Optimization of production conditions of lipase from B. licheniformis 
MTCC-10498

Sharma C.K. Sharma P.K. and Kanwar S.S.
Department of Biotechnology, Himachal Pradesh University, Shimla-171 005, INDIA

Available online at: www.isca.in
Received 23rd March 2012, revised 5th May 2012, accepted 8th May 2012



Abstract 
The initial broth containing 0.3 (%; w/v) yeast extract, 0.1% sodium nitrate (w/v) etc. were calibrated to a final pH of 7.5 to determine cumulative effect of all the selected components on lipase production by B. licheniformis MTCC 10498. The MB broth was autoclaved at 1.1 bar for 20 min. at 121°C.  This broth was inoculated with 10% (v/v) of 36 h old seed culture and incubated under shaking at 55ºC for 72 h. The inoculated MB broth was harvested at 72 h by centrifugation (10, 000 X g for 20 min. at 4ºC; Sigma 3K30, Germany). The supernatant was filtered through Whatman filter paper no. 1. This enzyme preparation was termed as crude lipase. The lipase produced by B. Licheniformis MTCC-10498 in various batches was recorded. The pH was adjusted to 8.0 ± 0.2 and the final volume was made to 1000 ml with sterile distilled water. This crude lipase was subjected to lipase assay and finally 2.0 U/ml activity were recorded.

Keywords: B. licheniformis, optimization, yeast extract, fatty acids, spectrophotometer.


Introduction
Lipases have emerged as one of the leading biocatalysts with proven potential for contributing to the multibillion dollar underexploited lipid technology bio-industry and have been used in ex situ lipid metabolism and ex situ multifaceted industrial applications1,2. A number of lipases have been studied and the majority of them originate from microorganisms such as Achromobacter, Alkaligenes, Arthrobacter, Bacillus, Burkholdria, Chromobacterium and Pseudomonas3. Thus the finding of new producers of lipases and the optimization of process/production strategy for enzyme are important aspects of current research impetus.

Material and Methods
Chemicals: NaNO3, K2HPO4, KCl, MgSO4, FeSO4.7H2O, (NH4)2SO4 Celite-545 (S. D. Fine-Chem. Ltd., Hyderabad, India); yeast extract and gum acacia (Hi-Media Laboratory, Ltd., Mumbai, India); sucrose, KCl, KI, KNO3, isopropanol, ammonium persulphate, 2- mercaptoethanol, HCl, bromophenol blue and molecular sieves (3Å X 1.5 mm; Merck-Ltd., Mumbai, India); p-nitrophenyl formate (p-NPF), p-nitrophenyl acetate (p-NPA), p-nitrophenyl benzoate (p-NPB) p-nitrophenyl caprylate (p-NPC),  p-nitrophenyl laurate(p-NPL), p-nitrophenyl palmitate (p-NPP) from Alpha-aesar, Heysham, England. n-Hexane, Silica gel (0.040-0.063 mm, 230-400 mesh) acetic acid and triton-X100, tween-20, 40 and 80 (Qualigens Chemicals, Mumbai, India); phenyl methylsulphonyl fluoride (PMSF), sodium dodecyl sulphate (SDS), sodioum lauryl sarcosine (SLS), acrylamide, bisacryl amide (N,N-methylenebisacrylamide) glycerol, glycine and Tris (2-hyroxymethyl-2-methyl-1, 3-propanediol) (Sigma Chemicals Co., USA). All chemicals were of analytical grade and were used as received.
       
Microorganism: For the present study, water samples were collected from hot-spring of Tattapani (Mandi District, Himachal Pradesh, India) in sterile containers. For the isolation of lipolytic microorganisms, 100 µl of water samples were plated on triolien and tributyrene agar plates. The formation of clear zone around the colony on the plate was considered as lipolytic microorganisms. In total, 101 isolates were found. Microorganisms which formed large clear zone around the colony were assayed for lipase activity4. The microorganism which shows highest lipase activity was designated as BTS-20. The microbial culture was  maintained by repeated sub-culturing at 55oC on a mineral based  (MB) medium, supplemented with 0.5% (w/v) sucrose and 1.0% (v/v) of cottonseed oil as a sole carbon source (pH 7.5). Further, identification and biochemical characterization was done by Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh. It was identified as Bacillus licheniformis MTCC 10498. Glycerol stocks were prepared (25%; v/v) and stored at -20 oC till further use.
       
Assay and Unit of lipase activity: Lipase assay were performed by a colorimetric method4. The absorbance of p-nitrophenol released were measured at 410nm (Lab India, UV/ visible spectrophotometer, India). The enzyme activity was defined as ?mole (s) of p-nitrophenol released per min by one ml of free enzyme or per g of immobilized enzyme (weight of matrix included) under standard assay conditions. Specific activity was expressed as ?mole (s) of the p-nitrophenol released per min per mg of protein.
Preparation of inoculum of Bacillus licheniformis MTCC-10498: A loopful of Bacillus licheniformis MTCC-10498 culture taken from the MB-agar slant was asceptically transferred into 50 ml (250 ml Erlenmeyer's flask) of MB broth supplemented with sucrose (0.5%, w/v). The seeded broth was incubated at 55°C in a shaking incubator at 150 rpm up to 36 h. This inoculum was used to prepare glycerol stocks or to inoculate the production broth in the experiment performed.  
       
Standardization of parameters for lipase production: Different parameters for production conditions viz. inoculum size, production temperature, pH, carbon, nitrogen sources, oil, surfactant, salt concentration, buffer system etc. were optimized as given below:
       
Optimization of inoculum size: Inoculum size was optimized by inoculating the production broth by using 36 h seed culture of varying size 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, and 12% (v/v). The sterile broth was inoculated with 10% v/v (5.2×106 cells/ml; A660=2.0) of seed culture (36 h old). Lipase activity as well as growth of batch culture was estimated. The broths taken in 250 ml flasks (pH 7.5, 50 ml each flask) were incubated at 55oC for 36 h under shaking at 150 rpm. Thereafter, the harvested cell free broth (centrifuged at 10,000 X g for 10 minute at 4oC) was assayed for lipase activity.
       
Effect of incubation temperature and pH on lipase production: Most favorable production temperature was studied by incubating the production broth at different temperature of 45, 50, 55, 60 and 65°C and lipase activity was assayed in the cell-free broth after 72 h. For the production of pH, the production broth of varying pH viz. 7.0-10.0 were inoculated with seed culture and incubated at 150 rpm in a shaking incubator. Lipase activity was assayed by colorimetric method after 72 h incubation. 
       
Effect of agitation rate and volume of lipase production: To optimize agitation rate for the enzyme production, the broth (50 ml in 250 ml Erlenmeyer's flask) was inoculated with 10% (v/ v) inoculum at different rpm viz. 60, 90, 120, 150, and 180 rpm. The broth was harvested after 72 h and was assayed for lipase activity by colorimetric method. Most favorable production volume was studied by incubating the production broth at different volume viz. (25, 50, 100 and 150 ml). The broth was harvested 72 h and was assayed for lipase activity by a colorimetric method. 

Effect of carbon and nitrogen sources on lipase production: Various carbon sources viz. (Mustard oil, Cotton seed oil, and Olive oil and Soyabean oil) were supplemented in the production broth. Each of the oil was emulsified with gum acacia (0.5%; w/v). Lipase activity was determined in cell-free broth after 72 h. The production broths having different concentrations (1.0, 2.0, 3.0 and 4.0%; v/v) of cotton seed oil were prepared and inoculated with the seed culture of bacterial isolate. After an incubation period of 72 h, the cell-free broth was assayed for lipase activity. Each of the various nitrogen sources (yeast extract, beef extract, urea, peptone and tryptone) were individually added to production broth at concentration of 0.5 (%;w/v). The lipase activity was assayed after 72 h incubation in cell-free broth. A 36 h old seed culture (10%; v/v) was inoculated in sterile broth containing anyone of the selected inorganic nitrogen sources that included ammonium nitrate, ammonium chloride, urea, sodium nitrate, and ammonium sulphate (0.3%; w/v) along with yeast extract (1.0%; w/v) and cotton seed oil (1%, v/v) in each of the flask. The flasks were incubated at 55± 2 ºC under continuous shaking (120 rpm). At periodic intervals 4 ml of culture broth was aseptically withdrawn to assay protein and lipase activity. To check the effect of different concentration of yeast extract on lipase production, the production broth was prepared with concentration (0.1, 0.3, 0.5 and 0.7%; w/v) of yeast extract in the production broth and the cell-free broth harvested after 72 h incubation at 55oC to determine the lipase activity. 

Effect of surfactant on lipase production:	The effect of each of the selected surfactant (viz., Sodium dodecyl sulphate, sodium lauryl sarcosine, tween-20, tween-40, tween-60, tween-80 and triton X-100 was separately evaluated by incorporating (0.5%; v/v) the surfactant to the production broth which was inoculated with 10% (v/v) of 36 h old inoculum.

Effect of carbohydrate on lipase production: To evaluate the effect of various carbohydrates (cellobiose, lactose, fructose, ribose, mannose, xylose, dextrose and sucrose) each of the carbohydrates (1%; w/v) was added into the production broth. The amount of lipase produced after 72 h was assayed in each case.

Effect of fatty acid on lipase production: The effect of various fatty acids as inducers (5 mM) each of the palmitic acid, triolein, tristearin, stearic acid, myristic acid, oleic acid and lauric acid) for lipase produced by B. licheniformis MTCC-10498 was studied by including separately each of the selected compound in the broth before sterilization. The broth dispensed in the flasks was seeded with 10% (v/v) of the 36 h old inoculum and flasks ware incubated under optimized temperature and pH. The amount of lipase produced in each case was determined at 72 h post inoculation.

Cumulative effect of optimized components on lipase production by B. licheniformis -MTCC 10498: The optimized broth containing 0.5 (%; w/v) yeast extract, 0.3% sodium nitrate and tween 80 (0.5%, w/v) was calibrated to a final pH of 7.5 to determine cumulative effect of all the selected components on lipase production by B. licheniformis MTCC 10498. The MB broth was autoclaved at 1.1 bar for 20 min. at 121°C.  This broth was inoculated with 10% (v/v) of 36 h old seed culture and incubated under shaking at 55 ºC for 72 h. The inoculated MB broth was harvested at 72 h by centrifugation (10, 000 X g for 20 min. at 4ºC; Sigma 3K30, Germany). The supernatant was filtered through Whatman filter paper no. 1. This enzyme preparation was termed as crude lipase. The lipase produced by B. licheniformis MTCC-10498 in various batches was recorded. The pH was adjusted to 7.5 ± 0.2 and the final volume was made to 1000 ml with sterile distilled water.

Growth and lipase profile of Bacillus licheniformis MTCC-10498: The seed culture (36 h old) at 10% (v/v) concentration was inoculated in the production broth (50 ml final volume taken in 250 ml Erlenmeyer flask). This flask was incubated at 55ºC in an incubator shaker (120 rpm; Genei shaker, Bangalore). At 4 h intervals, 2 ml of inoculated broth was aseptically sampled up to 120 h post inoculation. A660 value of each of the sample was recorded to determine the growth of the bacterial strain and the same was plotted against time. At the same time intervals, 2 ml of culture broth was separately withdrawn aseptically and cell-free broth (centrifugation at 10, 000 rpm for 10 minutes at 4ºC) was assayed at A410 to determine lipase activity with respect to the time post inoculation.

Lipase production under optimized conditions: The extracellular lipase production by B. licheniformis MTCC-10498 in the optimized medium broth was studied by inoculating the sterile broth with 10% (v/v) of 36 h old inoculum. The seeded broth was incubated at 55 ± 1 ºC for 72 h under continuous shaking (120 rpm). The lipase production in different batches (n = 5) were recorded. The culture broth was further processed to obtain purified lipase. The lipase activity was assayed using p-nitrophenyl palmitate (p-NPP), a chromomeric substrate. Lipase activity of crude lipase, purified or matrix bound lipase was assayed employing a modified colorimetric method (Winkler and Stuckmann, 1979). The stock solution (20 mM) of p-NPP was prepared in HPLC grade isopropanol. The reaction mixture contained 80 µl of p-NPP stock-solution, 20 µl of lipase and tris buffer (0.05 M, pH 8.5) to make final volume 3 ml. The reaction mixture was incubated at 55 °C for 10 min in a water bath (Banglore Genei Pvt. Ltd., Banglore). Further lipase reaction was stopped by chilling at -20°C for 5 min. Control containing heat inactivated (5 min in boiling water bath) enzyme was also incubated with each assay. The absorbance (A410) of heat inactivated lipase was subtracted from the absorbance of the corresponding test sample. The absorbance A410 of the p-nitrophenol released was measured and expressed in millimoles (mM). The unknown concentration of p-nitrophenol released was determined from a refrence curve of p-nitrophenol (2-20 µg/ml in 0.05 M Tris buffer pH 8.0) (Appendix 1). Each of the assays was performed in triplicate unless otherwise stated and mean values ± S.D. were presented. Stock solutions of various p-nitrophenyl esters p-NPF, p-NPA, p-NPC and p-NPL were also prepared for use in the some of the experiments. Following two methods were used for the estimation of protein in the sample:

Spectroscopic method: Absorbance of protein sample was measured at 280 nm against the experimental buffer as blank using a spectrophotometer during column chromatography experiments5 to determine protein concentration in the fractions.
Lowry's method: A standard quantitative assay for determining the protein content in a solution was used6.

Results and Discussion 
The bacterium strain used to produce an extracellular lipase in the present studies was characterized as an aerobic thermophillic, rod shaped of the genus Bacillus (figure 1 and figure 2) the strain was maintained by repeated sub-culturing on MB medium containing 1% (v/v) cotton seed oil. Culture stocks of B. licheniformis MTCC 10498 were prepared in glycerol (25% v/v; culture) and stored at -20 °C till further use. 


Figure-1
Showing single colony of B. licheniformis MTCC-10498 on MB agar plate

Optimization of production temperature:  The B. licheniformis MTCC-10498 in the MB broth produced and accumulated maximum activity at 55 °C, reaching a highest enzyme activity of 0.327 ± 0.05 U/ml after 48 h post inoculation. A lower or higher cultivation temperature caused a decline in lipase production (table 1). In one liter of MB, NaNo3 1.0 g , yeast extract 3.0 g, sucrose 1.0 g, cotton seed oil 1% (v/v) was used.
       
Table-1
Effect of production temperature
Temperature  (°C)Lipase activity (U/ml)Relative activity (%)450.255 ± 0.0778500.301 ± 0.0492550.327 ± 0.05100600.228 ± 0.0169.7650.209 ± 0.0163.9
Effect of pH on lipase production: Lipase production has been shown to be markedly dependent on pH in different species of microorganisms. The microorganism was grown in 50 ml of production medium with a pH range of 7.0-10.0 at 30°C under shaking conditions for 36 hours. Maximum lipase production was observed at pH 7.5 (~0.4 U/ml). The enzyme production decreased with increase in pH after 7.0 as shown in table 2.

Table-2
Effect of pH on lipase production
pHLipase Activity (U/ml)Relative activity (%) (%)7.00.349 ± 0.0589.77.50.389 ± 0.021008.00.356 ± 0.0391.58.50.350 ± 0.0289.99.00.327 ± 0.0584.09.50.221 ± 0.0257.510.00.109 ± 0.0328.0
Effect of agitation rate on lipase production: The agitation rate was studied by varying rpm (60-180). A maximum lipase activity of 0.402 U/ml was recorded at rpm 120. Lipase activity decreased on either side by increasing or decreasing rpm (table 3).  

Table-3
Effect of agitation rate on lipase production
Agitation rate (rpm)Lipase activity (U/ml)Relative activity (%) 600.150 ± 0.0537900.309 ± 0.01781200.402 ± 0.041031500.394 ± 0.01981800.389 ± 0.0296       
Optimization of inoculum concentration: When a 5% (v/v) inoculum of 36 h seed culture was employed in the production broth, 0.481 ± 0.05 U/ml of enzyme was produced at 55 ± 0.01 °C. After 5% inoculum concentration there is slight increase in lipase activity, however it is not economic (table 4). Inoculum contained nitrogen (NaNO3 1 g/L and yeast extract 3 g/L as well as the carbon sources (sucrose 5 g/L and cotton seed oil 1%; v/v).

Table-4
Optimization of inoculum concentration
Inoculum size  (%; v/v)Lipase activity (U/ml)40.402 ± 0.0450.481 ± 0.0560.488 ± 0.0470.512 ± 0.0580.523 ± 0.0190.535 ± 0.02100.541 ± 0.01110.535 ± 0.01120.519 ± 0.01       
Optimization of chemical parameters for lipase production: Optimization of lipidic carbon sources: Amongst various carbon sources used in this study, the presence of cotton seed oil produced a relatively higher (0.882 U/ml) lipase activity in comparison to control (sucrose used as a sole carbon source), while addition of olive oil gave minimum activity (table 5). All potable oils were initially emulsified with 0.1% gum acacia. Control consisted of sucrose instead of oil in lipase production broth.

Table-5
Optimization of lipidic carbon sources
Lipidic carbon source
(%; v/v)Oil concentration vs. Lipase activity (U/ml)Nil0.5%1.0%1.5%2.0%Cotton seed oil (Ginni)0.101 ± 
0.010.672 ± 
0.010.882 ± 
0.050.702 ± 
0.010.512 ± 
0.01Mustard oil0.101 ± 
0.010.517 ± 
0.010.602 ± 
0.020.585 ± 
0.020.412 ± 
0.01Mineral oil0.101 ± 
0.010.312 ± 
0.020.350 ± 
0.030.313 ± 
0.040.262 ± 
0.02Olive oil0.101 ± 
0.010.261 ±
 0.010.252 ± 
0.020.212 ± 
0.010.212 
± 
0.01       
Effect of nitrogen sources on lipase production: Diverse nitrogen sources (both organic and inorganic) were tested individually by supplementing each of the selected N- sources used at 1.0% (w/v) in the production broth, yeast extract produced highest amount (0.982 U/ml) of lipase after 72 h of incubation at 55 ± 1°C. However, the control broth (broth without NaNO3 and yeast extract resulted in much lower activity (0.12 U/ml; table 6)). The broth containing 0.5% (w/v) YE was further supplemented with 1% additional YE as stated concentrations.

Table-6
Effect of organic N- sources on lipase production
N-sources (0.5%)Lipase activity (U/ml)Relative activity (%)Beef extract0.101 ± 0.0210.2Casein0.112 ± 0.0211.4Peptone0.092 ± 0.019.3Tryptone0.075 ± 0.027.6Yeast extract0.982 ± 0.02100Control (none)0.120 ± 0.0212.2

Table-7
Effect of 1% additional yeast extract on lipase production
Yeast extract  (%, w/v)Additional YE  (1%,/v)Lipase activity (U/ml)Relative activity (%)0.11.11.112 ± 0.02113.20.31.31.123 ± 0.01114.30.51.51.157 ± 0.01117.80.71.71.178 ± 0.02119.90.91.91.198 ± 0.01121.91.02.01.208 ± 0.03123.02.03.01.103 ± 0.011123.04.01.056 ± 0.02107.5Control0.50.982 ± 0.05100
In a separate experiment when the amount of yeast extract was gradually increased above 0.5%, w/v by supplementation of additional yeast extract to the broth, it was noticed that additional presence of yeast extract promoted the production of lipase produced by B. licheniformis MTCC-10498 (table 7). Yeast extract at a final concentration of 3.0% (w/v) resulted in 1.103 U/ml lipase in the production broth. Since there is minor increase in promoting the yield of lipase by additional yeast extract, it is economical to use 0.5% (w/v) in subsequent experiments.
       
Effect of supplementation of inorganic nitrogen sources on lipase production: Among various inorganic N-sources that were additionally supplemented in the product broth (containing yeast extract 0.5%, w/v), additional supplementation of the NaNO3 enhanced the lipase production to 1.062 U/ml (table 8). Optimal activity of combined nitrogen source was considered 100%.
Table-8
Effect of supplementation of inorganic nitrogen sources
Additional inorganic source (0.3%; w/v)Lipase activity
(U/ml)
Relative activity
(%)NaNO31.062 ± 0.05100NH4NO30.355 ± 0.0133.4(NH4)2SO40.542 ± 0.0251.0NH4Cl0.612 ± 0.0357.6Urea0.512 ± 0.0148.2None (only YE)0.672 ± 0.0263.2
Effect of salts in lipase production: Addition of K2HPO4, KCl, MgSO4 and FeSO4 in the concentration of 0.1%, 0.05%, 0.05%, and 0.01% (w/v) enhanced the lipase activity up to 1.108, 1.128, 1.144 and 1.168 U/ml respectively. When any of the salts was not added lipase activity declined (table 9).
Table-9
Effect of addition of salts in lipase production
Salt (w/v, %)Lipase Activity (U/ml)Lipase activity without saltK2HPO4 (0.1 )1.108 ± 0.050.608KCl (0.05)1.128 ± 0.050.577MgSO4 (0.05)1.144 ± 0.020.712FeSO4 (0.01)1.168 ± 0.050.633
Effect of carbohydrates as inducers on lipase production: To evaluate the effect of carbohydrates as inducer in lipase production in the broth, various carbohydrates were individually tested at a concentration of 1.0%. As evident all carbohydrates used in experiment induced the lipase activity (table 10). The control contains emulsified cottonseed oil (1.0%; v/v) and all optimized component till this step.

Table-10
Effect of carbohydrates as inducer
Carbohydrate (1.0%; w/v)Lipase activity (U/ml)Relative activity (%)Cellobiose1.364 ± 0.01116.7Lactose1.216 ± 0.02104.1Fructose1.106 ± 0.0994.6Ribose1.196 ± 0.05102.3Mannose1.182 ± 0.01101.1Xylose1.219 ± 0.01104.3Dextrose1.369 ± 0.01117.2Sucrose1.412 ± 0.04120Nil1.168 ± 0.05100
Effect of surfactants on lipase production: Effect of supplementation of each of surfactants (0.05%; v/v) was studied using broth lacking both yeast extract as well as NaNO3 (table 11). Most of the surfactant resulted in a decline in the amount of the lipase that varied from 0.1 to 0.3 U/ml in comparison to the control. However, the presence of tween-80 resulted in 1.808 U/ml lipase activities in the presence of 1.0% sucrose and 1.414 U/ml in the absence of sucrose (1.0%). Increasing or decreasing the concentration of tween-80 resulted in decline in activity. Lipase production broth was supplemented with sucrose (1%, w/v).
Table-11
Effect of surfactant on lipase production
Surfactant (0.5%; v/v)Lipase activity (U/ml)Relative activity (%)SDS0.712 ± 0.0960.9Sodium lauryl sarcosine (SLS)0.636 ± 0.0154.4CTAB0.767 ± 0.0265.6Tween 201.008 ± 0.0186.3Tween 600.810 ± 0.0269.3Tween 801.808 ± 0.02154.7Triton -X1001.126 ± 0.0196.4Control (None)1.168 ± 0.05100
Effect of fatty acid (s) on lipase production:  The inductive effect of presence of the fatty acids on lipase production by B. licheniformis MTCC-10498 was studied. Lipase activity is enhanced by each of the fatty acid when used in the production broth. Highest increase (189.5%) in the lipase activity was recorded when tri-olein was used (table 12). Control lacking in cotton seed oil.
Table-12
Effect of fatty acid (s) on lipase production
Fatty acid (5mM)        Lipase activity (U/ml)        Relative activity (%)Palmitic acid1.918 ± 0.01164.2Myristic acid1.451 ± 0.01124.2Oleic acid1.656 ± 0.03141.7Stearic acid1.628 ± 0.04139.3Lauric acid1.817 ± 0.02155.5Triolein2.004 ± 0.01171.5Tristearin2.001 ± 0.01171.3Tripalmitin2.001 ± 0.02171.3Control 1.168 ± 0.02100
Cumulative effect of optimized medium constituents on the production of lipase by B. licheniformis MTCC-10498: Optimized medium containing 1.0% (w/v) yeast extract, 0.3% (w/v) NaNO3, 5mM trioein, 1% (w/v) sucrose, 0.5% tween -80 was adjusted to under optimized pH 8.0  and optimized temperature 55 ± 1ºC (table 13). * Cotton seed oil emulsified with 0.1% gum acacia was added to the sterile production broth. **Sucrose (1.0%, w/v), tween-80 (0.5% v/v) and triolein were added in the final concentration of 5mM in optimized broth.

Table-13
Comparison of initial MB and Optimized MB for lipase production
ConstituentsInitial MB (g/L)Optimized Broth (g/L)Cotton seed oil*----10 mlYeast extract3.05.0NaNO31.03.0K2HPO40.011.0KCl0.250.5MgSO40.250.5FeSO4.7H2O0.0010.001Additives**Nil---
Table-14
Batch wise yield of lipase
BatchVolume (ml)Lipase activity (U/ml)Specific activity U/mg1.5002.001 ± 0.021.012.5001.999  ± 0.041.023.5002.001 ± 0.021.034.5002.002 ± 0.021.005.5002.006 ± 0.011.006.5001.998 ± 0.021.00Average5002.001 ± 0.021.01Growth and lipase profile of B. licheniformis MTCC-10498: The production broth when inoculated with 10% (v/v) of 36 h old seed culture produced optimal amount of lipase activity (2.0 U/ml), specific activity (1.01 U/mg) and protein (1.88mg/ ml) at 72 h post inoculation (figure 2) under optimized conditions. The culture broth rendered cell free by centrifugation at 10,000 x g at 4°C for 10 min was filtered through Whatman filter paper no. 1 and assayed for lipase activity using p-NPP.
       

Figure-2
Growth and lipase profile of B. licheniformis MTCC-10498 using 5% inoculum.

In the present study, a thermotolerant bacterial isolate Bacillus licheniformis MTCC-10498 that produce a novel lipase was exploited to obtain the this enzyme in its purified form. To enhance the lipase production in broth by B. licheniformis MTCC-10498 effect of inducers fatty acids and carbohydrates etc. are studied. It was obvious that different microorganism possess different modes of carbohydrate metabolism. Production is significantly influenced by other carbon sources, such as sugars, alcohols, polysaccharides, whey, casamino acids and other complex sources7.

Generally, microorganisms provide high yields of lipase when organic nitrogen sources are used, such as peptone and yeast extract, which have been used as nitrogen source for lipase production by various Bacillus spp. viz., B. alkalophilus, B. coagulans, B. cereus, and B. licheniformis strain H18-12. R. glutinis seems to require organic nitrogen sources (e.g., yeast extract and tryptone), an inorganic nitrogen source such as ammonium phosphate appears to favor lipase production14. Production of the extracellular and cell bound enzymes were reported to depend on the carbon and nitrogen composition of the medium14,15. Inorganic nitrogen sources such as sodium nitrate have also been reported to be effective in some microbes16.

Ca2+ stimulated17 the lipase activity and most of the metal ions (Mg2+, Fe2+ Cu2+ and Mn2+) had strong inhibition on lipase production from Burkholderia sp. Ions of calcium, magnesium and sodium were reported to greatly enhance the lipase production from different type of microorganisms. For instance, lipase production by B. multivorans was enhanced18 in the presence of Ca2+; improvement of lipase production was done by P. fluorescens NS2W in the medium containing19 Mg2+ and Ca2+; stimulation of lipase production using Burkholderia sp. GXU56 by addition of Mg2+ was observed20; and, addition of 1.5% NaCl to the medium induced high level of lipase production21 by P. fluorescens NS2W.

Lipases from Bacillus stearothermophilus SB-1, B. atrophaeus SB-2 and B. licheniformis SB-3 are active over a broad pH range22 (pH 3-12). Bacterial lipases generally have temperature optima in the range23 30-60°C. A highly thermotolerant lipase has been reported from B. stearothermophilus, with a half-life24 of 15-25 min at 100°C. In another similar study with metal ions (1 mM) and chelating agents, P. pseudoalcaligenes F-111 lipase activity was 60%  inhibited by Fe3+ but not by Ca2+, Hg2+, Zn2+, Mn2+, Cu2+,  Mg2+,  Co2+,  Cd2+ and  Pb2+ , metal chelators (EDTA, o-phenanthrolin) did not significantly affect the alkaline lipase activity25.

Non-specific reversible inhibitors are compounds that do not  act  directly  at  the  active  site  but  inhibit  lipase  activity  by  changing  the conformation  of  lipase  or  interfacial  properties. Surfactants, bile salts26 and proteins belong to this group of inhibitors. However, surfactants and bile salts activate the enzyme in some cases. Previously, a moderately thermophillic bacteria B. coagulans MTCC 6375 exhibited an extracellular lipase activity27.

Conclusion
Bacillus licheniformis MTCC-10498 lipase showed optimal activity by manipulating various physical and kinetic parameters (i.e., temperature, pH, agitation rate, carbon, nitrogen, inoculum size, salt concentration etc.).   

Acknowledgments 
The financial assistances in provided by University Grants Commission, New Delhi, India to Chander Kant Sharma and the funds provided by Department of Biotechnology, Government of India to Department of Biotechnology, Himachal Pradesh University, Shimla (India) are thankfully acknowledged.

Reference 
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