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Bacteria have given a lot of advantage to human being especially when it comes to the research and commercialized product. A lot of study can be developed through its life cycle and metabolism with bacteria. Like human, bacteria also secrete enzyme to undergo basic reaction of life. Enzyme from bacteria have been developed as a tool for various types of process in industry such as in detergent industry, foods and beverages industry, textile industry and also leather industry (Kirk et al.,2002). In the industrial applications, lipase has developed as the one of the most important enzyme that has been used widely. For example lipase is used for hydrolysis of fat from milk. The free fatty acids released acts as precursor for the synthesis of other flavour compound such as acetoacetate and flavour esters (Romano et al., 1996).
Unfortunately, the production of enzyme from bacteria has decreased due to the expression system problems faced by the scientists. Luckily, the transformation and cloning process now can depend on the yeast. In yeast, a few advantages have been utilized to enhance the production of enzyme. Yeast can grow with high cell densities in the inexpensive media. It is also secrete the protein to the media that help in the purification process. In yeast expression system itself, it has a vector that can be manipulated easily. Since so many techniques have been manipulated, scientists are now more preferable to develop their new vector for themselves. With this vector, the production can be controlled and maximized. But for the main reason, the development of new vector is to enhance the production of enzyme from the yeast.
Considering many advantages of using yeast as an alternative system to express lipase, thus, this research is conducted to with the following objectives:
To clone thermostable lipase gene in locally isolated yeast.
To optimize thermostable lipase expression in local yeast.
Lipases (EC 18.104.22.168) also known as triacylglycerol acylhydrolases are participating in hydrolysis of lipid into glycerol and free fatty acid. Uniquely, they possess different characters of work by catalyzing the lipid conversion in aqueous and non aqueous interface which lead them differrent from esterase (Verger, 1997; Schmidt and Verger, 1998). Lipases are different from esterase because they are only activated when adsorbed to an oil-water interface (Martinelle et al., 1995). There are a lot of lipase sources such as in plants, animal and microorganisms. Nowadays, researches are more depending on lipases in microbes due to its stability and can be produced in bulk with lower costs (Vakhlu and Kour, 2006).
Sources of lipases
It is known that lipases sources are widely distributed in nature but only microbial lipases were known to have the commercial value (Sharma et al, 2001). These microbial lipases contribute 2% from the sources of enzyme in the microorganisms' world (Hasan et al., 2006). Even on that percentage, the scientists still cannot fully use the advantage for the research that is being done. They still have little knowledge in understanding the mechanism for each lipase they have known. Since lipases are now being more demanding in the market, scientists usually insert the gene into certain prokaryotes such as E. coli to produce more lipases. This is due to their short life cycle, high stability and rapid growth on inexpensive media (Wiseman, 1995).
Applications of lipases
Lipase already makes a big impact through its availability to function in many ways. Clinically, lipases has found as indicator to determine of lipids in biosensors which are can be electronic or chemical in nature (Pandey et al.1999). In food and beverages industry, lipase was used as substance to improve the aroma of the products. It is also used as moisturizer and emulsifiers in the cosmetic product (Vulfson, 1994). In chemistry, lipase has been developed as a catalyst for ester formation. Then the esters can act as flavoring agents in food industry (Vulfson, 1994).
Lipases have many different types coming from many sources. Nowadays the most interesting lipase comes from thermophilic microorganisms. Not only that, certain mesophiles and psycrophiles also secrete this type of enzyme (Demirijan et al., 2001). Thermostable lipase is a unique enzyme because the reaction can be performed at higher temperature in comparison with the normal mesophiles (Li and Zhang 2005). Furthermore, this type of lipase also gives more advantage such as higher stability, higher reactivity and higher process yield. These characteristics really give a lot of advantage for biotechnology applications because it helps in further or rare research applications (Maugeri et al., 2001). The enzyme also has low viscosity and fewer contamination problems. In addition, it gives benefit to the industry due to lower cost of production.
Yeast is unicellular, non filamentous fungi that usually in spherical or oval in shape. Most of the yeast is classified as ascomycota and they can be found widely in nature. Yeast can reproduced either sexually or asexually. In asexual reproduction, they will undergo simple cell division or by pinching off from mother cell like Saccharomyces cerevisiae. For sexual reproduction, they will form asci or basidia (Campbell and Reece, 2002).
The most famous yeast use in industrial application is baker's yeast, Saccharomyces cerevisiae. This yeast has already become a tool for production of many products for years such as beer, wine and bread (Saloheimo, 2004). According to Macrae and Hammond (1985), a company from Japan has used yeast from Candida rugosa to hydrolyze castor bean and produces fatty acid from it.
2.3 Yeast expression vector
In cloning technique, a few tools are needed to complete the process. The most important are DNA fragment, host cell, restriction enzyme and also a vector. The vector itself is built from the DNA sequence. It is needed because its function to transfer the foreign genetic material from other sources to the host cell. Theoretically, despite the concept is quite simple and easy, there are certain cases that the gene is not successfully expressed. It is also not guarantee that high level of protein will be express in the host because the process is so complex and must be perfected to produce it (Romanos et al, 1992).
So, a good vector system is required to produce high level expression of gene of interest. Plasmid cloning vector has been the most common vector that scientists used to make interact between the yeast and bacteria (Sikorski and Hieter, 1989).
Yeast expression system
For years, studied on yeast has been developed for production of better results in research. Before that, the manipulations of genes in organisms only focus on bacteria especially Escherichia coli. This is due to its short life cycle, easy to grow and has a good expression of genes. Despite all of this, there are few problems in its expression system such as misfolded or insoluble protein. In yeast, this problem can be ignored because yeast generally can form disulfide bond that is important to form tertiary structure of protein. It is can also undergo glycosylation process which make proteins more applicable and usable. Another good reason for gene expression in yeast is that they have a strength promoter, efficient in secretion and of course high cell density when grown on media (Romanos et al, 1992). For example in Pichia pastoris, it has a very strong and tightly regulated promoter known as alcohol oxidase I gene (AOX1). Moreover, its secretion of protein to the media helps other researchers to purify protein more easily.
Cloning of lipase gene in yeast expression system
Lately, the cloning of lipase gene in yeast has become a trend in biotechnology world. Starting at prokaryotic cell for example Escherichia coli, the new research is now depending on the expression from the yeast because of it unique expression platforms (Boer et al., 2007). Not only lipase, the other gene also have been expressed using yeast as a host. For instance, the first gene expressed in yeast is insulin, expressed from Saccharomyces cerevisiae (Melmer, 2005). Table 1 shows example of lipases those have been cloned in various yeast expression systems.
Table 1: Lipases expression in yeasts system
Type of yeast
Type of lipases
Level of expression
glyceraldehyde-3-phosphate dehydrogenase (GAP) & alcohol oxidase (AOX)
Synthetic lipase gene from F. heterosporum
Native Hansenula polymorpha
(Nicaud et al., 2002)
Certain type of yeast has a very good promoter that can be easily manipulated. In yeast, the expression system is based on shuttle-vector system. Plasmid in Pichia pastoris is designed to be integrated in yeast genome namely (yeast integrated plasmid) YIp, which offers higher expression and better stability compare to Escherichia coli (Pfeffer, 2008).
2.4.1 Transformation of vector into yeast host
Generally, transformation is the process where foreign DNA was inserted into the cell to form recombinant DNA. There are several methods can be used to transform recombinant plasmid into yeast host such as electroporation, chemically competent, spheroplast, polyethylene glycol (PEG) and alkaline cation. These methods, give different efficiency by showing numbers of transformants frequency. Table 2 shows the general characteristics between these four methods.
Table 2: General characteristics of transformation methods (Cregg and Russell, 1998)
Transformation frequency, per Âµg
As shown, the most powerful method can be used is electroporation which offer greater efficiency and faster compare to other methods. In this technique, external electric field was applied to the cell and it is believed that the phospholipid bilayer will be induced and permeation site will be stabilized to allow the introduction of macromolecules of interest (Hendricks and Jesuthasan, 2007). Table 3 shows the parameters for electroporation using selected instruments.
Table 3: Parameters for electroporation (Cregg and Russell, 1998)
Cuvette gap, mm
Sampe volume, uL
Charging voltage, V
Field strength, kV/cm
Pulse length, ~ms
Electroporator II (Invitrogen)
2.4.2 Optimization of enzyme expression
Optimization is required to produce amount of enzyme that can express in industrial scale. The parameters normally are media of the grown culture, temperature, and also pH. Changing one parameter at one time is the common method that used to identify the best condition for the host (Lim et al, 2007). Media optimization usually involved the Carbon and Nitrogen sources.
While all the parameters are manipulated, the production of the expressed gene also must be observed continuously. For certain type of yeast such Pichia pastoris, the expression is controlled or induced by methanol and inhibited by other types of carbon sources such as glucose and ethanol (Damaso et al; 2003).
Screening of lipase activity in locally isolated yeasts (S4, S5, R1, R2, WB)
Selection of yeasts as potential host
Transformation of recombinant lipase in newly constructed vector into yeast host
Expression of thermostable lipase
Optimization of the lipase production
SIGNIFICANCE OF PROJECT
Lipase has already become an important and demanding enzyme nowadays due to its variable applications. So, this research is not only to study about the production of lipase but also for the listed target.
A newly constructed vector can be used to express recombinant protein.
Locally isolated yeast can be introduced as a new expression system for commercialization
Thermostable lipase can be commercialized by using new develop expression system