Microorganism and inoculum preparation
The extremely halotolerant Halomonas elongata MM-5 (Accession No: MT039456.1) isolated from the salt pan was used in this investigation. The pure culture of Halomonas elongata MM-5 was maintained on slants on media consisting of glucose - 5.0, L-glutamine-5.0, Na2HPO4- 6, KH2PO4-3, MgSO4 ; 7H2O- 0.49, CaCl2- 0.01, NaCl-50, Agar-20 g/L of pH-8. The slants after streaking were incubated at 37°C for 72 h in static condition for the growth of isolate and were kept at 4°C in the refrigerator until further use. The development of inoculum was done in 250 ml Erlenmeyer flask containing 100 ml of sterile aforementioned medium (pH-8) and incubated at 37°C.
Collection of substrates
Abundantly available various agro-industrial byproducts including rice husk (RH), bengal gram husk (BGH), green gram husk (GGH), red gram husk (RGH), safflower oil cake (SOC), groundnut oil cake (GOC), black gram husk (BgH), groundnut skin (GS) and wheat bran (WB) were collected from the local market and processed.
Screening of various substrates for L-glutaminase production under SSF
10gms of each processed substrate as exclusive production medium was added individually in 250ml Erlenmeyer flask, moisturized with distilled water (60%), mixed thoroughly, sterilized by autoclaving at 121°C for 20 minutes, cooled at room temperature (RT) and inoculated. After inoculation, the flasks were kept for fermentation at 37ºC in an incline position to provide a large surface area (Athira et al. 2014).
The extraction of the crude enzyme from the fermented substrates was carried out by means of simple contact method (Sathish et al. 2008). In 1:5(w/v) ratio, 0.1 M phosphate buffer of pH 7.0 was added to the fermented solid medium, mixed properly and kept at 150 rpm for 30 min on a rotary shaker. Later to recover the leachate the contents were squeezed in cheesecloth. The process was carried out twice, the extracts were combined and centrifugation was carried out for 15 min at 10,000 rpm in a cooling centrifuge after it collected the supernatant and used as the crude enzyme.
Nesslerization method was used to determine the activity of L-glutaminase (Imada et al. 1973). In the assay, 500 μl of crude enzyme was allowed to react with 500 μl of 0.04 M L-glutamine, 500 μl of distilled water and 500 μl of phosphate buffer of pH 7.2 and kept for incubation for 30 minutes at 37ºC. The reaction was terminated by adding 500 μl of 1.5 M trichloroacetic acid (TCA). Reaction mixture (100 μl) was added to 3.7 ml of distilled water. Nessler’s reagent (200 μl) was added, allowed to incubate for 15 minutes and optical density was recorded at 450 nm. One international unit of the enzyme was defined as the amount of L-glutaminase that releases 1 μmole of ammonia under standard assay conditions. L-glutaminase yield was expressed as international units per gram of dry substrate (IU/ gds).
Screening of mixed substrates
For optimization of the substrate composition different combinations of the substrates were taken in the percents as below.
1) Red gram husk: safflower oil cake (60:40)
2) Red gram husk: groundnut oil cake (60:40)
3) Red gram husk: bengal gram husk (60:40)
4) Red gram husk: groundnut skin (60:40)
5) Red gram husk: safflower oil cake: bengal gram husk (60:20:20)
6) Red gram husk: groundnut skin: bengal gram husk (60:20:20)
7) Red gram husk: wheat bran: bengal gram husk (60:20:20)
8) Red gram husk: groundnut skin: wheat bran: bengal gram husk (40:20:20:20) and
9) Red gram husk alone (100%).
10gms of each above combination of substrates were added separately in 250ml Erlenmeyer flasks, moisturized with distilled water (60%), mixed thoroughly and sterilized. After allowing to cool at room temperature (RT), these flasks were inoculated and kept for incubation at 37ºC in the incline position.
Optimization of various parameters
The different parameters optimized in the present study were fermentation time, pH, temperature, moisture content (%), concentration of L-glutamine, effect of carbon sources, nitrogen sources and effect of inoculum volume. The method utilized for the optimization of different process parameters affecting L-glutaminase production under SSF was to assess the influence of a single parameter (Sandhya and Lonsane 1994; Mohammed et al. 2019a).
The influence of the incubation period on L-glutaminase synthesis under SSF was investigated by incubating the flasks after inoculation for 7 days period and measuring the L-glutaminase production regularly after 24 h up to 7 days.
Initial pH of the medium
The influence of initial pH on L-glutaminase synthesis ability of Halomonas elongata MM-5 was investigated by adjusting the pH of the solid medium in the range 5–10 with different buffers.
The influence of temperature on L-glutaminase synthesis ability of Halomonas elongata MM-5 under SSF was studied. The active culture of Halomonas elongata MM-5 was inoculated in solid medium and incubated at different temperatures (10– 60°C).
Initial moisture content
To investigate the influence of the initial moisture content of the solid substrate medium on L-glutaminase yield the substrates were moisturized with different moisture levels in the range of 40–200%. This was carried out by changing the volume of buffer added to moisturize the solid substrates. The prepared flasks were autoclaved, inoculated, mixed thoroughly and kept for incubation at 40°C (VivekBabu and Lingappa 2004).
Effect of carbon sources
To study the influence of carbon sources on L-glutaminase production various carbon sources including glucose, xylose, fructose, maltose, dextrose, lactose and sucrose were screened. These carbon sources were supplemented at 1% concentration in solid substrate medium (Bhosale et al. 2015).
Effect of nitrogen sources
The influence of various organic and inorganic nitrogen sources such as beef extract, yeast extract, malt extract, tryptone, skimmed milk, urea, ammonium chloride and ammonium sulphate at 1% concentration on the yield of L-glutaminase was examined.
Effect of L-glutamine concentration
The effect of L-glutamine concentration on L-glutaminase synthesis under SSF by Halomonas elongata MM-5 was examined by adding L-glutamine in different concentrations (0.5 – 3.0 % w/w) in the solid medium.
The amount of inoculum which may provide more L-glutaminase yield was analysed. The inoculum size selected for this study ranged from 0.5% to 5% (Mohammed et al. 2019b).