CHAPTER 1 : INTRODUCTION

1.1 Background of Study

Many compounds are currently produced through reactions that use chemical catalysts or expensive experimental conditions. However, this practice is aggressive to the environment, generating effluents with high cost of treatment, and consuming too energy. Enzymatic catalysis appears thus to reduce the energy demand contributing to a reduction in by-product formation. However, the acquisition cost of enzymes is still high, making difficult the access of biocatalyst to industries. An alternative to this financial barrier is immobilize the enzyme (ZHENG et al., 2012). Constant efforts are being made to improve the enzyme’s activity, efficiency, reproducibility and stability during industrial processes (Wang et al. , 2010).

Enzyme immobilisation is confinement of enzyme to a phase (matrix/support) different from the one for substrates and products. Inert polymers and inorganic materials are usually used as carrier matrices. Apart from being affordable, an ideal matrix must encompass characteritcs like inertness, physical strength, stability, regenerability, ability to increase enzyme specificity/ activity and reduce product inhibition, nonspecific adsorption and microbial contamination (Singh, 2009). Immobilization generates continous economic operations, automation, high investment/capacity ratio and recovery of product with greater purity (D’Souza, 1998). There are several factors affecting the immobilization processes such as adsorption, covalence bound, entrapment and cross- linking.

During the initial years of the development in the field of immobilized enzymology, researchers used to find only the advantage of the immobilized enzymes in comparison to their soluble / free counterparts. Advantages of immobilized versus soluble enzymes included comparative studies in pH profile, various denaturing agents organic solvents, and temperature. Now recently during the last couple of decades, immobilized enzyme technology has advanced into and ever- expanding and multidisciplinary fields to analyze clinical, industrial and environmental samples. Examples of the recent developments and used of immobilized enzymes in different fields such as in medicine, antibiotic production,drug metabolism,food industry, biodiesel production and bioremediation.

1.2 Problem Statement

The use of enzymatic route for production of biofuels is growing up due the mild reaction conditions. In fact of that, we must find a way to reduce the cost to ensure the experimental that are highly cost can be carried out and despite of that, immobilize enzyme is the best way that can be used. In addition, it also can be use multiple or repetitive in a single batch of enzyme. It become more economics as it also has the ability to stop the reaction rapidly by removing the enzyme from the reaction solution. By finding this solution, so that there are no problem arise to continuously used it as a biocatalyst.

1.3 Objectives

1. To provide an alternative way to reduce the experimental cost by using immobilized enzyme
2. To study the differences on the enzyme activity when using pH and mass media
3. To develop the easiest method that can be use by the others as it does not need a professional qualitification.

CHAPTER 2 : LITERATURE REVIEW

2.1 Enzyme

First and foremost, enzymes are the active components in the cells, where they induce chemical transformations. Besides, enzymes are large, complex macromolecules, consisting largely of protein and usually contain a prosthetic group (one or more metal atoms). It also acts as biocatalyst that catalyzes all chemical conversions needed for the system’s survival and reproduction (Buchholz et al., 2005; Idris et al., 2008).

Besides, enzyme also act as catalyst to boost up the rate of chemical reaction by factor up to more than and result to the reduction of activation energy compared to uncatalyzed reaction (Buchholz et al., 2005) as shown in figure 3.1. On top of that, enzymes have unique characteristics for instance high activity, selectivity, and specificity which permit their performance in their complex process.

Figure 2.1: Free energy diagram for an uncatalyzed and enzyme-catalyzed reaction (Buchholz et al. , 2005)

Enzymes are specific to the types of reactions that they catalyze. In addition, they are more stable and easier to handle than the original microorganisms from which they were isolated. On top of that, enzymes play an important role in biochemical analysis and it is widely used in many industrial processes especially in food production and many more.

On the other hand, properties of most enzymes are dependent on pH of their environment. Dependence of catalytic activity on the concentration of hydrogen ions in solution is given by protonable groups. These groups are the parts of active site of enzymes and also are present in substrate molecules. Thus, the reaction between the enzyme and the substrate is dependent on the degree of protonation. That is the reason why majority of enzymes are catalytically active in specific interval of pH values. If the pH value is higher or lower than pH optimum of certain enzyme, catalytic activity of enzyme decreases. pH optimum is value of pH at which the activity of an enzyme reaches the maximum. Changing of pH values of reaction medium is one way how to regulate the activity of enzyme (Vodrazka et al. , 2001).

Even though enzymes give benefits in many application, there are some restrictions should be stated. Enzymes are excellent reactions catalysts but without any improvement thus in pure state, they are not very suitable for use in reactors in industrial scale. On the other hand, enzymes may be instable due to spontaneous oxidation, self digestion, or denaturation and they work properly only on natural substrates and under physiological conditions. In addition, they are soluble in water and also in organic solvents and can be strongly inhibited by excess amount of substrates, product formed during enzyme-catalyzed reaction and however by certain by-products of the reaction.

2.2 Immobilized Enzyme

Enzyme immobilization can be described as the attachment of free or soluble enzymes to different types of support. This process turns the soluble enzyme into insoluble form by interaction with specific type of support. As a result, the mobility of the enzyme is reduced or lost (Khan et al . , 2010). Immobilized means enzyme has been confined or localized so that it can be reused continuously (Ramachandra et al . , 2002). Immobilized enzyme are currently the subject of considerable interest because of their advantages over soluble enzymes or alternative technologies, and the steadily increasing number of application for immobilized enzymes (Tisher et al. , 1999). Numerous methods for achieving the immobilization of lipases are available, each involves a different degree of complexity and efficiency. There are various methods used to date are adsorption, ionic bonding, covalent bonding, cross linking, entrapment, and encapsulation (Ramachandran et al. , 2002).

2.3 Support

Chen et al (2011) studied the effect of hydrophobicity of membranes used as support for the immobilization of lipase by covalent bond in the activity and stability of the enzyme, and obtained better results when used more hydrophobic membrane. Studies claim that the region surrounding the active site of lipases is hydrophobic, and because of that, they recognize hydrophobic surfaces as similar to their natural substrate and undergo interfacial activation (FERNANDEZ- LAFUENTE et al. , 1998). Zhou (2012) showed that, in general, substrates with high specificity had greater surface area for adsorption capacity, while the more hydrophobic again, the best results are attributed to the improved interfacial activity of the lipase. It is possible to predict, taking into account the objectives in view, the selected substrate should have high surface area, be thermally stable, chemically durable, resistant to contamination and reasonable cost (KANDASAMY et al. , 2010). Because of all these characteristics was chosen support material, the MCM41, whose family is characterized by having a hexagonal arrangement of uniform pores and well- defined size, with linear channels constructed with a silica matrix (KRESGE et al. , 1992).

2.4 Methods of Immobilization

Many review and books on the immobilization of enzyme have been published during the last two decades (Bahulekar et al. , 1993 Kennedy & Cabral, 1983). In the course of the last decades, numerous methods of immobilization a variety of different materials have been developed. However, different types of immobilization may have different effects on the enzyme activity or stability, is not always predictable at forehand (Arroyo et al. , 1999; Cao, 2011). Therefore, advantages and disadvantage have been described in Table 3.1 below. On the other hand, it is known that any type of immobilization method has the potential to stabilize the enzyme relative to their native form (Cao, 2011).Basically, there are four ways to immobilize enzyme onto surfaces as explain below :

• Adsorption

This method immobilization is the connection between enzyme and support without any chemical modification. Besides, this is the most used method for immobilization due to some advantages such as low cost, no chemical additives required and high activity (Fukuda et al. , 2011).

• Covalence

This method of immobilization is based on covalent bound between enzyme and carrier. During the catalyst action, the interaction between enzyme and support is very strong which makes the enzymes very stable. Instead having an advantage, covalent binding also has some disadvantage as well like the reaction conditions are complicated, there is a high risk of loose of enzyme activity during the process, and some coupling reagents are toxic (Fukuda et al. , 2011).

• Entrapment

This method of immobilization is process of capture of the enzyme into the inner hollows of some specific matrix or into microencapsules of polymer. Thus, an enzyme inside of the matrix is not attached to the polymer and its free diffusion is only restrained (Fukuda et al. , 2011).

• Cross Linking

This method of immobilization is the interaction between enzyme, coupling reagent, and carrier will form the three dimensional network structures. This method lead to strong interaction between the protein and the carrier, but activity of the immobilized enzyme is low. That is the reason why cross linking is often combined with adsorption to achieve higher immobilization efficiency (Fukuda et al. , 2011).

Table 2.1 :The advantages and disadvantages of immobilization method

2.5 Advantages of Immobilized Lipase

Although lipases presently account for no more than 3% of all enzymes produced worldwide, the use of immobilized lipases for the modification of melted fats and oils is currently a subject of expanding interest (Ramachandran et al. , 2002). This interest is due in part to the fact that the use of lipases has the potential to be more cost effective when enzyme are employed in immobilized rather than in free form (Ramachandran et al. , 2002).

Besides, there are several reasons to use immobilized enzymes such as easy separation of enzyme from the product and reuse of the enzyme. Easy separation of the enzyme from the product simplifies enzyme applications and permits reliable and efficient reaction technology. Enzyme reuse provides a number of cost advantages (Tisher & Kasche, 1999). Furthermore, the use of immobilized lipases leads to a decrease in potential for contamination of the product via residual lipases, thus avoiding the need for downstream thermal treatment (Ramachandra et al. , 2002). The immobilization of enzyme is a useful tool to meet cost targets and has a number of technological advantages (Tisher & Kasche, 1999).

Immobilization also permits multiple uses of the lipases and often enhances its thermal and chemical stability, thus leading to predictable decay rates. It also enhance oppurtunities for better control of both the process and product quality. In fact the increment costs of using an immobilized biocatalyst in a continuos process are more than 20 times lower than with a traditional one (Ramachandra et al. , 2002). Owing to the world wide variety of properties of individual enzyme species and the varying requirements of reaction technology for the target compounds, it is necessary to exploit the wealth of methods and techniques of immobilization (Tisher & Kasche, 1999).

The main disadvantage of immobilization can be loss of activity due to immobilization, limitation in substrate’s diffusion, possible leakage of the biocatalyst from the support (Ramachandra et al . , 2002).

2.6 Classification of Enzyme

Enzyme can be classified into six categories according to the reaction catalyzed. Every enzyme is defined by a unique set of 4 numbers. The first number denotes the reaction they catalyze, the second number indicates chemical structure that are changed in the process, the third shows the properties of the enzyme involved in the catalytic reaction and the forth implies the running number (Buchholz et al. , 2005). Table 3.2 shows the six group of enzyme and the type of reaction catalyzed (Knez et al. , 2001).

Table 2.2: Classification of enzyme and the reaction catalyzed (Knez et al. , 2001)

2.7 Lipase from Candida Rugosa

Lipase from Candida rugosa sp. is one of the most attractive commercially available lipases for complete hydrolysis of triacylglycerols because it has ability to liberate all types of acyl chains, despite of their positions in the triacylglycerols (Virto et al. , 1994). Candida rugosa sp. is one of the most extensively studied microorganisms by biotechnologists due to its powerful lipase ( E.C. 3.1.1.3) production capacity. In addition, Candida rugosa sp. lipase has extensive substrate specificity which provides successfully used in a variety of hydrolysis and esterification reactions.

Furthermore, it is possible used in synthesis of several pharmaceuticals (Benjamin & Pandey, 1998) due to its high stereoselectivity and regioselectivity. The yeast of Candida rugosa sp. secreted several extracellular lipases and they differ in terms of molecular weight, carbohydrate content, isolectric point specificity (Pernas et al. , 2000). Besides, it is commonly used for several streoselective esterification reactions in organic medium under mild reaction conditions (Abdul Rahman et al. , 2005).

CHAPTER 3: MATERIALS AND METHODS

1. Materials and Equiment

Table 3.1: List of chemicals and equipment

2. Experimental design

3.2.1 Preparation of Buffer solution

In conducting this experiment, at pH 7.0 and 8.0, three buffer solutions of 50 mM of Potassium phosphate at pH 6.0 are made.

2. Preparation to remove organic part on support

1 g of MCM 41 support are mixed with 10 mL of 10% HNO3 v/v and are strirring for 30 minutes. The solution are filtrate and the successive washes of water and buffer solution are removed.

3.2.3 Dilution for the p-NP (p- nitrophenol)

0.0014g of the reagent are weighed and are dissolve in a buffer to complete 100mL in volumetric flask. Dilutions of p-NP are made in the same buffer and are analyze on a spectrophotometer with absorbance 410nm using blank reaction buffer solution.

4. Determination of enzymatic activity

The substrate are using lipase and p-NFL (p-nitrophenillaurate). 0.018 g of the substrate are dissolve in 1 mL of DMSO and buffer are add until 100 mL flask are complete. For preparing the lipase solution, 0.1 g of the enzyme solution are dissolve in a 100mL of the buffer solution and subsequently performing various dilutions. Lipase solution with substrate are place in 2mL of cuvette at 40s for contact time. The pH variation is due to buffer at pH 6, 7 and 8.

5. Immobilization process

A beaker containing 20mL of p-NPL is place on magnetic stirrer is use for immobilization. To know the influence of the mass of support for immobilization, different mass of MCM 41 are add. The samples is collect and is being analyze by spectrophotometer at interval 10 minutes.

CHAPTER 4 : EXPECTED RESULTS

The expected results for this study are:

1. The increasing of the enzyme activity as the concentration of p- NF concentration is add and at what concentration it direct to to the enzymatic activity.
2. By using the enzyme concentration ( lipase enzyme), the curve of enzymatic activity is expect to fall.
3. The time for adsorption between the enzyme and support at pH 7 is just short if smaller mass support is use.
4. The pH 7 is most convenient than pH 8 for the adsorption of the mass support of MCM 41.

CHAPTER 5 : CONCLUSION

The use of enzymatic route for the production of biofuels can be overcome by using the immobilization of enzyme. The influence of pH and mass media used will effect enzyme activity. The experiments that carry out at pH 6 will show a fall absorbance with the increasing of the enzyme concentration. This experiments also show that the lipase is best at pH 7 and pH 8 where the results are better at pH 7.0. As the optimal immobilization at a very short time where it shows high affinity of the enzyme for support.The mass support also shows best in a very small amount of adsorbed enzyme. This also shows that the cost benefits with the use of support, which will be smaller.