Isolation and Characterization of a Novel Thermo-solvent-stable Lipase from Pseudomonas Brenneri and its Application in Biodiesel Synthesis

Abstract Pseudomonads are one of the most studied species of bacteria as they display remarkable metabolic and physiological versatility. This enables them to colonize a wide variety of terrestrial and aquatic habitats, generating biotechnologically interesting enzymes. Here, the partial purification and characterization of a novel, extracellularly-produced, lipase from Pseudomonas brenneri is described. The partially purified lipase was active over a broad pH range (5.0–9.0) and was stable at 70 °C for 45 min. The lipase displayed significant stability, and in some cases activation, in the presence of organic solvents with log P ≥ 2.0. Such stability characteristics indicated that this lipase could potentially be useful as a biocatalyst for biodiesel production. This was subsequently demonstrated through the facile production of Fatty Acid Methyl Esters in the presence of olive oil and methanol. Possible applications for this novel, stable lipase include the bioremediation of oil in the environment.


Introduction:
Enzyme biotechnology has progressed from catalysis in an aqueous medium to synthesis in nonaqueous media. Since non-aqueous biocatalysis is preferred for many processes, a significant research effort in recent decades has been devoted to establishing platforms for its enhancement of non-aqueous biocatalysis (Illanes, 2016). However, many enzymes are either inactivated or denatured in organic solvents which limits their usefulness (Kumar et al., 2016). Physical (immobilisation), chemical (protein modification) and genetic methods (protein engineering) have been employed to tailor enzymes in order to increase their activity, stability and selectivity in nonaqueous environments (Villeneuve et al., 2000). However, nature provides a vast natural microbial pool of novel enzymes which can be screened by exploring the diversity of the microbiome (Adrio and Demain, 2014). Solvent stable lipases are key biocatalysts in non-aqueous solutions and can catalyse various reactions (such as esterification, alcoholysis, acidolysis and interesterification; (Sharma and Kanwar, 2014)). Due to their wide ranging applications, lipases have long been subjected to intensive study. Pseudomonas sp. are a known source for the isolation of lipases. For example, a lipase from P. cepacia has been used for the synthesis of cinnamyl propionate (Badgujar, Pai and Bhanage, 2016) while lipases from P. fluorescens and P. stutzeri have been used in the cosmetic, oral care and pharmaceutical industries (Vescovi et al., 2017;Cao et al., 2012). In this laboratory, we have previously identified lipases with potential in organic synthesis (Priyanka et al., 2019).
In this study, a novel extracellular lipase was identified in a Pseudomonas brenneri culture isolated from soil sample. The lipase was partially purified and characterised following production optimisation. This thermo-organo stable novel lipase was employed in the production of Fatty Acid Methyl Esters (FAMEs) as a proof of application exercise. Based on initial characterisation and this application, the novel lipase will be of further interest for various biocatalytic and biotechnological uses in non-aqueous media, as well as bioremediation of oil in the environment (Kumar, 2020).

Chemicals and materials:
All chemicals used were of analytical grade and purchased from Sigma-Aldrich. GE Healthcare supplied the Q-Sepharose high performance (HP) resin. The GC column for the analysis of FAME(s) was purchased from Bruka™.  (bacteriological peptone, yeast extract, tryptone, ammonium sulfate, Urea, L-Lysine, L-Arginine, Glutamic acid, Glutamine and Asparagine respectively) were used as a substitute for 50 g/L peptone in the basal lipase production medium, as described in previous work (Priyanka et al., 2019).

2.5.
Lipase partial purification Following fermentation using the optimized conditions (pH 6.8), cell free supernatant was harvested by centrifugation at 4 °C, 5000g for 20 min. This was then initially filtered through a 1.2 μm pre-filter and, subsequently, a 0.45 μm filter. The filtered cell free supernatant was dialyzed in a 14 kDa cut-off dialysis membrane (Sigma-Aldrich) at 1:20 ratio in 10 mM Tris HCl, pH 8.5, for 4 h at room temperature under continuous stirring using a magnetic stirrer. After 4 h, the buffer was replaced with fresh buffer and dialysis was continued overnight (15 h) at 4 °C. After dialysis, the crude extract was alkaline precipitated for 30 min at pH 9.0 at room temperature. After centrifugation at 5000g for 20 min at 4 °C and filtration through 0.45 µm filter; the alkaline precipitated filtrate was adjusted to pH 8.5 using HCl. This filtrate was loaded onto an anion exchange Q-Sepharose HP resin pre-equilibrated with 10 mM Tris-HCl pH 8.5. Partially purified lipase was collected from the anion exchange chromatography resin when a step elution with 750 mM NaCl in 10mM Tris-HCl pH 8.5 was performed.
Following dialysis, the protein sample was transferred to an air-tight container and was stored at -80 °C for 12 h. The frozen sample was later freeze dried at -54 °C, 0.002 milli bar. The freezedried protein sample was reconstituted based on requirements for characterization and application.

2.6.
Stability studies/characterisation All stability studies were carried out using 30IU/ml of lipase with a specific activity of 3.18 IU/mg. Following enzyme incubation at 60 °C for 4 h, the lipase thermal half-life (T1/2) was estimated.
The partially purified lipase was analysed for stability in a variety of organic solvents by gently mixing the selected solvent and the enzyme at both 28 °C and 40 °C in screw cap glass vials. The effect of modifiers such as metal ions, enzyme inhibitors and surfactants on the lipase, at 28 °C and 40 °C, were explored in a similar way. For organic solvent and modifier stability studies, a percentage residual activity value was measured relative to control (i.e. the enzyme solution without any solvents/additives). A Lineweaver-Burk plot was employed to estimate Michaelis-Menten steady state kinetic constants of Km and Vmax. In all cases, with the exception of substrate specificity, lipolytic activity was measured using the standard spectrophotometric assay employing p-NPP as substrate.

2.7.
FAME synthesis using olive oil 500 IU of lipase (specific activity of 3.18IU/mg) was used for the transesterification of 1gm olive oil using methanol (molar ratio 9:1 for methanol: oil). The reaction mixture was maintained at 40 °C and 120 rpm for 72 h in a shaker water bath. After transesterification, the sample was centrifuged at 5,000g for 10 min and the solvent layer (top layer) containing FAME was carefully pipetted into a clean sealed glass container. FAMEs generated were subsequently analysed by TLC and GC. TLC detection of FAME post transesterification was carried out as per Kim and colleagues (2014). In brief, a 90:10 (v/v) n-hexane:diethyl ether solvent mix was used as the mobile phase and after full development of TLC plate, the FAME spots were visualized using a 10 % (v/v) ethanoic phosphomolybdic acid spray, followed by drying at 105 °C for 5 min.
A Scion-436GC (comprising a GC column BR-SWax, 0.25 mm x 30 m, with FID detector) was used for FAME GC analysis. FAMEs generated by transesterification of olive oil were identified by comparing their RT (Retention Time) with the RT of a standard FAME mix (Sigma-Aldrich; Religia and Wijanarko, 2015). The GC method published by Agilent (David, Sandra and Vickers, 2005) was used for all the FAME analysis.

Isolation and identification of solvent tolerant lipase producing strain
Two lipolytic cultures isolated from soil samples (GPS location 53°00'12.4"N, 6°20'47.9"W; a woodland in a national park) were stable in cyclohexane, ethanol, n-hexane, heptane and methanol by plate overlay method (Patel, Nambiar and Madamwar, 2014). 16S rRNA sequencing of these strains identified one of the lipolytic cultures as Pseudomonas reinekei (see Priyanka et al., 2019) while the other isolate was identified as Pseudomonas brenneri. The stability of the extracellular lipase produced by Pseudomonas brenneri in the presence of n-hexane, as observed by plate overlay method, is shown in Figure 1.

Lipase production
Extracellular microbial lipases are useful for biotechnological applications as they can be easily recovered from the fermentation broth. However, optimization of media and growth conditions are vital for the successful development of a productive fermentation process (Padhiar, Das and Bhattacharya, 2011). Media composition, as well as factors like initial inoculum and fermentation temperature and time, are known to effect the extracellular production of lipases (Andualema and Gessesse, 2012). In this study, a one-factor at a time approach was employed to optimise these influencing variables for P. brenneri production (Ayinla, Ademakinwa and Agboola, 2017).

Fermentation time and inoculum percentage
At a suitable inoculum size, the nutrient and oxygen levels are appropriate for bacteria growth and therefore, maximum lipase production. Conversely, if the inoculum size is too small, insufficient biomass will lead to reduced levels of lipase secreted over the culture period. For example, for P.
In the current study, maximum lipase activity for P. brenneri lipase was obtained with 2.5 % (v/v) inoculum after 2 days of fermentation in basal lipase producing medium (see Figure 2). The rate of lipase production is related to the organism, however, generally, extracellular lipases are produced in the late logarithmic or stationary phases of microbial culture (Gupta et al., 2016).
Various cultivation periods, ranging from 5 h to 168 h, have been described as optimal for different lipase producing organisms. Lipases from P. reinekei (Priyanka et al., 2019) and

Nitrogen Source
Tryptone and peptone are common organic sources of nitrogen used for microbial culture and enzyme production. For the lipase from P. brenneri, a 1 % (w/v) bacteriological peptone supplement resulted in a significant (p≤0.05, t-test; see Figure 3) increase in lipase production to 0.91 IU/mL and was the best nitrogen source of those explored. It has been reported that peptone is an inducer of lipase production as it provides NH4 + ions, which stimulate bacterial growth and increases enzyme production (Kumar et al., 2012). A 1% (w/v) peptone supplement was found to be the best nitrogen source for lipase production by Pseudomonas gessardii (Ayinla, Ademakinwa and Agboola, 2017) and a 0.5 % (w/v) peptone level was optimal for lipase production in ). Therefore, it was important to explore different amino acids supplements to examine their effect on lipase production.

Partial purification
Partial lipase purification was accomplished by a two-step process (see Table 1). The initial, and little used, purification step of alkaline precipitation (pH 9.0) removed some contaminant proteins from the lipase preparation. A second purification step of anion exchange chromatography (pH 8.5) eluted partially purified lipase, in an elution buffer containing 750 mM NaCl at pH 8.5.
Increasing the pH to pH 9.0 triggered precipitation of the dialysate, which could not be used for purification on chromatographic resins. Hence, to avoid precipitation and to permit the use of the dialysate after alkaline precipitation for chromatography, the pH of dialysate was lowered to 8.5.
Following the purification procedure; a lipase with specific activity of 3.18 IU/mg was achieved, with an overall yield of 56.89%.

Effect of pH
The lipase from P. brenneri showed maximum stability (>90% relative activity) between pH 5.0 to 8.0 (see Figure 4). However, at pH 3.0, 4.0 and pH 10.0; a significant loss of activity was seen (p≤0.05, t-test). The optimal pH of this lipase was 6.0-8.0, as is the case for many other ). An optimal pH of 6.0-8.0 makes this lipase ideal for detergent formulations, as well as for flavour synthesis and bioremediation (Salihu and Alam, 2015).

Thermostability
Lipases from many Pseudomonas species are known to have optimum temperatures from 4°C to 90°C (11,26,28,29). P. brenneri lipase showed a broad temperature range for optimum lipolytic activity. The lipase showed >80 % residual activity at 70 °C after 45 min of incubation (   Table 2).  Table   3). The lipase was found to be stable in 10 % (v/v) ethanol after 24h of incubation. However, increased concentrations of both methanol and ethanol generated a statistically significant loss in activity. Conversely, non-polar organic solvents with higher log P possess a reduced ability to strip the essential water from the enzyme structure. Hence, lipases tend to be more stable and active in high log P solvents (cyclohexane, n-hexane, n-heptane etc.). Enhanced lipolytic activity was

Substrate specificity
The substrate specificity of lipases provides useful information for the rapid selection of enzyme/substrate partnerships to catalyse desired reactions. A preference for long chain fatty esters is a desirable characteristic for lipases (Li et al., 2014). P. brenneri lipase demonstrated a wide substrate specificity range (from medium length to long chain phenyl esters i.e. C8:0 to C16:0).

Enzyme Kinetics
Km values as low as 0.037 mM, and a Vmax as high as 188.6mmoL/L/min, using p-NP hydrolysis have been reported for lipases from Pseudomonas aeruginosa SRT 9 (Borkar et al., 2009). For the P. brenneri lipase the Vmax and Km values were 5.17±0.12mmoL/min/mg and 0.37±0mM respectively. The kinetic parameters were studied using pNP-palmitate as the substrate at 28°C.
Kinetic parameters were estimated from a Lineweaver Burk plot (see Figure 8).

Application of P. brenneri lipase in FAME generation using olive oil
As the lipase from P. brenneri (H3) was stable towards various organic solvents and showed specificity towards long-chain esters, it was considered suitable for biodiesel production.
Methanol is widely used as the alcohol for transesterification reactions due to its widespread availability and solubility (Musa, 2016). Generally, for complete conversion of oil to methyl esters (also known as biodiesel or FAME), at least a 3-fold molar ratio of alcohol to oil is required in the reaction mixture (Bose and Keharia, 2013). A higher molar ratio of alcohol to oil is required to increase the contact between oil and alcohol and to drive the reaction equilibrium towards biodiesel synthesis. Increased alcohol to oil ratios result in enhanced biodiesel yield in a shorter time (Helwani et al., 2009). Therefore, in this study, a molar ratio of 1:12 for olive oil:methanol was used for transesterification reaction. After 72h of incubation at 40°C, P. brenneri lipase generated FAME(s) as detected by TLC (see Figure 9), with a five-batch average yield of 85 % (±5 %). This yield is comparable to commercially available immobilised enzymes commonly used in biodiesel production; Novozym435 (>90%) and Candida sp. 99-125 (87%) (Tan et al., 2010). The composition of the crude FAMEs synthesized was compared to the standard FAME mix (Sigma-Aldrich) using GC (Figure 10; Eder, 1995).

Conclusion
In the current study, a novel lipase secreted by Pseudomonas brenneri was identified, isolated, characterised and applied to FAME synthesis. The results of this study indicate that this lipase is may be a metalloenzyme which is thermo-and organic solvent stable and is activated in the presence of organic solvents with log P>2.0. The lipase displayed excellent operational lipolytic activity between 10°C and 60°C towards p-NPP as substrate. The lipase was employed in the synthesis of crude FAMEs from olive oil, in the presence of methanol, as a proof of application.
This initial work indicates the potential application of this enzyme in biodiesel synthesis using diverse oil sources, as the enzyme displayed specificity towards a wide range of esters. Additional work will be required to extend the application of this novel enzyme in biodiesel synthesis, with the possibility of enhanced biocatalysis through novel strategies for immobilisation and stabilisation (Kumar, 2019).     FAMEs MAGs/DAGs Figure 10: GC chromatogram of crude FAMEs mixture synthesized from the transesterification reaction of olive oil using lipase from P. brenneri. Peak 1: Solvent and Peak 6: Oleic Acid. Peak identification was carried out by comparing the Retention Time of the FAME with Retention of FAME 37-component standard FAME mix.  Table 2: The effect of various metal ions and effector molecules/chemicals (10mM) on the stability/activity of P. brenneri lipase was investigated and reported by the spectrophotometer assay. The residual activity (%) was calculated relative to that of enzyme solution in the absence of any additive, after 24h of incubation at 28°C. The data represented are the mean of three independent experiments and the standard deviations are noted (*P ≤ 0.05, **P ≤ 0.01, ****P < 0.0001 represents significant, very significant and extremely significant difference based on t-test)  Ol31.DATA µV