Production of Commercially Suitable

LIST OF FIGURES

Figure 2.1.	The important steps of enzyme production	4
Figure 2.2.	Phase diagram for water	8
Figure 2.3.	Typical profile for ammonium sulphate precipitation	10
Figure 3.1.	Deesterification of pectin by PME	16
Figure 4.1.	The reactions of polyphenol oxidases	25
Figure 4.2.	The nonenzymatic reactions during formation of dark colored melanins	25
Figure 4.3.	Some of the good substrates of PPO in plants	26
Figure 5.1.	Standart curves for protein determination	42
Figure 6.1.	Change of PME activity in orange peels during season	43
Figure 6.2.	Effect of NaCl concentration on PME activity extracted from peels	46
Figure 6.3.	Effect of extraction period on PME activity extracted from peels	46
Figure 6.4.	Effect of heating for 30 min at different temperatures on PME activity	47
Figure 6.5.	Effect of heating at 50 ^oC on PME activity	48
Figure 6.6.	Effect of different CaCl2 concentrations on PME activity	49
Figure 6.7.	Stability of the prepared PME during storage at + 4 ^oC in liquid form	50
Figure 6.8.	The effect of prepared orange peel PME on film forming ability of pectin	51
Figure 6.9.	Monophenolase activity of PPO from mushroom stems	52
Figure 6.10.	Diphenolase activity of PPO from mushroom stems	53
Figure 6.11.	Temperature profiles of monophenolase activity of PPO partially purified with different procedures from different acetone powders	57
Figure 6.12.	The effect of pH on monophenolase activity of PPO partially purified with different procedures from different acetone powders	58
Figure 6.13.	pH stabilities of monophenolase activity of PPO partially purified with different procedures from different acetone powders	59
Figure 6.14.	Optimum temperature for monophenolase activity of PPO	60
Figure 6.15.	The stability of monophenolase activity of PPO in acetone Powders	60
Figure 6.16.	The stability of monophenolase activity of PPO in different lyophilized forms stored at -18 ^oC	62
Figure 6.17.	The stability of diphenolase activity of PPO in different lyophilized forms stored at -18 ^oC	62
Figure 6.18.	The effect of lyophilization with dextran on heat stability of monophenolase activity of PPO	63
Figure 6.19.	The effect of lyophilization with dextran on pH stability of monophenolase activity of PPO	64
Figure 6.20.	The phloridzin oxidation of PPO lyophilized with dextran	65

LIST OF TABLES

Table 2.1.	Different methods for cell disruption	5
Table 2.2.	The main chromatographic techniques and their separation principles	14
Table 3.1.	Some properties of PMEs from different fruits and vegetables	18
Table 4.1.	Some properties of PPOs from different fruits and vegetables	28
Table 6.1.	The PME activities obtained by applying different extraction procedures	44
Table 6.2.	Distribution of PPO monophenolase and diphenolase activities in mushrooms	54
Table 6.3.	Results of partial purification with ammonium sulfate and acetone precipitation of PPO monophenolase activity from mushroom stems	55

CHAPTER 1
INTRODUCTION
Recently, extensive studies have been conducted related to the use of plant enzymes in food industry. For example, Ridgway et al (1997) extracted and used apple polyphenol oxidase (PPO) successfully for biosynthesis of antioxidant compounds 3-hydroxyphloridzin and 3-hydroxyphloretin from phloridzin and yellow/orange colored dimerized oxidation products of phloridzin that may be used as food colorants. Ridgway and Tucker (1999) also developed a procedure for the partial purification of commercially suitable PPO from apple leaf. However, since PPO in apples can not oxidize the p-diphenols (laccase activity) and lacks monophenolase activity it may only be used for some specific applications.

The monophenolase activity of PPO is essential for many different applications such as the production of plant pigments such as red-violet betalains and gold colored aurones (Strack and Schiliemann 2001) and biosynthesis of antioxidant compounds such as hydroxytyrosol (Espin et al. 2001). Thus, with its hydroxylation and high oxidation capacity PPO in mushrooms is quite suitable for many food applications including fermentation of cocoa and tea (Ridgway et al. 1997, Selamat et al. 2002), removal of undesirable odors caused by volatile sulfur compounds (Negishi et al. 2001, Negishi et al. 2002) and enzymatic cross-linking of proteins (Thalmann and Lötzbeyer, 2002). The ability of mushroom PPO to oxidize sinapic acid was also reported by Choi and Sapers (1994). Thus, as done by Lacki and Duvnjak (1998) with Trametes versicolor PPO, removal of sinapic acid and increase of the nutritional values of canola meal and canola protein concentrates may be conducted by mushroom PPO. One of the most interesting features of mushroom PPO is its ability to inhibit the attachment of some bacteria. For example, Cowan et al (2000) demonstrated that by oxidizing the critical tyrosine residues of glucan binding lectin and glucosyltransferases, PPO prevents the attachment of Streptococcus sobrinus, bacteria responsible from the formation of oral cavities, to glucans deposited on the tooth surface. Kolganova et al (2002) also showed that the PPO reduces the adhesion of some viruses and pathogenic bacteria to buccal epithelial cells, while unaffecting the attachment of probiotic bacteria. These findings are quite interesting and may open the way of using PPO in foods such as gums and confectionaries to increase the tooth health of people. Out of food industry, mushroom PPO may also be used to remove undesirable phenols from wastewaters (Ikehata and Nicell 2000) and produce biosensors for the detection and quantification of phenolic compounds (Rubianes and Rivas, 2000, Climent et al. 2001). Several clinical applications such as using PPO as a catalyst to produce L-DOPA, a drug for the treatment of Parkinson`s disease (Sharma et al. 2003), a marker of vitiligo, an autoimmune disease and a tumor suppressing and prodrug therapy agent (Seo et al. 2003) also attracts considerable interest.

Another enzyme, attracting great attention in food processing is pectin methylesterase (PME). The fungal PME is now extensively used in beverage industry for fruit juice extraction and clarification (Alkorta et al. 1998, Bhat, 2000, Demir et al. 2001, Cemeroğlu and Karadeniz 2001, Kashyap et al. 2001, Sarıoğlu et al. 2001). The use of enzyme in the modification of pectin, oil extraction, firming of fruit and vegetables and peeling of fruits becomes also very popular (Ralet et al. 2001, Schmelter et al. 2002, Degraeve et al. 2003, Suutarinen et al. 2000, Suutarinen et al. 2002, Pszczola, 2001, Kashyap et al. 2001, Vierhuis et al. 2003, Pretel et al. 1997, Janser, 1996). Currently, the plant PME is not used in food industry. However, there are some successful experimental studies related to the use of commercial citrus PME for the firming of fruits and vegetables and modification of pectin (Suutarinen et al. 2000, Massiot et al. 1997).

In the production of commercial enzymes, microorganisms are the primary sources. However, 15 % of the enzyme production is still provided by extracting from animal or plant sources. In this study, the main objectives were; (1) to develop some simple and effective extraction and/or partial purification procedures for PPO and PME enzymes from agro-industrial wastes and (2) to process the extracted enzymes to commercially suitable preparations. The waste materials used in the study were mushroom stems and orange peels for the PPO and PME enzyme, respectively. These, materials are known as rich sources of the indicated enzymes (Moore and Flurkey 1989, Ratcliffe et al. 1994, Seo et al. 2003, Cemeroğlu et al. 2001, Nielsen and Christensen 2002, Johansson et al. 2002, Cameron et al. 1994, Cameron et al. 1998). The enzymes obtained from edible plant materials mostly need no toxicity tests for food applications. Also, because of the non-complex nature of the plant extracts these enzymes may easily be used as crude or partially purified preparations. Moreover, the use of waste materials in enzyme extraction may provide an extra income to factories and reduces the costs of waste material treatments.

CHAPTER 2
ENZYME PRODUCTION FOR INDUSTRIAL APPLICATIONS
2.1. Extraction of Enzymes
In the production of commercial enzymes, microorganisms are the primary sources. Currently, 50 % of the commercial enzymes are obtained from fungi and yeast and 35 % are obtained from bacteria. The remaining enzyme production, on the other hand, is conducted by extraction from plant or animal sources (Rolle, 1998). A good example for commercial animal origin enzymes is rennet, whereas papain may be an example for the plant origin enzymes (Gölker, 1990).

Following the production of enzymes by microbial fermentation or providing suitable plant or animal sources the first step to obtain industrial enzymes is extraction. In this process, the enzymes are extracted from a source or from a fermentation media and then in the second step the extracts are purified by further processing. According to the mode of application, the degree of purity may range from raw enzymes (sometimes relatively crude preparations in the form of plant executes, chopped fruits, leaves and pounded grains) to highly purified forms. The overall steps for the preparation of enzymes from different sources were given in Figure 2.1.

For the extraction of an enzyme from a plant material (root, stem, grain, nuclear sap, etc.), the material is first ground or minced with different crushers or grinders. At this step, processes such as peeling, removal of seeds, etc. may also be applied. After that, desired enzymes can be extracted with water and/or suitable buffer solutions. On the other hand, when animal organs are used in the extraction, they must be transported and stored at low temperatures for short times to retain the enzymatic activity. Most animal proteins are present in specific muscles or organs surrounded with a fatty layer that often interacts with subsequent purification steps. Thus, before freezing operation fats and connective tissues should be removed. The enzyme containing frozen organ or tissue can be cut, minced or homogenized with a blender, grinder or mill (for hard tissues) for producing a cell paste. Then, the enzyme is extract with an appropriate buffer solution.

Figure 2.1. The important steps of enzyme production (Gölker, 1990)

For the production of microbial enzymes, cultivation and then fermentation are applied after selection of a suitable microorganism. When enzyme is extracellular, there is no other process required at this step. However, if the enzyme is intracellular, for releasing the enzyme, different cell disruption techniques are applied (Gölker, 1990; Temiz, 1998).
2.1.1. Cell disruption methods
There are different methods for cell disruption based on the cell type, and the nature of the intracellular product. However, only a few of these methods are used in large scale production. These methods can be classified as mechanical and non-mechanical methods (Table 2.1).
2.1.1.1. Mechanical methods
Among the mechanical methods, high pressure homogenization is the most common one working well on laboratory scale. The principle of this method is based on the subjection of the cell suspension to high pressure by extruding through a valve to atmospheric pressure and disruption of the cells by shearing forces and simultaneous decompression.

On large scale processes, disruption with ball mills is used frequently. This method is based on breaking of cells with balls generally having a diameter of 0.2 to 1 mm. Ultrasonic vibrators are also sometimes used for the disruption of cell wall and membrane of bacterial cells (Gölker, 1990, Shuler and Kargi 2002).

Table 2.1. Different methods for cell disruption (Gölker, 1990).

Mechanical methods	Nonmechanical methods
High pressure homogenization	Drying (freeze-drying, organic solvents)
Grinding (ball mill)	Lysis (Physical: freezing, osmotic shock; Chemical: detergents, antibiotics; Enzymatic: lysozyme)
Ultrasound

2.1.1.2. Nonmechanical methods
Drying of microorganisms and preparation of acetone powders are applied to alter the cell wall and to permit subsequent extraction of the cell contents. To lyse the cell walls, different methods are applied by physical, chemical and enzymatic means. With osmotic shock or freezing, the cell wall and membrane can be disturbed to release the enzyme easily into the extraction media. Different detergents can also be used to dissociate proteins and lipoproteins from the cell membranes. Another alternative is the application of lytic enzymes such as lysozyme. However, this treatment is too expensive (Gölker, 1990, Salusbury, 1995, Shuler and Kargi 2002).
2.2. Clarification of Enzyme Extracts
During purification of enzymes, clarification is applied to remove particulate material (e.g. cells, organelles, debris or precipitated macromolecules) from the surrounding liquid (e.g. fermentation medium or buffer). The size, shape, and resistance to shear of the bulk should be taken into account for the selection of the most suitable clarification technique. Because of the small size of bacterial cells and slight difference between the density of fermentation medium and these cells, clarification is very difficult in microbial enzyme production. However, large cells such as yeast cells can be collected by decantation. In contrast, animal or plant residues can easily be separated with centrifugation or filtration (Gölker, 1990, Whittington, 1995).
2.2.1. Centrifugation
The principle of this method is based on the application of radial acceleration to a particle suspension by rotational motion. As a result, particles denser than the bulk fluid move outwards and separated. Generally, centrifugation is applied to separate particles that size is between 100 and 0.1 μm. There are different types of centrifuges such as tube centrifuges and continuous flow centrifuges used at the laboratory scale and plant scale, respectively.
2.2.2. Filtration
The filtration is defined as the separation of solid particles from liquid or gaseous streams. This is the most cost-effective method for the separation of large particles and cells from fermentation broth. There are different types of filters such as drum filter, rotary vacuum filter, ultrafilter, microfilter, and plate and frame filter. Continuous rotary vacuum filters are the most widely used types in the fermentation industry.
2.2.3. Flocculation and flotation
The principle of this method is based on the agglomeration of coagulated dispersed colloids in the medium with the addition of flocculating agents such as polyelectrolytes or CaCl₂. The agglomerates formed can be removed by filtration or centrifugation. However, when there are no stable agglomerates formed, particles can be removed by flotation. In this method, the particles adsorbed onto gas bubbles and accumulated at the top of the extract (Gölker, 1990).
2.3. Concentration of Enzymes
After clarification, the enzyme concentration of the obtained extract is often very low. Thus, a concentration step is generally required in order to aid subsequent purification steps. The major concentration procedures that do not inactivate enzymes are; (1) addition of a dry matrix polymer; (2) freeze-drying (lyophilization); (3) ultrafiltration; (4) precipitation, (5) aqueous two-phase partitioning, and (6) removal of salts and exchange of buffers
2.3.1. Addition of a dry matrix polymer
The principle of this method is based on the addition of a dry inert matrix into the protein solution. The pores of these matrixes are quite small. Thus, this allows the absorption of water and small molecules but not the proteins. To separate matrix, the extract was then centrifuged, filtered or settled by the gravity (Harris, 1995).
2.3.2. Freeze-drying (Lyophilization)
The principle of this method is based on the removal of water from a frozen solution by sublimation under vacuum. The phase diagram showing the sublimation of water was given in Figure 2.2.


Figure 2.2. Phase diagram for water
Freeze-drying can be applied both for concentration and preservation of proteins by preparing their dehydrated form as dry powder. There are different freeze-dryers, which may differ in their specifications according to the model and manufacturer. However, in general, the process occurs first by the removal of bulk water from the frozen protein solution by sublimation under vacuum (primary drying) and then the removal of remaining bound water from the protein by controlled heating (secondary drying). As a result, the protein was prepared as a dry powder with a moisture level lower than 1 %. Freeze-drying is a time consuming process and require high capital and running costs. However, it is very suitable for high value biomolecules like enzymes (Fagain, 1996, Shuler and Kargi 2002).
2.3.3. Ultrafiltration
The principle of this method is based on the transport of a protein solution through a filter medium or membrane in the ultrafiltrate and retention of the solute behind the membrane in the retentate. The driving force for the separation is the pressure difference across the membrane. The diameters of the membrane pores range between 1 and 20 nm that may separate proteins in the range of 5.000-500.000 Daltons.

2.3.4. Precipitation
Enzymes are very complex protein molecules having both hydrophobic and hydrophilic groups. The solubility of a protein molecule in an aqueous solvent is determined by the distribution of these groups onto its surface. Protein precipitates may be formed due to aggregation of protein molecules by adjusting the system temperature, pH, ionic strength and dielectric constant. Currently, precipitation is usually used as a crude separation and partial purification step. Also, it can be used for concentrating proteins prior to analysis or a subsequent purification step. After the precipitation, the precipitate is collected by filtration or centrifugation, dissolved in a suitable buffer or water, desalted if necessary and used in the subsequent purification steps (Harris, 1995).
2.3.4.1. Precipitation by increasing the ionic strength (salting out)
This method is related with the hydrophobic nature of the protein surface. The solubility of proteins in an aqueous solvent is determined by distribution of hydrophobic and hydrophilic groups on their surfaces. In proteins, most of the hydrophobic groups exist in the interior parts whereas some exist at the surface, often in patches. In solution, the surface of proteins is surrounded by a water jacket that prevents the protein-protein interactions by shielding the hydrophobic areas. After the addition of high concentrations of salts to a protein solution, the added ions interacted with water strongly, remove water surrounding the protein and expose hydrophobic patches. In such a case, the contact of different proteins’ hydrophobic groups causes aggregation. The aggregates formed are collected by centrifugation and redissolved in fresh buffer or water. In practice, this is the most commonly used method for fractionation of proteins.

The optimum salt concentration required to obtain the desired precipitation changes according to the properties of protein, type of salt used, and the method of contact. Experimentally, the optimum salt concentration is determined by finding the concentration of total protein and concentration and/or activity of target protein at different salt concentrations. Figure 2.3 shows the typical profile of precipitation with ammonium sulphate, the most commonly used salt for protein precipitation. The ammonium sulphate precipitation may be applied simply by bringing the salt concentration to optimum precipitation conditions of the target protein. Also, before the precipitation of the target protein, the solution may first be brought to a percent saturation that precipitates only the proteins other than the target protein. In such a two step precipitation process, a higher purity is obtained for the target protein.

Figure 2.3. Typical profile for ammonium sulphate precipitation
2.3.4.2. Precipitation by decreasing the ionic strength (salting in)
The principle of this method is based on the reduction of ionic strength in the medium. In this case, ionic interactions between protein molecules increase leading to aggregation and precipitation. However, since low ionic strengths can only be achieved by addition of water, the application of this method decreases the concentration. This form of precipitation may occur at later stages of purification (when removing salts by gel filtration, dialysis, or diafiltration) (Harris, 2001).
2.3.4.3. Precipitation by organic solvents
This type of precipitation is based on decreasing the solubility of proteins, by reducing the dielectric constant (solvating power) of the protein solution. A reduction in dielectric constant causes the formation of stronger electrostatic forces between the protein molecules and enhances protein-protein interactions that resulted with agglomeration and subsequent precipitation.

Acetone and ethanol are the most commonly used solvents. Generally, an equal volume of acetone but four volumes of ethanol is used for precipitation. Thus, because of the little amounts needed for the precipitation acetone is more preferable. Optimum organic solvent concentration can be determined experimentally with a similar method like in salting-out. Precipitation by organic solvents should be applied at or below 0 ^oC. At higher temperatures, the protein may change its conformation, enabling the solvent to access the protein interior and disturb the hydrophobic interactions at these locations. After collecting the pellet by centrifugation, the remaining solvent can be removed by dialysis, gel-filtration or evaporation at reduced pressure.

2.3.4.4. Precipitation by alteration of pH
This method is based on adjusting the pH of the extract around the isoelectric point (pI) of the target protein. At this pH, the negative charges of one protein molecule attract the positive charges of the other. Thus, as occurs in the precipitation by solvents, electrostatic attraction causes the aggregation. The protein aggregates may be collected by centrifugation and redissolved in suitable buffers or water.
2.3.4.5. Precipitation by organic polymers
The mechanism of this method is similar to the method of precipitation with organic solvents but this one requires lower concentrations. The most widely used organic polymer for this kind of precipitation is polyethylene glycol (PEG).
2.3.4.6. Precipitation by denaturation
Precipitation by this method can be applied if the contaminating proteins, but not the target protein, are denatured by changes in temperature and pH, or addition of organic solvents. During denaturation, the tertiary structure of proteins is disrupted and the random coil structures formed cause aggregation. Aggregate formation is highly influenced by the pH and ionic strength. Close to the pI of the protein and at lower ionic strength the aggregation accelerates considerably.

2.3.5. Aqueous two-phase partitioning
The principle of this method is based on the extraction of soluble proteins between two aqueous phases containing incompatible polymers, or a polymer and a high ionic strength salt. Due to the incompatibility, when the polymers are mixed, large aggregates formed tend to separate due to steric exclusion. Most soluble and particulate matter partition to the lower, more polar phase, whilst proteins partition to the upper, less polar phase. After that the separation of phases can be performed by decantation or centrifugation.

Separation of proteins among each other can be achieved by manipulating the partition coefficient by altering the average molecular weight of polymers, the type of ions in the system, ionic strength, or presence of hydrophobic groups. In this method, by application of sequential partitioning steps or alternatively by mixing of polymers to yield more than two phases, a higher degree of purification can be achieved (Andrews and Asenjo 1995, Shuler and Kargi 2002).

2.3.6. Removal of salts and exchange of buffers
During purification of proteins, it is necessary to change the buffer of extract or to remove salts from the extract. For this purpose, different methods can be applied such as dialysis, diafiltration, and gel filtration.
2.3.6.1. Dialysis
Dialysis is a membrane separation technique. In this method, the protein solution is placed in a special semi-permeable membrane bag usually made from cellulose and the bag is placed in a selected solvent. Small molecules can pass freely across the membrane until their concentration is same on the both sides of the membrane whilst large molecules are retained. The driving force for diffusion of the salts across the membrane is the relative concentration of the salts in the two solutions. The solvent, which is normally water or buffer, is replaced with a fresh one at regular intervals. During dialysis, the medium should be stirred continuously.

2.3.6.2. Diafiltration
In diafiltration, ultrafiltration membranes may be used to separate small molecules like salts, sugars, or alcohols from the protein solutions. The driving force for the filtration is pressure and the protein of interest is retained on the filter. The process, applied by the addition of water or buffer to the protein solution, is continued until the ionic strength of the filtrate reaches to that of the added water or buffer.
2.3.6.3. Gel filtration
This method is a molecular sieving process that can be used to fractionate molecules according to size. The sieving medium is a gel, which has pores of a fixed diameter, smaller than the protein molecules. Thus, when protein solutions are applied to a column, filled with this gel, small molecular weight substances such as salts are retained by the gel particles while protein molecules are excluded. The elution of column with a buffer or water is enough to elude and collect the salt free proteins from the column.
2.4. Purification
Partial purification is sufficient for many industrial applications. However, for analytical purposes and medical use enzymes must be highly purified. The most widely used technique for this purpose is chromatography. The general principle of this method is based on differential separation of sample components between a mobile phase (passing fluid mixture) and a stationary phase (a bed of adsorbent material). The four mainly used chromatographic techniques are ion exchange, affinity, gel filtration and hydrophobic interaction chromatography. The type of the chromatographic technique to be used depends on the properties of the protein and the aim(s) of the purification process (Table 2.2).

Hydrophobic chromatography is applied when the aqueous phase is at high ionic strength. Gel-filtration chromatography can be used for purification, buffer exchange, desalting or molecular weight determination.

Table 2.2. The main chromatographic techniques and their separation principles

Chromatographic technique	Properties of protein affecting separation
Ion-exchange	Charge
Affinity	Specific binding
Gel-filtration	Molecular size
Hydrophobic interaction	Exposed hydrophobic groups at the surface

For a complete purification, multiple types of chromatographic techniques are applied following to each other. Among these techniques ion-exchange and affinity chromatography are the most widely applied methods for purification of proteins from bioprocesses.

2.5. Product Formulation
The storage time of enzymes may vary from a few days to more than one years depending on the nature of enzyme and storage conditions. Different factors like proteolysis, aggregation, and certain chemical reactions cause loss or deterioration of enzymes’ biological activities. Considering their stability during transport and storage, the produced enzymes are formulated in different ways.

In liquid enzyme formulations, to prevent the microbial contamination, different antimicrobials can be added to enzyme extract. Alternatively, to avoid the addition of antimicrobials, enzyme solutions can be filtered through a 0.22 m filter that excludes all bacteria.

To stabilize the activity of enzymes, the addition of low molecular weight substances like glycerol or sucrose can be used frequently. Salts like ammonium sulphate can also stabilize proteins in solution by forcing the protein molecules to adopt a tightly packed, compact structure by salting out hydrophobic residues.

In some cases, enzymes only can be stored by storage in refrigerator at 4-6 ^oC in 50 % glycerol or as slurries in approximately 3 M ammonium sulphate. However, the proteins that loose their activity at refrigerator temperatures require lower temperatures such as –18 or –20 ^oC.

Application of drying is another alternative for stable storage of enzymes. The drying conditions of the enzyme are selected according to the heat sensitivity, physical properties and desired final moisture content of the product. For example, freeze-drying can be used for drying of heat sensitive enzymes. Other drying alternatives for heat stable enzymes are vacuum tray dryers, rotary vacuum dryers, spray dryers, and pneumatic conveyor driers (Fagain, 1996, Gölker, 1990, Shuler and Kargi 2002 ).

Another application for enzyme stabilization is the immobilization of enzyme on a solid carrier. Sharma et al (2003) studied the storage stability of immobilized tyrosinase by two different immobilization methods and found that in any case the storage stability of immobilized enzyme derivatives is higher than the storage stability of soluble enzymes.

CHAPTER 3
PECTIN METHYLESTERASE
3.1. Pectin methylesterase and Other Pectinases
Pectin methylesterase (PME, E.C. 3.1.1.11) is a member of pectinases, a group of enzymes that use pectic substances as substrate. According to their action on pectic substances, pectinases are classified as deesterifying and depolymerizing enzymes (Alkorta et al. 1998, Cemeroğlu et al. 2001). PME catalyzes the deesterification of pectin and produces liberated carboxyl groups and methanol as product (Figure 3.1). Other members of pectinases such as polygalacturonase (PG) and pectate lyase (PL) are depolymerization enzymes acting on different forms of pectin.

Figure 3.1. Deesterification of pectin by PME (Micheli,2001)

Pectin, is the principal polysaccharide of the middle lamella and it forms approximately 30 % of polysaccharides constituting the cell wall in higher plants. Pectins contain a backbone of smooth homogalacturonan regions, (14) linked α-D-galacturonic acid units that may be methyl esterified at C 6 position, and hairy rhamnogalacturonic regions that interrupt the galacturonic acid residues with (12) linked α-L-rhamnopyranosyl residues carrying neutral sugar side chains (Ralet et al. 2001, Johansson et al. 2002, Wicker et al. 2003, Giovane et al., 2004).
3.2. Sources of PME
PME exists in all higher plants, but it is particularly abundant in citrus fruits (Johansson et al. 2002). The enzyme is also produced by some bacteria and fungi that are pathogenic to plants (Johansson et al. 2002, Giovane et al. 2004). In plants, by working in coordination with other pectinases such as PL and PG, PME prepares and modifies the pectin. Also, it was reported that the enzyme plays some important roles in cellular adhesion, stem elongation, development, dormancy breakage, fruit ripening, seed and pollen germination, pH regulation, and defense mechanisms of plants against pathogens (Pimenta-Braz et al. 1998, Micheli, 2001, Johansson et al. 2002).

PME is a cell wall bound enzyme that two or more isoforms were detected in higher plants differing in molecular weight, biochemical activity and physical and chemical characteristics (Cameron et al. 1998, Corredig et al. 2000). The action pattern of the PME on pectic substances and the kinetic properties of the enzyme are highly affected from the source. For example, plant PMEs and bacterial PMEs from Erwinia chrysanthemi generally remove methyl ester groups of pectin linearly (single chain mechanism). In this action pattern, the binding of enzyme is followed by conversion of all subsequent substrate sites on the polymer chain. In contrast, the fungal PMEs (from Aspergillus niger) act on pectin with the multiple chain mechanism. In this type of action pattern, for each enzyme-substrate complex formation the enzyme catalysis the limited number of substrate sites randomly. It was reported that the random deesterification is conducted by acidic PMEs whereas linear deesterification is conducted by alkaline PMEs (Denes et al., 2000). Recently, it has also been reported that the mode of deesterification depends also on the initial degree of methylesterification of pectins and pH of medium (Micheli, 2001).

3.3. The Effects of PME on Food Quality
As indicated above, pectin acts as a cement material among the plant cells and plays an important role for the texture of fruits and vegetables. In unripe fruits, the pectin is insoluble and bound to cellulose microfibrils in the cell walls. During ripening, processing and storage, naturally occurring PME catalyzes the specific demethylation of pectin at C6 position of galacturonic acid units. The demethylized pectin is a good substrate for PG, and other depolymerization enzymes that degrade pectin and cause the loosening of cell walls. This kind of softening, mediated by PME, is a great problem in fruit and vegetable processing industry (particularly in freezing and canning industry). Thus, PME from different fruits and vegetables such as sweet cherry (Alonso et al. 1996); carrot (Ly-Nguyen et al. 2002); acerola fruit (Aparecida de Assis et al. 2002);
Table 3.1. Some properties of PMEs from different fruits and vegetables

Source	Optimum pH	Optimum temperature (^oC)	Temperature stability^a	References
Sweet cherry (Isoforms, I1 to I4)	I1 : 7.0 I2 : - I3 : < 5.2 I4 : 5.8	-	I1 and I3 are thermolabile I2 and I4 are thermostable 100 % (1 min , 60-70 ^oC)	Alonso et al (1996)
Carrot	7.3-7.4	48.5	50 % (10 min, 55 ^oC) 5 % (10 min, 60 ^oC)	Ly-Nguyen et al (2002)
Acerola fruit	9.0	-	125.5 % (90 min, 98 ^oC)	Aparecida de Assis et al (2002)
Strawberry	6.9-7.0	59-60	-	Ly-Nguyen et al (2002)
Mandarin	9.0	-	20 % (1 min, 70 ^oC)	Rillo et al (1992)
Navelina Orange	7.0	50	-	Christensen et al (1998)
Valencia orange peel	9.0 (6.0 at 1.2 % NaCl)	-	-	Savary et al (2002)
Commercial orange PME	9.0 (6.0 at 1.2 % NaCl)	55-60	50 % (5 min, 55 ^oC) inactivated at 65 ^oC	Savary et al (2002)
Green bean pod isoforms (P1 and P2)	P1: 6.5-9 P2: 7-9	-	P1: inactivated (4 min, 80 ^oC) P2: inactivated (8 min, 60 ^oC)	Laats et al (1997)
Apple	6.5-7.5	-	100 % (1 min, 40 ^oC) inactivated (1 min, 90 ^oC)	Castaldo et al (1989)

^a % retained activity after the indicated heating

strawberry (Ly-Nguyen et al. 2002); mandarin (Rillo et al. 1992); Navelina Orange (Christensen et al. 1998); Valencia orange peel (Savary et al. 2002); green beans (Laats

et al. 1997); apple (Castaldo et al. 1989) has been extracted, purified and characterized (Table 3.1).

Because of its close relation with the cloud loss of fruit juices, the enzyme PME is also attracts a great attention in fruit juice industry. Particularly in citrus juices, that cloud stability is an important quality criteria, the inactivation of enzyme by heat treatment is essential (Giovane et al. 2004). If the enzyme can not be inactivated immediately after fruit juice extraction, it demethylates the pectin rapidly. The low methoxy pectin cross-links readily with divalent ions such as Ca⁺⁺ and Mg⁺⁺ and forms some insoluble pectates. This induces the loss of cloud stability by precipitating pulp-particles and separating fruit juice serum. Such products are not attractive for consumers. Also, during concentration of such fruit juices, the gelation occurred causes problems during reconstitution, pumping and blending operations (Cameron et al. 1998).

The PME in citrus juices is more heat stable than the microorganisms in the juice. Thus, the heat treatment applied to these fruit juices (90 ^oC for 0.5-1 min) for pasteurization is more severe than that desired for microbial inactivation (Cemeroğlu and Karadeniz, 2001).
3.4. Industrial Applications of PME
Pectinases have been utilized in the commercial sector for wine and fruit juice industry since 1930. Commercial pectinase preparations, account for about 25 % of the global food enzymes, are produced generally from fungal sources (from Aspergillus niger) and used in many applications in food industry without purification. According to the specific objective(s) of the processes, the enzyme PME is used alone or in combination with other enzymes, mostly with other pectinases. The processes that PME involved are introduced below.
3.4.1. Clarification of fruit juices
In fruit juices, pectic substances are the main substances responsible for the cloudiness. Thus, in the production of clear fruit juices the pectin in juices should be degraded by the application of pectinases. The degradation of pectin reduces also the viscosity of fruit juices and eases their filtration and concentration. To degrade pectin, PME is used in combination with other pectinases such as PG and PL. Following the degradation of pectin, the viscosity of fruit juice drops and suspended cloud particles lost their stability and precipitated.

In the clarification of fruit juices, the PME enzyme may also be used alone. However, such a treatment needs the addition of CaCl₂ to fruit juice. This removes pectin in fruit juice as insoluble calcium pectates and causes the clarification (Massiot et al. 1997, Alkorta et al. 1998, Wicker et al. 2002).

In fruit juice industry, pectinases may also be used to increase the yield of fruit juice extraction. The treatment of mash with pectinases causes the degradation of cell wall and middle lamella of plant cells. This releases the fruit juice and increases yield. Another application of pectinases in fruit juice industry involves liquefaction of fruit mashes. In such an application, the pectinases are combined with cellulases and hemycellulases for the complete disruption of the cell walls. This method can alternatively be used for the production of fruit juices from tropical fruits (e.g. banana) that can not be processed with classical methods (Cemeroğlu and Karadeniz, 2001; Demir et al. 2001). In all these enzymatic treatments, degradation of pectin facilitates pressing and increase juice yield. Also, the aromatic quality of fruit juices increases and amount of waste material reduces (Sarıoğlu et al. 2001).

In industry, the use of pectic enzymes in fruit juice processing has traditionally been conducted in batch reactors using soluble enzymes. This operation causes the loss of enzyme. Also, the presence of enzyme in the final product and alteration of organoleptic properties are inevitable (Demir et al. 2001). Thus, different studies have been conducted for the immobilization of pectinases. For example, Demir et al. (2001) studied the use of immobilized commercial pectinase named ‘Pectinex Ultra SP-L’ for mash treatment of carrot puree. This treatment maintains almost 93 % of the enzyme after the fifth treatment. However, in practice the application of pectinases are still conducted by the classical methods.

Pectinases are also used in wine-making industry. The utilization of these enzymes influences the methanol concentration in musts and wines. However, their use increases the volume of free-run juice (by breakdown of polysaccharides and solubilization of middle lamella), extraction yield of polyphenols, and color and aromatic compounds (Pimenta-Braz et al. 1998, Rogerson et al. 2000, Kashyap et al. 2001).
3.4.2. Firming of fruits and vegetables before processing
Texture, an important quality attribute of fresh and processed fruits and vegetables, is closely related with the structural integrity of the primary cell walls and middle lamella, which are mainly composed of pectic substances. In industry, most of the processes such as blanching, freezing, dehydration, pasteurization and sterilization applied to preserve fruits and vegetables cause irreversible physical damages on cellular tissues. However, the negative effects of processing on texture can be overcome by applying different processes, which involve the use of PME enzyme. For example, the application of PME and CaCl₂ by vacuum infusion is now used for firming fruits and vegetables (Degreave et al. 2003). The mechanism of firming in this method is based on the demethylation of naturally occurring pectin in plant tissues by the action of PME and the chelation of the added or natural calcium with the free carboxyl groups generated in pectin molecules. Such a chelation causes the formation of networks among pectin molecules (the egg box model), stabilizes pectin and increases the firmness of plant tissues. Some of the successful applications of enzymatic firming by use of PME involves, firming of strawberries before jam making and freezing (Suutarinen et al. 2000, Suutarinen et al. 2002) and firming of apples, strawberries, and raspberries before pasteurization (Degreave et al. 2003).

Besides commercial PMEs, the in situ PME in the product may also be used for firming of fruits and vegetables. In this method, the in situ PME should be activated by low temperature blanching. The free carboxyl groups produced then were cross-linked with divalent ions in the medium or CaCl₂ added to form a stable network. Firming by this method is suitable to apply for whole or sliced potatoes to be processed to French fries (Yemenicioğlu, 2002).

3.4.3. Modification of pectin

Pectin produced from apple and orange peels has widespread application in food industry as gelling, thickening, and stabilizing agents. The degree of esterification of pectin molecule greatly influences its functional properties. The degree of esterification (DE) of pectin may range between 0 and 100 % and on the basis of DE, pectins are divided into two groups; high methoxylated (HM) pectins with a DE higher than 50 %, and low methoxylated (LM) pectins with a DE lower than 50 %. Pectin can be modified by PME to obtain the required DE value (Morris et al. 2000, Schmelter et al. 2002). Pectins with low DE are particularly useful to obtain gels without using sugar and acid.

3.4.4. Production of low sugar jams, jellies, and compotes

Traditionally, HM pectin is used for the preparation of jams and jellies. The gelling mechanism of HM pectin is based on hydrophobic interactions and dehydration at low pH (< 4.0). In such gels, the presence of high concentrations of sugar (> 60 %) is essential for gelling. In contrast, LM pectin forms gels by ionic interactions in which calcium or other divalent cations interact with free carboxylic acid of two adjacent chains, and give rise to cross-linking of these chains. In such a gelling mechanism, the sugar concentration is not very important. Thus, LM pectins are suitable for the production of low sugar (diabetic) jams and jellies (Cemeroğlu and Acar, 1986).

3.4.5. Other applications
Pectinases, including PME, can also be used in some different industrial applications. One of these applications is oil extraction. Oils from coconut germ, sunflower seed, palm kernel, rape seed are industrially extracted with organic solvents. The most commonly used solvent is hexane, which is a potential carcinogen. Thus, alternatively, cell wall degrading enzymes, PME and other pectinases are used in combination to extract oil in different crops by liquefying the structural components of their cell walls. Pectinase preparations (such as Olivex) are also used in olive oil industry to increase the oil extraction output and to improve certain olive oil quality indicators (Kashyap et al. 2001, Vierhuis et al. 2003).

Another application of combinational use of PME, other pectinases and cellulases is the peeling of fruits. Peeling of fruits has traditionally been applied by hand or treatment with steam, boiling water, acid or alkali. But these methods sometimes cause poor product quality (losses of fruit juice and/or disintegrations at the fruit surface) in delicate fruits. Also, in chemical methods, the disposal of the used peeling solution is a great problem. Therefore, application of pectinases and cellulases by vacuum infusion can be used as an alternative method for peeling of delicate fruits. For example, Pretel et al (1997) applied a commercial preparate (Rohament PC) containing pectinases and cellulases to remove peels and skins of oranges and to obtain whole fruit segments.

CHAPTER 4