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The production of BLA can be enhanced using two approaches which are the media manipulating and genetic engineering. Media manipulating involves changing the composition of the culture media and the surrounding condition for the fermentation of bacteria. It is of a better choice as compared to genetic engineering of the bacteria as the production of amylase depends on the survival of B. licheniformis which is influenced by the culture physical and chemical conditions (Abdel-Fattah et al., 2013). Besides that, media manipulating is also cheaper due to lower energy and cost input as the sources are readily available and easy to be obtained. Also, media modification allows the interaction between the parameters to be studied through statistical models (Sumrin et al., 2011). In contrast, genetic engineering can be used to make alterations on the coding or control regions of the bacteria amylase gene sequence to increase its expression. This method is time and cost consuming as it involves gene extraction, cloning of libraries, PCR, DNA assembly, sequencing and transformation (Leguia et al., 2013). Also, wastage may happen due to higher chance of uncontrollable mutations.
There are two methods for media optimization which are the one-variable-at-a-time (OVAT) method and the response-surface-methodology (RSM). For OVAT, only one factor is manipulated without changing any other factors (Soni et al., 2012). This method is less practically suitable as it neglected the interactions of different factors. For instant, the bacteria may need a combination of factors such as certain degree of temperature, pH and concentration of ions in order to produce higher amount of BLA. By changing only one factor, this method is also less efficient as the real optimum condition for the culture cannot be determined (Abdel-Fattah et al., 2013). This may prevent the bacteria from secreting maximum amount of BLA. Furthermore, OVAT is time consuming as more experimental trials have to be performed.
In contrast, the RSM implements a set of experimental strategies, mathematics and statistical analysis which explain the interactions of different parameters (Abdel-Fattah et al., 2013). RSM is performed by carrying out experiments designed with one or more variables being manipulated and the results are fit into mathematical models to estimate the coefficients which allow the amount of BLA produced (response) to be estimated (Sumrin et al., 2011). By repeating the experiment trials, the most suitable and optimum conditions to obtain higher BLA yield can be determined. RSM can be carried out in different design models such as Plackett-Burman design (PBD), Central Composite (CCD) and Box-Behnken design (BBD) where PBD is more suitable for primary screening the key parameters while CCD and BBD are used for true determination of optimum level for the parameters identified (Sharma & Satyanarayana, 2011). RSM is an alternative to OVAT as it enables the effect of interactions between each parameter to be studied. Also, it saves time since fewer experimental trials are needed.
The production of BLA is greatly affected by the media conditions and has to be optimized. One of the parameters that have to be considered in optimizing the production of BLA is the nutrient supplement such as carbon and nitogen. Nutrients are essential for the survival of B. licheniformis as it determines the level of amylase produced. This is supported by Kumar et al. (2012) who claimed that the presence of carbon sources such as glucose, lactose and starch or nitrogen sources such as ammonium nitrate and ammonium dihydrogen phosphate in culture media will stimulate the BLA production. Carbon is the main constituent of cellular materials such as cell wall and it supplies energy through glycolysis which is required for metabolic activities (Chen et al., 2008). It is essential in maintaining the survival of. On the other hand, nitrogen is needed for DNA replication and binary fission of the bacteria as the nitrogen makes up the nucleic acid backbone of DNA and RNA. Although carbon and nitrogen sources can aid in increasing BLA production, they are expensive for commercial uses. Hence, more economic and environmental-friendly sources should be used to supply nutrients for the bacteria to production amylase. Chandrashekhar et al. (2012) suggested that banana wastes can be used in the fermentation medium for the production of BLA as they contain high amount of sugar.
The level of BLA produced from fermentation is also affected by the incubation temperature. According to Mansilla et al. (2004), plasma membrane is made up of proteins and phospholipid bilayers with saturated or unsaturated fatty acids. At low temperature, the hydrocarbon tails of fatty acids undergo trans- formation to become closely packed (Mrozik et al., 2004). This decreases the fluidity, causing the plasma membrane to be more rigid and there will be lesser trans-gauche isomerization (Mrozik et al., 2004). Consequently, the transport across the membrane is reduced or prevented due to reduced permeability, leading to a lower amount of BLA produced. High temperature will unfold the 3D structures of transport proteins embedded in plasma membrane, causing them to be denatured as the non-covalent interactions such as hydrogen bonds, hydrophobic and van der Waals interactions are broken (Uversky, 2009). The phospholipid will also melt, causing the plasma membrane to rupture and lead to the bacteria cells death. As a result, the level of BLA decreased. As stated by Zare et al. (2012), the optimum temperature for BLA production is 37ËÅ¡C and reduction in BLA yield is observed for any other temperature below or above 37ËÅ¡C. However, this result is opposed by Oziengbe and Onilude (2012) who claimed that the maximum the optimum temperature for B. licheniformis to grow is 60ËÅ¡C. Thus, a more suitable system should be established to find out the most suitable temperature for both the bacteria to grow and to secrete more BLA.
Another parameter that has to be considered in optimizing the BLA production is the pH of the fermentation. Sankaralingam et al. (2012) reported that maximum production of BLA was observed at pH 7 and acidic or alkaline media will reduce the amount of BLA secreted. pH influences the growth of B. licheniformis by causing morphological changes in the bacteria (Kathiresan & Manivannan, 2006). Also, it will affect the solubility of nutrients which is required for bacterial growth, ions bindings and metabolic activities such as synthesis of ATP which determine the survival of bacteria (Slonczewski et al., 2009). Besides, pH will affect the amylase stability in the media. Amino acids are zwitterions and they are connected through non-covalent interactions such as hydrogen bonds, disulphide bridges, hydrophobic interaction and van der Waals interaction to form a 3D structure of BLA. When there is a decrease in pH, the concentration of hydrogen ions is higher and these hydrogen ions will bind to the carboxyl group of the amino acid, causing conformational changes in BLA as the interactions are disrupted. This appears to be the same for alkaline condition where the hydroxide ions will cause the amino group to lose a hydrogen ion. Consequently, the BLA is denatured leading to a lower amount of yield obtained.
In conclusion, it is very important to optimize the fermentation of B. licheniformis in order to obtain maximum yield of BLA. Media manipulation is better in optimizing BLA production as it is cheaper and simpler compared to genetic engineering which is more expensive due to complex procedures and instrumentation and it is time consuming. Media optimization can be carried out using OVAT and RSM methods but RSM is more preferred as it allows the true optimum condition to be determined from the interactions of different parameters. In order to increase the amount of BLA produced, availability of carbon and nitrogen sources, incubation temperature and the pH of the media have to be considered as these parameters influence the survival of bacteria and final amount of BLA. However, future studies have to be carried out to further investigate the optimum conditions for BLA production as there are still limitations on present methods.