Chlorophyllase

Other Names

AtCLH1AtCLH2BoCLH1CaCLHChlaseChlase1chlorophyll chlorophyllidohydrolasechlorophyll hydrolasechlorophyll-chlorophyllido hydrolasechlorophyllase 1chlorophyllase1CLHCrCLH1CyanoCLHEC 3.1.1.14

Synopsis

Chlorophyllase: A Comprehensive Reference Article

1. Identity and Chemical Characterization

Systematic Name, Classification, and Synonyms

Chlorophyllase (commonly abbreviated Chlase or CLH) carries the systematic name chlorophyll chlorophyllidohydrolase. It is a membrane protein classified under the Enzyme Commission number EC 3.1.1.14. Recognized synonyms in the scientific literature include chlase, atclh1, boclh1, atclh2, crclh1, chlorophyllase 1, cyanoclh, caclh, and chlase1. The gene products that encode chlorophyllase activity are referred to collectively as CLHs.

Natural Sources

Chlorophyllase is one of the earliest discovered enzymes present in plants and green algae. Chlase activity exists in almost all tested plants, diatoms, and green algae. The enzyme can be found in the chloroplast, thylakoid membrane, and etioplast of at least higher plants such as ferns, mosses, brown and red algae, and diatoms. Enzymes of this class are apparently universal in green plants and photosynthetic bacteria. The enzyme occurs in especially high concentrations in the leaves of certain plants, among them Heracleum spondylium, Datura stramonium, and Beta vulgaris.

The first plants from which the genes encoding chlorophyllase were isolated were Citrus sinensis and Chenopodium album. Several CLH genes have subsequently been isolated from different sources, including Citrus species, wheat, Ginkgo biloba, Brassica oleracea, Chenopodium album, and Arabidopsis thaliana. More CLH genes were identified from different species, including plants, green algae, and even cyanobacteria.

Common Forms and Preparations

Chlorophyllase is not typically sold as a discrete dietary supplement in its enzymatic form. Its primary relevance to human health and commerce arises through two pathways: (1) as an endogenous plant enzyme whose activity transforms dietary chlorophyll into its metabolites in vivo and during food processing; and (2) as a biocatalytic tool used in research and industry to produce chlorophyll-derived compounds. Several applications have been described for chlorophyllase, including removal of chlorophylls from vegetable oils, use in laundry detergents, and production of chlorophyllides. Industrial processes exist in which chlorophyll contamination can be reduced in compositions such as plant oils by treatment with chlorophyllase; the water-soluble chlorophyllide produced is also green in color but can be removed by aqueous extraction or silica treatment.

Chlorophyllase used in industrial or research preparations may be from any convenient source — plant, algal, bacterial, or fungal — and is formally classified as Enzyme Commission class EC 3.1.1.14. Recombinant chlorophyllase expressed in heterologous systems such as Escherichia coli has been immobilized on various supports, including magnetic iron oxide nanoparticles, for potential industrial use.

2. History of Discovery

Since it was discovered in 1912, Chlase has been extensively investigated. The splitting of chlorophyll into its two main components — phytol and the color component chlorophyllin — was found to take place through the action of an enzyme occurring in the leaves, which Willstätter called "chlorophyllase," allowing him to elucidate the nature of crystallized chlorophyll. The esterification of chlorophyllide was first reported in 1911, when Willstätter and Stoll discovered hydrolysis of chlorophyll to chlorophyllide in crude extracts of Heracleum leaves and named the enzyme responsible for this reaction chlorophyllase. Richard Willstätter was subsequently awarded the Nobel Prize in Chemistry in 1915, in part for this discovery.

During the course of his work on chlorophyll, Willstätter found that in the presence of ethanol, an enzyme present in plant tissue (chlorophyllase) catalyzes the transesterification of phytol by ethanol. Chlorophyllase was first identified by Willstätter and Stoll; the scope of its activity was subsequently studied by Fischer and Lambrecht. In aqueous acetone or ether, chlorophyllase catalyzes the hydrolysis of 7-propionic acid esters; in aqueous methanol or ethanol, it catalyzes transesterification.

Chlorophyllides a and b, methyl chlorophyllides, or ethyl chlorophyllides can be isolated directly from plants rich in chlorophyllase by first allowing the leaf meal to digest with aqueous acetone, methanol, or ethanol; the leaf meal of plants rich in the enzyme can be used repeatedly to catalyze hydrolysis or esterification of chlorophyll derivatives.

The first CLHs to be cloned and characterized were reported in 1999. The enzyme, which catalyses the hydrolysis of chlorophyll to derive chlorophyllide, was discovered by Willstätter and Stoll (1913), who suggested that removal of the phytol chain could be the first step in chlorophyll catabolism. Evidence accumulated in more recent years points to the critical role of Chlase in the initial step of chlorophyll catabolism.

3. Key Constituents and Active Compounds

Enzyme Structure

The crystal structure of chlorophyllase reveals the dimeric architecture of the enzyme, the arrangement of catalytic residues, an unexpected divalent metal ion-binding site, and a substrate-binding site that can accommodate a diverse range of pigments. Like related enzymes, chlorophyllase contains at least seven α-helices and nine β-strands and lacks a lid domain; the actual location of the catalytic residues in the tertiary architecture of chlorophyllase more closely resembles the pancreatic lipases.

The deduced amino acid sequence of the first cloned chlorophyllase (CaCLH from Chenopodium album) had a lipase motif overlapping with an ATP/GTP-binding motif (P-loop). Interestingly, two disulfide bonds are evident in the structure of chlorophyllase. The first disulfide bond is found between Cys220 and Cys264, linking two loops of the structure together, partially folding over the active site, and sitting approximately 7 Å away from the catalytic Ser residue in the active site. An additional disulfide bond links Cys270 from each of the monomeric subunits together.

The oxidation state of the Cys residues is imperative to the activity and stability of chlorophyllase, illuminating a biochemical trigger for responding to environmental stress. Bioinformatics analysis of the chlorophyllase enzyme family reveals widespread conservation of key catalytic residues and the identified "redox switch" among other plant chlorophyllase homologs.

Reaction Products: Chlorophyllide and Phytol

The two primary products of the chlorophyllase-catalyzed reaction are chemically and biologically distinct compounds:

Chlorophyllide (Chlide): Hydrolysis of the phytol chain by endogenous chlorophyllase results in chlorophyllide, which has spectral properties similar to native chlorophyll. Chlorophyllide retains the green porphyrin ring structure and serves as an intermediate for downstream catabolism.
Phytol: Phytol is a microbial metabolite of chlorophyll A; primary food sources in Western diets are nut skin for phytol and lipids in dairy, beef, and fish for its metabolites. Phytol and its metabolites have gained interest as dietary compounds for cancer prevention because, as natural ligands of peroxisome proliferator-activated receptor-α and -γ and retinoid X receptor, they have provided some evidence in cell culture studies and limited evidence in animal models of anti-carcinogenic, anti-inflammatory, and anti-metabolic-syndrome properties at physiological concentrations.

Gene Expression and Regulatory Control

Phylogenetic analyses have shown that CLH genes cluster into two groups: the first group (including Arabidopsis AtCLH1 and Citrus sinensis CsCLH) is characterized by ethylene and methyl jasmonate-regulated expression; while the second group (Arabidopsis AtCLH2 and C. album CaCLH) is expressed at low, constitutive levels. Ethylene and methyl jasmonate, which are known to accelerate senescence in many species, can enhance the activity of the hormone-inducible form of this enzyme.

4. Mechanisms of Action

Catalytic Mechanism

Chlorophyllase is the first enzyme involved in chlorophyll degradation and catalyzes the hydrolysis of the ester bond to yield chlorophyllide and phytol. It reacts via transesterification or hydrolysis of a carboxylic ester in which its natural substrates are 13-OH-chlorophyll a, bacteriochlorophyll, and chlorophyll a. Hydrolysis of chlorophyll starts with the attack of a carbonyl group of chlorophyll by the oxygen of the hydroxyl group of the crucial serine residue of the chlorophyllase. This attack forms a tetrahedral transition state. The double bond of the attacked carbonyl reforms and the serine is then esterified to chlorophyllide. The phytol group consequently leaves the compound and replaces the serine residue on the chlorophyllase enzyme. The addition of water to the reaction cleaves the phytol off the enzyme.

The enzyme is also known to function in the esterification of chlorophyllide and transesterification. The enzyme functions optimally at pH 8.5 and 50°C. Recombinant studies have found optimum conditions can vary by organism: the optimal pH and temperature for recombinant OaCLH activity are 7.0 and 40°C, respectively, and recombinant OaCLH has hydrolysis activities against chlorophyll a, chlorophyll b, bacteriochlorophyll a, and pheophytin a, preferring chlorophyll b and chlorophyll a as substrates.

Role in Chlorophyll Catabolism and Senescence

Chlorophyll catabolism is dramatically visualized during leaf senescence and fruit ripening but also occurs at a basal level during natural turnover, which is affected by environmental conditions such as excessive light, leading to photoinhibition. The breakdown serves as a prerequisite in the detoxification of the potentially phototoxic chlorophyll and chlorophyll intermediates during leaf senescence. Rapid degradation of chlorophyll and its intermediates is therefore necessary to prevent cell damage due to the potential phototoxicity of chlorophyll.

Chlorophyllase was long considered to be the first enzyme involved in chlorophyll breakdown, while strong evidence has since shown that it is not involved in chlorophyll breakdown during leaf senescence. On the other hand, it is possible that CLH is involved in chlorophyll breakdown during fruit ripening. Citrus CLH is located in chloroplasts, responds to ethylene, and is involved in chlorophyll degradation during fruit ripening, while plastidic AtCLH1 functions in protecting young leaves from long-term photodamage by regulating PSII repair.

The two CLHs of Arabidopsis have been shown to be dispensable for leaf senescence. Instead, a novel esterase, pheophytinase (PPH), which specifically dephytylates pheophytin (but not chlorophyll), is required for chlorophyll breakdown in Arabidopsis and rice. In some instances, such as during fruit ripening in Citrus, CLHs have been proposed to be involved in dephytylation. At present, the possibility that CLHs play a role in fruit cannot be excluded.

Defense and Stress Response Roles

It was recently discovered that Arabidopsis CLH1 is located in developing chloroplasts but not in mature chloroplasts, and plays a role in protecting young leaves from long-term photodamage by catalyzing chlorophyll turnover in the photosystem II (PSII) repair cycle. The true involvement of CLH in chlorophyll breakdown has been questioned, since not all isolated genes have a chloroplast transit peptide, suggesting alternative pathways occurring outside of the chloroplast or involvement of enzymes other than CLH. The inducibility of certain CLH genes by methyl jasmonate — a wound and defense signaling compound — has led to the hypothesis that chlorophyllase may also participate in plant defense responses against herbivores and pathogens.

5. Traditional and Historical Use

Chlorophyllase as an isolated or purified enzyme has no documented traditional or ethnomedical history of direct human use. The historical record regarding this enzyme is exclusively scientific, beginning with laboratory investigations in the early twentieth century. The broader context of chlorophyll-containing plants in traditional medicine is, however, relevant, since chlorophyllase activity in those plants contributes to the chemical transformations that may underlie some traditional uses.

Since its discovery in 1912, Chlase has been extensively investigated. In the early stages, research on Chlase was mainly focused on its purification and properties from different plant species and algae.

The use of chlorophyll-rich plant materials — from which chlorophyllase activity was later identified as a key determinant — has deep historical roots. Research in the 1940s indicating that chlorophyllin solutions slowed the growth of certain anaerobic bacteria in the test tube and accelerated the healing of experimental wounds in animals led to the use of topical chlorophyllin solutions and ointments in the treatment of persistent open wounds in humans. During the late 1940s and 1950s, a series of largely uncontrolled studies in patients with slow-healing wounds, such as vascular ulcers and pressure ulcers, reported that the application of topical chlorophyllin promoted healing more effectively than other commonly used treatments. It should be noted that the active agents in these traditional and early clinical applications were chlorophyllin derivatives, not chlorophyllase itself.

Chlorophyllin has been used orally as an internal deodorant and topically in the treatment of slow-healing wounds for more than 50 years without any serious side effects. In the late 1950s, chlorophyllin was added to papain and urea-containing ointments used for the chemical debridement of wounds in order to reduce local inflammation, promote healing, and control odor. Chlorophyllin-containing papain/urea ointments are still available in the US by prescription.

6. Scientific Evidence by Area of Use

Critical prefatory note: Chlorophyllase, as an endogenous plant enzyme, is not itself a marketed dietary supplement. Its relevance to human health derives from its role in generating biologically active metabolites — primarily chlorophyllide and phytol — from dietary chlorophyll, and from its industrial use in producing chlorophyll derivatives for pharmaceutical and food applications. The vast majority of health-related evidence concerns chlorophyll, chlorophyllin (sodium copper chlorophyllin, SCC), or phytol, not chlorophyllase itself. The following sections address each area, clearly delineating the connection to chlorophyllase activity.

6.1 Chemoprotection and Anticarcinogenesis

In vitro and animal research suggest that chlorophyll and its derivatives may possess antioxidant, anti-inflammatory, and anti-cancer properties. Chlorophyllide (the product of chlorophyllase-mediated hydrolysis) has received some investigational attention as a potentially bioactive compound, as have the parent chlorophyllins more broadly.

In animal models of aflatoxin B1 (AFB1)-induced liver cancer, administration of sodium copper chlorophyllin (SCC) at the same time as dietary AFB1 exposure significantly reduces AFB1-induced DNA damage in the livers of rainbow trout and rats and dose-dependently inhibits the development of liver cancer in trout. Natural chlorophyll has also been found to inhibit AFB1-induced liver cancer in the rat. Collectively, this evidence supports a role for SCC and/or chlorophyll itself in limiting cancer initiation. In contrast, data suggest a limited role for SCC in influencing cancer progression: one rat study found that SCC did not protect against aflatoxin-induced liver damage when given after tumor initiation.

Although in vitro and in vivo studies suggest anticancer effects, evidence of efficacy in humans remains scarce. A chemoprevention trial in humans using SCC revealed that daily ingestion of SCC tablets (300 mg/day) resulted in the absorption of Cu-chlorin e4 ethyl ester into the bloodstream, providing the initial evidence that chlorophyll derivatives can be absorbed by the human gastrointestinal tract. Evidence strength in this area is characterized as preliminary for cancer-specific applications.

6.2 Wound Healing and Dermatology

Chlorophyllase produces chlorophyllide, which retains the porphyrin chromophore structure of chlorophyll. Early applications of chlorophyllin-based products in wound care form the primary evidence base.

Research in the 1940s indicating that chlorophyllin solutions slowed the growth of certain anaerobic bacteria in the test tube and accelerated the healing of experimental wounds in animals led to the use of topical chlorophyllin solutions and ointments in the treatment of persistent open wounds in humans. These early studies were largely uncontrolled.

6.3 Internal Deodorization

The US FDA has published a monograph on chlorophyllins for internal use in human beings, addressing deodorant drug products for internal use for over-the-counter human use. This monograph covers chlorophyllin-based products, the production of which may involve chlorophyllase-catalyzed reactions during raw material processing. The evidence for internal deodorizing effects of chlorophyllin is mixed and largely based on older studies.

6.4 Gastrointestinal and Gut Microbiota Effects

Chlorophyll and its derivatives appear to support glucose regulation primarily through actions in the gastrointestinal tract. They modulate gut microbiota, improve glucose tolerance, reduce inflammation, and alleviate obesity-related markers. These findings are based on in vitro and animal studies; robust human clinical trial data are not yet available.

6.5 Diabetes and Metabolic Health

Chlorophyllin has demonstrated significant potential in mitigating diabetes-induced hepatic damage. In studies involving streptozotocin-induced diabetic mice, sodium-copper chlorophyllin supplementation resulted in notable improvements. Recent research highlighted that chlorophyll and its derivatives may beneficially influence glucose metabolism and oxidative stress, key factors in diabetes. All available mechanistic and efficacy evidence remains at the preclinical (animal or cell culture) level for diabetes-specific claims.

6.6 Phytol-Mediated Health Effects

Phytol and its metabolites have gained interest as dietary compounds for cancer prevention because, as natural ligands of peroxisome proliferator-activated receptor-α and -γ and retinoid X receptor, they have provided some evidence in cell culture studies and limited evidence in animal models of anti-carcinogenic, anti-inflammatory, and anti-metabolic-syndrome properties at physiological concentrations. However, there may be a narrow range of efficacy, because phytol and its metabolites at supra-physiological concentrations can cause in vitro cytotoxicity in non-cancer cells and can cause morbidity and mortality in animal models.

Phytol is a component of chlorophyll with reported anticancer and immune-enhancing effects. It has been reported that phytol enhances the activity of natural killer cells that detect and remove cancer cells and promotes macrophage functions in immunity. These reports are based on preclinical laboratory studies.

6.7 Food Science and Postharvest Applications

The role of chlorophyllase in food quality is well-established. Comparison of in vitro chlorophyllase catalytic constants from greened rye seedlings and in vivo rate constants from whole tissue studies suggests that the conversions of chlorophyll to chlorophyllide and pheophytin to pheophorbide in coleslaw, cucumbers, and brined olives were the result of chlorophyllase activity. Low-temperature withering can slow chlorophyll degradation in postharvest tea leaves via significant inhibition of enzyme activity and gene expression of Mg-dechelatase, chlorophyllase, and pheophorbide a oxygenase.

7. Body Systems and Health Areas of Association

Although chlorophyllase itself has not been clinically studied in humans as a dietary supplement, the compounds it produces and the broader class of chlorophyll derivatives with which it is associated have been investigated across several physiological systems:

Hepatic system: Animal studies suggest protective effects against chemically induced liver damage through chlorophyllin and related derivatives.
Gastrointestinal tract: Chlorophyll derivatives modulate gut microbiota and reduce GI inflammation in preclinical models.
Integumentary system (skin): Historical use of topical chlorophyllin in wound care; small modern studies on acne and wound healing.
Metabolic system: Preclinical evidence for effects on glucose tolerance and obesity markers.
Immune system: Preclinical reports of phytol enhancing natural killer cell activity.
Photosynthetic/plant biology: The enzyme's primary documented role remains in plant cell biology — chlorophyll turnover, PSII repair, fruit ripening, and defense signaling — rather than in mammalian physiology.

8. Dosage Forms and Reported Dosages

Chlorophyllase is not available as a direct supplement in standardized dosage forms for human consumption. The following dosages pertain to chlorophyll-derived products — whose production and bioconversion may involve chlorophyllase activity — as reported in the cited scientific literature:

Chlorophyll and chlorophyllin supplements are most often taken in a liquid or capsule form at dosages ranging from 100 to 300 mg daily. More studies in humans are needed to determine what the effective dose of chlorophyll (or a particular chlorophyll derivative) is.
A chemoprevention trial in humans used SCC tablets at 300 mg/day, which resulted in detectable absorption of Cu-chlorin e4 ethyl ester into the bloodstream.
Another study conducted on human volunteers detected the presence of pheophytin and pheophorbide derivatives in the blood three hours after the ingestion of 1.2 kg of freshly boiled spinach.
Most dietary supplements contain the chlorophyll derivative sodium copper chlorophyllin, rather than natural chlorophyll, due to its increased stability.

Recombinant chlorophyllase immobilized on supports is described as potentially useful for the industrial production of chlorophyll derivatives, with specific enzyme units and immobilization yields reported in research papers, but these are industrial rather than human therapeutic quantities.

9. Safety Considerations and Interactions

Safety of Chlorophyll and Derivatives

Dietary supplements containing chlorophyll and chlorophyllin are available and generally considered safe, with no reported adverse side effects over several decades of human use. Skepticism about their effectiveness persists, however, due to the lack of robust scientific evidence supporting their claimed health benefits.

Phytotoxicity Risk from Chlorophyll Metabolites

A key safety consideration arising from chlorophyllase activity relates not to the enzyme itself but to the potential accumulation of its intermediate products. The chlorophyll degradation process is essential to leaf senescence and fruit ripening due to the much needed elimination of chlorophyll and their derivatives to avoid accumulation of phototoxic pigments. In mammalian contexts, chlorophyll breakdown products such as pheophorbide have phototoxic potential if they accumulate, which is why controlled catabolism (including chlorophyllase-initiated steps) is physiologically important.

Phytol Toxicity Considerations

There may be a narrow range of efficacy for phytol and its metabolites, because at supra-physiological concentrations they can cause in vitro cytotoxicity in non-cancer cells and can cause morbidity and mortality in animal models. This indicates a dose-dependent risk profile for phytol — one of the two products of chlorophyllase catalysis — at doses far exceeding those encountered through normal dietary chlorophyll intake.

Industrial Handling Considerations

As proteins, chlorophyllase enzymes are easily susceptible to inactivation and are usually restricted by a lack of long-term operational stability; also, it is difficult to recycle and reuse the enzyme without immobilization or stabilization strategies. In industrial settings, these properties require controlled handling.

Evidence Limitations

Despite years of use as a dietary supplement [for chlorophyll-related products], there is very little human clinical research exploring the potential benefits of chlorophyll. There is not enough information available to know definitively how chlorophyll might work [in humans]. The evidence that does exist for health benefits of chlorophyll derivatives is largely preclinical (in vitro or animal). Chlorophyllase as an isolated entity has no human clinical trial record as a therapeutic agent.

10. Current Research Directions and Outstanding Questions

There remain important unanswered questions related to CLH. These include the precise subcellular localization of different CLH isoforms, the identity of the core dephytylation enzyme during fruit ripening, and the exact interplay between chlorophyllase and alternative catabolic enzymes such as pheophytinase (PPH).

The oxidation state of the Cys residues is imperative to the activity and stability of chlorophyllase, illuminating a biochemical trigger for responding to environmental stress. Bioinformatics analysis reveals widespread conservation of key catalytic residues and the identified "redox switch" among other plant chlorophyllase homologs.

Recombinant chlorophyllases are being explored as biocatalysts to produce chlorophyllide derivatives that have potential as drugs for the biotechnology and pharmaceutical industry. Immobilized chlorophyllase preparations have demonstrated retained residual activity of approximately 60% after 17 cycles in a repeated-batch operation, meaning immobilized preparations can be used repeatedly to lower cost and are potentially useful for the industrial production of chlorophyllide and phytol.

References

Health Conditions

Health conditions that Chlorophyllase may help support.

No conditions available.

Body Systems

Body systems that Chlorophyllase may help support.

No body systems available.