Thermostable Lipolytic Enzymes Research Articles

Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase

The extracellular expression of enzymes in a secretion host such as Bacillus subtilis is a useful strategy in reducing the cost of downstream processing of industrial enzymes. Here, we present the first report of the successful extracellular expression in Bacillus subtilis WB800 of Geobacillus stearothermophilus lipase (T1.2RQ), a novel industriallydesirable thermostable lipolytic enzyme which has an excellent hydrolytic and transesterification activity. Signal peptides of α-amylase, extracellular protease, and lipase A, as well as two different promoters, were used in the secretion and expression of lipase T1.2RQ. Lipase activity assay using p-nitrophenyl laurate showed that all three signal peptides directed the secretion of lipase T1.2RQ into the extracellular medium. The signal peptide of lipase A, resulted in the highest extracellular yield of 5.6 U/ml, which corresponds to a 6-fold increase over the parent Bacillus subtilis WB800 strain. SDS-PAGE and zymogram analysis confirmed that lipase T1.2RQ was correctly processed and secreted in its original size of 44 kDa. A comparison of the expression levels of lipase T1.2RQ in rich medium and minimal media showed that the enzyme was better expressed in rich media, with up to an 8-fold higher yield over minimal media. An attempt to further increase the lipase expression level by promoter optimization showed that, contrary to expectation, the optimized promoter exhibited similar expression levels as the original one, suggesting the need for the optimization of downstream factors. The successful extracellular secretion of lipase T1.2RQ in Bacillus subtilis represents a remarkable feat in the industrial-scale production of this enzyme. How to cite: Ridwan E, Suwanto A, Thenawidjaja M. Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase. Electron J Biotechnol 2021;53. https://doi.org/10.1016/j.ejbt.2021.07.003

Application of Bacterial Thermostable Lipolytic Enzymes in the Modern Biotechnological Processes: A Review

Review focuses on the modern applications of bacterial lipolytic enzymes in biotechnology and covers the scope of their properties including their activity and functional stability at different temperatures, pH, substrate specificity, and activity in the presence of different chemicals. The recent data on the production of genetically engineered strains producing the bacterial lipolytic enzymes and approaches to improving their productivity are presented. The applications of bacterial lipases in biotechnological processes used in the production of biofuel, chemicals and detergents, in the food industry, and in wastewater treatment are considered.

Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs

BackgroundAmong the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc.ResultsIn order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5–70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98–99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins.ConclusionsThe results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

Cell surface and cellular debris-associated heat-stable lipolytic enzyme activities of the marine alga Nannochloropsis oceanica

Microbial surface display of lipases can be effectively employed for the development of whole-cell biocatalysts for industrial bioconversions. In the present work, we report for the first time the presence of thermostable lipolytic enzyme activities against p-nitrophenyl laurate, both on the cell surface and the cellular debris fraction of the marine microalga Nannochloropsis oceanica (strain CCMP1779). Whole cell-associated lipolytic activity (WCLA) shows a 2.5-fold stimulation after heat treatment at 100 °C for 60 min, while the activity of the respective cell debris is retained for 15 min. In contrast, heat treatment renders the soluble fraction of the disrupted cells inactive. The progress curve of cellular debris-associated lipase activity is biphasic and levels off very fast. Treatment with the surfactants SDS, Triton X-100 and CHAPS, which are known to inhibit lipase activity in various degrees, results in a loss of both cell bound and cell debris lipolytic activities (CDLA). The highest whole cell lipase catalytic efficiency was observed against p-nitrophenyl butyrate and the optimum pH for hydrolysis was determined at pH 7.0. Both unheated and heated undisrupted whole cell biocatalysts are also catalytically active against olive oil. High-salt concentrations (1M NaCl) lead to about 50% whole cell enzyme inhibition whereas the activity of heated cells increases. These findings offer novel insight into the biocatalytic properties and the biotechnological applicability of microalgal lipases from N. oceanica.

Thermus thermophilus as a Source of Thermostable Lipolytic Enzymes.

Lipolytic enzymes, esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3), catalyze the hydrolysis of ester bonds between alcohols and carboxylic acids, and its formation in organic media. At present, they represent about 20% of commercialized enzymes for industrial use. Lipolytic enzymes from thermophilic microorganisms are preferred for industrial use to their mesophilic counterparts, mainly due to higher thermostability and resistance to several denaturing agents. However, the production at an industrial scale from the native organisms is technically complicated and expensive. The thermophilic bacterium Thermus thermophilus (T. thermophilus) has high levels of lipolytic activity, and its whole genome has been sequenced. One esterase from the T. thermophilus strain HB27 has been widely characterized, both in its native form and in recombinant forms, being expressed in mesophilic microorganisms. Other putative lipases/esterases annotated in the T. thermophilus genome have been explored and will also be reviewed in this paper.

Biochemical Characterization of A Novel Thermophilic Esterase Isolated from Shewanella sp F88

The main objective of this study was to purify and characterize an esterase from Shewanella sp F88. The enzyme was purified 41-fold and an overall yield of 21 %, using a two-step procedure, including ammonium sulfate precipitation and Q-sepharore chromatography. Molecular weight of the enzyme was 62.3 kDa according to SDS-PAGE data. The enzyme showed an optimum activity at pH 6.5 and 58 ˚C. Evolution of substrate specificity demonstrated that this thermostable enzyme had the highest activity towards para-nitrophenol acetate (pNPA, C2). Michaelis-Menten constant (Km) and maximum velocity (Vmax) of pNPA-hydrolyzing reaction were 12.6 mM and 550 U.mg-1, respectively. Enzyme activity was declined in the presence of metal ions (2 and 5 mM), including Fe2+, Ca2+, Cu2+, Zn2+, Mg2+ and Mn2+. The half-lives of purified esterase was 70 and 31 min at 60 °C and 80 °C, respectively. In conclusion, the enzyme is a novel thermostable lipolytic enzyme characterized from Shewanella species.

Scaling-up the production of thermostable lipolytic enzymes from Thermus aquaticus YT1

The lipolytic enzymes synthesized by Thermusaquaticus YT1 present extremely interesting properties of thermostability (more than 70% of activity after 12 days at 80°C and a half-life time of 1 h at 95°C), which point out the interest of proposing efficient strategies to successfully tackle the scale-up of the production process. In this study,viable scaling-up of the production process was implemented,and relevant aspects affecting the enzyme synthesis, such as the mineral composition of the culture medium, the aeration and the agitation have been evaluated.A strategy combining the modification of the culture medium and the aeration degree was also approached by adding perfluorocarbons, compounds which improve the availability of oxygen in the culture medium. An opposite response of biomass and lipolytic activity to the aeration conditions was found between scales (about 600 U L(-1) at high aeration levels in flask vs. 150 U L(-1) at high aeration rates in reactor), which further demonstrates the important role of the hydrodynamic conditions on the suitable development of the biological process. In all cases, the cultures were kinetically characterized and the Luedeking and Piret model turned out to be a valuable tool to conclude that the produced lipolytic enzyme is a growth-associated metabolite, no matter the medium and the scale.

Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k (cat)/K (m)) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.

On the hyperthermostability of lipolytic enzymes from Thermus aquaticus YT‐1: exploring their application to polymer degradation

AbstractBACKGROUND: The investigation of enzymes from extremophilic microorganisms has generated great scientific interest in recent decades owing to their ability to operate in harsh conditions. Since lipolytic enzymes are one of the three groups most commercially demanded, the study of novel thermostable lipolytic enzymes is of much interest.RESULTS: Lipolytic enzymes produced by Thermus aquaticus YT1 have been investigated. Very interesting properties against thermal inactivation, with a half‐life time of 1 h at 95 °C were found. The use of compounds such as polyols is a strategy used to further improve the thermostability of these enzymes, and the modelling of the deactivation process with a series‐type equation, allows one to quantitatively ascertain the free energy of deactivation. Finally, the application to dibutyl adipate hydrolysis was investigated, as an indicator of the potential usefulness of the enzymes in polymer biodegradation.CONCLUSION: The crude lipolytic enzymes possess outstanding properties of thermostability (high lipolytic activity after 12 days at 80 °C, and a half‐life time of 1 h at 95 °C), and they can be significantly improved by adding polyols (addition of glycerol entailed activity losses of just 3% after 1 h at 95 °C). These enzymes are able to catalyze ester cleavage, showing a zero‐order kinetics. Copyright © 2011 Society of Chemical Industry

Biochemical Analysis of a Novel Lipolytic Enzyme YvdO fromBacillus subtilis168

The predicted amino acid sequence of Bacillus subtilis yvdO exhibits similarity to that of the proteins belonging to the patatin family of lipolytic enzymes. In the present study, YvdO was overproduced in Escherichia coli and purified, and its enzymatic properties were determined. YvdO hydrolyzed p-nitrophenyl fatty acid esters. The enzyme was specific to middle-chain fatty acids, and its optimum pH was approximately 7.5. It maintained 86% of its initial activity after incubation for 30 min at 80 degrees C, and its secondary structure was retained at up to 80 degrees C. Free myristic acid was detected as the product of the reaction with YvdO and 1-myristoly-2-lyso-sn-glycero-3-phosphocholine, while YvdO did not hydrolyze 1,2-dimyristoly-sn-glycero-3-phosphocholine. These results suggest that YvdO is a novel thermostable lipolytic enzyme that has the ability to hydrolyze lysophospholipids.

Thermostable lipolytic enzymes production in batch and continuous cultures of Thermus thermophilus HB27

Several studies in laboratory-scale bioreactors are undertaken in order to verify the beneficial effect of thermal spring water in the culture medium of Thermus thermophilus HB27. Two bioreactor configurations, stirred tank and airlift, are investigated to determine the most suitable one to develop a continuous process. Water mineral composition affects the lipolytic enzyme secretion and growth of T. thermophilus HB27 in both bioreactor configurations. Furthermore, the lipolytic activity is strongly enhanced when stirred tank bioreactor is used. Moreover, operation in a stirred tank at an agitation rate of 650 rpm leads to the highest total lipolytic activity (intra- and extracellular enzyme) around 280 U/L after 32 h. Continuous cultures operating in the optimised conditions determined in batch cultures are carried out. It is noticeable that the stirred tank bioreactor was able to operate in a continuous flow mode without operational problems. In addition, the lipolytic activity obtained is about 2-fold higher than that attained in batch cultures.

Assessment of Relevant Factors Influencing Lipolytic Enzyme Production by Thermus thermophilus HB27 in Laboratory‐Scale Bioreactors

AbstractNowadays, there is not much information on the large‐scale production of thermostable lipolytic enzymes by thermophilic organisms. Therefore, in this study the lipolytic enzyme production by Thermus thermophilus HB27 in laboratory‐scale bioreactors was undertaken. In order to determine the most suitable bioreactor, two configurations were investigated: stirred‐tank and airlift bioreactor. It was demonstrated that the stirred‐tank configuration led to the highest lipolytic extracellular activities, about 2.3‐fold higher than those found in the corresponding cultivation in the airlift bioreactor. On the other hand, the influence of several factors such as culture nutrients concentration, aeration, and agitation rate on the production of thermophilic lipolytic enzymes in a 5‐L stirred‐tank bioreactor was assayed. At reduced nutrients concentration (50 % with respect to the basal medium), a higher product/biomass yield was attained, without any operational problems. From the relationship between mass transfer coefficient (KLa), aeration, and agitation rates it was concluded that there is a lesser dependence on the aeration than the agitation rate. In addition, the mechanical stirring of the bioreactor could tear the membranes that contain the rotund bodies often found in cultures of thermophilic microorganisms, thus increasing the extracellular enzyme production.

Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme. Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities at alkaline pH, of 7–9 and 8–10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 50–75 °C, with optimal activity at 70 °C and were stable at 70 °C for at least 120 min. Both PLP and Est1 exhibited high V max toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C 4 and C 5) and long chain (C 14 and C 16) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which usually show no activity for substrates longer than C 10. We suggest that PLP and EstA enzymes are novel and have a; b potential use in industrial applications.

A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: Purification and characterization

A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at 60-65 ( composite function)C and retained 100% activity after incubation at 60 ( composite function)C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation at 70 ( composite function)C and 23 min incubation at 80 ( composite function)C. Catalytic function of lipase was activated by Mg(++) (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA, DEPC, betaME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K ( m ) and V (max) of 0.5 mM and 0.139 microM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active site and antigen-binding site of enzyme do not overlap.

Effects of lipolytic enzymes Pseudomonas fluorescens on liberation of fatty acids from milk fat

Effects of thermostable lipolytic enzymes Pseudomonas fluorescens 66 ZB in pasteurized milk on concentration of free fatty acids (VMK) in milk were studied in selected milk samples. Identical bulk milk samples were analysed by the method specified in previous papers (Vyletělová et al. 1999a, b, 2000). Reference milk samples (without bacterial strains) and the experimental ones (containing Ps. fl. 150 th. CFU/ml and 2800 th. CFU/ml, resp.) were stored at 6.5°C and 14°C and analysed at regular time intervals (24 h) – Table 1. An extractive-titric method (Kadlec et al. 1996; Table 2 and Fig. 2) was used for monitoring of fatty acid (MK) liberation. Precise analyses of MK and VMK were made by the chromatographic method (Figs. 1, 3 and 4). Medium-chain fatty acids (C12–C16) are liberated first of all; short-chain acids (C6–C10) were found sporadically or in very small quantities (Table 2). Dissociation constant of the specific fatty acid liberated from milk fat affects principally relationships between pH and free fatty acid concentration. The predominating proportion of long-chain acids in liberated fatty acid formation is associated with lower reduction of pH as compared to the predomination of fatty acids with shorter chains associated with more substantial reduction of pH. In our study, a rapid decrease of pH was noted before 168 h (Table 24); this corresponds to low concentrations of short-chain free fatty acids. Vyletělová et al. (2000) found significant relations between pH and contents of VMK (measured by the extractive-titric method); in some samples, correlation coefficients amounted to r = –0.93*** (P £ 0.001). The extractive-titric method analysing VMK concentrations (mmol/kg milk fat) provides results characterized by a systematic rise (e.g., 32.0 mmol/kg instead of 13.0 mmol/kg in raw milk). According to Kratochvíl (1992) 20 mmol VMK/kg milk fat signalized the starting point characterizing flavour degradation of milk caused by activities of fatty acids C12–C14 above all; the transformed value (respecting specifics of the extractive-titric method) amounts to 49 mmol/kg. In case of higher storage temperature a significant break is found after 144 h; in case of lower temperature this break is after 192 h (Table 2). Limits determining potential lipolytic modifications of milk flavour (RLZCHV) as related to specific samples and temperatures at VMK levels amounting to 49 mmol/kg or 20 mmol/kg are outlined in Fig. 2. Milk samples No. 5 and No. 6 stored at higher temperature surpassed this risk limit at 56 h and 64 h, respectively (Table 2, Fig. 2). On the contrary, milk samples stored temperatures corresponding to the standard storage temperature (storage of raw milk, transport, storage of pasteurized milk) surpass the mentioned risk level after 90 h and 140.5 h. Obtained results document the predominant role of storage temperature in the whole complex (production and processing of milk as a raw material or an intermediate product); evident differences in contamination rates (105 an 106) can be characterized as secondary effects in this case (Table 2). As related to practical conditions, the mentioned facts imply immediate processing of raw milk and pasteurized milk. This postulate must be respected namely by dairy plants producing delicate milk products. Vyletělová et al. (2000) found a notable VMK increase/24 h (7–11 mmol/kg milk fat) under specific temperatures and microbial contamination.

Microbial growth and lipolytic activities of moderate thermophilic bacterial strains

Nine bacterial strains grew at 50°C on a mineral medium containing Tween 80 as sole carbon source and three of them produced significant lipolytic activities. The strains showed optimal growth at 50°C and thus, they are moderate thermophiles. Lipolytic activity were maxima at 57–67°C suggesting high thermostability. Lipolytic activity production was promoted by the addition of Tween 80 in culture medium and inhibited by glucose addition. These strains are convenient sources of thermostable lipolytic enzymes for biotechnological applications. © Rapid Science Ltd. 1998