Enhanced Production of Novel Glutaminase Free Recombinant L-Asparaginase II of Erwinia Carotovora Subsp. Atroseptica Scri 1043 in Escherichia Coli BL21 (DE3) in a Batch and Fed-Batch Culture

Research Article

Austin J Biotechnol Bioeng. 2015;2(1): 1034.

Enhanced Production of Novel Glutaminase Free Recombinant L-Asparaginase II of Erwinia Carotovora Subsp. Atroseptica Scri 1043 in Escherichia Coli BL21 (DE3) in a Batch and Fed-Batch Culture

Rachna Goswami1,2, Venkata Dasu Veeranki1* and Krishnamoorthy Hegde1

1Department of Biotechnology, Indian Institute of Technology Guwahati, India

2Department of Bioscience, AP IIIT Nuzvid, Rajiv Gandhi University of Knowledge Technologies (RGUKT), India

*Corresponding author: Venkata Dasu V, Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.

Received: November 06, 2014; Accepted: December 03, 2014; Published: January 29, 2015

Abstract

L-asparaginase (E.C.3.5.1.1) is used for treatment of acute lymphoblastic leukemia. It is also used as a processing aid for reducing the formation of acrylamide in starchy foods preparations. In this study, the effects of glucose, controlled pH and Dissolved Oxygen Concentration (DOC) level on cell growth and production of novel glutaminase free recombinant L-asparaginase II of Erwinia carotovora subsp. atroseptica SCRI 1043, expressed in Escherichia coli BL21 (DE3) were investigated in a batch bioreactor using Taguchi experimental design technique. At optimum circumstances of glucose (1.5 g/l), controlled pH (7.0) and DOC (40%), the maximum dry cell weight and production of recombinant L-asparaginase II was found to be 1.84 g/l and 24.57 U/ml, respectively. Fed-batch culture is used frequently to increase expression of heterologous recombinant proteins in Escherichia coli. Therefore, the production of recombinant L-asparaginase II was performed in fed batch culture. In fed-batch fermentation, 7.35 g/l of dry cell weight and 96.78 U/ml of recombinant glutaminase free L-asparaginase II were achieved, corresponding to about four fold increase in dry cell weight and production as compared with the batch culture.

Keyword: Erwinia carotovora subsp. atroseptica SCRI 1043; Recombinant L-asparaginase II; Taguchi’s method; Batch and Fed batch

Introduction

L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been widely used for the treatment of acute lymphoblastic leukemia and non-Hodgkin’s lymphoma [1]. It is also used in food industry for acrylamide free food production [2] model enzyme for the development of new drug delivery system [3] and L-asparagine biosensor for leukemia [4]. The antileukemic effect of L-asparaginase is postulated to result from the rapid and complete depletion of the circulating pool of L-asparagine, as most of the cancer cells are dependent on an exogenous source of this amino acid for survival. However, normal cells are able to synthesize L-asparagine and thus are less affected by its rapid depletion due to treatment with this enzyme. The L-asparagine deficiency rapidly impairs the protein synthesis and leads to a delayed inhibition in DNA and RNA synthesis and hence an impairment of cellular functions, resulting in cell death [5,6]. Studies on the molecular structure [4], catalysis [7], clinical aspects [5], genetic determinants involved in regulation [8] and stabilization to enhance biological half-life [9] of L-asparaginase have been reported. Several gram-negative bacteria contain two L-asparaginases, a low affinity cytoplasmic enzyme and a highaffinity periplasmic enzyme. In Escherichia coli and several other bacteria, the synthesis of cytoplasmic asparaginase I is constitutive, while expression of periplasmic asparaginase II is activated during anaerobiosis. It has been suggested that the latter one probably has a special function in anaerobic fumarate respiration by providing aspartate, which is then converted to fumarate. Further, only the type II enzyme has shown substantial antitumor activity [10]. The various side effects of this drug are mainly due to the presence of partial glutaminase activity [11]. Hence, for successful clinical studies, glutaminase free L-asparaginase is highly desirable.

The production of L-asparaginase has been studied in Serratia marcescens [12,13], Erwinia carotovora [14], E. coli [15], Enterobacter aerogenes [16], Pseudomonas aeruginosa [17], and Bacillus subtilis [10] with various carbon and nitrogen sources. The synthesis of L-asparaginase by gram negative bacteria is regulated by environmental and nutritional factors. The results are contradictory in terms of the effect of glucose [12,15] and oxygen [16] on the production of this enzyme. On the other hand, the production of L-asparaginase from wild strain is very limited. Therefore, recombinant L-asparaginases were developed to increase the expression levels [5,8,9,18]. The interest in E. carotovora L-asparaginase II arose from its significantly lower glutaminase activity as compared to that exhibited by E. coli and E. chrysanthemi enzymes [5].

The production of any metabolite varies from shake flask to bioreactor fermentation. This might be possible due to uncontrolled pH, agitation pattern and aeration in shake flask fermentation. The strategy of studying one variable at a time and keeping all others at a predetermined level is very ineffective in many cases and also a time consuming technique [19,20]. Factorial designs are not preferred to optimize variables in bioreactors, as the experiments cannot be carried out in blocks. Additionally, they need more information to design parameter levels [21]. Taguchi’s method has been used in industrial process design, mostly in developmental trials. This method is used to produce adequate process information to set up the screening and optimal conditions of parameters for particular process using a minimum number of experiments possible. The basic principle of this technique serves as screening filters, which evaluate the effects of various process variables and identify those factors which have main effects on the process using less number of experiments [22].

Escherichia coli have been used most widely as a host system for the expression of recombinant proteins, as it was characterized in terms of its physiology, molecular genetics and expression systems [23]. The High Cell Density Culture (HCDC) techniques have been developed using E. coli systems for the production of recombinant proteins with high productivities [24-26].

The selection of nutrient feeding strategy plays a vital role in high cell density cultivations as it affects the metabolic pathway fluxes, and consequently influences the the specific productivity of recombinant proteins, formation of by-products (acetic acid) and cell growth. A significant relation was observed between the post-induction glucose feeding strategy and recombinant protein production [27,28]. The feed rate of glucose would be adjusted to control the formation of acetate in fed-batch cultures. To date, there is no rule established for selecting a particular feeding strategy to achieve maximum productivity for a given recombinant protein. Prior to carbon source, the nitrogen source is also very much important for extending the cell growth and improving protein production [29,30].

In this work, we investigated the influence of initial glucose concentration, controlled pH and Dissolved Oxygen Concentration (%) (DOC) level on the production of glutaminase free recombinant L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043 in E. coli BL21 (DE3) using Taguchi’s experimental technique in a batch bioreactor. Recombinant L-asparaginase II production was also performed in fed batch culture for enhanced productivity.

Materials and Methods

Chemicals

Isopropyl- ß-d-Thiogalactopyranoside (IPTG) and ampicillin were purchased from Sigma, India. Culture media and their constituents, L-asparagine, ammonium sulfate, were procured from Hi-Media, India. Nessler’s reagent was purchased from Loba Company, India. All chemicals were procured from Sigma unless otherwise stated and were of the highest quality.

Bacterial strains and plasmid

Erwinia carotovora subsp. atroseptica SCRI 1043 was kindly provided by Dr. Paul Birch, Scotland Crop Research Institute (SCRI), Scotland. Escherichia coli BL21 (DE3) and plasmid pET 22b(+) were purchased from Novagen, USA. The gene encoding L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043 was amplified by PCR, and introduced between BamHI and XhoI restriction sites of pET 22 b(+) in the downstream of the T7 promoter and resultant recombinant construct was transformed in E. coli BL21 (DE3) for recombinant L-asparaginase II expression [20]. The over-expression of cloned gene was controlled by T7 polymerase responsive promoter. The positive clone was confirmed by restriction digestion analysis and colony PCR (data not shown). It was maintained in 20% sterile glycerol at -80oC.

Inoculum development

A loopful of frozen glycerol stock culture (kept at -80°C) was streaked on a LB-agar plate containing ampicillin (100 μg/ml) and incubated at 37°C for 14–16 h. A single colony was isolated and transferred in 20.0 ml of sterile LB medium containing ampicillin (100 μg/ml) in Erlenmeyer flask (100 ml) on a rotary shaker at 37°C and 200 rpm for 6–8 h and this pre-inoculum was transferred at a rate of 2.34 % (v/v) to the inoculum medium (tryptone: 14.50 g/l, yeast extract: 5.30 g/l and NaCl: 4.03 g/l).

Optimization methodology for enhanced production of recombinant L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043 in batch bioreactor

In order to minimize the number of experiments and for medium components optimization, shake flask experiments were carried out by exploiting central composite design (data not shown). It is well accounted that production of any metabolite varies from shake flask to bioreactor fermentation. This might be because of uncontrolled pH, agitation pattern and aeration in shake flask fermentation. It is reported that on addition of glucose less than 0.05%, the plasmid stability and protein yields improves [20,31,32]. Therefore, Taguchi’s method was applied to know the parameters, which significantly affect the recombinant L-asparaginase II production in a batch bioreactor. Three parameters (glucose, controlled pH and DOC) in nine experiments were used to evaluate the influence of variables on recombinant L-asparaginase II production according to Taguchi’s orthogonal array. The parameters and their levels employed in Taguchi’s experimental design are mentioned in the Table 1. The glucose concentration and levels of controlled pH and DOC changed according to the experimental plan given in the Table 2. MINITAB® Release 15.1, PA, USA was used in the current study. In each experimental run, the result was recorded as the recombinant L-asparaginase II production (U/ml) and corresponding signal-tonoise (S/N) ratio was calculated by Eq. 1 with an overall objective for estimating the effects of a range of parameters on recombinant L-asparaginase II production, where a large S/N ratio is desired. S N =10log[ 1 y 2 n ] MathType@MTEF@5@5@+=feaaguart1ev2aqatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLnhiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr4rNCHbGeaGqiVCI8FfYJH8YrFfeuY=Hhbbf9v8qqaqFr0xc9pk0xbba9q8WqFfeaY=biLkVcLq=JHqpepeea0=as0Fb9pgeaYRXxe9vr0=vr0=vqpWqaaeaabiGaciaacaqabeaadaqaaqaaaOqaamaalaaabaGaam4uaaqaaiaad6eaaaGaeyypa0JaeyOeI0IaaGymaiaaicdaciGGSbGaai4BaiaacEgadaWadaqaamaalaaabaWaaSaaaeaacaaIXaaabaGaamyEamaaCaaaleqabaGaaGOmaaaaaaaakeaacaWGUbaaaaGaay5waiaaw2faaaaa@438C@