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  • Published on: 7th September 2019
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2.1. Solid-state fermentation

In nature, several microorganisms secrete enzymes for their own needs. Many of these enzymes have high added-values, since they can be employed in processes in the food industry, which leads to the isolation of these microorganisms for this purpose. The SFF is an industrial-scale production method of enzymes by isolated microorganisms in an economically and environmentally friendly manner, which makes this process widely endorsed by the industry (Thomas et al., 2013). This technique is based in a simple principle: the cultivation of an isolated microorganism in controlled conditions of certain parameters (i.g. temperature, relative humidity, oxygen concentration) on a solid substrate  in absence of free water or near to it . Therefore, the substrate must contain all the water and nutrients necessary for the microorganism development or it must be an inert material that serves as a support for the nutrients (Singhania et al., 2009). This method does not cause a sudden change in the microorganism habitat, since it represents almost their native growth condition. As a result, the production level of enzymes is higher compared to other method such as the Submerged Fermentation (SmF), reaching the necessary level to meet the industrial demand. Moreover, by using this production method, industries mitigate problems inherent to its production process, such as waste destination. In SSF, industrial wastes can be used for the production of commercially important enzymes, adding value to these compounds which would generate costs to be disposed of properly in addition to environmental problems related to its disposal (Nigam et al., 2009). This is one of many reasons why several studies have been conducted in order to improve and optimize SSF. For instance, fungi and yeast are most suitable for use at the expense of bacteria due to the physicochemical conditions that are found in SSF fermentation, as moisture and water available for use. However, with the advancement in the understanding of the nutritional needs of microorganisms, the composition of the substrates, how is the interaction between them and advances in bioreactors itself, it is possible to overcome certain barriers, as shown by Sharma and Satyanarayana , (2012),   with the production of α-amylase from a strain of bacteria on a SSF system.

Several examples arise in this promising scenario for cost reduction and utilization of industrial waste for production of enzymes. Rosés and Guerra, (2009), performed surface response methodologies and empirical modeling to reach at optimal conditions of temperature, pH, moisture and the physical form that the substrate should be (granulated with specific particle size) for the cultivation of the filamentous fungus Aspergillus nidulans U0-01 in order to produce α-amylase. The substrate used was sugarcane bagasse, which is an important industrial waste found in countries that produce ethanol from sugarcane. Currently, the bagasse does not have many industrial applications apart from being burned to generate electricity. Rosés and Guerra, (2009), showed that the optimum conditions for α-amylase production in that case was: sugarcane bagasse particle size of approximately 7 mm, ph 6.0 and temperature around 30ºC. In these conditions, they produced 457.82 EU/g of dry support.  Another example was elucidated by Coradi et al. (2013) in which the lipase production system for the filamentous fungus Trichoderma harzianum in a SSF system was for the first time described. Coradi et al. (2013) tested various industrial residues for lipase production in a SSF and reached the conclusion that a substrate consisting of a mixture of sugarcane bagasse and castor oil cake  in a 1:2 ratio supplemented with some other nutritional sources generated the best rate of production of this enzyme by the fungus T. harzianum in comparison with the other substrates tested. At that time, the vast majority of the studies focused on production of this class of enzymes by other microorganisms in other fermentation system to be discussed in the next section. Another example also involving lipases production was published by Salgado et al. (2013). The Aspergillus niger strains MUM 03:58, Aspergillus ibericus MUM 03:49, and Aspergillus uvarum MUM 08:01 were selected for lipase production by SSF with the main residue found in the olive mill industry, called Two-phase olive mill waste (TPOMW). It was observed that the highest amounts of lipase were produced by A. ibericus on a mixture of TPOMW  , urea, and exhausted grape mark. The optimal condition for lipase production by this species was found using 0.073 g urea/g   and 25% of exhausted grape mark resulting in 18.67 U/g of lipolytic activity. Finally, knowing that the use of industrial waste is both an economically and environmental solution, Muthulakshmi et al. (2011) tested the production of proteases by SSF using different industrial waste, as seed cotton, rice straw and bran wheat. They found that the bran wheat residue generated better results with respect to enzyme production. Therefore, further studies were made in order to optimize fermentations parameters. The cultivation was done at pH 5.0 for 7 days at 30 °C with a supplement of 3% KNO3 as the nitrogen source, inoculum size 3% and substrate concentration 3% that yielded in 170 U/mg of  the protein using the filamentous fungus Aspergillus flavus.

2.2. Submerged Fermentation

The SmF is performed in a liquid nutrient medium, where the microorganism is submerged. Because it is held in liquid medium and not solid, some microorganisms such as bacteria end up being favored because they require greater degree of moisture to survive that is not present in solid fermentation (Singhania et al., 2009; Thomas et al., 2013) . Furthermore, the extraction of the final product, whether it be enzymes or secondary metabolites of interest, is easier and less costly than in comparison with the SSF, in which it is necessary to extract the final product from the cultured microorganisms therein. However, the use of industrial waste for production of enzymes by SmF is not as easy as to SSF. Several studies have been published comparing the production of certain enzymes by these two methods of fermentation, suggesting that in each case, a preliminary study is required to determine the most suitable method. A study published by Hashemi et al. (2013) showed significant differences between the production of α-amylases from Bacillus ssp. by SSF and SmF. It was observed that for solid-state fermentation, the optimum pH for enzyme activity was 4 and the temperature was 70 °C whereas for the enzyme produced by SMF theoptimum pH was around 3.0 at 60 °C. Furthermore, it was observed that for the enzyme produced by SSF, pH and temperatures led to lower enzyme activity in 45.8 ºC of temperature and pH 5.5, while for the enzyme produced by SmF lowered activities were found in 74.1 °C of temperature and pH 5.5. This study shows the importance of studying the enzyme production by fermentation processes because each has specific characteristics that may be advantageous or not.

2.3. Enzyme’s expression systems and engineering

Regarding productivity and production cost, with the steady growth in the use of enzymes in various industrial processes as a sustainable solution (Adrio et al., 2014), there is the need to create production systems that are compatible with the requirements of industrial processes. Due to advances in biotechnology, it was possible to create enzymatic production systems based on the DNA technology of cloning genes of interest in microorganisms with specific characteristics such as: protein expression, resistance to inhibitors and resistance to temperature changes and pH. This new technology  would allow the production of large-scale enzymes, because until then, these enzymes were taken from the native organism such as plants and animal cells that do not have the essential features for use in large scale production (Fernandes et al., 2010). There are many different microorganisms that can be used for this purpose, so it is important to understand the differences between then. The bacterium Escherichia coli has been widely used for enzyme production because it has the ability to grow to high cell densities in a short time, has the ability to accumulate about 50% of its dry weight in heterologous enzymes,  it grows in cheap culture media and it is easy to handle. These characteristics make it a good microorganism for the industrial production of enzymes, however, there are some disadvantages of using this bacterium as a host such as it inability to perform post-translational modifications, which are responsible for correct protein folding and therefore relates to the enzyme activity (Adrio et al., 2014). Because of this, it is important to evaluate the enzyme amino acid sequence to make sure that E. coli will be able to produce it in the active form. According to Morrow, (2007), an alternative to the use of E. coli is the yeast Picchia pastoris, which also has the ability to produce high levels of heterologous enzymes and may reach 30 g/L of it. Picchia pastoris is also capable of performing post-translational modifications, ensuring the correct folding of the heterologous enzymes. Other widely used yeast is Saccharomyces cerevisiae, which like P. pastoris, also carries out post-translational modifications and has high levels of enzyme production.Nevertheless, P. pastoris offers several advantages over S. cerevisiae such as a responsive promoter that allows methanol tight control of heterologous protein expression and greater capacity to integrate exogenous DNA fragments to the genome, facilitating the construction of stable strains capable of producing the enzymes, among other . (Adrio et al., 2014).

 One example of the utilization of P. pastoris as a industrial enzyme production system is with the enzyme laccase. Laccases are used in the food industry for wine and beer stabilization and therefore require a suitable production system that is capable of supply the industrial needs. Kittl et al. (2012) enabled the production of a laccase from Botrytis aclada by cloning its gene in P. pastoris, reaching the production of 0.495 g /L of the protein in the extracellular medium, which was at that time the highest concentration of this protein obtained from a heterologous expression system with P. pastoris. Lipases, enzymes used, for example, for the manufacture of margarine from fat-oil blends, were produced with P. pastoris  under the control of  the methanol responsive promoter at high concentrations of 20 U/mL in the extracellular medium (Shi et al., 2010).

Due to advances in genetics and biotechnology, it is now possible not only to improve the production of the enzymes as described above, but it is also possible to improve various characteristics of the enzymes itself such as stability, which are intrinsically related to the amount of enzyme used in the process, ability to act in a higher pH range without losing activity and greater specificity for certain substrates, among other features. Two approaches can be employed in order to improve enzymes features.The first approach is (a) directed evolution and second (b) rational protein design. The first is based on a set of techniques that allow the insertion of random mutations and recombination in the genome of the host to be selected, aiming the different clones of this microorganism that obtained the best mutations that somehow improved the protein that was meant to be improved . In this case, it isn’t necessary a vast knowledge of the enzyme itself to be able to improve it, as the technique is based on the random insertion of mutations that are inserted into the sequence of the enzyme and subsequently selected, making this a versatile technique (Fernandes et al., 2010). An example of this technique is with glucoamylase, an enzyme responsible for starch processing. The starch is liquefied at 105 °C in the presence of α-amylases but has to be cooled to 60 °C so that the glucoamylase may be used. In order to eliminate this cooling step which is costly for the industry, researchers applied directed evolution technique in this enzyme for increasing its termoestability. The result was an enzyme with an increase of 5.12 kJ/mol in the free energy of thermal inactivation without losing its activity (Wang et al., 2006). Another example is the application of the enzyme glucose isomerase derived from Thermotoga neapolitana for production of sweeteners. This enzyme has optimum activity in media with neutral pH and at temperatures around 100 °C, however  the isomerization of glucose occurs in industry in an alkaline media and in a temperature  of50 °C.. To solve this problem, Sriprapundh et al. (2003), applied the direct evolution technique and produced a mutated enzyme which has the optimum activity around 60ºC in alkaline pH and is more thermostable in comparison with the parental one, showing the efficiency of this technique. .. Finally, the second approach used, the rational protein design, was only possible thanks to advances related to the understanding of protein structures and their functions, which was accompanied by the development of bioinformatics. This technique is based on the rational insertion of changes in the protein aminoacid sequence. To do that, previous bioinformatics analyzes has to be done to indicate which changes can improve the characteristics of that enzyme. Unlike the first approach of directed evolution, is necessary to have thorough knowledge of the amino acid sequence of the protein, the structure and functionality of each part of the enzyme that will be modified (Adrio et al., 2014; Fernandes et al., 2010). An example of this technique was the improvement of other glucose isomerase which the rational insertion of two point mutations (G138P and G274P) that was responsible for a higher specific activity of the enzyme, increased half-life compared to the parental and greater thermostability (Zhu et al., 1999). Although these two techniques could generate improved enzymes, both are not mutually exclusive and can be used in a complementary manner (Adrio et al., 2014).

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