L-Asparaginase is an enzyme found in a wide range of organisms including plants, microbes, animals, and in the serum of certain rodents but not in human beings. This enzyme has been considered as a therapeutically important antitumor drug due to its antileukemic properties. This well-known chemotherapeutic agent used in combination with other drugs is especially used in the treatment of acute lymphoblastic leukemia (ALL) – mainly in children-. (Batool et al., 2015).
Cancer is the ranks among the first four in most common causes of death in children in Turkey (Uzunhan et al., 2012). Acute lymphoblastic leukemia (ALL) is the most common form of leukemia in 80% of children and adolescents with leukemia. However, only the asparaginase from Escherichia coli and Erwinia chrysanthemi was approved to be used as part of a multiagent chemotherapy to treat ALL. But, the drug, which is obtained from prokaryotic microorganisms usually presents some problems such as hypersensitivity and immune inactivation. Within this context, eukaryotic microorganisms such as filamentous fungi and yeasts have been investigated for this enzymes production, due to better compatibility with the human system. (Cachumba et al., 2016). Thus, the fungal enzyme could provide an alternative to the bacterial enzymes as an antitumoral agent as it presents stability and optimum pH near physiological conditions (Jozalaa et al., 2016). In recent studies, L-Asparaginase from isolated Aspergillus terreus showed a great carcinostatic effect on the static tumor with no glutaminase activity and no cytotoxicity (Loureiro et al., 2016; Doriya et al., 2016).
The alternatives to L-asparaginase are not available in many parts of the world, including Turkey. Major restraining factors for global L-asparaginase market are its side effect in human body. Due to the side effects, developing new production strategies (e.g. using eukaryotic organisms instead of prokaryotes) has become important issue. In addition, cost of L-asparaginase production is another important point. Using of efficient, renewable, economical and easily available carbon sources is necessary to lower the prices. In this way, it will be possible for anyone with the ALL to have a chance to be treated.
In this research, A. terreus has been selected as an effective alternative for the production of L-asparaginase due to its previously mentioned properties. Additionally, the low cost production process of the therapeutic drugs is as very important as the effective treatment. In this study, effects of different lower cost industrial by-products on L-asparaginase production potential and enzymatic activity will be investigated. For this purpose corn steep liquor, molasses and cheese whey which are easily available wastes in Turkey have been chosen as the substrates to be used. As a result of this study, it was aimed to produce L-asparaginase with low immunogenicity using low cost substrates.
Key words: L-Asparaginase, acute lymphoblastic leukemia, corn steep liquor, molasses, cheese whey.
In recent years using enzymes for therapeutic purposes, especially in cancer treatments has gained great importance. L-asparaginase is one of these enzymes, which is mainly used in combination with other drugs for the treatment of certain malignancies such as acute lymphoblastic leukemia (ALL) – mainly in children- , Hodgkin’s disease, acute myelocytic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma (Batool et al., 2015).
Especially in ALL treatment, there is no effective alternative to L-asparaginase. The enzyme catalyzes the deamination of L-asparagine to L-aspartate and ammonia. L-asparagine is an essential amino acid for many tumor cells for protein synthesis and cell growth. Certain sensitive malignant cells cannot synthesize L-asparagine unlike normal cells due to the low expression or absence of the L-asparagine synthetase gene, therefore they obtain required L-asparagine from external sources for optimal growth. In the presence of L-asparaginase, tumor cells get deprivated and cannot survive, this is the main target of L-asparaginase treatments since tumor cell required a huge amount of asparagine to keep up their rapid growth (Souza et al., 2017).
Wide range of bacteria, yeast, fungi, algae, actinomycetes and higher plant are used as source of L-asparaginase but the microorganisms are considered as effective sources for the production of therapeutic enzymes since they are easy to manipulate. Also it facilitates economic production, consistency, ease of process optimization, and purification (Vimal et al., 2017). There are two different types of bacterial asparaginases. Type I L-asparaginases are cytoplasmatic enzymes that show low affinity to asparagine, while type II L-asparaginases are located in the periplasmic space with high affinity to substrate. Only type II asparaginases have been used as therapeutic agent, because the enzyme’s antitumor activity is related to their affinity for L-aspargine. The L-asparaginases from only Escherichia coli (in its native form or as a pegylated enzyme) and Erwinia chrysanthemi was approved to be used as part of a multiagent chemotherapy to treat ALL (Doriya et al., 2016, Souza et al. 2017). Unfortunately, there are some side effects associated with existing therapy like anaphylaxis, coagulation abnormality, thrombosis, liver dysfunction, pancreatitis, hyperglycemia, and cerebral dysfunction. These side effects are developed either due to the production of antiasparaginase antibody in the body or due to L-glutaminase activity of L-Asparaginase enzyme used (Vimal et al., 2017). In general, main reason of these problems is indicated that using enzymes derived from prokarytic source. Instead, being eukaryotic and evolutionarily closer to human can minimizes the chances of immunological reactions. Thus, eukaryotic microorganisms have been considered for this enzymes production, due to better compatibility with the human system. (Cachumba et al., 2016). Therefore, the fungal enzymes could provide an alternative to the bacterial enzymes as an antitumoral agent since human beings are more closely related to fungi. And immunological reaction chance is less than bacterial enzymes. (Batool et al., 2015)
Fungal asparaginase has acquired importance because of that it is produced extracellularly, and it is a very easy to purify extracellular enzyme. Wide range of fungal strains are being reported as efficient producers of L-asparaginase (Table 1).
L-Asparaginase from isolated filamentous fungi,Aspergillus terreus showed a great carcinostatic effect on static tumor. As a result of Loureiro et al., (2012), it was observed that L-Asparaginase from A. terreus showed anti proliferative effects on two leukemic cell line RS4; and HL60, with a molecular weight similar to that of E. coli asparaginase with no glutaminase activity. Also, L-asparaginase from A. terreus was show no cytotoxicity against normal human cells.
Cancer is the one of the most common causes of death in children in Turkey (Uzunhan et al., 2012). Acute lymphoblastic leukemia (ALL) is the most common form of leukemia in 80% of children and adolescents with leukemia. This rate is almost one third of the cancer diseases seen in children and adolescents. According to the German Child Cancer Data Bank in Mainz, acute lymphoblastic leukemia is detected in approximately 500 children and adolescents aged 0-14 years in Germany each year. ALL disease can be seen at any age, so it can also be seen in adults. Although L-asparaginase is one of the key drugs in this treatment, the total costs of the intensification course of 30 weeks is approximately between $57,893 and $113,558 (Tong et al., 2013). Even though, L-Asparaginase treatment has up to 80% success, the high cost of treatment causes many patients to be unable to complete the treatment. In this case, reducing the cost of the treatment by reducing the cost of production will be the effective method. In industrial processes, using industrial by-products as a substrate is a common approach for lowering the costs. But in the medical productions, one of the important parameter is that using by-products which are not harmful effects on patient. In order to L-asparaginase production, some agricultural wastes such as corn steep liquor, groundnut oil cake, wheat bran, coconut oil cake, soya bean, rice bran, sesame oil cake, orange peel have been reported as a substrate (Zia et al., 2012, Dias et al. 2015, Kumar et al. 2012, Vimal et al., 2017). Corn steep liquor (CSL), cheese whey (CW), molasses are easily available wastes in Turkey, however, there is no study of L-asparaginase production from A.terreus by using these agroindustrial residues. CSL is a by-product of starch manufacturing from maize and is rich in carbohydrates, nitrogen, vitamins, and minerals which are the main compounds for fungal growth. The typical composition of CSL is given in the additional documents (Okafor 2016). Main carbohydrates in CSL are lactose and glucose, which are effective carbon sources for A.terreus L-asparaginase specific activity (Farag et al., 2015). Also, it is an excellent source of organic nitrogen, which is influential in the fungal growth. Molasses is a by-product of the sugar industry, and therefore is an important carbon source. Two types of molasses are available in Turkey; sugar cane and beet. Compositions of them slightly differ, but both are quite rich in sugar (more than %50 (w/w)). Sucrose, glucose and fructose are the main carbon components of molasses. Detailed information of compositions is given in the additional documents (Okafor 2016). Whey is one of the most important by-products of milk technology. In general, the remaining yellowish green colored liquid is called cheese whey. The characteristics and composition of the whey depend on the cheese production technology and the quality of the milk used in cheese production. The run-off water contains approximately 93% water according to the average composition, and the rest is lactose, amino acids and peptides (Yerlikaya O. ,2010).
Corn is grown in many parts of Turkey, and so has a significant production volume of starch and starch based sugar production (Poyrazoglu 2007). Which means that the CSL waste produced will be proportional to the volume of production. Similarly, 843 thousand ton molasses are produced by the sugar industry in 2016, and it is expected that this value will increase in the next years (Turkseker 2016). According to statistical analysis, in 2016, more than 450 thousand ton cheese whey was produced in Turkey (USK, 2016). Each of these byproducts has significant components of L-asparaginase production, and they are easily available in Turkey market. Due to these advantages they are chosen for this project.
In addition, leukemia is the most common childhood cancer worldwide. Among the major types of leukemia, acute lymphoblastic leukemia (ALL) contributes to 80% of all leukemia cases. Today, North America dominates the global Asparaginase market in terms of value, owing to a significant revenue share of the region in the pharmaceutical industry. But there is still high demand for asparaginase in regions such as North America, Europe and Asia Pacific. When considered these situations asparaginase production will be an important investment for the Turkey.
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In this study, the effects of three different industrial by-products will be investigated to the L-asparaginase production potential and activity. A fungi, A.terreus, will be used as a producer. For this purpose, each substrate will be used at different concentrations in Czapek Dox’s liquid medium. Medium without byproduct will be used as a control. Also, using of whey instead of water will be investigated.
After optimum concentrations which are showed maximum enzyme activity are determined, anti-proliferative effects of enzymes on The human leukemia cell lines HL-60 (pre-myeloid leukemia) and RS4; (leukemia with lymphoid and myeloid characteristics) will be investigated.
The corn steep liquor, molasses and cheese whey will be used as substrate. They will be provided from corresponding production plants. The concentration of them will be varies from 1% to 6% in Czapek-Dox medium.
A loopful of A. terreus culture was transferred into modified Czapek Dox medium for preparation of fungal spore suspension. The medium contained glucose 2 g/L, L-asparagine 10 g/L, KH2PO4 1.52 g/L, KCl 0.52 g/L, MgSO4.7H2O 0.52 g/L, CuNO3, FeSO4. 7H2O and ZnSO4.7H2O in trace amounts and was kept in shaking condition at 120 rpm for four days to obtain homogenous spore suspension (1×10-6-10-8 spores/mL).
Fermentation and Harvesting of L-Asparaginase
Substrate screening will be carried out by inoculating 7% v/v freshly prepared Aspergillus terreus suspension at different concentrations of Czapek dox medium. Prior to use, the media will be sterilized at 121 °C for 15 minutes and the medium pH was maintained at 6.2. The inoculated flasks will be kept at 30 °C at different time periods in shaking incubator at 120 rpm. Harvesting of samples will be carried out after four days. The media will then centrifuged at 9000g for 10 minutes at 4 °C to obtain the clear supernatant containing L-asparaginase enzyme. The crude extract will then used for further analytical studies.
L-Asparaginase Enzyme Assay
Nesserlization method was used to check the enzyme activity. L-asparaginase hydrolyzes the L-asparagine to L-aspartic acid and ammonia. Ammonium liberated in the reaction will be determined by the Nesseler reaction. L-asparaginase unit of activity is defined as the amount of enzyme that liberated 1 mmol ammonia/min at 37 °C.
According to this method, the reaction mixture contained 1.7 mL of 0.01 M L-asparagine and 0.1 mL enzyme. The addition of 0.01 mL trichloroacetic acid (TCA) will be used to stop the reaction after 30 minutes of incubation at 37 °C. The amount of ammonia released will be determined calorimetrically at 480 nm by adding 0.5 mL Nessler Reagnet to tubes containing 0.5 mL supernatant and 7.0 mL distilled H2O mixture after centrifugation at (10,000 rpm, 5 min). The content in the tubes will be vortex and left to stand for 10 min. at room temperature. The control that contained only TCA without the enzyme will be used as the control. Protein will be estimated using the Biuret method with bovine serum albumin as the standard.
Cells will be seeded in 96-well plates at 1 × 104 cells per well. After 24 h, L-asparaginase will be added at concentrations of 12.5 ?g/mL, 25 ?g/mL, 50 ?g/mL, 100 ?g/mL and 200 ?g/mL. At different time points (48, 72, and 96 h) of continuous drug exposure, 10 ?l of XTT dye (3 mg/mL) will be added to each well. The plates were incubated for 2 h at 37°C and the formazan product will be measured at 450 nm by using an iMark microplate reader (Bio-Rad Laboratories). Cell survival will be calculated by subtracting the background absorbance of media alone and then dividing the absorbance of test wells by the absorbance of the control (untreated) wells.
The first step, fermentation and hervesting of enzyme, will be carried out at Bioprocess Laboratory of Marmara University Bioengineering Department. The filamentous fungus Aspergillus terreus MTCC 1782 used in the present study will be ordered from Institute of Microbial Technology, Chandigarh, India.
Second step, cell proliferation assay, will be carried out at Industrial Biotechnology and Systems Biology Laboratory of Marmara University Bioengineering Department. The Cell lines HL-60 and RS4 will be purchased from the American Type Culture Collection.
Expected Results and Benefits
The main objective of this study is that produce L-asparaginase with low immunogenicity using low cost substrates. For this purpose corn steep liquor, molasses and cheese whey which are easily available wastes in Turkey have been chosen as the substrates to be used. At the end of this work it is aimed to find a cheap carbon and nitrogen source for L-asparaginase production. It is aimed to provide a supply of the country’s economy, with exports of product which are in great demand in the world. This work is intended to be a reference to industrial production later.