Linamarase Enzyme Activity Lyophilisation Lyoprotectant Cryoprotectant Compounds Biology Essay

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Cassava Roots are an important carbohydrate source in many developing countries, due to its high starch content and tolerance to extreme environment. Many traditional dishes in Africa are made with it. However, consuming some cassava root without proper treatment may lead to poisoning, since linamarase (enzyme) and the linamarin (cyanogenic potential compound) contained in the cell wall and the cytoplasm in cassava root cells respectively react and produce free cyanide.

Drying and peeling are the example of helping detoxifying, but the effectiveness is unknown.

One of the most common methods for testing the toxicity of cassava root is to test the concentration of the linamarin by reacting with partially purified exogenous linamarase and comparing the result to standard calibration curve. The activity of the linamarase determines the accuracy of the test. 3EU/ml is its minimum required activity, otherwise the toxic effect of the sample is underestimated.

Current practice, lyophilisation is used for preserving the linamarase. Cryoconservation is the processes of free-thaw of rehydrate linamarase. However, structure of protein may be altered in the lyophilisation and cryoconservation process, which could reduce its activity. Protectants compounds, such as sucrose and trehalose, could reduce the change in lyophilisation and free-thaw process upon proteins


Cassava is believed that it origins form 2000BC in Amazon (Vaughan & Geissler, 2009) and used by tribal group. It can grow up to 24 Meter high. Its roots grow horizontally and shallowly below the ground. Feeder root is used for the storage of food and it can grow to as deep as 10- 100 cm. The size of the roots is wide ranged. The length varies from 10 cm to120 cm and the weight varies from 1 kg to 8 kg. If it grows on a newly prepared forest land, no fertilizer is required.(Grace, 1977).

Cassava can tolerate and grow in extreme environments, for example limited nutrient soil, humid and dry environment (Cardoso et al. 2005). It can normally grow with rainfall from 500mm per year to 5000mm per year. Intercropping it with other food plants is a good way to reduce economic lost in unpredictable weather. Also it is a good food source to against famine (Onabolu et al.1998; Vaughan & Geissler, 2009). Many people depend on it to survive (Yeoh et al. 2001), so that its safety affect many people health. The scope of this review is to identify the importance of the safety of cassava root and the factor affecting accuracy of the toxicity tasting method, especially preserving the activity of linamarase.

Importance of cassava in developing countries

In 2000, Cassava was estimated to be an important carbohydrate source for about 500 million in many developing countries (Yeoh et al. 2001). There was 34million tonnes cassava is produced around 2006 (Sanni et al. 2006). It is widely spread over the tropical and sub-tropical areas (Vaughan & Geissler, 2009). Approximate 70% of the cassava root in the world is produced by Nigeria, Indonesia and Congo Democratic Republic. Amount them, Nigeria is the largest production country (Onabolu et al. 1994).

More important, it is essential for farmers who lack resources could cultivate it as a food and economic source (Egan et al. 1997), due to its high tolerance to the extreme environment mentioned in introduction.

Nutritional value

On average, cassava contains about 70.3% moisture, 21.5% starch, 5.13% sugars, 1.12 % protein, 0.5%fats, 1% fiber, 0.5% Ash, (Grace, 1977). Due to its high moisture content, it is highly perishable (Onabolu et al. 1994). In addition, the harvest and the nutrient of cassava root depend on the cultivating environment. As a result, the harvest and the nutrient of cassava root vary from place to place.

The application of cassava

The applications of cassava root are wide ranged, from human and animal consumption to industrial utilities. For human and animal consumption, it is consumed directly or consumed after processed, such as flour, starch and syrup. Many dishes, such as meat balls, French fries, could be made by cassava original ingredients (Sanni et al. 2006). For industrial utilities, it is processed and become coal mining and paint industry (Sanni et al. 2006).

Toxicity and health

According to Cardoso et al. (2008), cyanide poisoning includes "vomiting, nausea, dizziness, stomach pains, weakness, headache and diarrhoea and occasionally death". These above are the acute effect. Konozo is a cyanide poisoning disease with short onset time. Most of the cases happened on the children and women of child bearing age in southern Africa (Cardoso et al. 2005). According to Essers et al. (1993), an "irreversible paralysis" may be resulted. However, Tropical ataxic neuropathy (TAN) is another cyanide poisoning disease with long onset time. Most of the case happened on the elder people in West Africa (Cardoso et al. 2005). According to Essers et al. (1993), "loss of vision, ataxia of gait, deafness and weakness" may be resulted.

Cyanogenic potential (CNP) of cassava roots

Linamarin and Lototaustralin (methyl linamarin) are two types of glucosides containing in cassava root. The ratio content of linamarin to lotaustralin is about 97 to 7 (Tivana et al. 2009).

There are two types of cassava roots, better taste and sweet taste. The tastes mainly depend on the concentration of linamarin (Cardosoa et al. 2005). Linamarin is a cayanogenic compound, which produce hydrogen cyanide though a chain reaction.

The growing environment is one of the main factors concentrations of glycoside (Vaughan & Geissler, 2009). There are normal practice methods to remove the toxicant formed in cassava root, such as peeling, washing, boiling, toasting and fermentation (Vaughan & Geissler, 2009)

According to Onabolu et al. (1994), on one hand, many cassava root processing factories are built in Africa, but the process of the detoxification of it is not carried out properly. On the other hand, the factories of the Thailand are better developed. The cassava roots are processed in an industry level (Onabolu et al.1994). Thus, different safety levels of cassava products are sold.

Location of linamarin and linamarase

linamarase and linamarin contained in the cell wall and the cytoplasm in cassava root cells respectively react and produce free cyanide. (Tivana et al. 2009)

Principal ways of processing / detoxifying cassava

The main theory of detoxifying cassava is that allowing the reaction between the linamarase and linamarin to product hydrogen cyanide (Unung et al. 2006), which evaporates to air, thought a chain reaction. By peeling, washing, sun- drying and simple wetting method can reduce the cyanogenic potential of cassava root. Increase the surface area and reduce the moisture content facilitate the removal of hydrogen cyanide (Bradbury & Denton, 2009; Essers et al. 1995; Unung et al. 2006).

Principal method used to assay cassava CNP

There are two main methods are used in testing the toxicity of cassava root, steam distillation and enzyme assay. For steam distillation method, it is an American Organization for Analytical Chemistry (AOAC) method, which is a universal standard. HCN is isolated by steam distillation and then followed by a titration to determainate the cyanogenic potential. For the Enzyme assay method, the cassava root is homogenization in acid and then the solution is filtered and hydrolysis by exogenous enzyme. The cyanogenic potential of resulted solution is determined by the spectrophotometer (Essers et al.1993).

Importance of part-purified linamarase enzyme

The accuracy of testing cyanogenic potential of cassava root by reacting with partially purified exogenous linamarase hinges on the activity of the linamarase. If the activity of linamarase is lower than 3EU/ml, then the result shown underestimates the toxic effect of the sample (Essers et al.1993).

Lyophilisation and cryoconservation

Lyophilisation contains two main steps, freezing the solution and drying the frozen solution in a vacuum condition. Primary and secondary drying steps are contained in the drying process. Frozen water is dried in the primary step, while the 'bound' water is dried in the secondary step. (Bhatnagar & Denton, 2007; Wang, 2000) cryoconservation is a freeze-thaw process toward subjects.

In cyonoservation, protein activity may lose, for example lactate dehydrogenase, due to freezing. Since water is crystallized, available water decreases. Then, pH decrease as available water decreases. Finally protein may be altered, cue to the change in protein. (Wang, 2000)

Why lyophilisation is necessary

Lyophilisation (freeze drying) is a common method for preserving protein, from a liquid stage to a solid stage. Lyophilised proteins, in general, have a longer shelf life. (Carpenter et al. 1997; Wang, 2000). In aqueous stage, proteins usually undergo alteration in a short time, because of marginal stabilities. Amorphous mixture is achieved to increase the shelf life of protein products by lyophilisation or spaying drying (Chang & Pikal, 2009; Katayama et al. 2008). A careful designed lyophilisation can protect the structure of protein from alterlation (Meister & Gieseler, 2008).

Potential damage that lyophilisation can do

Within lyophilisation, different kinds of stresses are developed on the unprotected protein compounds and denature the protein. According to Wang (2000), ''these stresses include low temperature, formation of dendritic ice crystals, increase in ionic strength, pH changes, phase separation, and removal of the protein hydration shell.''.

There are several reasons that account for the alternation of protein in lyophilisation. They include covalent and noncovalent aggregation, deamidation, oxidation and maillard reaction (Chang & Pikal, 2009).

Types of lyoprotectant

Although the principle of how the protective compounds is inconclusive, there are different types of protectants are commonly used. (Chang & Pikal, 2009). When these protectants are added to protein, hydrogen bonds between protein and protectants are formed to replace the hydrogen bonds formed between protein and water molecules. Thus, the lost of drying protein is reduced (Chang & Pikal, 2009). One or more stabilizer is essential in drying process to achieve a long shelf life. (Meister & Gieseler, 2008).

Sugar/ polyols

Among all type of sugar and polyols, disaccharide give the best protection in lyophilisation.

Sucrose ad trehalose are the most common protectant used in stabilizing protein. However, an essential quantity must be added. In some suggestion the ratio of a molar stabilizer / protein ranges from 300/1 to 500/1 (Meister & Gieseler, 2008).


In the testing of the stability of lyophilized recombinant human serum albumin by Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis, protein aggregations are found in some mannitol formulation, but the aggregation decrease in the formulation with mannitol and sucrose formulation. (Han et al. 2007)


Trehalose is a good lyoprotectant. In protecting phosphofructokinase activity, there is more than 60% recovery at 400mM trehalose, while 5% recovery in glucose in the same concentration (Carpenter et al. 1993).


Polymers, protein subject, non-aqueous solvents, surfactants, amino acids and miscellaneous excipients can also act as a protective compound (Wang, 2000).

Types of cryoprotectants


0.025M and 0.050M sucrose is added before cooling of frozen bull sperm, the 67% and 66% of the sperm damaged, there is 1-2 % lower than that of the control with 0.05 confidence level. (Chen et al. 2009)


Trehalose shows it ability in protecting L. acidophilus. in freeze- thaw cycle. 94% of the frozen cells survive in 20-30% trehalose after 12 successive freeze thaw. Comparing to its control experiment, 99% of CFU is reduced (Doung et al. 2006).

The protecting effect of trehalose also depends on the concentration. In protecting frozen ram semem, trehalose at 50mOsm has the same protecting effect as 100mOsm. However, when trehalose has a concentration excess 200mOsm, the frozen ram has a higher morbidity than that of the control sample (Aisen et al. 2002).

Freeze-thaw damage and the need for cryoprotectants

Activities of some proteins, such as Lactate dehydrogenase, are reduced in free- thawing cycle. Since the protecting mechanism in the lyophilisation is different from that in the freeze- thawing cycle, a good lyprotectant does not necessary mean that it is also a good protectant in free- thawing cycle of protein (Wang, 2000).

Lyophilisation of linamarase enzyme

Although partially-purified linamarase enzyme is commercially available, it is very expensive and therefore unobtainable for developing-country scientists. There is no evidence in the scientific literature of any work regarding protective compounds for the lyophilisation of this enzyme.


In conclusion, the fact in the litterateur has shown the importance of cassava in developing countries. It is one of the main carbohydrate sources for them and the negative impact to the health of consumers, from chronic disease to death. Also, the process of developing and diminishing the toxic effect in cassava root is clearly stated.

Secondly, the significant of preserving linamarase activity in testing the cyanogenic potential in cassava root is demonstrated. It is a main factor to determine the quality of the test.

In addition, the application of lyophilisation in preserving linamarase is shown. However, it has limitation. Stresses are produced on the linamarase in the process. Also, the freeze- thaws also produce stress on rehydrated lyophilised linamarase.

Finally, the ability of lyoprotectant and cryotectant compound in protecting the structure of product, including protein, in lyophilisation and freeze- thaw respectively is recognised.

However, the number of researches in applying lyoprotectant and cryotectant in the lyophilisation of linamarase are limited and more research is needed.