Literary Review Introduction

Literary Review
Introduction:
This project will focus mainly on the enzyme pectinase and its biochemical characteristics. The biochemical characteristics will be tested and conditions will vary to determine the optimum condition for the enzyme pectinase. This enzyme is plays a major role in the fruit and vegetable processing industry. They are natural and eco-friendly biological enzymes. They allow companies to develop practices and increase the yield of their products in a safe and productive way.
Pectinase is a complex enzyme consisting of pectolyase, pectozyme and polygalacturonase activities. Pectinases are a large group of enzymes that break down pectic substances into simpler molecules. 1
The commercial pectinase being used is Novozyme Pectinex. It is produced from a selected strain of Aspergillus niger. It consists of a mixture of enzymes which are pectintranseliminase, polygalacturonase and pectinesterase. 2
This commercial enzyme will undergo various tests to determine how varying different characteristics may alter the enzyme activity. The optimum conditions for pectinase will be determined. This will provide key information for producing the maximum amount of product for the quantity of enzyme provided.
Pectinase: Background
In 1886, Debary put forward the important role of pectinases in the decomposition of plant cell wall pectin . Pectinase is a complex enzyme consisting of pectolyase, pectozyme and polygalacturonase activities. Pectinases are a large group of enzymes that break down pectic substances into simpler molecules. Pectinases belong to the hydrolase family of enzymes. These enzymes break down the carbohydrate pectin. Pectin is a polysaccharide found in plant cell walls and is a jelly-like glue that holds plant cell walls together. 1.

Pectinases were used as food processing aids at domestic level and their first use in the industrial sector was observed in the year 1930. Pectinases have also been used in wine production since the 1960’s.
For this enzyme to work best, it requires optimum temperature and ph. A commercial pectinase is typically activated between 45? and 55? and works best between ph 3.0 to ph 6.0.

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40690806267450Figure SEQ Figure * ARABIC 1- Chemical structure of Pectinase
Figure SEQ Figure * ARABIC 1- Chemical structure of Pectinase

Classification of Pectinase:
Pectinase can be divided into four different groups;
Protopectinase.

Polygalacturonase.

Pectin Lyase.

Pectinesterase.

Protopectinase:
This enzyme encourages the degradation of pectin. It is identified as a catalytic mechanism and is divided into 2 groups which are known as Type A pectinase and Type B pectinase. Type A mainly acts on the inner polygalacturonic acid region of protopectin. Type B predominantly acts on the outer polysaccharide chains of the galacturonic acid chains and cell wall components.

Polygalacturonase:
Polygalacturonase is a pectinase which demonstrates the hydrolysis of galacturonic acid chain in an underwater environment. According to the different mechanisms of hydrolysis, it can be divided into two types:
Fungal exogalacturonase results in the final product as a monomeric galacturonic acid.

Bacterial exogalacturonase, the resulting product is a dimeric galacturonic acid.
Pectin Lyase:
This type of pectinase separates pectic polymers by action of trans-elimination. The pectin lyase enzymes breaks the glyosidic bond at the C-4 position and eliminates the H atom from the C-5 position. This results in the product of this reaction becoming unsaturated. Pectin lyase can be divided further groups due to the different substrates present and catalytic mechanism. These sub-groups are endogalacturonase, exogalacturonase, endopolymethylgalacturonase and exopolymethylgalacturonase.

Pectinesterase:
Pectinesterase can remove the methoxy group and promote the demethylation of pectin. Pectinesterase catalyses the de-esterification of methyl-esterified D-galactosiduronic acid yielding substrates for depolymerising enzymes, particularly acidic pectins and methanol. 4
Pectinase: Mode of Action

27031953697605Figure SEQ Figure * ARABIC 2- Pectinase: Mode of action
Figure 2- Pectinase: Mode of action
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Pectinase are identified as a heterogenous group of enzymes.
They are classified into three groups Polygalcturonase (PG), Pectinesterase (PE), and pectin lyase (PL) based on their mode of action on the substrate.
The polygalacturonase mode of action is based on hydrolysis.

The pectinesterase mode of action is based on trans-elimination.
The pectin lyase reaction is based on a de-esterification reaction.
There are two main enzymes which hydrolyse glyosidic linkages.

Polygalacturonase is a pectin depolymerising enzyme which acts on the ? 1?4 glycosidic bond of the main chain of polygalacturonic acid. This results in chain being hydrolysed by the production of a water molecule.

Polymethylgalacturonases (PMG) are enzymes which catalyse the hydrolytic cleavage of ? (1?4) glycosidic bonds.

Pectinesterases also known as pectin methyl hydrolase are involved in the de-esterification reaction. These enzymes catalyse de-esterification of the methoxyl group of pectin and yields pectic acid. 5
Substrate: What is Pectin?
4614545124777500Pectin is polysaccharide found in plant cell walls and is a jelly like glue that holds plant cell walls together. It is considered a structural carbohydrate. This substrate is usually located in the primary cell wall and the middle lamella of plant tissues. Pectins can vary in chain length and complexity. The characteristic structure of pectin is a linear chain of alpha (1-4) linked D- galacturonic acid that forms the pectin backbone. This pectin back-bone is known as homogalacturonan.

4684883163579Figure SEQ Figure * ARABIC 3- Chemical Structure of Pectin.

00Figure 3- Chemical Structure of Pectin.

This type of D-galacturonic is a reducing sugar and is present in polymerised forms in Pectin. These acid residues in pectin become esterified which causes the formation of methyl esters.

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Figure SEQ Figure * ARABIC 4- Pectin in powder form.

Commercially Pectin is categorised according to their methoxy content and its ability to form gels quickly or slowly.
Pectin can be split in to high methoxy pectins which are more than 50% esterified and low methoxy pectins which are less than 50% esterified.
Fruits High in Pectin Content Fruits Low in Pectin Content
Apples Blueberries
Oranges Raspberries
Grapes Strawberries
Cranberries Cherries
Plums Peaches
Blackberries Pears
Currants Rhubarb
Gooseberries *Citrus peels also have a high pectin content* Health Benefits of Pectinase.

Pectinase is a useful digestive aid because pectin is a significant component in the human diet. Pectin is used as a thickener and gelling agent in many prepared foods such as jellies and jams. Additional enzymes that are useful for breaking down plant-based foods include cellulase and hemicellulose. These enzymes work together to help to increase the nutritional value of these foods.

Pectinase mainly benefits the digestive system, but it also contributes to supporting intestinal bacteria, colon function as well as seasonal conditions.
Digestive Support:
It increases the bioavailability of sugar in food.

Colon Function:
Provides energy required for proper function of colon. Pectin is broken down and produces fatty acids that are essential to maintain a healthy level of intestinal activity.
Bacteria Balance:
Helps support a healthy level of bacteria in intestines and maintains optimum pH for growth. Bacteria are essential for proper digestion of food.
Seasonal Conditions:
Pectinase reduces intestinal absorption of certain materials in food that may carry harmful bacteria due to seasonal conditions. 6
.5Pectinase Applications
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Pectinase has mainly applications in Industry. Microbial pectinases account for 25% of the global food and industrial enzyme sales. They are extensively used in juice extraction, waste water treatment, fruit juice clarification, refinement of vegetables and as an analytical tool in assessment of plant products.
Pectinase in Fruit Juice Industry:
Fruit juice extraction is carried out by using mechanical grinding of fruits that result in jelly like products. Due to complex pectin structure of fruits, it is difficult to extract all the juice present in this highly viscous jellified pulp. Pectinase acts on the substrate pectin and breaks the glycosidic bonds present between the galacturonic acid monomers. This action decreases the water holding capacity of pectin. And allows for a higher yield of juice to be obtained.
Studies have been carried out and published stating that the juice yield is significant increased when fruit is treated with pectinase enzyme.

Different fruit juices need different types of enzyme treatment depending on their pectin concentration and the consumer preferences. To obtain the desired product, process parameters are needed to be optimised for example like enzyme concentration, enzyme treatment duration, incubation temperature and pH of solution.

As seen in Table 1 previously, fruits such as apples, grapes and berries require an intense pectinase treatment. 5
Pectinase in Oil Extraction Industry:
Oils from rape seed (Canola), coconut germ, sun- flower seed, palm, kernel and olives are traditionally produced by extraction with organic solvents. The most commonly used organic solvent is Hexane.
Pectinase is used in the extraction of vegetable oil in an aqueous process by liquefying the cell wall component of the oil containing plant. Other methods include, addition of pectinase enzyme during mechanical grinding process of olives. This allows oil to be released easily in the following separation techniques.
Enzyme treatment hence causes an increase in yield of olive oil. 7
Pectinase in Coffee and Tea fermentation Industry
Pectinase plays a significant role in the coffee and tea fermentation industry.
Fermentation of coffee using pectinase enzyme is done to remove the mucilage coat from the coffee beans. Pectic enzymes are sometimes added to remove the pulp layer of the bean which contains pectin. Cellulases are also present in the enzyme preparation which aid the digestion of the mucilage. A diluted commercial enzyme preparation is sprayed on to the beans at a dose of 2±10 g per ton at 15±20°C. The fermentation stage of coffee processing is accelerated and reduced to about 20 h by enzyme treatment. Largescale treatment of coffee with commercial pectinases is costly and not economical, inoculated waste mucilage is used as a source of microbial pectic enzymes.

Fungal pectinases are a used in the manufacturing of tea. Enzyme treatment accelerates tea fermentation, but the enzyme dosage must be altered carefully to avoid damage to the tea leaf. The addition of pectinase improves the foam-forming property of instant tea powders by destroying pectins in tea. 7
Pectinase in Textile Industry:
Pectinase has been introduced to the textile industry as an eco-friendly way of carrying out the scouring process. Bio-scouring is an eco-friendly method for removal of non-cellulosic impurities from the fibres with specific enzymes. It reduces the risk of severe pollution and doesn’t not require high energy.
This process makes the surface of the textile fibre more hydrophilic as well as preventing fibre damage.
Alkaline pectinases have been considered the most suitable enzyme for cotton scouring because the degradation and elimination of pectin facilitates the removal of loosened waxes. Different enzymes are used depending on the fabric being treated and the results desired. 8
Pectinase in Wine Industry:
The main purpose of using pectinase enzymes in the wine making industry is to support the extraction process and maximise juice yield. It also contributes to the filtration process and flavour and colour.
Wines treated with these enzymes proved to be more stable and had a reduced filtration time in comparison untreated wines. Treatment of macerated fruits with enzymes before the addition of inoculum resulted in improved characteristics of wine.
Pectinase treatment increased the yield of juice when it was treated 0.6 % enzyme concentration and fermentation was conducted at 30 °C and pH 4.5 for a 12 hour period.
The addition of pectinase enzymes in the wine making process increases the levels of methanol in wine due to the activity of Pectin Esterase. Methanol is toxic and its maximum concentration in wine should be regulated meaning pectin esterase should be at low concentrations in commercial mixtures. 8
Pectinase in Animal Feed.

The use of pectinases in production of ruminant feed decreases the feed viscosity and increases the absorption of nutrients by ruminants.

It releases nutrients by enzymatic action which reduces the amount of faeces. The specific enzyme preparations have become a valuable tool for economically improving the digestive processes in the ruminants. Ruminants diet was supplemented by cocktail of enzymes containing xylanases, pectinases and cellulases. These enzymes increase the digestibility of organic matter and improves animal performance by contributing to ruminal fibre digestion which allows increased digestible energy intake.

The animal feed is treated by spraying feed with pectinase enzyme before feeding. This process improves digestibility of feed, improvements in palatability and changes in gut viscosity. 8
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Factors that Affect Enzyme Activity
Temperature
PhEnzyme concentration
Substrate Concentration

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746199/
References
Pedrolli DB, Monteiro AC, Gomes E, Carmona EC. Pectin and pectinases: production, characterization and industrial application of microbial pectinolytic enzymes. Open Biotechnology Journal. 2009 Jul 21:9-18.

Reading, U. o., 2018. Pectinase. Online Available at: http://www.ncbe.reading.ac.uk/materials/enzymes/pectinex.html.

Amin F, Bhatti HN, Bilal M. Recent advances in the production strategies of microbial pectinases—A review. International journal of biological macromolecules. 2018 Sep 11.

Creative Enzymes, Pectinase, Date Accessed: 09/11/2018
Available at: https://www.creative-enzymes.com/similar/Pectinase_533.htmlHeena V, Lokesh K N, Jyoti S J. Pectinase: A Useful Tool in Fruit Processing Industries. Nutri Food Sci Int J. 2018; 5(5): 555673.

Xtendlife. Health benefits and Uses of Pectinase. No date.

Available at: https://www.xtend-life.com/blogs/supplement-ingredients/pectinaseKashyap DR, Vohra PK, Chopra S, Tewari R. Applications of pectinases in the commercial sector: a review. Bioresource technology. 2001 May 1;77(3):215-27.

Garg G, Singh A, Kaur A, Singh R, Kaur J, Mahajan R. Microbial pectinases: an eco-friendly tool of nature for industries. 3 Biotech. 2016 Jun 1;6(1):47.