Production of Tumor-Inhibitory L-Asparaginase by Submerged Growth of Serratia marcescens1

Abstract

Production of a tumor-inhibitory asparaginase by submerged fermentation with Serratia marcescens ATCC 60 was studied to ascertain optimal nutritional conditions for large-scale production leading to enzyme purification studies. Five strains of S. marcescens were screened in shake-flask studies and were found to produce 0.8 to 3.7 IU/ml 48 hr after inoculation. The requirements for asparaginase production with S. marcescens ATCC 60, the high producing strain, included the following: 4% autolyzed yeast extract medium (initial pH 5.0), an incubation temperature of 26 C, and limited aeration for a zero level of dissolved oxygen during the fermentation. Addition of various carbohydrates to the fermentation medium did not enhance yields. The peak cell population in the fermentation medium and the maximal asparaginase yields occurred simultaneously. Highest enzyme yields were found when the pH of the fermentation cycle rose to approximately 8.5. Yields of 4 IU of asparaginase/ml of cell suspension have been obtained consistently in 40 to 42 hr from 10-liter volumes (500 ml/4-liter bottle) produced on a reciprocating shaker. Scale-up to a 60-liter fermentor yielded 3.1 IU/ml in 35 hr.

Effect of chemical and physical parameters on the production of l-asparaginase from a newly isolated Serratia marcescens SK-07

“…In general, glucose was regarded as a repressor for l-asparaginase production in bacteria (Heinemann and Howard 1969;Barnes et al 1977;Mukherjee et al 2000). In the current investigation, the production of l-asparaginase was enhanced in the presence of l-asparagine.…”

Effect of Vitreoscilla hemoglobin on production of a chemotherapeutic enzyme, L‐asparaginase, by Pseudomonas aeruginosa

“…Glucose has been generally regarded as a highly potent repressor for the L-asparaginase production. Repression of L-asparaginase synthesis by glucose has been shown in bacteria such as Serratia marcescens [7], E. coli [15], and E. aerogenes [3], whereas opposite observations have also been seen for S. marcescens [26]. In line with these findings, glucose showed a slightly inhibitory effect on L-asparaginase synthesis by P. aeruginosa.…”

“…L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) is an enzyme of high therapeutic value due to its use in treatment of leukemia and other lymphoid malignancies [2]. Although it has been isolated and characterized from various microorganisms, the enzyme with the potential described above has only been determined in a few gram-negative bacteria [3][4][5][6][7]. In general, these bacteria produce two types of this enzyme, L-asparaginase I and L-asparaginase II.…”

“…Whereas L-asparaginase I is a constitutive cytoplasmic enzyme and its synthesis is almost unaffected by the growth conditions, L-asparaginase II is an inducible periplasmic enzyme, and its amount in the bacterial cell varies greatly depending on growth conditions [4]. Only L-asparaginase II, however, has substantial antitumor activity and is a successful drug in the treatment of acute lymphoblastic leukemia, leukemic lymphosarcoma and lymphosarcoma [7][8][9]. The term L-as-paraginase used in this study refers to type II enzyme unless otherwise indicated.…”

“…L-Asparaginase was reported to be synthesized during anaerobiosis, due to induction by the fumarate nitrate reduction protein, and positive regulation by the cAMP receptor protein [14]. Commercially, L-asparaginase is mainly produced by submerged or solid-state fermentation [7,16]. The importance of oxygen supply for microbial growth and product formation by microorganisms in these types of fermentations is well known.…”