Microbial enzymes are identified to play a central role as metabolic catalysts, leading to their usage in various productions and applications , the end usage market for industrial enzymes is tremendously wide-spread with frequent industrial profitable applications (Adrio, et al., 2005). Over 500 industrial products are being complete using enzymes (Kumar, and Singh, et al., 2013). The request for industrial enzymes is on a incessant rise driven by a growing necessity for maintainable solutions.
Microbes have helped and continue to serve as one of the major and useful sources of numerous enzymes (Demain, and Adrio, et al., 2008). Many industrial processes, including chemical synthesis for manufacture of chemicals and pharmaceuticals . Furthermore, enzymes can be designated genetically and chemically-modified to improve their main properties: stability, substrate specificity and specific activity (Johnson, et al., 2013).
L-Glutaminase (L-glutamine amidohydrolase EC 3. 5. 1. 2) is known for its use as enzyme from diverse sources differ greatly in their possessions such as anticancer activity, immunogenicity, cytotoxicity, activity at physiological circumstances, plasma clearance rate, substrate specificity, temperature, pH and salt tolerance etc., there is a incessant search for L-glutaminase with properties appropriate for its use as antileukemic and/or flavor enhancing agent which plays a important role in the cellular nitrogen metabolism of together prokaryotic and eukaryotic cells (Sivakumar, et al., 2006).
In recent years, L-glutaminase has been deliberate due to their sole biotechnological flexibility and their ability to catalyst a wide spectrum of bioconversion responses of flavour compounds, L-glutaminase can be resulting from plant as well as animal foundations, microbial enzymes are commonly used for industrial determinations (Prakash, et al., 2009).
Another significant application of L- glutaminase is in biosensors for monitoring glutamine stages in mammalian and hybridoma cell cultures without the essential of distinct measurement of glutamic acid, L Glutaminase is usually observed as a key enzyme that controls the wonderful taste of fermented foods such as soy sauces (CruzSoto, et al., 1994).
On an industrial scale, L-glutaminases are produced largely by Aspergillus and Trichoderma sp. (Balagurunathan, et al., 2010). From an industrial opinion of view, filamentous fungi are chiefly stimulating as producers of L-glutaminase because they excrete significantly greater quantities of glutaminolytic enzymes into an extra cellular culture medium than bacteria or yeasts, industrially significant enzymes have conventionally been produced by submerged fermentation (SMF), But in recent years, SSF processes have been progressively used for the production of these enzymes. Interesting fact in SSF which added renewed notice from researchers in assessment of its economic and engineering advantages are cheap agro-industrial residues (Khandeparkar, et al., 2006). L-glutaminase has involved much care with respect to proposed applications in together pharmaceuticals and food. A diversity of microorganisms, comprising bacteria, yeast, filamentous fungi and molds have been described to produce L glutaminase (Iyer and Singhal, et al., 2008) of which the greatest potent producers are fungi (Balagurunathan, et al., 2010). L-glutaminase has recognized attention as a therapeutic in inconsistency of cancer and HIV (Rajeev Kumar, et al., 2003).
Other term of L-glutaminase Phosphate-activated glutaminase PAG is measured to be a mitochondrial enzyme, while PAG activity has also been create in nuclei in mouse tissue (Campos-Sandoval, et al., 2007).
For the production of field chemicals like theanine by c-glutamyl transfer reactions and as a flavour garnish in food industry (Renu, et al., 2003), the use of L-glutaminase as a flavour- garnish agent in Chinese foods has substituted the usage of monosodium glutamate which performances as an allergen for individuals (Jeon, et al., 2009).
1- History of L-glutaminase
The investigation work on L-glutaminase enzyme was originally started in the year 1956. The significance of L-glutaminase enzyme was accidently found when Alexander B. Gutman and Tsai-Fan, et al., (1963) were employed on the measurement of the total uric acid-N15 enrichment and the N15 profusion of each of the four uric acid nitrogen’s, originate that an abnormality of glutamine metabolism in primary gout is disguised. It is recommended that the abnormality in glutamine metabolism may have meaning for the pathogenesis of primary gout. Thus, from then the education on this enzyme was focused. In the following year, the outcome of a number of sulphonamide byproducts was studied on the combination of urea and on the activity of the enzyme L-glutaminase in vitro in liver slices and in isolated mitochondria .
Further (Goldstein, and Schooler, et al., 1967) found that variations in activity as well as level of L-glutaminase are significant in the regulator of ammonia synthesis in the kidney. In 1964 (Greenberg, et al., 1964) projected the anticancer activity of L-glutaminase. Later (El-Asmar and Greenberg, et al., 1966) noticed that L-glutaminase of Pseudomonas sp. capture the initial growth of numerous murine carcinomas but have slight effect on the survival time of animals.
Roberts, et al., (1970) found that L-glutaminase of a Gram negative rod-shaped bacterium repressed Ehrlich ascites carcinoma (one type of breast carcinoma). Imada, et al., (1973) extensively deliberate the distribution of L-asparaginase and L-glutaminase in several microorganisms. The authors confirmed 464 bacterial strains, 1326 yeast strains and 4185 fungal strains for L-glutaminase activity. They experiential that Bacillus sp presented L-asparaginase and L-glutaminase activity. Streptomyces and Nocardia genera have only intracellular amidases . In fungal strains Fusarium sp. and Pencillium sp. are ability to products the asparaginase, whereas Moniliaceae sp. produced L-glutaminase only without asparaginase activity. The ascomycetous fungi informed to be manufacturing either L-asparaginase or L-glutaminase built on substrate.
Among I326 yeasts, L-asparaginase or L-glutaminase followed frequently in certain serological groups of yeasts. Numerous strains of Cryptococcus and Rhodotorula group retain L-glutaminase and L-asparaginase. L-Glutaminase alone was designed in many strains of Candida scottii and Cryptococcus albidus, both of which are associated to Basidiomycetes from soya mash. Constructed on the therapeutic and food industry use of L-glutaminase many investigators worked on the several aspects of L-glutaminase. Still a lot of study exertion is going on in the world wide.
The structure of L-Glutaminase has been resolute using X-ray deflection to a resolution of up to 1.73 Å. There are 2 chains having 305 residues that formation the length of this dimeric protein. On both strand, 23% of the amino acid contented, or 71 residues, are found in the 8 helices. 21%, or 95 residues, make up the 23 beta sheet strands (Hashizume, et al., 2010) , that is in figure 1.
Figure 1: x-ray of Crystal structure of protein-glutaminase (Hashizume R, et al., 2010).
3- Mode of action of L-glutaminase
The family of amidohydrolase that catalyze the deamination of glutamine has two classes. The first class contains glutaminase, which is very specific for glutamine and catalyzes the hydrolysis of glutamate to glutamine in figure 2 (Elshafei, et al., 2014) , and catalyzes the hydrolysis of glutamine to glutamic acid (Wakayama, et al., 2005),figure 3. The second class has the enzyme that is fewer specific and catalyzes the hydrolysis of glutamine to glutamic acid and asparagine to aspartic acid with like efficiency and extensive substrate specificity, This is a class of amidohydrolase that consumes received significant attention as particular of them are being used in the management of leukemia mainly acute lymphocytic leukemia (ALL) (Sabu, et al., 2003).
Figure2: Schematic representation of mechanism of action of L- glutaminase (Elshafei, et al., 2014).
Figure 3: Schematic representation of mechanism of action of L- glutaminase (Wakayama, et al., 2005).
A main share of recent investigation on glutaminase has fixated on mammalian glutaminase, their biochemistry, regulation and genetic make-up, justly due to the character of glutaminase in mammalian metabolism. However, this situation is varying as a number of new trainings are attempting being devoted to attain an in depth information of regulatory, structural and biochemical features as well as the gene expression of glutaminases from many microbial sources. Still, there is sufficiently of room for examination on glutaminases including the isolation of salt-and thermo-tolerant enzymes, which would meaningfully enhance their applications in the food industry. Furthermore, a detailed sympathetic of the guideline of gene expression based on molecular methods and other incomes would contribute vastly towards developing successful approaches for strain enhancement which is a requirement for any industrially significant enzyme (Sarada, et al., 2013).
One of the furthermost significant roles of glutaminase is found in the axonal stations of neurons in the central nervous system. Glutamate is the most plentifully used excitatory neurotransmitter in the CNS. After being out into the synapse for neurotransmission, glutamate is quickly taken up by near astrocytes, which convert it to glutamine. This glutamine is then complete to the presynaptic terminals of the neurons, where glutaminases change it back to glutamate for loading into synaptic vesicles. Although together “kidney-type” (GLS1) and “liver-type” (GLS2) glutaminases are expressed in brain, GLS2 has been described to exist only in cellular nuclei in CNS neurons (Olalla, et al., 2002).
4- Characteristics of L-glutaminase Enzyme
4.1-Ion and Metals
The basic extracellular L-glutaminase produced by Pseudomonas aeruginosa strain CG-T8- II.1. performed optimally at pH 7,0 and unchanging at 37-45 °C. The enzyme could accept NaCl concentration up to 16% and 20%, and loosing the activity by 21% and 25.88%, respectively. The effect of metal ions Mg2+, Co2+ and Mn 2+ as Cl 2 salt increased the activity, while addition of Ca 2+ , Fe 3+ , and Zn 2+ reduced the activity (Al Hammed, and Jassim, et al., 2011).
4.2- Molecular weight
There has been varied difference in the molecular weight of L-glutaminases from diverse sources, which could series between 40 and 148 kDa. Based on intelligences the higher molecular weight (148 kDa) L-glutaminase was purify from P. aurantiaca (Imada, et al., 1973). The 40 kDa molecular weight L-glutaminase by P. nitroreducens (Tachiki, et al., 1996) is the lowermost molecular weight enzyme produced by the microorganisms. Most of the L-glutaminases are monomers, however, certain of the bacterial L-glutaminases are described as dimers and tetramers.
The optimum activities of L-glutaminase from Pseudomonas aeruginosa were at pH of 7.5-9.0 and 8.5 respectively. L-glutaminase from Pseudomonas species was stated to be active finished a broad range of pH 5-9 with an optimum nearby pH 7.0. An intracellular L-glutaminase from Cryptococcus albidus chosen an optimum pH of 5.5-8.5 (Sarada.K. V,. et al., 2013).
L-glutaminase had presented a wide difference in its temperature stability. L-glutaminase from Pseudomonas species presented maximum activity at 37°C and was not stable at high temperature, where as the enzyme from Clostridium welchii reserved activity at 60°C (Kozlov, et al., 1999). Glutaminase from Cryptococcus albidus taken 77% of its activity at 70°C even later 30 minutes of incubation. Glutaminase I and II from Micrococcus luteus had temperature ideals of 50°C and the attendance of sodium chloride (10%) increased the thermo-stability. But numerous of the L-glutaminases have described both an optimal and stable temperature of around 28-50°C.
L-glutaminase also varied in their capability towards L-glutamine and also has diverse iso-electric points. Many substances and heavy metals inhibit the enzyme, L-glutaminase activity (Sarada, et al., 2013).
4.5- Salt tolerant
Therefore, salt-tolerant glutaminase may show potentially important roles in food fermentation process that need high-salt environments. The two isozymes of Micrococcus glutaminase (I and II) were found to be highly salt tolerant. Glutaminase I was stable and exhibited about 1.3-folds developed activity in the presence of 8 – 16% NaCl than in the absence of NaCl, whereas glutaminase II was not stabilized and activated under the similar condition (Yoshimune, et al., 2010), Table 1 view different kind of microorganisms produce L-glutaminase and display their salt tolerant.
Table 1: Salt tolerance of L-glutaminases produced by various microorganisms
Activity % NaCl
Bacillus subtilis 90 16 Yokotsuka et al., 1987
Aspergillus oryzae 20 18 Yano et al., 1988
65 18 Shimizu et al., 1991
Pseudomonas fluorescens 75 18 Shimizu et al., 1991
Cryptococcus albidus 65 18 Shimizu et al., 1991
06 18 Shimizu et al., 1991
Micrococcus luteus I 130 16 Moriguchi et al., 1994
Micrococcus luteus II
100 16 Moriguchi et al., 1994
86 16 Wakayama et al.,
5- Production of L-glutaminase
5.1- Microbes for the production of l-glutaminase
Practically all living cells produce L-glutaminase but only convinced microbial strains have the probable for industrial production of this enzyme. It is universal from the presence point of opinion in plants, animals and microbes together in prokaryotes and eukaryotes. Among some well deliberate genera in microbes value mentioning from study viewpoint are E. coli, Pseudomonas sp., Brevibacterium sp., Vibrio costicola, Streptomyces gresius, Hypocrea jecornea, Streptomyces rimosus, Streptomyces avermitilis and Streoptomyces labedae, Zygosaccharomyces sp., Bacillus sp. and Acinetobacter species, Hansenula, Cryptococcus, Candida, Aspergillus oryzae and Beuveria bassiana ,Micrococcus luteus k, (Yoshimune, et al., 2010).
5.1.1- Advantages of microbial production of enzymes
Little fermentation time, cheap media, comfort of developing simple screening events, fast growth of microbes, biochemical variety, enzyme concentration may be amplified by environmental and genetic operation, flexibility of excellent of fermentation conditions, higher production rate (Thongsanit, et al., 2008) .
5.2- Production method of L glutaminase
Diverse methods of fermentation technology can be practical for the production of L-glutaminase. Commercially, L-glutaminase has been produced by submerged fermentation technique, but in fresh years, it is also presence produced under solid state fermentation technique, using usual (E.g. brans, husks, oil cakes etc) and inactive solid materials, e.g., polystyrene beads (Sarada, et al., 2013).
5.2.1- Submerged fermentation (smf)
Submerged fermentation (smf) involves of the cultivation of microbial cells in liquid media under measured conditions, in large vessels called bioreactors, for the production of required metabolites. (smf) suggestions advantages such as easy online monitoring of process parameters and process automation. Many of the culture medium components are common cheap substances containing nitrogen sources (e.g., ammonium salts, sodium nitrate) and carbon (e.g., glucose, sucrose, maltose), minerals and vitamins, and that can be supplied in uniform quantity. Table 2 shows the various bacteria, yeast and fungus used for the production of the L-glutaminase. The highest yields of enzyme are gained when cells are grown aerobically in a basal synthetic medium composed of L-glutamic acid, trace minerals, , phosphate buffer and ammonium sulfate. The writers experimental that the temperature between 15 to 25 oC is favourable to the organism growing and enzyme production.
Table 2: Various fermentation parameters for the production of L-glutaminase in submerged fermentation
Organism Carbon and nitrogen source Reference
Achromobacteraceae L-Glutamic acid 2.0% and
ammonium sulfate 0.4 % Roberts, et al.,
D-Glucose 3.0% and yeast Extract
0.5% Sato et al., 1999
Beauveria bassiana L-Glutamine 1.0 %, yeast extract
1.0% Keerthi, et al.,
L-Glutamine 2.0 % and D-glucose-
1.0 % Kumar and
Chandrasekaran, et al.,
Maltophilia L-Glutamine 1.0 %
Wakayama, et al.,
L-Glutamine 1.0%, Glucose 1.0%
and Malt extract 1.0% Sivakumar, et al.,
rouxii Sucrose 1.78%, yeast extract 4.8%
and glutamine 0.5 % Iyer and Singhal, et al.,
Providencia sp Glucose 1.0 % and urea 0.5% Iyer and Singhal, et al.,
rouxii Sucrose 1.78%, yeast extract 4.8%
and glutamine 0.5 % Iyer and Singhal, et al.,,
5.2.2- solid state fermentation (ssf)
The authors detected that solid-state fermentation was desirable to (smf) for L- glutaminase production in terms of produce efficiency, since 25 to 30 fold increase in enzyme production was found under (ssf). For the production of L-glutaminase in (ssf) several agro industrial materials were used as solid provision. Table 3 shows the various bacteria, yeast and fungus used for the production of the L-glutaminase. Many authors described that wheat bran was found to be a better support for enzyme production (Sayad, et al., 2009). Separately from the wheat bran, copra cake powder , rice bran, sesamum oil cake and ground nut cake powder were used as solid substrates for enzyme production (Prabhu and Chandrasekaran, et al., 1996). However impregnated with mineral salts ,Polystyrene beads, and glutamine were used as solid substrate for glutaminase production. Renu and Chandrasekaran, et al., (1992) observed that Pseudomonas fluorescens, Vibrio cholerae, and Vibrio costicola, from among the strains partitioned from marine environments of Cochin, produced L-glutaminase extracellularly in copious amounts. Process conditions for large-scale production of this enzyme were optimized in solid-state fermentation. L-Glutaminase was found to be encouraged by L-glutamine.
The capacity to adsorb onto polystyrene appears to be a basic property of marine bacteria. In their natural environment, numerous species of marine bacteria exist only lower than adsorbed conditions on debris or solid substrates. In another search by (Prabhu and Chandrasekaran, et al., 1997), the finest process parameters inducingL -glutaminase production by marine V. costicola in solid-state procedure using polystyrene as an inert sustenance were optimized. Maltose and potassium dihydrogen phosphate improved enzyme yield by 23% and 18%, respectively, while nitrogen sources had an inhibitory result. As in the earlier training, leachate with high L-glutaminase exact activity and low viscosity was improved. In the research by (Sabu, et al., 2000) the probable of Beauveria sp. for L-glutaminase making using polystyrene as solid support under solid-state method was assessed. In recent trainings by (Sayed, et al., 2009) found that wheat bran was the greatest solid substrate for initiation of the L-glutaminase by Trichoderma koningii.
Table 3: Various fermentation parameters for the production of L-glutaminase in solid state fermentation
Organism Solid Substrate pH Medium
Polystyrene beads 7 35 Prabhu and
Chandrasekaran, et al.,
BTMFS 10 Polystyrene beads 9 27 Sabu, et al.,
rouxii Wheat bran and sesame
oil cake normal 30 Kashyap, et al.,
elegans Soya bean curd normal 25 Han, et al., 2003
oligosporus Soya bean curd normal 35 Han, et al., 2003
koningii Wheat bran 7 30 EL-Sayed, et al., 2009
220.127.116.11- Effect of moisture content
Primery moisture contented is a main factor in the L-glutaminase enzyme production, Moisture optimization can be used to control and to modify the metabolic action of the microorganism (Pandey, et al., 1999), this could be proficient by the quicker growth of microorganism at higher moisture content and the following first initiation of the enzyme production.
18.104.22.168- Effect of nitrogen sources
Since first biotechnological route is likely to be based on basic enzymes, choice of best nitrogen sources is significant to increase their activities in the culture supernatants. As far-off it is concerned, nitrogen can be an important regulating factor in the microbial production of enzymes. (El-Sayed, et al., 2009).
22.214.171.124- Effect of metal ions
When diverse metal ions were used in the (ssf) medium, the maximum enzyme activity was obtained in minimum at KCl (24 U/g) and NaCl (52 U/g). Later the enzyme production could have resulted in well utilization of metal ions, which enhanced the L-glutaminase production. Similar outcomes of inhibition of glutaminase activity by the adding of metal ions were described by (Prabhu and Chandrasekaran, et al., 1996) .
126.96.36.199- Effect of additional carbon and nitrogen sources
Effect of several carbon sources (Glucose, Fructose, Galactose, Lactose, Maltose, Xylose, Sucrose, Mannitol), mixtures of carbon sources and inorganic nitrogen (Ammonium sulphate, ammonium nitrate and ammonium chloride) sources at diverse concentrations and organic nitrogen sources (peptone, corn steep liquor beef extract, yeast extract and soya bean meal) at 0.5% concentration were calculated on the production of l-glutaminase by Pseudomonas sp. KLM9 (Nathiya, et al., 2012).
188.8.131.52- Economical and industrial advantages of ssf
Solid state fermentation (ssf) offers several advantages over other conventional fermentations, such as (smf) etc. (ssf) was lead for the production of L-glutaminase using different agro- industrial byproducts including wheat bran, groundnut residues, sesamum oil cake, rice hulls, soya bean meal, corn steep, cotton seed residues and lentil industrial remains as solid substrates. Wheat bran was the greatest substrate for induction of L-glutaminase (Chanakya, et al., 2010). The major advantages include higher product yields, lesser capital and recurring expenditure, minor waste water output/less water need, reduced energy requirement, nonappearance of foam formation, simplicity, high reproducibility, humbler fermentation media, Smaller fermentation space, absence of rigorous regulator of fermentation parameters, economical to usage even in smaller scales, easier control of pollution, applicability of using fermented solids directly, storage of dried fermented matter, minor cost of downstream dispensation (EL-Sayed, et al., 2009).
184.108.40.206- Disadvantages of ssf
Problems commonly associated with solid state fermentation are heat build-up, bacterial contamination, scale-up, biomass development estimation and control of process parameters (Sarada, et al., 2013) .
5.2.3- Immobilized cells
Immobilization of cells can be defined as the attachment of cells or their inclusion in a distinct solid phase that permits exchange of substrates, products, inhibitors, and so forth but at the same time separates the catalytic cell biomass from the bulk phase containing substrates and products. Cells entrapped in either sodium alginate or agar are widely used (Kashyap, et al., 2002).
6- Sources of L- glutaminase
L-glutaminase activity is widely distributed in plants, animal tissues and in microorganisms counting bacteria, yeast and fungi. Over a insufficient decades, considerable research has been undertaken with the microbial production of extracellular L-glutaminase. The major advantage of using microorganisms for the production of L-glutaminase is the economical bulk production capacity and also microbes are easy to manipulate to obtain enzymes of desired characteristics (Sarada, et al., 2013).
A variety of microorganisms, including bacteria, yeast, moulds and filamentous fungi have been reported to produce L-glutaminase (Iyer and Singhal, et al., 2008) of which the most potent producers are fungi (Balagurunathan, et al., 2010). On an trade scale, glutaminases are produced mostly by Aspergillus and Trichoderma sp. (Pallem et al., 2010). produced by actinomycetes (Sunil Dutt, et al., 2014). L-Glutaminase is ubiquitous and present in many animal tissue, plants and widely distributed in large number of microbes (Turner, and Mcgivan, et al., 2003).
Terrestrial microorganisms such as Escherichia coli, Pseudomonas species, Acinetobacter sp., Bacillus sp., Hansenula, Cryptococcus, , Aspergillus oryzae, Candida and Beuveria bassiana were earlier reported for L-glutaminase synthesis , Not only this even L-glutaminase activity was reported from marine microorganisms such as Micrococcus luteus Pseudomonas fluorescens, Vibrio cholerae and Beuveria bassiana , and also from marine actinomycetes (Balagurunathan, et al., 2010) that is show in table 4. L-glutaminase production from seaweed associated microbes. The main objective of this study is to isolate industrially important glutaminase enzyme from endophytic fungi of seaweed( Sabu, et al., 2003).
L-glutaminase is expressed and active in periportal hepatocytes, where it generates NH3 (ammonia) for urea synthesis, as does glutamate dehydrogenase (Van Noorden, et al., 2014). Amongst bacteria, E.coli glutaminases have been calculated in much detail. Some of the examples of bacterial strains producing L-glutaminase include E.coli, Pseudomonas P.aureofaciens; Acinetobacter sp, Klebsiella aerogens, Bacillus sp, Erwinia caratovora, Aerobacter aerogens etc. L-glutaminase from the members of Enterobacteriaceae family has been best characterized among the bacterial genera. Among the fungal species, Aspergillus oryzae, Aspergillus sojae, Beauveria sp, Tilachlidium humicola, Trichoderma koningii, Verticillium etc., have been described to produce L-glutaminase.Among yeast species of Hansenula Candida utilis, Torulopsis sp., Zygosaccharomyces rouxii and, Rhodotorula, Candida scotii, Crytococcus albidus, etc have been reported to produce the enzyme L-glutaminase (Sarada, et al., 2013).
Table 4: Microbial sources of L-glutaminase
Organism HABITAT REFRENCE
Terrestrial Holchenberg, et al., 1985
Terrestrial Cook, et al., 1981
Bacillus subtilis Terrestrial Shimazu, et al., 1991
Terrestrial Wade, et al., 1971
Marine Moriguchi, et al., 1994
Terrestrial Sabu, et al., 2000a
Pseudomonas aurantica Marine Lebedeva, et al., 1989;
Terrestrial Eremenkov, et al., 1975
Pseudomonas nitroreducens Terrestrial Tachiki, et al., 1996
Marine Kumar, et al., 2001
Iyer and Singhal, et al., 2009
Vibrio costicola Marine Nagendraprabhu and Chandrasekaran, et al., 1996
Actinomucor elegans Terrestrial Chou, et al., 1993
Terrestrial Lu, et al., 1996
Terrestrial Jones and Lovitt, et al., 1995
Terrestrial Sabu, et al., 2000
Terrestrial Kumar, et al., 2009
Aspergillus sp Terrestrial Van den Broek and Affolter, et al., 1999
Marine Sabu, et al., 2000
Terrestrial Sayed, et al., 2009
Terrestrial Sabu, et al., 2000
Terrestrial Kakinuma, et al., 1987
Terrestrial Fukushima and Motai, et al., 1990
Terrestrial Kakinuma, et al., 1987
Terrestrial Sato, et al., 1999
Rhodosporidium toruloides Terrestrial Ramakrishnan and Joseph, et al., 1996
Terrestrial Abdumalikov, et al., 1967
Terrestrial Sabu, et al., 2000
Torulopsis candida Terrestrial
Kakinuma, et al., 1987
Marine Iyer and Singhal, et al., 2010
7- Purification of L-glutaminase
Solid ammonium sulfate was slowly added to the crude enzyme filtrate with gentle stirring to bring 40% saturation (fraction I). The mixture was allowed to stand overnight at 4°C. It was centrifuged at 10,000 rpm at 4°C for 20 min to remove the precipitate while the resulting supernatant was subjected to the addition of ammonium sulfate until reached to the concentration 50% saturation (fraction II), then it was allowed to stand at 4°C and the resulting precipitate was obtained by centrifugation at 10,000 rpm at 4°C for 20 min (Kashyap, et al., 2002).The resulting supernatant was further subjected to ammonium sulfate precipitation to bring 80% saturation (Fraction III) in a sequential manner as previously described. The enzyme precipitate obtained from each saturation was dissolved in a minimal volume of 0.01M phosphate buffer (pH 8) and dialyzed against 0.01M phosphate buffer (pH 8) for 48-72 h at 4°C and the buffer were changed occasionally.
7.1- Ion-exchange chromatography
Anion-exchange DEAE-cellulose (Diethylaminoethyl-cellulose) chro-matography was performed for further purification of the L-gluta-minase enzyme obtained from the previous ammonium sulfate pre-cipitation (Kashyap P, et al., 2002).
DEAE cellulose chromatography recovery and Purification of L-Glutaminase Product recovery during any bioprocess is difficult since the diluted and labile products of interest are always mixed with macromolecules of similar properties. Based on the production technique, downstream processes can be designed. It is usually achieved by filtration, centrifugation, and precipitation, and also by various chromatographic procedures such as ion exchange, gel permeation, and attraction chromatography. To confirm purity of the enzyme and to determine molecular weight, electrophoresis is performed using denaturing polyacrylamide gel electrophoresis (SDS-PAGE) (Kashyap, et al., 2002).
7.2- Heat treatment
The crude enzyme extracts were heated at 50ºC for 20 min, the tube was immediately cooled in ice bath and the sediment formed was removed by cooling centrifugation at 5500 rpm (- 4ºC) for 10 min (Elshafei, et al., 2014).
7.3- Gel filtration
7.3.1- Sephadex G-100 gel filtration
The most active partially purified enzyme fraction from the previous step was applied on a Sephadex G-100 column (1.5 x 50 cm) that was pre-equilibrated with a 0.05 M boric acid borate buffer pH 8.0 at a course rate of 20 ml/hr. The fractions were collected and examined for enzyme activity and protein content, (Elshafei, et al., 2014). The most active fractions were pooled together, dialyzed against the 0.01 M boric acid borate buffer (pH 8.0), and intense by lyophilization (-50ºC).
7.3.2- Sephadex G-200 gel filtration
The purified fraction obtained from the previous step was loaded onto the pre-equilibrated Sephadex G-200 column (2.0 x 50 cm) with 0.05 M boric acid borate buffer (pH 8.0), at a flow rate of 10 ml/h. The fractions were collected and examined for L-glutaminase activity and protein content (Elshafei, et al., 2014).
7.4- Acetone Fractationation
Acetone and enzyme (both with glucose and without glucose) was retained for a day and future centrifuged (Anubrata Paul, et al., 2015) .
7.5- Ammonium Sulphate Precipitation
L-glutaminase having glucose got saturated at 80%, while the enzyme (without glucose) got saturated at 95% , the enzymes obtained later ammonium sulphate precipitation was further purified by membrane dialysis and then exposed to purification by ion exchange chromatography (Anubrata Paul, et al., 2015) .
8- Application of L-glutaminase
8.1- Clinical Application of L-glutaminase
8.1.1- L-Glutaminase in Acute Lymphocytic Leukemia (ALL)
Microbial L-glutaminase activity has been identified originally as an anti-cancer drug. Several old studies were oriented towards using a native or a modification of L-glutaminase in cancer therapy; especially against adult leukemia, such as acute lymphoblastic leukemia (Bülbül, and Karakuş, et al., 2013) ,L-glutaminase also exhibits an anti-leukemic activity.Unlike normal cells, leukemic cells does not depend on L-glutamine synthetase, they directly depend on the exogenous supply of L-glutamine from the blood for their growth and survival. Therefore, blood L-glutamine serves as a metabolic precursor for the nucleotide and protein synthesis of tumor cells .Consequently, L-glutaminase sources selective death to L-glutamine dependent tumor cells by blocking the energy route for their proliferation (Sarada, et al., 2013). The enzyme L-glutaminase inhibits the proliferation as well as causes the death of leukemic cells by depriving them from L-glutamine supply (Young, and Ajami, et al., 2010).
Successful in vivo inhibition of tumor growth had been reported by this enzyme , Crystallization of L-glutaminase from microbial origin also reported, This enzyme is used in acute leukemia treatment , In vivo safety evaluation studies of yeast glutaminase also found to be non toxic (Oshita, K. et al., 2000). Cytotoxic studies of L-Glutaminase were found to be toxic to tumor cells and shown antioxidant activity (Nathiya, K. et al., 2012).
8.1.2- L-glutaminase in HIV therapy
One of the most therapeutic applications of L- glutaminase is in the inhibition of melanoma and DNA biosynthesis in affected cells. Such an application is proposed for the treatment of human immunodeficiency virus (HIV) by administrating L-glutaminase from Pseudomonas sp. 7A. The result will be a substantial reduction of the reverse transcriptase activity in human serum, which helps to inhibit HIV replication in infected cells. This unique approach can be applied to other pathogenic viruses, depending upon the identification of nutritional requirements for viral (Sarada, et al., 2013 ).
8.1.3- Anticancer activity of L-glutaminase
In most tumours glutamine is the primary mitochondrial substrate, and cancer cells display an addiction to glutamine (Wise, et al., 2010), consuming 15 times the amount of glutamine consumed by other cells under hypoxic conditions (Anastasiou, et al., 2012). Though cancer cells consume more amounts of glutamine, they are incapable of producing their own glutamine de novo (Wise, et al., 2010). while normal cells can do so. Hence, a strategy that reduces blood glutamine levels using glutaminase would control the growth of cancer cells under hypoxic conditions. Besides its applications in cancer therapy.
The rapid growth, support cell mass buildup, nucleic acid biosynthesis and mitotic cell division of cancer cells is permitted by metabolic variations, particularly increased aerobic glycolysis and increased glutaminolysis, fatty acid synthesis (Vander, et al ., 2009). Many experiments showed that cancer cells depends on glutamine,which is an important amino acid, has been implicated in the control of the growth and the proliferation of the normal and tumor cells, also served as the carbon source for the TCA cycle, substrate for glutathione synthesis (Figure. 4) and as vesicle for the transport the nitrogen and carbon to different tissues of living organisms (Deberardinis, et al., 2008).
Figure 4: Metabolic of cancer cells compared to normal tissue (Yuneva, et al ., 2009)
8.2- Application of L-Glutaminase in Food Industry
L-Glutaminase is considered an important agent as flavor enhancers in the food industry, due to its involvement in the synthesis of L-glutamic acid, the main compound is responsible for the delicious taste or flavor and aroma of many fermented products like soy sauce, miso ,sufu and popular (Sabu , et al., 2004). Glutamic and aspartic acids are both known essential amino acids contributing not only fine taste of food, and a sharp sour taste but also is improved the functional properties—such as solubility, viscosity,gelation, fat emulsification, and foaming—by increasing the number of negative charges in the protein (Sarada, et al., 2013).
Several reports used the microbial L-glutaminases either immobilized L-glutaminase or whole cells of L-glutaminase producing microbes in food flavoring for continuous conversion of glutamine to glutamate in food preparations (Hamada, et al., 1991).
8.2.1- L-glutaminase in industrial process as salt tolerant
Salt tolerant L-glutaminases is the most important in the industrial processes that required a high salt environment (Sabu, et al., 2000). L-glutaminases from conventional sources (A. oryzae) are halo tolerant and markedly inhibited by high salt concentrations as demonstrated by (Yano, et al., 1988) therefore (Moriguchi, et al., (1994) have reported that the use of halophilic L-glutaminase from bacteria as a possible alternative, and allowing for the use of high salt concentrations.
Recently, many studies directed toward cloning, purification and characterization a novel salt-tolerant genes such as, glutaminase-encoding genes, CngahA, CagahA from Cryptococcus spp. and novel peptidoglutaminase a sparaginase from A. sojae, because it plays an important role in the production of fermented foods (Ito, et al ., 2012). for the production of specialty chemicals like theanine by c-glutamyl transfer reactions and as a flavor enhancer in the food industry (Sivakumar, et al., 2006). The use of L- glutaminase as a flavour-enhancing agent in Chinese foods has replaced the use of monosodium glutamate which acts as an allergen for individuals (Jeon, et al., 2009).
8.3- Analytical Applications
L-Glutaminases are used both in free or immobilized enzymes on membranes forms as biosensors for monitoring glutamine and glutamate levels of fluids (Villarta, et al.,1992). Kikkoman corporation company, Japan is produced L-glutaminase from Bacillus sp. and then used in clinical analysis for determination of glutamine in conjunction with L-glutamate oxidase and peroxidase. The L-glutaminase can be used as biosensor to determine the L-glutamine stages in mammalian cell culture media (Balagurunathan, et al., 2010).
A highly purified L-glutaminase enzyme from mammalian source is important in clinical diagnostics and health monitoring. It can be used to analyze L-glutamine and glutamate the levels in the body fluids (Sarada, et al., 2013).
Tachiki, et al., (1998) developed a method for producing theanine (γ-l-glutamyl ethylamide) from glutamate and ethylamine by using a combination reaction of bacterial glutaminases with baker’s yeast. Generally, theanine is one of the major components of Japanese green tea. It\’s synthesized by theanine synthetase in plants. Recently, attention has been increased towards the clinical physiological roles of theanine. This is because of its ability to suppress the stimulation by caffeine to improve effects of antitumor agents, and their role as antihypertensive agents (Li, et al., 2013). The continuous production of threonine is done by using the immobilization cells of P. nitreducens as a source of L-glutaminase (Abelian, et al., 1993).