Production , purification and characterization of lichenin from Bacillus licheniformis

SwetaSushmitaTigga, Rubina Lawrence and Ebenezer Jeyakumar. Department of Industrial Microbiology, Jacob Institute of Biotechnology and Bioengineering, Sam Higginbottom University of Agriculture, Technology and Sciences.Allahabad-211007, (U.P) INDIA. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Lichenin arebacteriocin produced by Bacillus licheniformis, with probiotic, fungicide and medical importance.Lichenin producing Bacillus licheniformis was isolated from soil sample, collected from research plots of SHUATS, Allahabad. All the gram positive rods was biochemical analyzed and advance bacterial identification Software showed 89 % similarity of isolated bacterial cultures with Bacillus licheniformis.The crude bacteriocin of Bacillus licheniformis, exhibited antagonistic activity against Staphylococcus aureus MCCB 0139 whereas no zone of inhibition ascertained against Escherichia coli MCCB 0017. Ten variables viz incubation temperature, incubation period, pH and medium components consisting sorbitol, lactose, yeast extract, peptone, NH 4 NO 3, K 2 HPO 4 and MgSO 4 , respectively was optimized from run number 1 to 76. Response surface data showed maximum bacteriocin production by run number 43 at pH 8.0, incubation temperature 50⁰C, and incubation period 12 hrs. Optimization illustrated that effect of increasing pH from 4.0 to 8.0, incubation temperature from 37.5⁰C and 50⁰C have significant effect on bacteriocin activity alongwith peptone concentration from 0.25 % to 0.75 % have significant effect on bacteriocin activity. The average value depicted in the top and bottom phase of the cube and left and right face of the cube, respectively showed sorbitol, peptone and MgSO 4 have significant effect on bacteriocin activity. Crude lichenin was partially purified by 80 % ammonium sulphate precipitation, dialysis and ion exchange chromatography followed by quantification and estimation by Lowry's method. SDS-PAGE characterized purified lichenin and revealed < 9 kDa of molecular weight.

Dialysis
The ammonium sulphate precipitated pellet was dissolved in 1X phosphate buffer (0.1M) and purified by 1 kDa dialysis membrane tube. 400 μl dissolved pellet was transferred in 1 kDa dialysis membrane and then dialysis tube placed in a beaker containing 1X phosphate buffer (0.1M, 7.0 pH) on a continuous stirring by magnetic stirrer at 4⁰C for overnight.The purified dialysed lichenin was collected and stored at -20⁰C for ion exchange chromatography (Henaet al., 2011)

Ion exchange chromatography
The purified dialysed lichenin was subjected to cation exchange chromatography using Sephadex resin. The lichenin was eluted with 0.1M to 1.0M NaCl gradient in 0.1M, phosphate buffer, pH 6.4 using flow rate of 0.5 ml/min. The collected purified lichenin fractions was estimated by U.V spectrophotometer at 280 nm (Dusane et al., 2013)

Estimation of lichenin concentration
The collected ion exchange purified protein fractions was exercised for quantitative protein estimation by Lowry's method. In 10 tubes, 1ml of purified lichenin was taken, 2 ml of Lowry's reagent was added, incubated at room temperature for 10 min, 0.2 ml of follins-ciocalteau reagent again added in all tubes, incubated at room temperature for 30 min and then absorbance at 660 nm by visible spectrophotometer were taken for lichenin estimation (Henaet al., 2011)

Molecular weight determination by SDS-PAGE
The purified lichenin was separated by SDS-PAGE as described by Schagger and Jagow (1987). The prestained protein ladder covering a wide range of molecular weight from 10 to 245 kDa (MolBio, Himedia) was used. Proteins bands was detected by silver staining (Dusaneet al., 2013).Silver staining was performed by a procedure of Blum (1987) with slight modification in protocol given by proteomic resource centre, the Rockefeller University, New York.

Statistical analysis
The data recorded during the evaluation of antagonistic activity of cell free supernatant of Bacillus licheniformis using variance (ANOVA) to calculate significant effect of crude lichenin (bacteriocin) on gram positive S. aureus MCCB 0139.To optimize the various parameters in order to study lichenin (bacteriocin) production in the media Doptimal design with suitable categoric factors in addition to continuous factors in RSM was employed. The points chosen in this design was algorithmically according to number of factors and desired model ( Table 1). The points 1451 chosen were not at any certain positions they are selected to meet the optimality criteria. Linear, quadratic, cubic and other higher order (upto 6 th ) order models can be fitted to create a good design RSM. This design adds constraints to exclude that particular area where response cannot measure. Those particular area where response cannot be measure can be excluded by adding constraints to the design. The design allows greater control over the level chosen for the design so that discrete factor levels can be specified for numeric factors. A linear equation calculated as proposed for the model to estimate the response of the dependent variable is given in equation 1.0 Where Y is predicted response, x1, x2, x3, x4, x5, x6, x7, x8, x9 and x10 are independent variables.

Isolation and identification of Bacillus licheniformisfrom the soil sample
Out of hundred soil sample, only fifty gram positive strain of Bacillus licheniformiswas isolated on Bacillus medium at pH 7, 37±1⁰C for 24-48 hrs. Total eight isolated Bacillus licheniformiswas identified by biochemical analysis and according to Bergey's Manual of Determinative Bacteriology. Positive growth at 45ºC, growth at 7% NaCl and no growth marked at 65ºC. Positive citrate utilization, nitrate reduction, utilization of urease, egg yolk, casein hydrolysis, starch hydrolysis, ONPG, esculin hydrolysis, motility and oxidase whereas negative in indole production, voges-prouskauer was observed. Acid without gas in sugar fermentation was produced from glucose and from a wide range of carbohydrate viz mannitol, mannose, starch, cellobiose, fructose, glycerol, maltose, sorbitol, melibiose, ribose, sucrose and trehalose and no acid production was marked from arabinose, salicin, xylose, mesoinnositol, lactose and rhamnose respectively. The biochemical observation implemented in Advanced Bacterial Identification (ABIS) software, showed 89% similarity of bacterial isolates and confirmed eight bacterial isolates as Bacillus licheniformis.

Evaluation antagonistic activity of lichenin against test organisms
The identified eight Bacillus licheniformis isolates was assayed for antagonistic activity against the test organisms. Out of eight only seven Bacillus licheniformis identified isolates are capable to inhibit the growth of S. aureus MCCB 0139 but none against E. coli MCCB 001, described in Table 2. The result showed that S. aureus was inhibited maximum by isolate no S41 forming a zone of 26 mm at the concentration of fifty μl followed by isolate no S13 (19 mm Lichenin (bacteriocin) is known to form pores in the bacterial membranes because of their strong hydrophobic natures. The broad-spectrum inhibitory activity of lichenin against a pathogenic microorganisms Staphylococcus aureus could be due to its amphipathic nature (as it contains both hydrophobic and hydrophilic residues) which could cause it to have surfactant-like activity on cell membrane, thereby disturbing cellular function (Pattnaiket al. 2001). Bacteriocins from gram positive are usually ineffective against gram negative bacteria because bacteriocin cannot penetrate the outer membrane (OM). The cytoplasmic membrane of Gram negative bacteria is protected by an outer membrane (OM) composed of a phospholipid bilayer, surrounded by a network of lipids and polysaccharides referred to as lipopolysaccharides. The lipopolysaccharide layer forms a tight shield (Raetz and Whitfield, 2002) and acts as a barrier to many compounds, including antibiotics, hydrophobic compounds, detergents and dyes (Vaara, 1992 Optimization of the physical and chemical parameters for lichenin production:-Optimization was carried out using different production parameters viz sorbitol, lactose, peptone, yeast extract (YE), MgSO 4 , NH 4 NO 3 and K 2 HPO 4, pH (4, 6 and 8), incubation temperature (37.5⁰, 25⁰ and 50⁰C) and incubation time (12 hrs, 66 hrs and 120 hrs). Table 3 indicates that maximum zone of inhibition (32 mm) was obtained in run number 43, corresponding to 8 and 19 run numbers at pH 6.0 and 8.0, temperature 37.5⁰C and 50⁰C, incubation 1452 period at 66 hrs and 12 hrs with substrate concentration amounting to sorbitol 1.0 % and 1.25 %, lactose 0.05 %, peptone 0.5 % and 0.25 %, yeast extract 3.03 % and 0.06 %, ammonium nitrate 0.85 % and 0.05 %, dipotassium hydrogen phosphate 0.2 % and 0.1 % and magnesium sulpahte 0.015 % and 0.005 %, respectively (Fig 3). The normal probability shows that our proposed model was fitting the observation with very high accuracy (Fig 4).
Residual vs Predicted graph shows that all the studentized residual were lying within the 3 σ limits which clearly indicate that the whole error part was insignificant (Fig 5). Residual vs Run graph shows that the residual part for each run was also non-significant (Fig 6). All the ten variables for optimization were plotted as Residual vs Factor. Residual vsFactor for pH (Fig 7), temperature (Fig 8), incubation period (Fig 9), concentrations of sorbitol (Fig 10), lactose (Fig 11), peptone (Fig 12), yeast extract (Fig 13), ammonium nitrate (Fig 14), dipotassium hydrogen phosphate (Fig 15) and magnesium sulphate (Fig 16) shows that the model fits well for the present experiment. Absence of outlier was also observed in the plot. It means the present model is good in comparison to prediction.
The Cook's distance plot depicts the fitting well of the model in this experiment (Fig 17). Cook's distance is useful for identifying outliers in the x value (observation for predictor variables). It also shows the influence of each observation on the fitted response value. The design for the study was also good since the leverage value falls far away from 1.0 (Fig 18). The Predicted vs actual graph that the model was having high concentration of points along the diagonal indicating the goodness of the model (Fig 19). Selvaraj et al., (2012) reported that the close correlation between the experimental and predicted data indicates the appropriatness of the experimental design. The Box-cox plot for the power transformation shows that the lambda (λ) value of design lies between 0.54 to 0.92 with the least value at 0.73 with the current lambda (λ) values of 1.0, which lies in between the given range. Square root transformation with λ = 0.5 was applied with a constant value of k = 0.032 was used to make response value positive (Fig 20).

Fig 21-30
Illustrate the effect of individual parameters on zone of inhibition. An increasing trend in bacteriocin activity was recorded with increase in temperature from 25⁰ to 50⁰C (Fig 21) and increase in MgSO 4 concentration from 0.01 % to 0.03 % (Fig 22).However the activity of bacteriocin was found to decreasing on increasing the pH from 4.0 to 8.0 (Fig 23) and peptone concentration from 0.25 % to 0.75 % (Fig 24). It have been reported that bacteriocin production by Bacillus sp. Sh10 was studied at different pH values ranging from 4-10, showing inhibitory activity in the acidic and alkaline pH ranges with optimal activity at pH 8. Bacteriocin production in alkaline conditions are now gaining more attention in food industries because several food products vary from natural to alkaline conditions (Shayesteh et al., 2014).It has been cited in literature that nisin is the only commercial bacteriocin used as a food supplement at acidic pH while it is unstable at alkaline pH (Liu and Hansen, 1990). Further, no significant effect on bacteriocin activity by other parameters viz incubation period (Fig 25), concentration of sorbitol (Fig 26), concentration of lactose (Fig 27), concentration of yeast extract (Fig 28), concentration of NH 4 NO 3 (Fig 29), and concentration of K 2 HPO 4 (Fig 30) was observed. No evident relationship was obtained between growth and bacteriocin production while using different concentrations of carbon and nitrogen source. Shayesteh et al. (2014) reported that bacteriocin production by Bacillus sp. Sh10 at different pH values ranging from 4-10, showing inhibitory activity in the acidic and alkaline pH ranges with optimal activity at pH 8 which was agreeable with the present study. In contrast it have been cited bacteriocin activity was not observed using lactose, starch and sorbitol. Similar studies were reported which investigated the level of lichenin  . Similar findings also reported that bacteriocin production by Bacillus licheniformisSN2 was observed at 30⁰C and as well as at 40⁰C (Sersyet al. 2009). Bacillus licheniformisare observed to survive at 37⁰C and also at 50⁰C; therefore, bacteriocin can be produced at optimum 37⁰C temperature and at high temperature of 50⁰C. Bacillus subtilisalso shows same features regarding bacteriocin production because B. licheniformisand B. subtilisare closely related bacteria. The chrsomosome of B. licheniformis has large regions that are similar to Bacillus subtilis and B. subtilisorthologs, it is considered as a part of the subtilis group. From the above study, it can be concluded that pH have a significant effect on bacteriocin production and also related to growth rate. Most of the report of bacteriocin production by Bacillus licheniformis showed its production at slighty acidic and alkaline pH. Lichenin (bacteriocin) activity noted at pH 4, is due to natural adaptation of B. licheniformis at acidic pH.On studying the effect of incubation period with other optimization parameters no significant effect of incubation period on the bacteriocin activity was observed as depicted in The interaction of incubation temperature and incubation period with concentration of other substrates selected for optimization studies revealed the effect of lactose (Fig 61), yeast extract (Fig 62), NH 4 NO 3 (Fig 63) and K 2 HPO 4 (Fig 64) to be active since significant difference was observed in the average values depicted on the left and corresponding right face of the cube. However the effect due to incubation temperature and incubation period was found to be inactive. Previous study reported that the concentration of K 2 HPO 4 exhibited positive effect on the production, specific activity and biomass as K 2 HPO 4 provides a buffering action for optimizing media (Kayalvizhi  et al., 2008). Higher concentrations of K 2 HPO 4 (2.0-10.0 g/l) repressed the bacteriocin activity of plantaricin ST31 et al. 2006). Similar research has been reported that a high as well as low concentration of yeast extract (3.03 % and 0.06 %) supplemented medium was marked for bacteriocin production (Anthony et al.

2009).
From the above study it can be concluded that less concentrations of K 2 HPO 4 (0.2 % and 0.1 %) used for bacteriocin production and buffering action for optimizing media. Lactose provide carbon source in bacteriocin production by lactic acid bacteria or other bacteriocin producing bacteria. Yeast extract provide large quantity of free amino acids, short peptides and more growth factors and NH 4 NO 3 are supportive ingredients of bacteriocin production by Bacillus licheniformis .   Fig 65-72 depicts the interaction between pH and incubation period with other optimization parameters. The high variation in the average values of temperature as observed in the left and right face of the cube and pH depicted in the top and bottom surface of the cube suggested the interaction due to these factors to be active. However incubation period had no significant effect on the activity of bacteriocin (Fig 65).Similar effect was observed on studying the interaction of pH and incubation period with sorbitol (Fig 66), peptone (Fig 67) and MgSO 4 (Fig 68).
On evaluating the effect of pH and incubation period on remaining optimization parameters revealed only the effect due to lactose (Fig 69), yeast extract (Fig 70), NH 4 NO 3 (Fig 71) and K 2 HPO 4 (Fig 72) to be active.
The interaction between pH, incubation temperature and other optimization parameters viz. incubation period ( Fig  73) and concentrations of sorbitol (Fig 74), lactose (Fig 75), yeast extract (Fig 76), NH 4 NO 3 (Fig 77) and K 2 HPO 4 (Fig 78) revealed that only the average pH values as depicted in the top and bottom surface of the cube did not differ significantly suggesting the effect to be inactive. However when the interaction between pH, incubation period with peptone (Fig 79) and MgSO 4 (Fig 80) was analysed, the overall interaction between all the three variables were found to be active. In contrast it has been investigated that incubation period has a significant role in bacteriocin production. Bacteriocin concentration increased to a maximum at the mid-stationary phase and started declining at the end of the phase indicating it is synthesized as a secondary metabolite. Production of bacteriocin is generally associated with primary kinetics (Shayesteh et al. 2014). In agreement with previous research noted cell growth reached the stationary phase after 12 hrs of cultivation and maximum bacteriocin activity was observed from 15 hrs (Oliveraet al. 2004).
According to this reported results the incubation period 12 hrs and 66 hrs had no significant effect on bacteriocin activity and on other optimizing parameters as it comes in a primary kinetics. Getting bacteriocin activity after 120 hrs may be due to the fact that bacteriocin are synthesized as a secondary metabolite that suppressed the growth of gram positive bacteria.

Purification and Characterization of Lichenin produced by Bacillus licheniformis:-Ammonium sulphate precipitation
The culture supernatant of Bacillus licheniformis was purified by ammonium sulphate precipitation. The best lichenin precipitation was noted at 80% ammonium sulphate precipitation.
Similar research reported that formation of frothy flocculation and pelicular layer observed with ammonium sulphate (80 % saturation) upon overnight storage at 4ºC (a refrigerator temperature) (Pattnaik et al. 2001). The precipitation of an active compound by B. licheniformis isolated from marine sediment, observed at 30-60% ammonium in sulphate precipitation (Smitha and Bhatt 2012). From the above cited results a reason behind of getting frothy flocculation at 80%, due to lichenin (bacteriocin) are able to reach saturation level and so that proteins are concentrating in a bulk precipitation. It's very necessary for a protein to reach a saturation level during ammonium sulphate precipitation process so that proteins can be easily collected as pellet after centrifugation. A protein is hydrophobic in nature as it dissolved with phosphate buffer.

Dialysis
One of the most common method in removing salt is that of dialysis. The main feature of dialysis is that, is porous, pore size is such that small salt ions can freely pass through membrane, larger protein molecules cannot that is they are retained. Dialysis proceeds by placing a high salt sample in dialysis tube and putting it into the desired low salt sample as stated in Blaber (1998).

Ion exchange chromatography
Lichenin was purified and distinguished on the basis of their net charge by a procedure called ion exchange chromatography (IEC) using sephadex resin performed as mentioned in Berg (2002). Lichenin (bacteriocin) fractions number 1 to 10 were collected by 0.1 M to 1.0 M NaCl gradient The overall lichenin yield was quantified by UV spectrometer at 280 nm as mentioned in Table 4 and Fig 81, showing elution profile of lichenin from a sephadex column.
It have been reported that bacteriocin from Bacillus subtilis H27 purified by Q-sepharose and sephadex column chromatographies (Kindoliet al. 2012).

Summary and Conclusion:-
Bacillus licheniformis was isolated from the soil sample and identified according to Gram's staining microscopic examination, biochemical identification was based on Bergey's Manual of Determinative Bacteriology and Advance Bacterial Identification Software (ABIS), confirmed isolates as Bacillus licheniformis. Evaluation of antagonistic activity of crude lichenin by agar well diffusion assay against standard microorganisms S. aureus MCCB 0139 and E. coli MCCB 0017 confirmed lichenin (bacteriocin) producing Bacillus licheniformis. The crude lichenin exhibiting maximum clear zone of inhibition are noted against S. aureus MCCB 0139 was proceeded for optimization of physical and chemical parameters for lichenin production. Different run numbers from 1 to 76, with different media component viz sorbitol, lactose, yeast extract, peptone, NH 4 NO 3 , K 2 HPO 4 and MgSO 4, incubation temperature (37.5⁰C, 25⁰C and 50⁰C), pH (4, 6 and 8) and incubation period (12 hrs, 66 hrs and 120 hrs), respectively was optimized. Run no 43 comprising medium components 1.25% sorbitol, 6.65 % lactose, 0.25 % peptone, 0.06% yeast extract, 0.05 % NH 4 NO 3 , 0.3 % K 2 HPO 4 and 0.025 % MgSO 4 , pH 8 at 50⁰C for 12 hrs exhibited 32 mm clear zone of inhibition against S. aureus MCCB 0139 and no zone of inhibition against E. coli MCCB 0017 indication of maximum lichenin (bacteriocin) optimized production medium from Bacillus licheniformis. Purification of crude lichenin was proceeded by 80% ammonium sulphate precipitation, dialysis, ionexchange chromatography. Finally purified lichenin fractions from ion exchange chromatography was characterized by SDS-PAGE.
In this present study, following observations made and conclusion were run number 43 exhibited maximum lichenin antagonistic activity against S. aureus MCCB 0139 alongwith maximum zone of inhibiton within pH 8, at 55⁰C for 12 hrs, medium components amounting sorbitol 1.25 %, lactose 6.65 %, yeast extract 0.25 %, peptone 0.06 %, NH 4 NO 3 0.05 % and MgSO 4, respectively are considered to be an optimized production medium composition for 1456 lichenin production.The effect of individual parameters on zone of inhibition revealed a decrease in lichenin (bacteriocin) activity on increasing the pH from 4.0 to 8.0. Further, no significant effect on bacteriocin activity by other parameters viz. incubation period, concentrations of sorbitol, lactose, yeast extract, ammonium nitrate and K 2 HPO 4 was observed. The 3D response surface and 2D contour plot depicts two variables exhibiting effect of incubation temperature (⁰C) and incubation period (hrs) with other optimization parameters viz. pH, incubation period, sorbitol (%), lactose (%), peptone (%),yeast extract (%), NH 4 NO 3 (%) and MgSO 4 (%). Among all parameters, only incubation temperature (⁰C) was found to have a significant effect on bacteriocin activity. The cube plot depicts three variables (incubation temperature (⁰C), pH and incubation period (hrs)) involved in the bacteriocin activity of isolated bacteriocin. The effect of incubation temperature (⁰C) and incubation period (hrs) on concentration of substrate shows that temperature and substrate concentration have a significant effect on bacteriocin activity as observed by the average value depicted in the top and bottom phase of the cube and the left and right face of the cube, respectively. However the effect due to incubation period had no significant effect on the activity of bacteriocin. Ion exchange eluted 1 to 10 lichenin fractions number was estimated by Lowry's method and quantified at absorbance 660 nm, revealed that NaCl gradient from 0.6 M, 0.8 M, 0.9 M and 1.0M contains 0.031mg/ml, 0.032 mg/ml, 0.097 mg/ml and 0.012 mg/ml lichenin concentration, calculated by obtained linear regression equation, y = 1.710+ 0.038.The maximum 0.097 mg/ml lichenin concentration was characterized by SDS-PAGE revealed < 9 kDa. RSM is one of the effective statistical and mathematical techniques used for developing, improving and optimizing the complex process in the experiment. It describes the effect of independent variables, alone or in combination in the process. Lichenin characterization by SDS-PAGE exhibited small < 9 kDa and justifying to be in class II bacteriocins include (0.77-10 kDa), which are ribosomally synthesized, nonmodified and linear peptide which are large heat and pH stable.
Lichenin characterization showed this small peptide to be a model for studying anaerobiosis-specific expression of antibacterial proteins and bacteriocins for studying bacteriocin structure-functions relationships, host-range interaction and the physiology of bacteriocin production and immunity among the obligatory and facultative anaerobic bacteria. However, in depth studies of lichenin are required to explain the mode of action of lichenin.