Exploring the effects of oil inducer on whole cell-mediated methanolysis for biodiesel production

Whole cell-mediated methanolysis of renewable oils for biodiesel production has drawn much attention in recent years since it can avoid the complex preparation procedures of traditional immobilized lipase. During the cultivation of Rhizopus oryzae whole cell catalyst, plant oils are usually added into the medium as inducer for lipase synthesis. It was found that oil inducer not only influenced lipase production, but also led to varied whole cell's catalytic performance. In this paper, the related mechanisms were explored and it was found that the higher unsaturated fatty acid (UFA) was contained in oil inducer, the higher intracellular lipase could be obtained. Different oil inducers also resulted in varied compositions of cell membrane, which was further found to be responsible for the operational stability of the catalyst. Cells with membrane enriched with saturated fatty acid (SFA) exhibited better stability than those enriched with UFA. And further study showed that after glutaraldehyde cross-linking treatment, the operational stability of both UFA enriched cells and SFA enriched cells were enhanced greatly and no loss in cell's catalytic activity was detected after being repeatedly used for 15 batches.     

Tween 80 influences the production of intracellular lipase by Schizochytrium S31 in a stirred tank reactor

Marine microorganisms are a potential source of enzymes with structural stability, high activity at low temperature and unique substrate selectivity. Thraustochytrids are marine heterotrophic microbes, well known for the production of omega-3 fatty acids. In this study the effect of Tween 80 as a carbon source was investigated with regard to biomass, lipase and lipid productivity in Schizochytrium sp. S31. Tween 80 (1%) and 120 h of incubation were the optimum condition period for biomass, lipid and lipase productivity in a stirred tank reactor. The yields obtained were 0.9 g L⁻¹ of biomass, 300 mg g⁻¹ of lipid and 39 U/g of lipase activity. Sonication was optimised in terms of time and acoustic power to maximise the yield of extracted lipase. The extracted lipase from Schizochytrium S31 was observed to hydrolyse long chain polyunsaturated fatty acids DHA and EPA.     

Improved catalytic performance of GA cross-linking treated Rhizopus oryzae IFO 4697 whole cell for biodiesel production

Whole cell-mediated methanolysis of renewable oils for biodiesel production has drawn much attention in recent years since it can avoid the complex procedures of isolation, purification and immobilization required for the preparation of immobilized lipase. It has been demonstrated that Rhizopus oryzae IFO 4697 whole cell could catalyze the methanolysis of renewable oils for biodiesel production effectively and glutaraldehyde (GA) cross-linking treatment on whole cell catalyst could improve its stability in the repeated uses. The catalytic performance of cells with GA cross-linking treatment was studied systematically in this paper. The results showed that the treated cells expressed higher methanol tolerance, and high catalytic activity could be maintained with higher ratio of methanol to oil; the operational stability of whole cell catalyst and methanol utilization rate were also considered in optimization of methanol addition strategy. A novel methanol addition strategy was proposed, with which the reaction time could be shortened significantly and a biodiesel yield of 94.1% could be obtained within 24 h reaction; it was also found that with this methanol addition strategy, GA cross-linked whole cell expressed rather good operational stability; the reason for stability improvement was also investigated and should be attributed to less lipase leakage.     

Catalytic amidolysis of amino acid p-nitroanilides using transition state analogue imprinted artificial enzymes: Cooperative effect of pyridine moiety

Enzyme-like polymer catalysts with the imprints of phosphonate transition state analogue (TSA) lined along with imidazole and pyridine moieties were synthesized using methacryloyl-l-histidine and 4-vinylpyridine as the functional monomers and phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate – the TSA of hydrolytic reaction as the template for the amidolysis of N-benzyloxycarbonyl-l-phenylalanine p-nitroanilide (Z-l-Phe-PNA). Polymers containing different functional groups can act together to provide catalytic activity and selectivity superior to what can be obtained from monofunctional analogues. The higher rate acceleration exhibited by the bifunctional polymer over the monofunctional polymers indicates cooperative catalysis of imidazole and pyridine moieties. The optimum catalytic competence is shown by the bifunctional polymer containing imidazole and pyridine moieties in 2:1 M ratio which may be due to alignment of the functional groups in proper H-bond distance. In addition to the non-covalent interactions like hydrogen bonding or π-stacking interactions between the functional groups of the polymer and the template, 3D-microcavities complementary to the geometry of the template are necessary for effective shape selective binding. Michaelis-Menten kinetics implies that only the catalysts with imidazole moieties act as enzyme-like catalysts and imidazole is the key catalytic function of the enzyme mimics.     

Comparative and bioinformatics analyses of pathogenic bacterial secretomes identified by mass spectrometry in <Emphasis Type="Italic">Burkholderia</Emphasis> species

Secreted proteins (secretomes) play crucial roles during bacterial pathogenesis in both plant and human hosts. The identification and characterization of secretomes in the two plant pathogens Burkholderia glumae BGR1 and B. gladioli BSR3, which cause diseases in rice such as seedling blight, panicle blight, and grain rot, are important steps to not only understand the disease-causing mechanisms but also find remedies for the diseases. Here, we identified two datasets of secretomes in B. glumae BGR1 and B. gladioli BSR3, which consist of 118 and 111 proteins, respectively, using mass spectrometry approach and literature curation. Next, we characterized the functional properties, potential secretion pathways and sequence information properties of secretomes of two plant pathogens in a comparative analysis by various computational approaches. The ratio of potential non-classically secreted proteins (NCSPs) to classically secreted proteins (CSPs) in B. glumae BGR1 was greater than that in B. gladioli BSR3. For CSPs, the putative hydrophobic regions (PHRs) which are essential for secretion process of CSPs were screened in detail at their N-terminal sequences using hidden Markov model (HMM)–based method. Total 31 pairs of homologous proteins in two bacterial secretomes were indicated based on the global alignment (identity ≥ 70%). Our results may facilitate the understanding of the species-specific features of secretomes in two plant pathogenic Burkholderia species.     

Effects of methanol on a methanol-tolerant bacterial lipase

Methanol is often employed in biocatalysis with the purpose of increasing substrates solubility or as the acyl acceptor in transesterification reactions, but inhibitory effects are observed in several cases. We have studied the influence of methanol on the catalytic activity and on the conformation of the lipase from Burkholderia glumae, which is reported to be highly methanol tolerant if compared with other lipases. We detected highest activity in the presence of 50–70 % methanol. Under these conditions, however, the enzyme stability is perturbed, leading to gradual protein unfolding and finally to aggregation. These results surmise that, for this lipase, methanol-induced deactivation does not depend on inhibition of catalytic activity but rather on negative effects on the conformational stability of the catalyst.     

Encapsulation in a sol–gel matrix of lipase from Aspergillus niger obtained by bioconversion of a novel agricultural residue

Lipase from Aspergillus niger was obtained from the solid-state fermentation of a novel agroindustrial residue, pumpkin seed flour. The partially purified enzyme was encapsulated in a sol–gel matrix, resulting in an immobilization yield of 71.4 %. The optimum pH levels of the free and encapsulated enzymes were 4.0 and 3.0, respectively. The encapsulated enzyme showed greater thermal stability at temperatures of 45 and 60 °C than the free enzyme. The positive influence of the encapsulation process was observed on the thermal stability of the enzyme, since a longer half-life t _1/2 and lower deactivation constant were obtained with the encapsulated lipase when compared with the free lipase. Kinetic parameters were found to follow the Michaelis–Menten equation. The K _m values indicated that the encapsulation process reduced enzyme–substrate affinity and the V _max was about 31.3 % lower than that obtained with the free lipase. The operational stability was investigated, showing 50 % relative activity up to six cycles of reuse at pH 3.0 at 37 °C. Nevertheless, the production of lipase from agroindustrial residue associated with an efficient immobilization method, which promotes good catalytic properties of the enzyme, makes the process economically viable for future industrial applications.     

Kinetics of lipase recovery from the aqueous phase of biodiesel production by macroporous resin adsorption and reuse of the adsorbed lipase for biodiesel preparation

A commercial macroporous resin (D3520) was screened for lipase recovery by adsorption from the aqueous phase of biodiesel production. The influences of several factors on the adsorption kinetics were investigated. It was found that the kinetic behavior of lipase adsorption by macroporous resin could be well described by pseudo-first-order model. Temperature had no significant effects on lipase adsorption, while resin-to-protein ratio (R) significantly affected both rate constant (k₁) and equilibrium adsorption capacity (Q_e). No lipase was adsorbed when mixing (shaking) was not performed; however, protein recovery reached 98% after the adsorption was conducted at 200 rpm for 5 h in a shaker. The presence of methanol and glycerol showed significant negative influence on lipase adsorption kinetics. Particularly, increasing glycerol concentration could dramatically decrease k₁ but not impact Q_e. Biodiesel was found to dramatically decrease Q_e even present at a concentration as low as 0.02%, while k₁ was found to increase with biodiesel concentration. The adsorbed lipase showed a relatively stable catalytic activity in tert-butanol system, but poor stability in solvent-free system when used for biodiesel preparation. Oil and biodiesel were also found to adsorb onto resin during transesterification in solvent-free system. Therefore, the resin had to be washed by anhydrous methanol before re-used for lipase recovery.     

The General Secretory Pathway of Burkholderia gladioli pv. agaricicola BG164R Is Necessary for Cavity Disease in White Button Mushrooms

Cavity disease in white button mushrooms is caused by Burkholderia gladioli pv. agaricicola. We describe the isolation and characterization of six mutants of the strain BG164R that no longer cause this disease on mushrooms. The mutations were mapped to genes of the general secretory pathway (GSP). This is the first report of the association of the type II secretion pathway with a disease in mushrooms. Phenotypes of the six avirulent mutants were the following: an inability to degrade mushroom tissue, a highly reduced capacity to secrete chitinase and protease, and a reduced number of flagella. Using these mutants, we also made the novel observation that the factors causing mushroom tissue degradation, thereby leading to the expression of cavity disease, can be separated from mycelium inhibition because avirulent mutants continued to inhibit the growth of actively growing mushroom mycelia. The GSP locus of B. gladioli was subsequently cloned and mapped and compared to the same locus in closely related species, establishing that the genetic organization of the gsp operon of B. gladioli pv. agaricicola is consistent with that of other species of the genus. We also identify the most common indigenous bacterial population present in the mushroom fruit bodies from a New Zealand farm, one of which, Ewingella americana, was found to be an apparent antagonist of B. gladioli pv. agaricicola. While other investigators have reported enhanced disease symptoms due to interactions between endogenous and disease-causing bacteria in other mushroom diseases, to the best of our knowledge this is the first report of an antagonistic effect.     

Design and synthesis of lipase nanogel with interpenetrating polymer networks for enhanced catalysis: Molecular simulation and experimental validation

A temperature-responsive lipase nanogel (denoted as CRL-IPN nanogel), in which lipase is encapsulated into an interpenetrating polymer matrix formed by polyacrylamide and poly(N-isopropylacrylamide) (PNIPAAm) has been designed and synthesized for an enhanced stability and activity in both aqueous and non-polar organic solvents. A three-step method, including acryloylation, polymerization with acrylamide and sequential polymerization with N-isopropylacrylamide, was established to fabricate enzyme nanogel with temperature-sensitive interpenetrating polymer network. It has been shown by an all-atom molecular dynamics simulation that above mentioned polymer matrix forms a more hydrophobic environment, as compared to that obtained with sole polyacrylamide, because of the penetration of N-isopropylacrylamide into the polymer acrylamide network via hydrogen bonding, which is further confirmed by the fluorescence spectrum. This favours the uptake of hydrophobic substrates and thus the overall rate of enzymatic catalysis. The enhanced stability and catalytic performance of this novel lipase nanogel in aqueous and non-polar organic solvent were demonstrated by using hydrolysis reaction of p-NPP in aqueous and esterification reaction of ibuprofen in isooctane. In aqueous solution, the residual activity of CRL-IPN nanogel maintains its 70% activity at 60 °C after 4 h, compared with that free lipase only has 30% at the same condition. In addition, the CRL-IPN nanogel can be reused for 10 cycles with no loss of its activity. In isooctane, CRL-IPN nanogel gave a 33% yield of esterification of ibuprofen, in comparison to 22% using free lipase and less than 5% using lipase encapsulated in a polyacrylamide matrix. The enhanced stability and activity make this CRL-IPN nanogel promising for enzymatic catalysis in non-polar solvents.     

Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica Lipase: An Efficient and Stable Biocatalyst for Biodiesel Synthesis

Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with –NH₂ groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available.     

Lipase immobilized microstructured fiber based flow-through microreactor for facile lipid transformations

A facile continuous flow-through Candida antarctica lipase B immobilized silica microstructured optical fiber (SMOF) microreactor for application in lipid transformations has been demonstrated herewith. The lipase was immobilized on the amino activated silica fiber using glutaraldehyde as a bifunctional reagent. The immobilized lipase activity in the SMOF was tested calorimetrically by determination of p-nitrophenyl butyrate hydrolysis products. The specific activity of the immobilized lipase was calculated to be 0.91 U/mg. The SMOF microreactor performance was evaluated by using it as a platform for synthesis of butyl laurate from lauric acid and n-butanol in n-hexane and n-heptane at 50 °C, with products identified by gas chromatography–mass spectrometry (GC–MS). Different substrate mole ratios were evaluated, with 1:3, lauric acid:n-butanol showing best performance. Remarkably, percentage yields of up to 99% were realized with less than ∼38 s microreactor residence time. In addition, the SMOF microreactor could be reused many times (at least 7 runs) with minimal reduction in the activity of the enzyme. The enzyme stability did not change even with storage of the microreactor in ambient conditions over one month.     

Molecular mechanism of deactivation of C. antarctica lipase B by methanol

The catalytic activity of Candida antarctica lipase B upon alcoholysis of a constant concentration of 15.2% vinyl acetate (vol/vol) and varying concentrations of methanol (0.7–60%) in toluene was determined experimentally by measuring the initial reaction velocity. The molecular mechanism of the deactivation of the enzyme by methanol was investigated by fitting the experimental data to a kinetic model and by molecular dynamics simulations of C. antarctica lipase B in toluene–methanol–water mixtures.     

Degron mediated BRM/SMARCA2 depletion uncovers novel combination partners for treatment of BRG1/SMARCA4-mutant cancers

Recent studies have highlighted that cancer cells with a loss of the SWI/SNF complex catalytic subunit BRG1 are dependent on the remaining ATPase, BRM, making it an attractive target for cancer therapy. However, an understanding of the extent of target inhibition required to arrest cell growth, necessary to develop an appropriate therapeutic strategy, remains unknown. Here, we utilize tunable depletion of endogenous BRM using the SMASh degron, and interestingly observe that BRG1-mutant lung cancer cells require near complete depletion of BRM to robustly inhibit growth both in vitro and in vivo. Therefore, to identify pathways that synergize with partial BRM depletion and afford a deeper response, we performed a genome-wide CRISPR screen and discovered a combinatorial effect between BRM depletion and the knockout of various genes of the oxidative phosphorylation pathway and the anti-apoptotic gene MCL1. Together these studies provide an important framework to elucidate the requirements of BRM inhibition in the BRG1-mutant state with implications on the feasibility of targeting BRM alone, as well as reveal novel insights into pathways that can be exploited in combination toward deeper anti-tumor responses.     

Expression and characterization of Ca2+-independent lipase from Bacillus pumilus B26

A lipase-producing Bacillus pumilus strain (B26) was isolated from a soil sample collected in Korea. The cloned gene showed that the lipase B26 composed of a 34-amino-acid signal sequence and a 181-amino-acid mature part corresponding to a molecular mass (M_r) of 19,225. Based on the M_r and the protein sequence, the lipase B26 belongs to the lipase family I.4. The optimum temperature and pH of the purified enzyme were 35 °C and 8.5, respectively. The lipase B26 showed a ‘Ca²⁺-independent thermostability and catalytic activity’. These are novel properties observed for the first time in lipase B26 among all bacterial lipases and correspond with the suggestion that this enzyme had no Ca²⁺-binding motif around the catalytic His156 residue. This enzyme seems to be a true lipase based on the experimental results that it could hydrolyze various long-chain triglycerides (C₁₄–C₁₈) and triolein (C_18:1) and that it showed a typical interfacial activation mechanism toward both tripropionin and p-nitrophenyl butyrate.     

Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis under vacuum

Pseudomonas lipase immobilized on CaCO₃ powder was used for the glycerolysis of n−3 polyunsaturated fatty acid ethyl esters to prepare nutritionally valuable glycerides. The initial ethyl ester contained 65 or 99% docosahexaenoic acid ethyl ester (DHAEE) which is very unstable and readily oxidized. The process performance was intensified by: (1) using Pseudomonas lipase which has good specificity for docosahexaenoic acid (DHA), (2) shifting the reaction equilibrium by evaporation of the resulted ethanol under vacuum and (3) enhancing the enzyme operational stability by immobilization on CaCO₃ powder. Under these conditions, over 90% conversion of DHAEE was achieved in 5 h and the oxidative deterioration of DHA was avoided. The final product contained 53% partial glyceride and, thus, had good emulsifying power. The catalyst was reused 5 times showing a very good stability in this system. Other lipases were tried for this reaction and different glyceride compositions were obtained depending on the enzyme specificity for the 1(3)-position of glycerol.     

Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis

We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50°C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70°C, and retained over 90% of the full activity after incubation at 50°C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.     

A convenient test for lipase activity in aqueous-based solutions

We proposed a convenient and accurate method for the measurement of lipase activity in a uniform aqueous-based substrate solution. In this work, lipase from Candida rugosa was used as the model lipase to test its catalytic ability toward p-nitrophenyl palmitate (p-NPP), which was suspended in a mixture of p-NPP ethanol solution and buffer. An ultraviolet–visible spectrophotometer was used to efficiently measure the liberated p-nitrophenol without extraction or centrifugation. Several factors that affected lipase activity were investigated, such as the ratio of p-NPP ethanol solution to buffer, the concentrations of p-NPP and lipase, as well as the temperature, reaction time, pH and agitation rate. Additionally, enzyme catalytic parameters such as K_m, V_m and “activation energy” were also assessed. We determined the optimal conditions for lipase in this homogeneous system and demonstrated lipase's catalytic performance in this condition followed Michealis–Menten kinetics.     

Multifunctional hydrolytic catalysis: II. Hydrolysis of p-nitrophenyl acetate by a polymer catalyst which contains hydroxamate and imidazole functions

A water-soluble polymer catalyst was prepared by radical polymerization of a protected hydroxamate monomer, 1-methyl-2-vinylimidazole and acrylamide, and by the subsequent NH₂NH₂ treatment of the polymer. The hydrolysis of p-nitrophenyl acetate by the bifunctional copolymer obeyed typical burst kinetics: rapid accumulation of acetyl hydroxamate group and its slow decomposition. The acetylation rate of the hydroxamate group was rather close to that of a polymer which does not contain the imidazole unit. However, the deacylation was markedly accelerated by the presence of the imidazole unit, and the difference in rate constants amounted to 60- to 80-fold at pH 8–9. These results indicate that the overall catalytic efficiency of the bifunctional polymer is enhanced due to the complementary action of the imidazole and hydroxamate functions.     

Multifunctional hydrolytic catalysis: VI. Catalytic hydrolysis of p-nitrophenyl acetate by N-(4-imidazolylmethyl)benzohydroxamic acid

A bifunctional catalyst, N-(4-imidazolylmethyl)benzohydroxamic acid, was synthesized from benzohydroxamic acid and chloromethylimidazole, and used for the hydrolysis of p-nitrophenyl acetate. The reaction proceeded via the formation of the acetyl hydroxamate and its subsequent decomposition. The deacylation step was shown to be general base-catalyzed by the intramolecular imidazole group on the basis of the deuterium solvent kinetic isotope effect of 2.0. The efficiency of water attack on the acetyl hydroxamate was enhanced 130-fold by the imidazole group. The catalytic process is compared with the reactions of related monofunctional compounds, and finally its significance as a model of the charge relay system is discussed.