy savings.
  • They do not generate toxic waste.

As you can see, enzymes are pretty powerful! The main problem is adapting these enzymes to an industrial setting, which usually means tweaking the structure of an already existing enzyme to give it new features and make it work under certain conditions.That’s why at Zymvol we work to expand the use of green chemistry by creating custom-made enzymes for different industries. With some time and effort, we can make the world become a little bit greener.

 


References:

American Chemical Society. Green Chemistry History. https://www.acs.org/content/acs/en/greenchemistry/what-is-green-chemistry/history-of-green-chemistry.html 

CompoundChem (September 24, 2015). The Twelve Principles of Green Chemistry: What it is & Why it Matters. https://www.compoundchem.com/2015/09/24/green-chemistry/

Nature. Chemical Synthesis. https://www.nature.com/subjects/synthesis 

European Environment Agency (March 25, 2021) Synthetic biology and the environment. https://www.eea.europa.eu/publications/synthetic-biology-and-the-environment 

Aatresh, A.; Cumbers, J. (September 22, 2019) Can Synthetic Biology Make Insulin Faster, Better and Cheaper?. Synbiobeta. https://synbiobeta.com/can-synthetic-biology-make-insulin-faster-better-and-cheaper/ 

Straathof, A.J.J.; Adlercreutz, P. (2000). Applied Biocatalysis (2nd Ed.). CRC Press.


What are the Colors of Biotech?

Modern biotechnology arose in the late 20th century and is currently proving to be one of the key solutions to today's problems, especially regarding health and the environment.

According to the UN Convention on Biological Diversity, Biotechnology is defined as any technical application that uses biological systems, living organisms or parts of them to make or modify products or processes with specific uses.

Giving that those products can be of many types, Biotechnology can cover a wide number of applications: from increasing the quality and resistance of farm crops, to keeping hospital patients healthy by keeping track of their vital signs -and, of course, engineering enzymes for industrial use.

The Colors of Biotech

As a way to structure this vast array of biotech possibilities, scientists started to categorize them by color. Each branch, a different color. That’s why you’ll often hear about the Rainbow Code of Biotechnology.

So what does each color represent in this biotech rainbow?

  • Blue Biotech covers the aquatic and marine fields, by using ocean resources to create products and industrial applications.
  • Green Biotech has to do with everything agriculture-related, focused on improving crops in an accurate, targeted way.
  • Red Biotech centers on Healthcare, by developing an advanced class of drugs and therapies
  • Yellow Biotech covers Food Production
  • Brown Biotech for when Deserts and dry regions are involved
  • Golden Biotech is focused on the use of Bioinformatics, Computational Science, Agile organization and analysis of biological data. We recently wrote a post about what is Golden Biotech in more detail.
  • Gray Biotech encompasses the Environment and biodiversity, environmental protection, maintenance of biodiversity and removal of pollutants
  • White Biotech is for Industrial processes and gene based technologies, as well as the use of enzymes and microorganisms to produce biobased products
  • Purple Biotech is reserved for the laws, ethics and philosophy revolving around biotechnology
  • Black Biotech, as you can imagine, focuses on a darker topic: Bioterrorism and biological warfare

 

At ZYMVOL we are part of the golden branch of biotechnology, since we use computational approaches to improve and enable the discovery of industrial enzymes. For that, we use technology based on computational molecular modeling, machine learning and other tools that allow us to fully understand and work with the chemical structure and interactions between enzyme, substrate and its environment.

We are golden, but our technology can help develop solutions in all other colors!

Discover how we help our customers in Pharma, Chemicals, Biotech and other industries here.

 


References:

Convention on Biological Diversity (2006). Convention Text. Article 2. Use of Terms. https://www.cbd.int/convention/articles/?a=cbd-02

Kafarski, P. (2012). Rainbow Code of Biotechnology. CHEMIK. 66(8), 811-816.


Golden biotech: what it is and why it matters

You might be wondering: “what is golden biotech?”. The answer is rather simple: a biotechnology field that uses computer science as a main driving force. But do you know why it’s often referred to as “golden”? Or what exactly does it entail when taken into practice?

The color code of biotech

First of all, Golden biotech is known as “golden” because of the Rainbow Code of Biotechnology: a way to divide biotechnology’s vast array of applications into different categories, each one defined by a color.

Through this code, we know that when someone is talking about red biotech, they’re referring to health and medical applications; and when they’re talking about white biotech, they’re mostly talking about industrial uses.

All colors of the Biotech Rainbow are important, but what sets Golden Biotech apart is that it revolves around computers. For a technology to be considered golden, it has to rely heavily on some form of computational technique.

Golden biotech is a fairly recent addition to the biotech spectrum, but due to increasing advances in computer technology, one with a lot of potential to keep on growing in the following years.

Some of the main areas included in golden biotech are:

  • Bioinformatics. Field that focuses on analyzing large sets of biological data.
  • Nanotechnology. Field that uses technology at a nanoscale, or in other words, in atomic, molecular and macromolecular levels.
  • Computational Biology. Although closely linked to Bioinformatics, Computational Biology consists of using computational methods to develop models for the study of biological systems. This means relying on technologies like Machine Learning, Algorithms, Big Data (to name a few) for building these models.

Zymvol: an example of Golden Biotech company

Now that you know the definition of golden biotech and the main technologies behind it, you might say: “ok, but what does it really look like taken into practice?”

Just take a look at us. At ZYMVOL, we are golden. And is not that we are pretentious: it’s because we work in the golden branch of biotechnology.

At our company, we use a computational approach to improve and enable the discovery of industrial enzymes. We perform what we call “in silico enzyme evolution”, that is, engineer enzymes in the computer through molecular modeling, machine learning and other computer driven technologies. This allows us to fully understand the chemical structure and interactions between the enzyme, the substrate and their environment.

Take a look at the following video. What we do at ZYMVOL in a nutshell:

 

 

Through computer simulations we reproduce the enzyme, its environment and the desired reaction (substrates that interact with the enzyme) to be carried out: we perform different strategic mutations (amino acid substitutions) along the enzyme’s sequence and test its performance, looking at variables such as stability, activity or selectivity.

Thanks to computer simulations, we came up with the best combinations to test in the lab. We provide to our customers the sequences of the top performing candidates, so they produce in the lab only what matters.

This, at large, is the heart of golden biotechnology!

 


References:

Brown, K. (2018) Gold Biotechnology. Wikitech. https://wikitech21.wordpress.com/2018/08/26/gold-biotechnology/

DaSilva, Edgar J. (2004). The Colours of Biotechnology: Science, Development and Humankind. Electronic Journal of Biotechnology, 7(3), 01-02.

 


All you need to know about enzymes: biocatalysts for a greener future

If you’ve followed us for a while, you may already know that at ZYMVOL we work primarily with enzymes, designing and optimizing them through computer simulations. Enzymes are applied pretty much everywhere: from food products, cosmetics, in the synthesis of pharmaceutical products, and -as the natural biomolecules they are- even the inside of your own body.

But despite the key importance of these molecules in our daily lives, not many people know what they do! That’s why in this post we want to break down some of the main points regarding enzymes, its industrial uses and why they are the key to a greener, more sustainable chemical industry.

What are enzymes?

Enzymes are proteins naturally found in living organisms. They work as biocatalysts, which means they help “catalyze” or accelerate chemical processes. Instead of waiting hours or even days for the reaction to be completed, enzymes have the power to speed it up and produce many reactions in less than a second!

Take for example the lactase enzyme. When we drink milk, there’s a protein whose sole purpose is to break down lactose so we can digest it better. However, people who are lactose intolerant don’t have that enzyme, or simply  don’t have enough of it, so they have a harder time digesting it (and suffer the consequences of it).

What does an enzyme look like?

There are around 20 different types of amino acids in Nature, and they are crucial in defining an enzyme’s characteristics. An enzyme is made up of a sequence of amino acids, varying greatly on number. Some may just have 50 amino acids, some may have more than 200.

Here, the variability is enormous: proteins can have different lengths, and can be formed only by some of the around 20 available amino acids. That’s why In Nature, there are millions of different proteins, each one with a particular amino acid sequence.

Also, they tend to fold into themselves, that’s why you’ll usually see images of enzymes represented like this:

 

However, the most important thing to know is that an enzyme’s amino acid sequence defines its shape. And its shape determines its function.

How does biocatalysis work?

As we mentioned previously, enzymes’ function is to catalyze chemical reactions. A catalyst is a molecule that increases the rate of a chemical reaction without being consumed by the reaction.

Enzymes catalyze all reactions that take place in living organisms, and these reactions can be of different types, like synthesis or degradation of products, among others.

To understand the process better, take a look at the following image, which represents the most accepted model of enzyme catalysis, the Induced Fit model, representing a synthesis reaction (formation of a product):

The enzyme-catalyzed reaction takes place in an inner region of the enzyme known as “active site”. The molecule that is bound in the active site is the “substrate”. Active sites are very specific, and only react with very specific types of “substrates”.

The “substrate” interacts with the “active site”, forming a transient “enzyme-substrate complex” that becomes an “enzyme-product complex” once the chemical changes take place.

Even if initially the substrate doesn’t adhere perfectly to the active site, the enzyme is flexible enough to adapt to the substrate. When the reaction finishes, the formed product is released and the free enzyme can bond to another substrate, starting the process again.

Examples of enzymes in our daily lives

As mentioned before, enzymes play a very important role by accelerating chemical reactions happening in our bodies, like breaking down lactose.

But this is just one example of the millions of possibilities that exist. Besides very technical uses, scientists have also applied these bio-molecules in the industry to create all kinds of products. Nowadays, they can be found in:

  • Pharmaceutical.
  • Chemical.
  • Food.
  • Animal Feed.
  • Cosmetics.
  • Cleaning.
  • Textile.
  • Recycling.
  • Pulp and paper products.
  • Flavors and Fragrances.

Industrial applications of enzymes

There’s a long history of enzymes being used to elaborate certain goods (for example, with alcohol fermentation) and, in the 20th century, they started to become more present in various industries. However, not all natural enzymes are valid for the overwhelming amount of different and highly specific chemical processes that take place in the current market.

Global trends on consumer needs and social change push companies to improve their products in different aspects, like  composition,  performance  or  manufacturing, while implementing sustainable production processes and reducing costs.

Therefore, protein engineering and enzyme improvement has gained popularity for companies who want to maintain their competitiveness, while also transforming their production to comply with green chemistry standards.

Some of the most popular industrial enzymes include:

  • Alcohol Dehydrogenases. Those that can reduce aldehydes into primary alcohols and ketones into secondary alcohols.
  • Oxidative Enzymes and Oxidoreductases. Those that cause or accelerate an oxidation reaction.
  • Lipases. Those that help disaggregate fat through hydrolysis.

Why enzymes are key for a sustainable future

There’s no denying that one of the biggest challenges the world faces nowadays is tackling climate change, which many environmental experts predict will have terrible consequences in the following decades. It’s no wonder that in 2015, the UN set a list of Sustainable Development Goals, with goals specifically focused on Climate related issues and Responsible Production.

What many people don’t know is that the use of enzymes in an industrial level can make a big difference in the way companies operate, and, therefore, have a significant and positive impact on the environment.

Why is that? Some advantages of using enzymes include:

  • Mild reaction conditions: enzymes usually do not require harsh working conditions such as high temperatures or use of solvents or other hazardous auxiliary chemicals.
  • Eco-friendliness: enzymes can substitute organic catalysts that require heavy metals that eventually are released to the environment.
  • Speed: enzymes are able to carry out chemical reactions in a extremely fast way
  • Efficiency: with proper reaction conditions, enzymes are able to process all present substrate and convert it into desired product
  • High product selectivity: enzymes are able to react with specific, targeted molecules, even in complex mixtures
  • Savings: with the advantages commented above, companies can save resources

Moreover, and according to the OECD, the potential of climate change mitigation coming from biotechnology processes and biobased products (in which improved enzymes applied to the chemical sector are included) “ranges from between 1 billion and 2.5 billion tons CO2 equivalent per year by 2030”.

Enzymes implemented as industrial biocatalysts are paving the way for a green chemistry revolution. As more industries start to embrace their use, we can get closer to reaching a true bioeconomy.

 


References:

Heckmann, C. M.; Paradisi, F. (2020). Looking Back: A Short History of the Discovery of Enzymes and How They Became Powerful Chemical Tools. ChemCatChem, 12(24), 6082-6102.

Lehninger Principles of Biochemistry, 5th Edition. D.L Nelson and M.M Cox (2008)

Neitzel, J. J. (2010) Enzyme Catalysis: The Serine Proteases . Nature Education 3(9):21

Nature Education eBooks chapters Essentials of Cell Biology, Unit 2.4 and Cell Biology for Seminars, Unit 2.4  © 2014 Nature Education https://www.nature.com/scitable/topicpage/protein-structure-14122136/

OECD.(2011). Industrial Biotechnology and Climate Change. Opportunities and Challenges. http://www.oecd.org/science/emerging-tech/49024032.pdf

Phillips, Rob; Milo, Ron; How many reactions do enzymes carry out each second? Cell Biology by the Numbers. http://book.bionumbers.org/how-many-reactions-do-enzymes-carry-out-each-second/