Menu

ENZYMES IN HYDROPONICS

 

About Enzymes

 

An enzyme is an organic complex made up of amino acids, proteins, or RNA (ribonucleic acid which plays a role in transferring information from DNA to the protein-forming system of a cell). The composition and structure of an enzyme depends on its function within the metabolic process. One of the key actions of enzymes is they catalyze chemical reactions, increasing metabolic activity at substantial rates, while also protecting biological organisms – e.g. plants – from disease and stress.

 

Therefore, enzymes function as a biological catalyst in the general process of converting minerals into absorbable food for plants, with a little help from various types of bacteria, in some cases. Without enzymes, plants cannot effectively use the minerals available to them. Enzymes are used in ALL chemical reactions in living things; this includes respiration, photosynthesis, and growth.

 

Enzymes and Substrates

 

In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving substrates. The chemical reaction catalyzed by an enzyme is done at a very specific location within a molecule. This location is known as the ‘active site’. In the case of a single substrate, the substrate bonds with the enzyme active site and an enzyme-substrate complex is formed. The substrate is transformed into one or more products, which are then released from the active site. The basic structure of this process is described as the ‘lock and key’ model. This model defines that the substrate (key) fits neatly into the enzyme (lock) to accommodate a chemical process; however, only one kind of key can fit into the lock. Therefore, only one form of substrate can fit into the active site of the enzyme.

 

To take a practical example, when discussing enzymes and plants, cellulose is a substrate for ‘cellulase’ enzymes. Cellulose is the basic structural component of plant cell walls. It occurs in combination with other materials, such as lignin and hemicelluloses. It is a complex carbohydrate, or polysaccharide, consisting of 3,000 or more glucose units. Cellulose comprises about 33 percent of all vegetable matter (90 percent of cotton and 50 percent of wood are cellulose) and it is the most commonly occurring organic compound on Earth. The main structural element of a plant’s root cells walls are cellulosic components.[1]

 

Cellulose is an extremely tough material that can take a very long time to break down. However, cellulase enzymes greatly speed up the process and convert (hydrolyze) cellulose into sugars and other compounds. At this point, the sugars become available to plant roots and rhizosphere microflora (as a source of energy/food).

 

Therefore, the process of cellulase enzymes interacting with cellulose can be described as: 1) the substrate enters (bonds with) the active site of the enzyme; 2) the enzyme and substrate form a complex; 3) the enzyme changes the substrate, forming ‘products’ and; 4) the products leave the active site of the enzyme. These products (sugars/carbohydrates) then become food (energy) for microflora and plants See following illustration…

 

 

 

 

Coenzymes

 

Coenzymes are substances that enhance the action of enzymes.

 

Coenzymes are small molecules. They cannot by themselves catalyze a reaction but they can help enzymes to do so. Coenzymes are organic non-protein molecules that bind with the protein molecule (apoenzyme) to form the active enzyme (holoenzyme).

 

A number of the water-soluble vitamins such as vitamins B1, B2 and B6 serve as coenzymes.

 

On Specific Enzymes

 

Because a particular enzyme catalyzes only one reaction, there are thousands of different enzymes in a cell catalyzing thousands of different chemical reactions. Just one example, is that at least five enzymes (e.g. Rubisco, ribulose-5-phosphate kinase) play a pivotal role in the Calvin (C3) cycle.

While there are currently about 2500 known types of enzymes, they can generally be classified into six categories. The ‘Oxidoreductases’ make oxidation-reduction (the process by which an atom loses an electron to another atom) possible. ‘Hydrolases” break down proteins, carbohydrates, and fats. They do this by adding a water molecule – thus the name hydrolases. ‘Tranferases’ catalyze the transfer of a chemical group, such as a phosphate or amine, from one molecule to another. ‘Lysases’ catalyze the formation of double bonds between atoms by adding or subtracting chemical groups. ‘Isomerases” catalyze reactions involving structural rearrangement of molecules. (And) ‘Ligases” catalyze the formation of a bond between two substrate molecules through the use of an energy source.

 

To generalize somewhat, the agriculturally important enzymes, are those that catalyze the digestion or “hydrolysis” of certain large organic molecules like starch, cellulose, and protein. The enzymes hydrolyze these complex molecules, accelerating their digestion and yielding simpler substances such as simple sugars. These substances (products) then become food (energy) for microflora and for plants.  Since the process of digestion is referred to as hydrolysis (where a water molecule is added), the enzymes that catalyze the process are considered to be “hydrolyzing enzymes” or “hydrolases”.

The hydrolyzing enzymes include:

(1) Amylases, which catalyze the digestion of starch into small segments of multiple sugars and into individual soluble sugars.

(2) Proteases, (or proteinase), which split up proteins into their component amino acid building blocks.

(3) Lipase, which split up animal and vegetable fats and oils into their component part: glycerol and fatty acids.

(4) Cellulase (of various types) which breaks down the complex molecule of cellulose (e.g. decaying root and plant matter) into more digestible components of single and multiple sugars.

(5) Beta-glucanase, (or gumase) which digest one type of vegetable gum into sugars and /or dextrins.

(6) Pectinase which digests pectin and similar carbohydrates of plant origin.

 

About Enzymes in Hydroponics 

 

Okay, this one is extremely complex (aghhhh!!! not again you say) so let’s go back to earlier information that we covered on bennies and their modes of action in pathogen control. One of these modes of action is mycoparasitism, where certain fungi and bacteria attack pathogenic microorganisms by excreting lytic enzymes (such as cellulases, proteases, glucanases and chitinases) that enable it to degrade the pathogens cell walls and utilize its nutrients.

 

While the secretion of lytic enzymes is common amongst bacteria, fungal chitinases (and possibly the genes encoding them) appear to be more effective than are enzymes from other sources in their ability to inhibit pathogenic fungi.[2] Studies have, in fact, shown that a synergism exists between beneficial bacteria and fungi, and that the lytic enzymes released by beneficial fungi, such as Trichoderma spp., enhance the ability of beneficial bacteria to control pathogens.  This is just one reason why I use a combination of bacteria and fungi, in my growing system, to control plant pathogens.

 

Many commercially available hydrolyzing/lytic enzyme products are produced using Trichoderma spp. fungi which release lytic enzymes. These lytic enzymes are then used by hydroponic manufacturers in their liquid enzyme formulations.

 

These enzyme formulations have two modes of action when used in situations where plants are being grown.

 

1) They degrade cellulose, turning it into sugars/carbohydrates which then become available for plant uptake and act as a source of energy for microflora. Because sugars/carbohydrates act as energy for microflora this promotes an environment in which beneficial bacteria and fungi can thrive.

 

2) They ‘lyse’ (cause the dissolution or destruction of cells) pathogenic fungi cell walls, making the pathogenic fungi more susceptible to destruction by beneficial bacteria and fungi. [3], [4], [5]

 

When considering that, firstly, lytic enzymes create an environment that promotes high numbers of beneficial microorganisms (through converting cellulose into food for bennies) and, secondly, they break down pathogen cell walls, making pathogens more susceptible to destruction by beneficial bacteria and fungi, adding lytic enzymes to biologically diverse growing systems, at least on paper, makes a great deal of sense.

 

However…..

 

The complexities of enzymes, mycoparasitism and effective pathogen control becomes pronounced when overlaying positive research findings with liquid beneficial enzyme products. To demonstrate, let me quote Haran et al (1996) when discussing Trichoderma spp. and their ability to release lytic enzymes.

 

(Quote)


“The antifungal activity of cell-wall-degrading enzymes has been studied by several authors. Lorito et al (1993) tested antifungal activity of purified endochitinase and exochitinase (chitobiosidase) produced by T. harzianum strain P1. Inhibition of spore germination and germ-tube elongation were used as bioassays to evaluate the level of antifungal activity against different fungal species. Both processes were inhibited in all chitin-containing fungi tested, except T. harzianum. The degree of inhibition was found to be proportional to the levels of chitin in the cell waIl of the target fungi. These chitinolytic enzymes appeared to be biologically more active than enzymes from other sources and more effective against a wider range of fungi. Combining the activities of the endochitinase and exochitinase (chitobiosidase) resulted in a synergistic increase in antifungal activity. The authors suggested that mixtures of hydrolytic enzymes with complementary modes of action may be required for maximum efficacy and that correct combinations of enzymes may increase in vitro antifungal activity. Lorito e t al. (1994) reported the purification of two additional cellwall- degrading enzymes from the same strain of T. harzianum : an NAcetyl-/βglucosaminidase and a glucan 1,3-P-glucosidase. Using the above bioassays, they found a synergistic inhibitory effect on B. cinerea spore germination and germ-tube elongation when two, three or four enzymes were applied together. The highest level of antifungal activity was obtained when a solution containing all four cell-wall-degrading enzymes was used.”[6]

 

(End Quote)

 

Don’t try to get your head around this particular quote.

 

The point really is that because enzymes are very specific, in order to be effective, multiple enzyme types (dependent on pathogen) are shown to be required to obtain a high level of antifungal/pathogen activity. The question then becomes, do liquid enzyme products provide these specific enzymes at sufficient levels and ratios to provide broad spectrum pathogen control?

 

Possibly/probably not! (Highly unlikely, given that science is yet to make sense of things!)

 

Other than this, there is also one other significant problem/dilemma that needs to be considered.

 

Enzyme Product/Additive Quality

 

Enzymes are biological molecules and subject to degradation and loss of activity.

 

In order for enzymes to be stored and remain viable for long periods they need to be “immobilized” or “stabilized”. .

 

Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme. The advantages of immobilized, over soluble, enzymes is due to their enhanced stability and ease of separation from the reaction media, leading to significant savings in enzyme consumption.

 

Immobilization methods range from binding to prefabricated carrier materials to packaging in enzyme crystals or powders.

 

While enzyme immobilization science is extremely complex, different enzyme types require different forms of immobilization in order to ensure; 1) viability; 2) shelf life/stability and; 3) the highest level of activity possible (i.e. some forms of immobilization reduce biological activity while others are shown to increase biological activity – dependent on enzyme type). Research shows that in many/most cases immobilized enzymes are “substantially” more stable than free enzymes.

 

There is a variety of methods by which enzymes can be immobilized, ranging from covalent chemical bonding to physical entrapment. Immobilization can be broadly classified as:

 

  1. Covalent bonding of the enzyme to a derivatized, water-insoluble matrix.
  2. Intermolecular cross-linking of enzyme molecules using multi-functional reagents.
  3. Adsorption of the enzyme onto a water-insoluble matrix.
  4. Entrapment of the enzyme inside a water-insoluble polymer lattice or semi-permeable membrane.
  5. Lyophilization – a stable preparation of a biological substance created by freezing and  vaporizing the ice/water away under vacuum conditions (freeze drying)

 

You will note that in all cases in order to best stabilize enzymes they must be made water free first (when added to the right catalyst they then become active). Here’s the bad news on this front…. technically speaking, enzymes in aqueous solutions are inherently unstable due to a variety of intramolecular and intermolecular chemical reactions including hydrolysis, aggregation, deamidation, oxidation, β-elimination, and changes in conformation that may result in a loss of biological activity.[7],  [8]  Immobilized” (stabilized) powders when added to solution remain viable (biologically active) in significant quantities, when stored correctly, for approximately 1 to 5 months (dependent on enzyme type/species and other factors).

 

While there are means to increase liquid enzyme product shelf lives (i.e. stabilizers such as glycerol and storage temperatures) basically liquid enzymes are inherently less storage stable than their granular counterparts. One study showed that where liquid enzyme products containing xylanase, amylase and cellulase were stored at below 5 °C shelf life was about 5 months. However, cellulase showed good stability while there was sharp decline in xylanase activity when stored at 4 °C or at 30 °C whether carried in water or glycerol.[1]

 

What this information tells us is that 1) when a liquid enzyme product is formulated correctly, the shelf life is reasonably long – this particularly applies to cellulase; 2) that some enzymes have longer shelf lives than others, and; 3)  liquid enzyme additives should be stored at below 5 °C with 4 °C being about the ideal.

 

However, a key problem with many liquid enzyme products that growers are currently purchasing through hydroponic stores is, arguably, in many cases, by the time they reach the end user the enzymes have become biologically inactive (inert), or exist at such low levels that the product is largely ineffective.

 

Or, as Advanced Nutrients has put it:

 

 [Quote]


“Our scientists visited a bunch of hydro stores and bought enzyme products made by our competitors.

Then they tested them for enzyme concentration, variety and viability.

The tests showed that our competitors’ products were dead in terms of enzymatic units of activity…”

“Enzyme products made by our competitors were found to contain far fewer types of enzymes and they had inferior manufacturing standards. To put it bluntly, their enzyme formulas were junk.”

 

[End Quote]

 

Not exactly the way I like to put things (see author’s note); however, Advanced Nutrients make a good point. In simple terms, the moment free or encapsulated enzymes are added to water they become unstable. As a result, their shelf life, dependent on enzyme type, is speculative at best (this also applies to ANs SensiZyme, regardless of claims to the contrary!).

 

Other than this, each enzyme species has its own narrow pH and temperature range, and the process of its reaction depends on those conditions along with substrate.

 

You’re perhaps beginning to see how complex things are when it comes to enzymes, and given that often liquid enzyme products, sold through the retail hydroponic industry, may have sat around on hydroponic store shelves for months before purchase and then are used in varying environmental situations (e.g. pH, temps, media) this raises serious concerns as to their viability in broad spectrum pathogen control in various growing situations.

 

One other significant problem presents and that is, to my knowledge, not one single credible scientific study has trialled a liquid enzyme formulation for pathogen control (or any other purpose) in hydroponics. This means that while, theoretically, enzyme liquid formulations/additives probably/possibly offer pathogen control potential, this is yet to be demonstrated in any peer reviewed research. Other than this, as we have seen, enzymes are very specific and in order for any enzyme additive to be effective, multiple enzymes species (dependent on pathogen) are shown to be required to obtain a high level of antifungal activity.

 

The question then becomes, does that liquid enzyme additive, purchased through a hydroponic store, contain adequate levels of biologically active enzymes, and are the right species of enzymes present to offer broad spectrum pathogen control? The answer to this is who really knows…manufacturers often don’t specify, among other things, what enzymes are in their formulations, at what levels, and in most cases there is no specified shelf life or use by date.

 

So what am I saying here?

 

Well, let’s dumb things down a bit and speak in layman’s terms. In short, many years ago when I first began growing in coco coir I experimented with both enzymes and bennies, and combinations of both. Based on these experiences I now use bennies alone (although one could also say bennies and enzymes due to bennies excreting lytic enzymes). Perhaps that answers the complexities of enzyme science more clearly.

 

My own view (I stress “my own view”) is certainly, the possibilities that enzymes offer for pathogen control look good – however, given the scientific complexities, the lack of credible research data on liquid enzyme products, stability issues and the very different cultural practices of hobby hydroponic growers I don’t think that liquid enzyme additives are the best option for now….I.e. further information/data is needed.

 

Let’s leave that one there.

 

 

Author’s note, re Advance Nutrients claims of “our competitors’ products were dead in terms of enzymatic units of activity”.  Technically, enzymes aren’t living things (they are chemical compounds) so they can’t be “dead”. However, they can be “biologically inactive”, or inert, or be present at such low levels that the liquid enzyme product is ineffective.

 


[1] Genet, M., Li, M., Luo, T., Fourcaud, T., Clement-Vidal, A. and Stokes, A., Linking carbon supply to root cell-wall chemistry and mechanics at high altitudes in Abies georgei. Ann. Bot., 2011, 107, 311–320.

[2] Lorito, M et al. (1993)  Antifungal, Synergistic Interaction Between Chitinolytic Enzymes from Trichoderma harzianum and Ecterobacter cloacae

[3] Brand, T. and Alsanius, B.W. (2004) Enzyme Activity in Nutrient Solution of Closed Hydroponic Systems with Integrated Slow Filters. Acta Horticulturae

[4] Woo S, Fogliano V, Scala F, Lorito M (2002) Synergism between fungal enzymes and bacterial antibiotics may enhance biocontrol

[5] Lorito, M et al. (1993)  Antifungal, Synergistic Interaction Between Chitinolytic Enzymes from Trichoderma harzianum and Ecterobacter cloacae

[6] Haran, S. Schickler, H and Chet, I. (1996) Molecular mechanisms of lytic enzymes involved in the biocontrol activity of Trichoderma harzianum. Otto Warburg Center for Agricultural Biotechnology, The Hebrew University of Jerusalem, Faculty

of Agriculture, Rehovot 76-100, Israel

[7] Costantino RH, Langer R, Klibanov M. Solid-phase aggregation of proteins under pharmaceutically relevant conditions. J Pharm Sci. 1994;83(12):1662–1669. doi: 10.1002/jps.2600831205.

[8] Winters MA, Debenedetti PG, Carey J, Sparks HG, Sane SU, Przybycien TD. Long-term and high temperature storage of supercritically-processed microparticulate protein powders. Pharm Res. 1997;14(10):1370–1378. doi: 10.1023/A:1012112503590.

[1] Mohamed Ahmed El-Sherbiny, Ghadir Aly El-Chaghaby (2011) Storage temperature and stabilizers in relation to the activity of commercial liquid feed enzymes: a case study from Egypt. J Agrobiol 28(2): 129–137, 2011