A REVIEW ON BIOCHEMICAL AND THERAPEUTIC ASPECTS OF GLUTAMINASE

A REVIEW ON BIOCHEMICAL AND THERAPEUTIC ASPECTS OF GLUTAMINASE

Rahamat Unissa ^*, M. Sudhakar, A. Sunil Kumar Reddy and K. Naga Sravanthi

Malla Reddy College of Pharmacy, Maisammaguda, Dhulapally, Secunderabad - 500014, Hyderabad. India.

ABSTRACT: One of the prime candidates in the treatment of debilitating human cancers includes a family of enzymes referred to as L- glutaminases. The antitumor activity of these enzymes found effective in countering Acute Lymphoblastic Leukemia (ALL a commonly diagnosed pediatric cancer.). Further it is found to inhibit four human tumour cell lines namely Hep-G2 [Human hepatocellular carcinoma cell line], MCF-7 [Breast cancer cell line], HCT-116 [Colon cell line] and A549 [Human lung Carcinoma]. Apart from therapeutic use, it is having a number of other applications like in food industry, analysis and in production of fine chemicals. This review, hence, mainly focuses on the biochemical aspects of L-Glutaminase production, aiming to comprehend the physiochemical characteristics, application of L-Glutaminase and its properties. Processes central to these biochemical aspects, including submerged fermentation and Solid state fermentation of L- glutaminase producing organisms are also discussed.

L-Glutaminase, Submerged fermentation, Solid state fermentation, Acute lymphoblastic leukemia, Human hepatocellular carcinoma cell line, Breast cancer cell line

INTRODUCTION: L-Glutaminase (L-glutamine amidohydrolase E.C 3.5.1.2) is the enzyme that catalyzes the deamidation of L-glutamine to L-glutamic acid and ammonia ^{1, 2}. This is an essential enzyme for the synthesis of various nitrogenous metabolic intermediates ³. L-Glutaminase has received significant attention since it was reported extensively as antileukemic agent ^4-6. Unlike normal cells, leukemic cell do not demonstrate the L-glutamine synthetase, thus it is dependent on the exogenous supply of L-glutamine for their growth and survival ⁷.

Tumour cells have an absolute requirement for glutamine as a growth substrate. Glutamine is required as a precursor for both DNA synthesis and protein synthesis and may also be used as a respiratory substrate.

In experiments where glutamine metabolism in tumour cells has been specifically compared with that in non-transformed cells of the same origin, glutamine metabolism in the tumour cells has been found to be considerably faster. This is true for human hepatocytes and hepatoma cells ⁸. L-Glutaminase has received significant attention recently, owing to its potential applications in medicine as an anticancer agent and in food industries ^9-10. Microbial glutaminase have found application in several fields ¹¹. It has been tried as therapeutic agent in the treatment of cancer ^{2, 10} and HIV ¹². It is also used as an analytical agent in determination of glutamine and glutamate ¹⁴.

However one of the major use of microbial glutaminase in the food industry as a flavour enhancing agent ¹⁵_. L- glutaminase is generally regarded as a key enzyme that controls the delicious taste of fermented foods such as soy sauce ¹⁶. The gamma glutamyl transfer reactions of L-glutaminase is also useful in the production of the high marketed value specialty chemicals like threonine ¹⁴. To our knowledge, the reports on production of L- glutaminase from P. expansum are scanty. In recent years, L-Glutaminase in combination with or as an alternative to L-asparginase could be used as in enzyme therapy for cancer particularly leukemia¹⁴. Several attempts were made to produce glutaminase through genetic engineering.

This review focuses on various conditions implemented for the production of L-Glutaminase, in sub-merged fermentation and solid state fermentation, physiochemical characteristics and applications of L-Glutaminase. Biochemical characteristics and purification aspects of the enzyme are dealt with briefly. The aim of the review is to give an overview on microbial production of L-Glutaminase, hitherto.

FIG. 1: SYSTEMIC REPRESENTATION OF MECHANISM OF ACTION OF L-GLUTAMINASE

Occurrence and Distribution: L-Glutaminase plays a major role in the nitrogen metabolism of both prokaryotes and eukaryotes ¹⁸. It is found to be widely distributed in plants, animal tissues and microorganisms including bacteria, yeast and fungi ^{19, 20}. L-Glutaminase synthesis have been reported from many bacterial genera, particularly from terrestrial sources like E. coli ²¹, Pseudomonas sp²², Acinetobacter ²³ and Bacillus sp ²⁴. Although glutaminase have been detected in several bacterial strains, the best characterised were from members of Enterobacteriaceae family.

Among them E. coli, glutaminase have been studied in detail ²¹. However other members such as Proteus morganni, P. vulgaris, Xanthmonas juglandis, Erwnia carotovora, E. aroideae, Serratia marcescens, Enterobacter coacae, Klebsiella aerogenes and Aerobacter aerogenes ^{25, 19, 26} were also reported to have glutaminase activity. Among other groups of bacteria, species of Pseudomonas, especially, P. aeruginosa ^{27, 28} P. aureofaciens ^19, P. aurantiaca ^{22, 29}, and P. jluorescens ²⁰ are well recognised for the production of glutaminase. All these strains have been isolated from soil.

Among Yeasts, species of Hansenula, Cryptococcus, Rhodotorula, Candida scottii ¹⁹ especially Cryptococcus albidus ^{19, 20, 30}.

Cryptococcus laurentii, Candida utilis and Torulopsis candida were observed to produce significant levels of glutaminase under submerged fermentation. Saccharomyces cerevisiae was also shown to produce glutaminase ³¹. Species of Tilachlidium humicola, Verticillum malthoasei and fungi imperfecti were recorded to possess glutaminase activity ¹⁹_. Glutaminase activity of soy sauce fermenting Aspergillus sojae and A. oryzae were also reported ³²_.

Marine Microorganisms as source of L-Glutaminase: Reports on the synthesis of extracellular L-Glutaminase by marine microorganisms are very limited to marine bacteria including Pseudomonas jluorescens, Vibrio costicola and Vibrio cholerae ^{33, 34} and Micrococcus luteus ³⁵ and marine fungi Beauveria bassiana ³⁶ only.

Biological Role of L-Glutaminase in Normal Cells and Tumor Cells: Cancer cells, especially acute lymphoblastic leukemia (ALL) cells cannot synthesize L-Glutamine and hence demand for large amount of L-Glutamine for its growth. The use of amidases deprives the tumor cells from L- glutamine and causes selective death of L-Glutamine dependent tumor cells. L-Glutaminase can bring about degradation of L-Glutamine and thus can act as possible candidate for enzyme therapy.

Cancer cells require a robust supply of reduced nitrogen to produce nucleotides, non-essential amino acids and a high cellular redox activity. Glutamine provides a major substrate for respiration as well as nitrogen for the production of proteins, hexosamines, and macromolecules. Therefore, glutamine is one of key molecules in cancer metabolism during cell proliferation.

The notion of targeting glutamine metabolism in cancer, originally rationalized by the number of pathways fed by this nutrient, has been reinforced by more recent studies demonstrating that its metabolism is regulated by oncogenes. Glutamine can exert its effects by modulating redox homeostasis, bioenergetics, nitrogen balance or other functions, including by being a precursor of glutathione, the major non-enzymatic cellular antioxidant.

Glutaminase (GA) is the first enzyme that converts glutamine to glutamate, which is in turn converted to alpha-ketoglutarate for further metabolism in the tricarboxylic acid cycle. Different GA isoforms in mammals are encoded by two genes, Gls and Gls2.

As each enzymatic form of GA has distinct kinetic and molecular characteristics, it has been speculated that the differential regulation of GA isoforms may reflect distinct functions or requirements in different tissues or cell states. GA encoded by Gls gene (GLS) has been demonstrated to be regulated by oncogenes and to support tumor cell growth. GA encoded by Gls2 gene (GLS2) reduces cellular sensitivity to reactive oxygen species associated apoptosis possibly through glutathione-dependent antioxidant defense, and therefore to behave more like a tumor suppressor. Thus, modulation of GA function may be a new therapeutic target for cancer treatment ³⁷.

Media Optimization for Production of Glutaminases: Many studies have been done to optimize cultural conditions for L- Glutaminase production both in batch and continuous fermentation. Production of this enzyme depends on various parameters like concentration of carbon and nitrogen sources, pH of culture medium, temperature, fermentation time and oxygen transfer rate. It has been observed that these parameters vary for different organisms.

Properties of various microorganisms are mentioned in Table 1. Glutaminase is mostly obtained by submerged fermentation.

Effect of Additional Carbon and Nitrogen Sources on L-Glutaminase Production: Many L-Glutaminase producing microorganisms utilizes L-glutamine as both carbon and nitrogen sources and supplementation of any other carbon and nitrogen sources altered the L-Glutaminase production in most of the L-Glutaminase production fermentations. However, it was reported that addition of glucose enhanced the enzyme production in C. nodaensis ³⁸, Pseudomonas sp. ³⁹, Streptomyces rimosus ⁴⁰, Providencia sp. ⁴¹, T. koningii ⁴² and Beauveria sp. ¹⁷ Contradicting report on glucose – mediated suppression of the L-Glutaminase production in Achromobacteraceae ⁴³, S. maltophilia NYW-81 ⁴⁵ was also reported. Sucrose (Z. rouxii) and sorbital (B. bassiana BTMF S10) also supported L-Glutaminase production in addition to glucose.

This could be confirmed based on the fact that addition of these carbon sources resulted in enhanced L-Glutaminase production; Iyer and Singhal ³⁶. Different carbon sources namely glucose, maltose, sucrose, fructose, lactose and soluble starch at 1% (w/v) were added to the basal solid state fermentative medium of A. oryzae and they have exerted a considerable effect on the biosynthesis of L-Glutaminase. The maximum enzyme production was promoted by glucose followed by lactose and maltose.

The enhanced production of L-Glutaminase by the incorporation of glucose to the medium may be attributed to the positive influence of glucose as a co-metabolic agent ⁴⁶ for enhanced enzyme biosynthesis. These results were similar to those reported by production by Vibrio costicola by Prabhu and Chandrasekaran ¹⁰. And by Beauveria sp by Sabu et al., ¹⁷ Trichoderma koningii by Ashraf et al. ⁴⁷

Most of the L-Glutaminase producing microorganisms utilize the complex organic nitrogen sources rather than inorganic sources for effective enzyme production. It was noticed that addition of yeast extract enhanced the L-Glutaminase secretion by C. nodaensis ³⁸, B. bassiana BTMF S10 ³⁶ and Z. rouxii ^{48, 49}. While Siva kumar et al., ⁴⁵ observed that S. rimosus prefers the malt extract for higher L-Glutaminase production. Apart from complex nitrogen sources, inorganic nitrogen compounds such as ammonium sulphate and urea enhanced the enzyme production in Achromobacteraceae and Providencia sp. respectively ⁴³. However, Prabhu and Chandrasekaran ¹⁰ and Sabu et al., reported that none of the additional nitrogen source enhances the L-Glutaminase production in solid state fermentation ^{13, 17}. Among the various nitrogen sources, sodium nitrate in the medium promoted enhanced growth of microorganism and consequently the L-Glutaminase production, followed by malt extract and yeast extract .

These results were in similar to those reported by Prashanth Kumar et al. ⁵⁰ Incorporation of additional nitrogen sources enhance glutaminase yield. Among various nitrogen sources tested ammonium acetate was the best nitrogen source promoted maximum yield 853.84 U/ml for the Mucor racemosus strain. The least enzyme yielding nitrogen source was found to be sodium nitrite with an enzyme production of 538.46 U/ml ⁵¹.

The supplementation of additional nitrogen sources (either organic or inorganic) such as ammonium nitrate, ammonium sulphate, sodium nitrate, malt extract, yeast extract, urea and peptone had shown a profound impact on the production of L-Glutaminase by A. oryzae ⁵². Among the various nitrogen sources, sodium nitrate in the medium promoted enhanced growth of microorganism and 45.19U/gds of L-Glutaminase production was observed. These results were in similar to those reported by Prashanth Kumar et al., (2009) ⁵⁰.

Effect of pH: The pH and temperature tolerance of glutaminase from various microorganisms differed greatly. While optimal activities of glutaminase A and B of P. aeroginosa were at alkaline pH of 7.5-9.0 and 8.5 respectively ⁵³, glutaminase from Pseudomonas sp was reported to be active over a broad range of pH (5-9) with an optimum near pH 7.0 ⁵⁴. Glutaminase of Pseudomonas acidovorans showed optimum activity at pH 9.5 and retained 70% activity at pH 7.4 ⁵⁵.

An intracellular glutaminase from Cryptococcus albidus preferred an optimal pH of 5.5- 8.5 ²⁰.Whereas, glutaminase 1 and 11 isolated from marine Micrococcus luteus were active at alkaline pH values of 8.0 and 8.5 respectively ³⁵.

Glutaminase from A. oryzae and sojae recorded pH optima of 9.0 and 8.0 respectively ⁵⁶. The intra and extracellular glutaminase from A. oryzae were most active and stable at pH 9.0 ⁹. Glutaminase isolated from Penicillium brevicompactum NRC 829 showed its maximal activity against L-glutamine when incubated at pH 8 ^57.

Effect of Temperature: Microbial L-Glutaminase production is generally noticed at mild incubation temperature conditions ranging from 25 to 37 ºC. The temperature stability of glutaminases also showed wide variation. Glutaminase from Pseudomonas showed maximum activity at 37 ºC and were unstable at high temperatures ⁵⁸, whereas, the enzyme from Clostridium welchii retained activity up to 60 ºC ⁵⁹. Glutaminase from Cryptococcus alhidus retained 77% of its activity at 70°C even after 30 minutes of incubation ²⁰.

Glutaminase I & II from Micrococcus luteus had a temperature optima of 50°C and the presence of NaCl (10%) increased the 16 thermo stability ³⁵. The optimum temperature for activity of both intra and extracellular glutaminases from A. oryzae was 45 °C while they became inactive at 55 °C ⁹. Glutaminase isolated from Penicillium brevicompactum NRC 829 showed its maximal activity at 50 ºC, further increase in temperature at 70 ºC retained its activity indicates its thermostable nature ^57. L-Glutaminase obtained from Aspergillus oryzae revealed optimum activity in a temperature range of 37 to 45 ºC ⁶¹.

L-Glutamine was highly deamidated at 60 ºC by glutamine amydohydrolase enzyme partially purified from Penicillium politans NRC 510 ⁶⁰. Glutaminase isolated from P. brevicompactum NRC 829 indicated that no significant enzyme activity was lost when it was pre incubated at 50 ºC to 60 ºC for 60 min. L-Glutaminase retained about 92% of initial activity after incubation (in the absence of substrate) at 70 ºC for 30 min. Moreover, L-Glutaminase was still retaining about 66% of the original activity, after incubation at 80 ºC for 5 min, which revealed the high thermal stability of L-Glutaminase.

These results indicate the thermophilic nature of the purified amidase enzyme produced by P. brevicompactum NRC 829 ⁵⁷. L-Glutaminase purified from Aspergillus oryzae is stable up to 45ºC but lost its activity completely at 55 ºC ⁶¹. Prusiner et al., ²¹ performed the E. coli L-Glutaminase stability studies at low temperatures and the authors observed that the exposure of enzyme to cold temperatures resulted in a reversible inactivation of enzymatic activity, while subsequent warming to 24 ºC restored the activity and no protein denaturation occurred during this process.

Effect of Sodium Chloride: Sodium chloride was found to influence the activity of glutaminase from both fungi and bacteria of terrestrial origin. Salt tolerant capacity of various microorganisms is given in Table 2. Glutaminase from E. coli, P. fluorescence, Cryptococcus albidus and A. sojae showed only 65, 75, 65 and 6% respectively of their original activity in presence of 18% NaCl ²⁰. Similar results were obtained with glutaminase from Candida utilis, Torulopsis candida and A. oryzae ³¹. Salt tolerant glutaminase have been observed in Cryptococcus albidus and Bacillus subtilis ^{62, 63}.

Glutaminase I and II with high salt tolerance was reported from Micrococcus Iuteus K-3 ³⁵. High salt-tolerance of L-Glutaminase produced by Lactobacillus rhamnosus was reported ^64,65 where the presence of 5% (w/v) salt increased L-Glutaminase activity almost two-fold and 90% of the initial activity still remained at 15% (w/v) salt. On the other hand, L- glutaminases from other sources (Aspergillus oryzae) are markedly inhibited by high salt concentrations as demonstrated by Yano et al and Sabu A. et al., 2000 ^{9, 17}.

Effect of Various Substances and Heavy Metals: Glutaminase activity was found to be inhibited by various substances and heavy metals. Cetavlon, while accelerating glutaminase of Clostridium welchii, E. coli and Proteus moranii in crude extracts and intact cells, inhibited the purified enzyme ⁶⁶. Glutaminase of E. coli was found to be sensitive to heavy metals ⁴ and Acinetobacter glutaminase-asparaginase was inactivated by glutamine analogue 6-diazo 5-oxo L-norleucine even at very low concentration while unaffected by EDTA, NH₃, L-glutamate or L-aspartate ⁴³.

Various investigations have shown that glutaminase from Pseudomonas was activated by certain divalent anions and cations while inhibited by monovalent anions and by certain competitive inhibitors like NH₃, D and L-glutamic acid and 6-diazo 5-oxo L-norleucine ⁵³. In the case of fungi both intra and extracellular glutaminase from Aspergillus oryzae were inhibited by Hg, Cr and Fe but were not affected by sulphydroxyl reagents. EDTA, Na₂SO₄, and p-c Woromercuribenzoate strongly inhibited the Micrococcus luteus glutaminase I while glutaminase II was inhibited by EDTA, HgCh, Na₂SO₄, CuCh and FeCh ³⁹.

In case of glutaminases isolated from   P. brevicompactum Among, considerable loss of activity was observed only with Hg²⁺ and Cu²⁺ while Na⁺ or K⁺ acting somehow as an enhancer . EDTA has no effect on enzyme activity which indicates that L-Glutaminase might not be a metalloenzyme. L-Glutaminase is neither inhibited nor activated by reducing agents compounds including 2-mercaptoethanol (2-ME) and reduced glutathione (GSH) or thiol group blocking (namely iodoacetate) which indicates the absence of evidence for the involvement of SH group(s) in the catalytic site of this enzyme ⁵⁷.

TABLE 1: PROPERTY OF L-GLUTAMINASES FROM VARIOUS MICROORGANISMS