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With the rapid development of biotechnological industry, there is an urgent need for exploiting new materials to immobilize enzymes to improve the performance of biocatalysts. In this paper, hierarchically porous magnetic microspheres (PFMMs) were prepared through solvothermal method and rapidly grafted with a novel rigid-flexible dendrimer first synthesized from monomers of trimesoyl chloride (TMC) and 1,6-hexanediamine (HDA) via interfacial polymerization process for covalent immobilization of Pseudomonas fluorescens lipase (PFL). The maximum PFL loading of the synthesized support reaches 87.5 mgprotein/gsupport, and 864% activity recovery of PFMMs-G3.0-PFL can be achieved at pH 9.0. Then, it was used to catalyze the resolution of (R,S)-1-phenylethanol with vinyl acetate. Under the optimized conditions, 50.0% conversion with 99.0% ees can be reached within 1.5 h. In addition, a conversion of 49.2% and ees of 96.9% can be retained after 10 batches of running, displaying an excellent operational stability. Importantly, a further investigation shows that the obviously improved reusability of the immobilized PFL is ascribed to the increased rigidity in comparison to fully flexible dendrimer. Thus, the newly constructed protocol for lipase immobilization exhibits a great prospect in biochemical engineering.

Alternative to the traditionally independent production of lipase, chemical synthesis of nano-carriers, and then preparing nanoimmobilized enzymes, we exploit a yeast genetically programmed virus biomimetic lipase nanoreactor in a sustainable manner. The nanoreactor biogenesis process integrated lipase production, protein component (coat-protein subunit and scaffold protein) production, self-assembly of protein components, and the encapsulation of lipase into protein nanocages using a simple process. It included overexpression of nanocage components, coat-protein subunits, and fused lipase-scaffold proteins and subsequent spontaneous self-assembly and encapsulation based on the specific interaction between the coat-protein subunit and the scaffold protein fused in the target lipase enzyme. The genetically programmable lipase nanoreactor showed improved stability under various harsh conditions, and was validated in fatty acid methyl ester synthesis with 86% yield at a high concentration of waste cooking oil (200 mM), which demonstrates the robustness and feasibility of the lipase nanoreactor in biodiesel production.

Rhizomucor miehei lipase (RML), a GRAS catalyst with wide applications, was overexpressed in Yarrowia lipolytica, also a GRAS unconventional yeast, via a combined strategy, optimization for promoter, gene dosage and fermentation process. The lipase activity of the recombinant strain was first increased from 19.5 to 26.9 U/mL via codon optimization of rml gene. Subsequently, a method was developed for constructing hybrid promoters harboring different copy number of upstream activation sequences fragment (UAS1B), and the recombinant strain Po1g/hp12d-rml 25# reached 38.9 U/mL. On this basis, expression vectors with different optimized rml gene copy numbers were constructed and introduced into Y. lipolytica Po1g. The recombinant strain Po1g/hp12d-2rml 14# carrying 12 copies of UAS1B in the upstream of pLEUmin and 2 copies of rml gene obtained the highest lipase activity of 59.6 U/mL. Moreover, in optimized shaking flask culture parameters: 5% (m/v) of d -Sorbitol, 2% (v/v) inoculation density, initial pH 7.0, and 30 mL initial culture medium, the RML activity of Po1g/hp12d-2rml 14# further reached 157 U/mL after 84-h of incubation at 28 ℃. Overall, RML activity was enhanced about 8-fold compared with the initial recombinant strain via the combined strategy, which provides a consolidated basis for the large-scale production of RML in Y. lipolytica to match urgent demand of the market.

This article describes the successful synthesis of a novel nanocomposite of superparamagnetic multi-walled nanotubes with a four-arm polyethylene glycol amine polymer (mMWCNTs@4-arm-PEG-NH2). This composite was then employed as a support for the covalent co-immobilization of Rhizopus oryzae and Candida rugosa lipases under appropriate conditions. The co-immobilized lipases (CIL-mMWCNTs@4-arm-PEG-NH2) exhibited maximum specific activity of 99.626U/mg protein, which was 34.5-fold superior to that of free ROL, and its thermal stability was greatly improved. Most significantly, CIL-mMWCNTs@4-arm-PEG-NH2 was used to prepare biodiesel from waste cooking oil under ultrasound conditions, and within 120 min, the biodiesel conversion rate reached 97.64%. This was due to the synergy effect between ROL and CRL and the ultrasound-assisted enzymatic process, resulting in an increased biodiesel yield in a short reaction time. Moreover, after ten reuse cycles, the co-immobilized lipases still retained a biodiesel yield of over 78.55%, exhibiting excellent operational stability that is attractive for practical applications. Consequently, the combined use of a novel designed carrier, the co-immobilized lipases with synergy effect, and the ultrasound-assisted enzymatic reaction exhibited potential prospects for future applications in biodiesel production and various industrial applications.

Nowadays, breast milk is considered as the ideal food for infants owing to the most common oleic acid-palmitic acid-oleic acid (OA-PA-OA) fatty acid distribution of the human milk fat (HMF). This study reports the synthesis of 1,3-dioleoyl-2-palmotoylglycerol (OPO)-rich human milk fat substitutes in a two-step enzymatic acidolysis reaction with Rhizomucor miehei lipase (RML) immobilized on magnetic multi-walled carbon nanotubes(mMWCNTs). The immobilized RML (RML-mMWCNTs) showed better thermal and pH stability, convenient recovery and reusability than the free soluble form. Under optimized reaction conditions (1:8 tripalmitin (PPP)/OA, 10%wt. enzyme, 50 °C, 5 h), PA content at the sn-2 position and OA incorporation at the sn-1,3 positions reached 93.46% and 59.54%, respectively. Comparison tests have also showed that RML-mMWCNTs has better catalytic activity and reusability than the commercial lipase Lipozyme RM IM. The results suggest that RML-mMWCNTs is a promising biocatalyst for the synthesis of OPO-rich TAGs with potential use in infant formulas.

1,3-dioleolyl-2-palmitate (OPO) is an important component of the human milk fat. Its unique fatty acid composition and distribution play an important role in proper infant growth and development. Owing to this, it has been attracting researchers and manufacturers to synthesize and commercialize OPO as an important human milk fat substitute added to infant formulas. In this review, the role of OPO in human milk, the benefits of OPO (sn-2 palmitate)-supplemented infant formulas over the conventional infant formulas on infant growth, and lipase-catalyzed synthesis of OPO are discussed. Over the last 20 years of research on the benefits of OPO (sn2 palmitate)-supplemented infant formulas are summarized. Similarly, studies carried out on lipase catalyzed production of OPO for the last 21 years (1999-2019) are also done focusing on the raw materials, sn1,3-regiospecific lipases, immobilization materials, and solvents used in the laboratory-scale experiments. In addition, OPO-based products currently in the market and future research trends are briefly covered in this review. PRACTICAL APPLICATIONS: This work focuses on lipase-catalyzed synthesis of 1,3-dioleoyl-2-palmitoylglycerol (the most abundant triacyl glycerol in human milk fat) and its benefits to infants when it is added in infant formulas. Over the last 20 years of published research from the literature are summarized and future research trends for efficient OPO synthesis are also covered. This will provide current and future researchers on the field with the necessary background information on OPO synthesis and design their research plans accordingly for cost-effective production of OPO and OPO-supplemented infant formulas.

Yeast surface display has emerged as a viable approach for self-immobilization enzyme as whole-cell catalysts. Herein, we displayed Candida rugosa lipase 1 (CRL LIP1) on the cell wall of Pichia pastoris for docosahexaenoic acid (DHA) enrichment in algae oil. After a 96-h culture, the displayed CRL LIP1 achieved the highest activity (380 ± 2.8 U/g) for hydrolyzing olive oil under optimal pH (7.5) and temperature (45 °C) conditions. Additionally, we improved the thermal stability of displayed LIP1, enabling retention of 50% of its initial bioactivity following 6 h of incubation at 45 °C. Furthermore, the content of DHA enhanced from 40.61% in original algae oil to 50.44% in glyceride, resulting in a 1.24-fold increase in yield. The displayed CRL LIP1 exhibited an improved thermal stability and a high degree of bioactivity toward its native macromolecule substrates algae oil and olive oil, thereby expanding its potential for industrial applications in fields of food and pharmaceutical. These results suggested that surface display provides an effective strategy for simultaneous convenient expression and target protein immobilization.

Genetic engineering of the eukaryotic microalga Chlorella protothecoides remains in the early stages of development. There is also an urgent need to develop cost-effective biodiesel production processes from wet and lipid-rich C. protothecoides. Malic enzyme is a potential key enzyme responsible for a rate-limiting step associated with lipid accumulation in some microorganisms. In this study, we genetically engineered C. protothecoides by overexpressing a key malic enzyme to improve lipid accumulation for direct biodiesel production from microalgal biomass. An open reading frame encoding 509 amino acid residues of a putative malic enzyme was obtained and identified. Cloning and overexpression of the malic enzyme gene allowed genetic engineering of C. protothecoides for lipid accumulation and further direct production of biodiesel without the need for lipid extraction steps. The total lipid accumulation in genetically engineered C. protothecoides increased 2.8-fold compared to the wild type. A dehydration step using methanol pretreatment coupled with H2SO4 or lipase catalyzed biodiesel production enabled 93–98% biodiesel yields directly from microalgal biomass without extraction of lipid under optimal conditions. The efficient direct dehydration and chemical and enzymatic transformation processes could significantly reduce the overall cost of biodiesel production from wet and lipid-rich microalgal feedstocks.

Fatty alcohol is considered as a promising clean fuel alternative to fossil fuel. In this study, we developed a facile and robust multiple enzyme complex of lipase (LIP), carboxylic acid reductase (CAR) and aldehyde reductase (AHR) based on cohesin-dockerin specific interaction and E. coli-ice nucleation protein (INP) surface assembly for highly efficient conversion of triacylglycerols (TAGs) to fatty alcohols. The proximity effect, customized ratio and positional arrangement of the three enzymes were readily genetically generated. The optimized cell associated three enzyme complex gave 73% of conversion yield, and showed significantly improved stability in higher temperature, acidic/alkaline environments, polar organic solvents, high concentration of substrate, and good recyclability in successive biotransformations. The three enzyme complex provides an efficient non-natural synthetic cascade route for fatty alcohol bioproduction alternative to complex de novo biosynthesis route.

Burkholderia cepacia lipase (BCL) was a potential catalyst in chiral resolution. Immobilization can be used to increase the enzyme activity and operational stability. In this work, novel melamine-glutaraldehyde dendrimer-like polymers were firstly grafted on the aminated magnetic nanoparticles to increase protein binding sites for the lipase to be immobilized. The dendrimer-polymer-modified nanoparticles could obviously protect the conformation of lipase and increase the contact chance between substrates and catalyzed center of lipase. The standard esterification reaction between lauric acid and 1-dodecanol was used to test the lipases activities. The activity of the obtained immobilized lipase was 58.0-fold than BCL powder, and 3.0-fold than the immobilized lipase without modified polymers. Then, the immobilized lipase was used to catalyze the chiral resolution reaction between 1-phenylethanol and vinyl acetate. Different organic solvents were selected for chiral resolution reaction. It was found out that hexane and heptane were the best reaction media. Thermal stability and organic solvent tolerance of the immobilized lipase were greatly improved than those of the lipase powder. The most significant discovery was the improvement in enzyme activity by bioimprinting. It could greatly shorten the reaction time from 3 h to 20 min. The conversion and substrate enantiomeric excess (ees) reached 49.7% and 98.8% in 20 min, respectively. The immobilized lipase could be recycled for 8 times without obvious loss in activity. The study provides a good strategy for preparation of efficient biocatalyst, showing great potential for future industrial application.

Various nanoflowers are synthesized as supports for different methods of enzyme immobilization; however, the activities of these immobilized enzymes are limited because of their confinement in the nanoflowers. In order to increase the performance of nanoflowers, in this study, different protein-phosphate hybrid nanostructures were successfully synthesized and further enhanced by carbon nanotubes (CNTs) under the same conditions. Only Cu3(PO4)2 complex nanostructures exhibited flower-like structures and showed excellent results after enhancement with CNTs in this framework. An esterification reaction between lauric acid and 1-dodecanol was used to test enzyme activity during immobilization, revealing that the Cu3(PO4)2/CNT/protein complex exhibited 68-fold higher activity relative to free lipase and 51-fold higher than that of Cu3(PO4)2/Burkholderia cepacia lipase hybrid nanoflowers in the absence of CNTs. All three hybrid nanostructures showed good performance and exhibited excellent reusability in resolution reactions between 1-phenylethanol and vinyl acetate. Additionally, the substrate enantiomeric excess (ees) reached 98% in only 10 min, and the corresponding Cu3(PO4)2/CNT/protein complex could be recycled eight times without obvious loss of activity. This approach involving nanoflowers enhanced with CNTs will be highly beneficial for decreasing mass-transfer resistance and providing enhanced enzyme loading along with promising potential for industrial application.

In order to evaluate the biodiesel (BD) quality in a batch and a packed-bed reactor (PBR), a comparison was carried out in terms of the kinematic viscosity. Waste vegetable oil (WVO) and Burkholderia cepacia lipase were utilized as the main substrate and enzymatic catalyst, respectively. In the batch reactor, a kinematic viscosity of 36.75 mm2.s−1 at 20 °C was obtained in the first 2.5 min and 24.78 mm2.s−1 after 3 h with the application of ultrasound, without stirring. In the PBR, the lowest kinematic viscosity value was of 7.88 mm2.s−1 at 20 °C, and 5.83 mm2.s−1 at 40 °C, after 5 doses of 30 mL of methanol per round. The BD in both reactors was mainly produced by esterification of free fatty acids. The final mixture in the PBR met the requirement of kinematic viscosity of ASTM D6751-10, GB/T 19147-2003, and GB/T 265-1988 standards, respectively. Upon comparing the performance in both reactors, despite the limited BD yields, a common pattern of fast reduction in the kinematic viscosities was observed at the first reaction stages (approximately 70–80% reduction). Then flat-shape values were measured in the last reaction stages. At applying further strategies in both reactors, higher BD concentrations could be obtained.

As a promising biocatalyst, Yarrowia lipolytica lipase 2 (YlLip2) is limited in its industrial applications due to its low thermostability. In this study, a thermostable YlLip2 mutant was overexpressed in Pichia pastoris and its half-life time was over 30 min at 80 &deg, C. To obtain a higher protein secretion level, the gene dosage of the mutated lip2 gene was optimized and the lipase activity was improved by about 89%. Then, the YlLip2 activity of the obtained strain further increased from 482 to 1465 U/mL via optimizing the shaking flask culture conditions. Subsequently, Hac1p and Vitreoscilla hemoglobin (VHb) were coexpressed with the YlLip2 mutant to reduce the endoplasmic reticulum stress and enhance the oxygen uptake efficiency in the recombinant strains, respectively. Furthermore, high-density fermentations were performed in a 3 L bioreactor and the production of the YlLip2 mutant reached 9080 U/mL. The results demonstrated that the expression level of the thermostable YlLip2 mutant was predominantly enhanced via the combination of these strategies in P. pastoris, which forms a consolidated basis for its large-scale production and future industrial applications.

As a promising biocatalyst, Yarrowia lipolytica lipase 2 (YlLip2) is limited in its industrial applications due to its low thermostability. In this study, a thermostable YlLip2 mutant was overexpressed in Pichia pastoris and its half-life time was over 30 min at 80 °C. To obtain a higher protein secretion level, the gene dosage of the mutated lip2 gene was optimized and the lipase activity was improved by about 89%. Then, the YlLip2 activity of the obtained strain further increased from 482 to 1465 U/mL via optimizing the shaking flask culture conditions. Subsequently, Hac1p and Vitreoscilla hemoglobin (VHb) were coexpressed with the YlLip2 mutant to reduce the endoplasmic reticulum stress and enhance the oxygen uptake efficiency in the recombinant strains, respectively. Furthermore, high-density fermentations were performed in a 3 L bioreactor and the production of the YlLip2 mutant reached 9080 U/mL. The results demonstrated that the expression level of the thermostable YlLip2 mutant was predominantly enhanced via the combination of these strategies in P. pastoris, which forms a consolidated basis for its large-scale production and future industrial applications.

Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy numbers of ROL gene cassettes, which were then transformed into P. pastoris GS115 to generate a strain with specific copy numbers of ROL. An optimized gene-dosage recombinant strain of GS115/pAOα-5ROL 11# harboring five copies of ROL was screened, revealing production of the highest activity (2700 U/mL), which was 8-fold higher than that of the strain harboring one copy. The activity of GS115/pAOα-5ROL 11# was then enhanced to 3080 U/mL in a shaking flask under optimized culture conditions. Subsequently, the endoplasmic reticulum-associated protein-degradation-related genes Ubc1 or/and Hrd1 were co-expressed with ROL to further increase ROL expression. The activities of the recombinant strains, GS115/5ROL-Ubc1 22#, -Hrd1 15#, and -Hrd1-Ubc1 1#, were 4000 U/mL, 4200 U/mL, and 4750 U/mL, which was 29.9%, 36.4%, and 54.2% higher, respectively, than that observed in GS115/pAOα-5ROL 11#. Using the combined strategy, ROL expression was improved 15.8-fold, with maximum GS115/5ROL-Hrd1-Ubc1 1# activity reaching 33,900 U/mL via a sorbitol/methanol co-feeding strategy in a 3-L fermenter and resulting in a 1.65-, 1.26-, and 1.14-fold enhancement relative to the activities observed in strains GS115/pAOα-5ROL 11#, GS115/5ROL-Ubc1 22#, and GS115/5ROL-Hrd1 15#, respectively. These results indicated that heterologous overexpression of ROL in P. pastoris using this combined strategy is feasible for large-scale industrialization.

Lipase is one of the most widely used classes of enzymes in biotechnological applications and organic chemistry. Candida rugosa lipases (CRL) can catalyze hydrolysis, esterification and transesterification with high regio-, stereo- and enantio-selectivity. However, thermal inactivation above 45 °C limits CRL's applications. Studies on improving the thermal stability of CRL are often limited by its slow-growing eukaryotic expression host, which is not suitable for large-scale screening. Identification of thermally stable mutants by rational design, regarded as an efficient substitution of experimental efforts, would provide a method for site-directed improvement of CRL. In this study, mutation-induced stability changes in CRL Lip1 were predicted by three rational design methods. Followed by conservative analyses and functional region exclusion, five mutants of a hot-spot, Asp457Phe, Asp457Trp, Asp457Met, Asp457Leu, and Asp457Tyr, were identified and prepared for enzymatic characterization. These five mutants increased the apparent melting temperature of Lip1 from 7.4 °C to 9.3 °C, with the most thermostable mutant, Asp457Phe, exhibiting a 5.5-fold longer half-life at 50 °C and a 10 °C increase in optimum temperature. Furthermore, pH stability of Lip1 was also enhanced due to the introduction of Asp457Phe mutation. The study demonstrates that thermally stable mutants of CRL could be identified with limited experimental efforts using rational design methods.

Lipases are scarcely exploited as feed enzymes in hydrolysis of lipids for increasing energy supply and improving nutrient use efficiency. In this work, we performed homologous overexpression, in vitro characterization and in vivo assessment of a lipase from the yeast Yarrowia lipolytica for feed purpose. Simultaneously, a large amount of yeast cell biomass was produced, for use as single cell protein, a potential protein-rich feed resource. Three kinds of low cost agro-industrial wastes were tested as substrates for simultaneous production of lipase and single cell protein (SCP) as feed additives: sugarcane molasses, waste cooking oil and crude glycerol from biodiesel production. Sugarcane molasses appeared as the most effective cheap medium, allowing production of 16420 U/ml of lipase and 151.2 g/L of single cell protein at 10 liter fermentation scale. In vitro characterization by mimicking a gastro-intestinal environment and determination of essential amino acids of the SCP, and in vivo oral feeding test on fish all revealed that lipase, SCP and their combination were excellent feed additives. Such simultaneous production of this lipase and SCP could address two main concerns of feed industry, poor utilization of lipid and shortage of protein resource at the same time.

A screening for lipase was carried out in the metalophile of Ralstonia solanacearum. The putative lipase gene (FW376093) was cloned and expressed using E. coli as host. Subsequently, the recombined enzyme was purified and its enzyme properties were further examined. The results indicated the lipase of FW376093 was able to catalyze hydrolysis of short-chain p-nitrophenyl esters (C2–C12). It showed the optimal temperature and pH were respectively 80 °C and 8.5. The enzyme could be significantly enhanced by Na+2, Ca+2, and Fe+2, but significantly repressed by Cu+2, Ni+2 and Mn+2. Nevertheless, the lipase of FW376093 exhibited high tolerance to detergents and organic solvents. Phylogenetic assay reveals that it belongs to family II. The unique properties of the lipase of FW376093, having metalophilicity and high tolerance to organic solvent, render it with prosperous prospect in practical applications.

Inspired by coordinated self-assembly of small biomolecules, self-assembly of Mn2+ induced l -cystine generated metal-matrix composites is constructed to immobilize enzymes to meet the need of advanced enzyme engineering. Morphology of bovine serum albumin (BSA) directed self-assembled system suggests that protein molecules can directly get involved in coordinated self-assembling process. Similarly, construction of a two-enzyme directed self-assembled system with lipase and soybean lipoxygenase (I type) instead of BSA is employed to perform the cascade reaction in fast transforming soybean oil into fatty acid hydroperoxides (HPOs), valuable intermediates with potential use in anti-cancer medicine. The bioconversion can yield 1527.0 μmol·HPOs/mL·oil within 3 h upon optimizing the operating conditions. Meanwhile, the major component of HPOs is 13-hydroperoxides of linoleic acid caused by pH-dependent mechanism of soybean lipoxygenase. Moreover, a relative yield of 84.7% can be retained at 9 batches of the recycled biocatalyst, indicating a good operating stability, which can further reduce industrial cost. Therefore, the newly designed strategy broadens horizon for the incorporation of self-assembly into enzyme engineering, and specially adapts to be integrated with more new catalytic units.

Biodiesel production using immobilized lipase as a biocatalyst is a promising process. The performance of immobilized lipase is mainly determined by supporting materials and immobilization method. To avoid the shortcomings of adsorption and covalent bonding methods, in this study, we developed a novel heterofunctional carrier of being strengthened anion exchange and weakened covalent binding to avoid activity loss and improve operational stability of the immobilized lipase. 2,3-epoxypropyltrimethylammonium chloride with epoxy and quaternary ammonium group and glutaraldehyde were grafted onto aminated magnetic nanoparticles (AMNPs) to generate a new matrix, named GEAMNP. Then Burkholderia cepacia lipase (BCL) was immobilized on GEAMNP via anion exchange and covalent bonding. The transesterification between soybean oil and methanol was used to test the activities. Activity recovery of the immobilized BCL was up to 147.4% and the corresponding transesterification activity was 1.5-fold than that of BCL powder. The immobilized lipase was further used for biodiesel production to confirm its feasibility. The fatty acid methyl esters conversion yield could reach 96.8% in the first 12 h. Furthermore, the immobilized lipase, BCL-GEAMNP showed markedly improved operational stability, better reusability and higher esters than BCL-GAMNP, where MNPs were only modified with (3-aminopropyl) triethoxysilane and glutaraldehyde.

As a kind of important biocatalysts, Pseudomonas lipases are commonly applied in various industrial fields. Pflip1, a new extracellular lipase gene from Pseudomonas. fluorescens Pf0–1, was first cloned and respectively expressed in Escherichia coli BL21(DE3) and Pichia pastoris KM71, the recombinant proteins Pflip1a and Pflip1b were later purified separately. Then Pflip1a was further characterized. The optimum pH of Pflip1a was 8.0 and its optimal temperature was 70 °C. After incubation at 70 °C for 12 h, Pflip1a could retain over 95% of its original activity. It showed the highest activity towards p-nitrophenyl caprylate. Moreover, its activity was profoundly affected by metal ion, ionic surfactants and organic solvents. Furthermore, the two obtained recombinant lipases were immobilized on the magnetic nanoparticles for biodiesel preparation. The GC analysis showed that for the immobilized lipases Pflip1b and Pflip1a, the biodiesel yield within 24 h respectively attained 68.5% and 80.5% at 70 °C. The activities of the two immobilized lipases still remained 70% and 82% after 10 cycles of operations in non-solvent system. These characteristics and transesterification capacity of the recombinant protein indicated its great potential for organic synthesis, especially for biodiesel production.

Pseudomonas lipase is a well-studied lipase. However, few studies have been conducted to examine the mechanisms underlying the regulation of the lipase expression. Hfq is a global regulatory protein that, among others, controls the expression of multiple genes, regulate bacterial peristalsis, and participates in the regulation of quorum-sensing (QS) system. In this study, the effects of Hfq on lipase expression were investigated by knocking out the hfq and rsmY genes or overexpressing of hfq and rsmY genes. We found that Hfq regulates the expression of lipA at both transcriptional and translational levels. The translational level was the main regulatory level of lipA. Hfq also regulates the expression and stability of rsmY. Additionally, using hfq/rsmY double gene knock-out, we showed that Hfq can directly bind to the rsmY to regulate lipA activity. In conclusion, our results indicate that Hfq regulates the expression of rsmY mainly at the translational level to influence the expression of lipA in Pseudomonas protegens Pf-5.

Pseudomonas lipases are well studied enzymes. However, few studies have been conducted to explore the mechanism underlying the regulation of lipases expression. AlgR, a global regulator, controls the expression of multiple genes, regulates bacterial peristalsis, and participates in the regulation of quorum-sensing (QS) system, and so on. In this study, the effect of AlgR on lipase expression was investigated by knocking out the algR and rsmZ genes or overexpressing them. It is found out that AlgR can regulate the expression of lipA at both transcriptional and translational levels, but the transcriptional level was dominant. AlgR is also able to regulate the expression of rsmX/rsmY/rsmZ. Additionally, using algR/rsmZ double gene knock-out, it showed that AlgR could directly bind to the promoter sequence of rsmZ to regulate lipA activity. In conclusion, this study for the first time indicates that AlgR directly binds to rsmZ to regulates the expression of lipA via regulating transcription of rsmZ, and mainly regulates the expression of lipA at transcriptional level in P. protegens Pf-5.

Three sub-group lipases of Burkholderia cepacia lipase (BCL), Rhizomucor miehei lipase (RML), and Candida rugosa lipase (CRL) were covalently immobilized on dendrimer functionalized magnetic carbon nanotube and used as catalysts to catalyze biodiesel production. The effects of imprinting molecule, organic solvent, water, methanol, temperature and time interval of methanol addition on the yield of biodiesel were optimized. The results showed that bioimprinting could greatly enhance catalytic performances of the three immobilized lipases. The obtained lipases were then employed to catalyze biodiesel production, and the achieved optimum conditions were: for BCL, water content 5 wt. %, reaction temperature 35°C, and with t-butanol as reaction medium, methanol : oil molar ratio 4:1, its highest biodiesel yield attained 96.4%; for RML, water content 10 wt. %, reaction temperature 50 °C, n-octane as the reaction medium, methanol : oil molar ratio of 5:1, the utmost biodiesel conversion rate was up to 96.2%; and ...

In East Asia, for thousands of years, the fruit of Chinese tallow tree ( Sapium sebiferum ) has been used for multiple purposes because of its chemical composition; the presence of high amounts of lipids is remarkable, showing potential to be used as substrate for biodiesel synthesis. Previously have been reported the use of alkaline and enzymatic catalysts, microwave technology and the use of ionic liquids as co-solvents with the lipids of this tree species to produce biodiesel. This study shows the results of the use of Burkholderia cepacia lipase as enzymatic catalyst for transesterification of Chinese Tallow Kernel oil (CTK), extracted from the fruit of Chinese tallow tree, into biodiesel, with the use of ultrasonic assisted technology and without the usage of solvents. The optimal operational parameters were determined and the reactions were developed in a batch reactor with the use of ultrasonic irradiation and emulsification to enhance the mass transfer. The scaled-up experiments, in an especially designed 3 L capacity reactor, showed promising results, obtaining 55.20% biodiesel and a kinematic viscosity of 10.31 mm 2 .s −1 in only 4 h, in comparison with previously published ( in vitro ) methods. The valorization of this non-edible source of oil represents an opportunity to use as an alternative source for bioenergy and also to tackle the uncontrolled expansion of this oleaginous tree species in some ecologically fragile ecosystems.

Increasing energy demands coupled with decreasing fossil fuel resources create an urgent need to switch over bio-based fuel, such as biodiesel. In a newly designed reactor, the production of biodiesel was conducted with soybean oil (SBO) in solvent-free system through transesterification by immobilized Burkholderia cepacia lipase under the influence of ultrasonic emulsification. Effects of operational parameters viz. methanol-to-oil molar ratio, water concentration, immobilized enzyme concentration, and temperature on fatty acid methyl esters (FAME) yield were investigated. Molar ratio and enzyme concentration showed a significant linear correlation with biodiesel yield (r = − 0.8 and 0.6; respectively). Linear regression analysis of both parameters later revealed a significant prediction ability for biodiesel with a yield of 78.8 and 31.7%, respectively. FAME yield was 30% at 7 h in the pretreatment reactor. Without ultrasonic emulsification, the yield was significantly enhanced to 64% for the same reaction time in the packed-bed reactor. However, the reaction time was further reduced significantly under the influence of ultrasonic emulsification coupled with immobilized lipase as a catalyst and resulted into higher biodiesel yield of 68.6% at 3 h. Clearly, ultrasonic emulsification-assisted immobilized lipase catalysis of SBO might be a potential alternative route to conventional methods of biodiesel production.

International audience; As an alternative to in vitro lipase dependent biotransformation and to traditional assembly of pathways in cytoplasm, the present study focused on targeting lipase dependent pathways to a subcellular compartment lipid body (LB), in combination with compartmentalization of associated pathways in other lipid relevant organelles including endoplasmic reticulum (ER) and peroxisome for efficient in vivo biosynthesis of fatty acid methyl esters (FAMEs) and hydrocarbons, in the context of improving Yarrowia lipolytica lipid pool. Through knock in and knock out of key genes involved in triacylglycerols (TAGs) biosynthesis and degradation, the TAGs content was increased to 51.5%, from 7.2% in parent strain. Targeting lipase dependent pathway to LB gave a 10-fold higher FAMEs titer (1028.0 mg/L) compared to cytosolic pathway (102.8 mg/L). Furthermore, simultaneously targeting lipase dependent pathway to LB, ER and peroxisome gave rise to the highest FAMEs titer (1644.8 mg/L). The subcellular compartment engineering strategy was extended to other lipase dependent pathways for fatty alkene and alkane biosynthesis, which resulted in a 14-fold titer enhancement compared to traditional cytosolic pathways. We developed yeast subcellular cell factories by directing lipase dependent pathways towards the TAGs storage organelle LB for efficient biosynthesis of TAG derived chemicals for the first time. The successful exploration of targeting metabolic pathways towards LB centered organelles is expected to promote subcellular compartment engineering for other lipid derived product biosynthesis.

In this study, we overcame the limitations of single-enzyme system catalysis by codisplaying Candida rugosa lipase 1 (CRL1) and Rhizopus oryzae lipase (ROL) on the cell surfaces of the whole-cell catalyst Pichia pastoris to produce biodiesel from tallow seed oil. We screened double antibiotic-resistant strains on tributyrin plates, performed second electroporation based on single-displayed ROL on GS115/KpRS recombinants and single-displayed CRL1 on GS115/ZCS recombinants and obtained an ROL/CRL1 codisplay on P. pastoris GS115 surfaces. The maximum activity of the codisplaying GS115/pRCS recombinant was 470.59 U/g dried cells, which was 3.9-fold and 1.3-fold higher than that of single-displayed ROL and CRL1, respectively. When self-immobilized lipases were used as whole-cell catalysts, the rate of methyl ester production from GS115/pRCS harboring ROL and CRL1 was 1.4-fold higher than that obtained with single-displayed ROL. Therefore, biodiesel catalysis by synergetic codisplayed enzymes is an alternative biodiesel production strategy.

A polyamidoamine (PAMAM) dendrimer was grafted onto magnetic multi-walled carbon nanotubes (m-MWCNTs) to combine magnetic properties with a large surface functionalized with amino groups. Based on three dimensional structural (3D) analysis of enzyme, oriented-immobilization of Rhizomucor miehei lipase (RML) on the obtained m-MWCNTs-PAMAM matrix was achieved. The recovery activity of the immobilized lipase was up to 2808% and the corresponding esterification activity was 27-fold higher than that of the free enzyme. The immobilized enzyme was employed to catalyze biodiesel production from waste vegetable oil in a tert-butanol solvent system. Biodiesel conversion reached 94% under optimal conditions. Moreover, the immobilized lipase could be easily recovered and there was no significant decrease in conversion rates after 10 cycles of reuse. The results suggested that the immobilized RML is a potential catalyst with high stability and excellent operational reusability for biodiesel production.

In this study, biodiesel production from Chinese tallow kernel oil catalyzed by Candida rugosa lipase (CRL) in ionic liquid was investigated. Biodiesel was prepared from Chinese tallow kernel oil. Nineteen kinds of ionic liquids were screened for CRL to enhance its catalysis ability for biodiesel synthesis. Interestingly, [Hmim][PF6], not only help to simplify the downstream processing, but also boost the efficiency of CRL from about 35% to 95.4%. In addition, the biodiesel mixed with various blending ratios of fossil diesel was used to fuel the conventional diesel engine to examine fuel and emission properties. Most of fuel characteristics of the biodiesel met the standards set by EN and ASTM. Its output power was found to be close to pure diesel. The emission products, including hydrocarbons (HC, 52.7% lower) and carbon monoxide (CO, 48.6% lower), decreased substantially with the exception of oxynitride (NOx) emission, which displayed a 12.8% increase. The results suggest that biodiesel production from woody oil catalyzed by CRL in [Hmim][PF6], is feasible in future application with a promising prospect.

In the present study, a new lipase SL-4 from Burkholderia ubonensis SL-4, was purified by 80% ammonium sulphate precipitation, Q Sepharose FF anion exchange and Superdex 75 gel filtration chromatography finally leading to 68.5-fold purification and 13.34% recovery. It had a molecular mass of ca. 33 kDa and the whole gene (1095-bp) was cloned by using degenerate primers. Amino acid sequence analysis revealed that lipase SL-4 is a new member of subfamily I.2 lipases. Lipase SL-4 exhibited optimum activity toward p-NP myristate (C14) at pH 8.5 and 65 °C with a Km of 0.72 mM, a kcat of 391.63 s−1 and a kcat/Km of 543.93 s−1 mM−1. It had good thermostability at 50 °C and pH 8.5, and could be activated strongly by Ca2+ and Mn2+, but inhibited by some transition metal ions and EDTA, PMSF, DTT and β-ME. Additionally, lipase SL-4 possessed non-ionic detergent stability and organic solvent stability. When preliminarily employed to catalyze soybean oil for biodiesel production, the liquid lipase SL-4 could attain a conversion ratio of 92.24% in a solvent-free system. These results demonstrate that the new thermo-solvent-stable lipase possesses an attractive potential for biotechnological applications as biocatalyst, especially for biodiesel production.

This study is dedicated to efficiently produce Rhizopus oryzae lipase (ROL) by optimizing the expression of multiple expression-related helper proteins in Pichia pastoris. A series of engineered strains harboring different copy numbers of the ROL gene and different copies of the chaperone Pdi gene were first constructed to examine the influence of Pdi gene copy number on ROL production. The results showed that multiple copies of Pdi gene did not significantly improve ROL expression. Then, the effect of the co-overexpression of 10 expression-related helper proteins on ROL secretion was investigated by screening 20 colonies of each transformants. The data from shaking-flask fermentation suggested that Ssa4, Bmh2, Sso2, Pdi, Bip, Hac1, and VHb had positive effects on ROL expression. Subsequently, Ssa4, Bmh2, and Sso2, which all participate in vesicular trafficking and strongly promote ROL expression, were combined to further improve ROL production level. ROL activity of the screened strain GS115/5ROL-Ssa4-Sso2-Bmh2 4# attained 5230 U/mL. Furthermore, when the helper proteins Pdi, Bip, Hac1, and VHb were individually co-expressed with ROL in the strain GS115/5ROL-Ssa4-Sso2-Bmh2 4#, lipase activity increased to 5650 U/mL in the strain GS115/5ROL-Ssa4-Sso2-Bmh2-VHb 9#. Additionally, the maximum ROL activity of 41,700 U/mL was achieved in a 3 L bioreactor for high-density fermentation via a sorbitol&ndash, methanol co-feeding strategy, reaching almost twofold the value of the initial strain GS115/pAO&alpha, 5ROL 11#. Thus, the strategies in this study significantly increased ROL expression level, which is of great potential for the large-scale production of ROL in P. pastoris.

This study is dedicated to efficiently produce Rhizopus oryzae lipase (ROL) by optimizing the expression of multiple expression-related helper proteins in Pichia pastoris. A series of engineered strains harboring different copy numbers of the ROL gene and different copies of the chaperone Pdi gene were first constructed to examine the influence of Pdi gene copy number on ROL production. The results showed that multiple copies of Pdi gene did not significantly improve ROL expression. Then, the effect of the co-overexpression of 10 expression-related helper proteins on ROL secretion was investigated by screening 20 colonies of each transformants. The data from shaking-flask fermentation suggested that Ssa4, Bmh2, Sso2, Pdi, Bip, Hac1, and VHb had positive effects on ROL expression. Subsequently, Ssa4, Bmh2, and Sso2, which all participate in vesicular trafficking and strongly promote ROL expression, were combined to further improve ROL production level. ROL activity of the screened strain GS115/5ROL-Ssa4-Sso2-Bmh2 4# attained 5230 U/mL. Furthermore, when the helper proteins Pdi, Bip, Hac1, and VHb were individually co-expressed with ROL in the strain GS115/5ROL-Ssa4-Sso2-Bmh2 4#, lipase activity increased to 5650 U/mL in the strain GS115/5ROL-Ssa4-Sso2-Bmh2-VHb 9#. Additionally, the maximum ROL activity of 41,700 U/mL was achieved in a 3 L bioreactor for high-density fermentation via a sorbitol–methanol co-feeding strategy, reaching almost twofold the value of the initial strain GS115/pAOα-5ROL 11#. Thus, the strategies in this study significantly increased ROL expression level, which is of great potential for the large-scale production of ROL in P. pastoris.

We develop an efficient and economic cascade multienzymes for fatty alkene bioproduction based on the lipase hydrolysis coupled to the P450 decarboxylation in the form of multiple enzyme complex. One step preparation of a multienzyme complex was based on a mixture of cell extracts including dockerin-enzyme fusions and one cohesin-cellulose binding module (CBM) fusion through high specific interaction of dockerin and cohesin. Simultaneously, the CBM was bound to cellulose carrier to form co-immobilized multienzyme. The key factors affecting overall efficiency of alkene bioproduction including substrate channeling of hydrolysis and decarboxylation, the ratio and position of two enzymes, stability were all addressed by genetically engineering of the synthetic CBM-cohesin fusions. The multienzymes exhibited more than 9.2 fold enhancement in initial reaction rate and much higher conversion yields (69%-72%) compared to mixture of free enzyme counterpart. The enzymatic cascade based multienzymes could efficiently convert renewable triglycerides to alkenes.

A hierarchical mesoporous zeolitic imidazolate framework (ZIF-8) was processed based on cetyltrimethylammonium bromide (CTAB) as a morphological regulating agent and amino acid (l-histidine) as assisting template agent. Burkholderia cepacia lipase (BCL) was successfully immobilized by ZIF-8 as the carrier via an adsorption method (BCL-ZIF-8). The immobilized lipase (BCL) showed utmost activity recovery up to 1279%, a 12-fold boost in its free counterpart. BCL-ZIF-8 was used as a biocatalyst in the transesterification reaction for the production of biodiesel with 93.4% yield. There was no significant lowering of conversion yield relative to original activity for BCL-ZIF-8 when continuously reused for eight cycles. This work provides a new outlook for biotechnological importance by immobilizing lipase on the hybrid catalyst (ZIF-8) and opens the door for its uses in the industrial field.

A hierarchical mesoporous zeolitic imidazolate framework (ZIF-8) was processed based on cetyltrimethylammonium bromide (CTAB) as a morphological regulating agent and amino acid (l-histidine) as assisting template agent. Burkholderia cepacia lipase (BCL) was successfully immobilized by ZIF-8 as the carrier via an adsorption method (BCL-ZIF-8). The immobilized lipase (BCL) showed utmost activity recovery up to 1279%, a 12-fold boost in its free counterpart. BCL-ZIF-8 was used as a biocatalyst in the transesterification reaction for the production of biodiesel with 93.4% yield. There was no significant lowering of conversion yield relative to original activity for BCL-ZIF-8 when continuously reused for eight cycles. This work provides a new outlook for biotechnological importance by immobilizing lipase on the hybrid catalyst (ZIF-8) and opens the door for its uses in the industrial field.

Dimer acid cyclocarbonate (DACC) is synthesized from glycerol carbonate (GC) and Sapium sebiferum oil-derived dimer acid (DA, 9-[(Z)-non-3-enyl]-10-octylnonadecanedioic acid). Meanwhile, DACC can be used for synthetic materials of bio-based non-isocyanate polyurethane (bio-NIPU). In this study, DACC was synthesized by the esterification of dimer acid and glycerol carbonate using Novozym 435 (Candida antarctica lipase B) as the biocatalyst. Via the optimizing reaction conditions, the highest yield of 76.00% and the lowest acid value of 43.82 mg KOH/g were obtained. The product was confirmed and characterized by Fourier transform-infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Then, the synthetic DACC was further used to synthesize bio-NIPU, which was examined by FTIR, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), indicating that it possesses very good physio-chemical properties and unique material quality with a potential prospect in applications.

Pseudomonas lipases are well-studied, but few studies have examined the mechanisms of lipase expression regulation. As a global regulatory protein, PmrA controls the expression of multiple genes such as the Dot/Icm apparatus, eukaryotic-like proteins, and secreted effectors. In this study, the effect of PmrA on expression of the lipase lipA in Pseudomonas aeruginosa PAO1 was investigated by knocking out or overexpressing pmrA, rsmY, and rsmA. PmrA regulated the expression of lipA at both the transcriptional and translational level although translation was the pivotal regulatory mechanism for lipA expression. PmrA also regulated the expression of rsmY. Using gel mobility shift assay and pmrA/rsmY double gene knock-out model, we showed that PmrA directly bound to the promoter sequence of rsmY to regulate lipA expression. Translation of lipA was activated by the PmrA/PmrB system via RsmA. Specifically, the Shine-Dalgarno (SD) sequence located at lipA mRNA was overlapped through combination between RsmA and the AGAUGA sequence, subsequently blocking the 30S ribosomal subunit to the SD sequence, leading to translational inhibition of lipA. Transcriptional repression of RsmY initiated translation of lipA through negative translational regulation of rsmA. In conclusion, this study demonstrated that in P. aeruginosa PAO1, PmrA mainly regulated rsmY expression at a translational level to influence lipA expression. RsmY primarily activated lipA translation via negative translational regulation of rsmA.

International audience; On the basis of Thermomyces lanuginosus lipase functionally displayed on the cell surface of Yarrowia lipolytica yeast, a new green, integrated, and sustainable bioprocess for fatty acid methyl esters (FAMEs) production was developed. It couples surface-displayed enzyme generation to enzymatic FAMEs production, in combination with simultaneous regeneration of in situ carbon source for yeast utilization. During FAMEs synthesis, in a one-pot fermentation flask containing growing cells displaying lipase, waste cooking oil, and methanol, the in situ formed byproduct glycerol was used as an alternative carbon source to support further cell growth and lipase generation. The partial consumption of glycerol pushed the reversible reactions (transesterification, esterification, hydrolysis) toward formation of FAMEs. In the growing cells integrated system, the production of FAMEs via hydrolysis–esterification or transesterification–esterification reactions was dependent on the time when methanol was added to the culture medium. Compared to the resting cells system with an 81% FAMEs yield after 16 h, the self-cycling of glycerol for cell consumption (allowing cell growth and lipase displaying for further FAMEs forming) in the growing cells system significantly increased FAMEs synthesis with a more than 91% yield obtained in less time (6–10 h). The cells recovered from growing cell systems still exhibited satisfactory reusability, showing more than 80% FAMEs yield during 12 successive batches.

In order to achieve the industrialization of lipase-catalyzed biodiesel production, economic and effective carriers for immobilized lipase need to be exploited. In this study, Rhizomucor miehei lipase (RML) was immobilized on different kinds of macroporous resins and NKA was found to be the best carrier. The immobilized RML was then employed to catalyze biodiesel production from soybean oil. 8 different organic solvents were used as media in the trans-esterification reactions. Isooctane was proved to be the optimal one. Subsequently, immobilization conditions and reaction conditions were further optimized through single factorial experiments and response surface methodology (RSM). Results were as follows: the recovery activity of the immobilized RML was up to 1531.7%; the corresponding specific activity could reach 73761.99 U/g protein, almost 15 fold than that of the free RML; the biodiesel yield attained 99.2% under the optimal conditions in isooctane system; the immobilized lipase can yield 64% product after being run for 4 cycles, much better than the commercial immobilized lipase, Lipozyme RM IM (remains only 31%). In conclusion, NKA is a promising carrier for the immobilization of RML and exhibits a good performance in biodiesel production compared with Lipozyme RM IM.

A kind of pH-responsive particle was synthesized using modified pullulan polysaccharide. The synthesized particle possessed a series of merits, such as good dispersity, chemical stability and variability of particle size, making it a suitable carrier for enzyme immobilization. Then, Burkholderia cepacia lipase (BCL), a promising biocatalyst in transesterification reaction, was immobilized on the synthesized particle. The highest catalytic activity and immobilization efficiency were achieved at pH 6.5 because the particle size was obviously enlarged and correspondingly the adsorption surface for BCL was significantly increased. The immobilization enzyme loading was further optimized, and the derivative lipase was applied in chiral resolution. Under the optimal reaction conditions, the immobilized BCL showed a very good performance and significantly shortened the reaction equilibrium time from 30 h of the free lipase to 2 h with a conversion rate of 50.0% and ees at 99.2%. The immobilized lipase also exhibited good operational stability; after being used for 10 cycles, it still retained over 80% of its original activity. Moreover, it could keep more than 80% activity after storage for 20 days at room temperature in a dry environment. In addition, to learn the potential mechanism, the morphology of the particles and the immobilized lipase were both characterized with a scanning electron microscope and confocal laser scanning microscopy. It was found that the enlarged spherical surface of the particle in low pH values probably led to high immobilized efficiency, resulting in the improvement of enantioselectivity activity in chiral resolution.

To improve the productivity of Candida rugosa lipase (CRL) and alleviate respiration limitations during high cell-density fermentation, codon-optimized CRL LIP1, and Vitreoscilla hemoglobin (VHb) were co-expressed in Pichia pastoris. The activity of the recombinant strain that expressed LIP1 and VHb from dual promoters, named GS115/9Klip1FZvgb-lip1 #1, toward olive oil reached 620 U/mL, which was 1.69-fold greater than that of the recombinant strain GS115/9Klip1 #139 (365 U/mL) which only expressed LIP1 from a single promoter, and 1.37-fold greater than that of the recombinant strain GS115/9Klip1FZlip1 #39 (450 U/mL), which only expressed LIP1 from two promoters, in shaking flasks. With FM22 as the basic medium and methanol/D-sorbitol (1:1, v/v) as an inducer, the maximum activity of GS115/9Klip1FZvgb-lip1 #1 reached 7490 ± 379.5 U/mL, which was 2.65-fold greater than that of GS115/9Klip1 #139 (2820 ± 112 U/mL) and 1.82-fold greater than that of GS115/9Klip1FZlip1 #39 (4100 ± 205 U/mL) in 10 L fer...

Industrialization of enzyme-catalyzed biodiesel production has been hindered by low conversion efficiency and long reaction time. As a representative example, Rhizopus oryzae lipase (ROL) exhibited a poor performance in biodiesel production. However, higher yields and shorter reaction time could be achieved by screening different lipases that could synergically catalyze the reaction with ROL. The results showed that Novozym 435 combined with ROL displayed the best performance, with a biodiesel yield that was 30% higher than that of ROL alone in 30 h. Hence, the use of a synergic strategy was investigated systematically. Under optimized conditions, the biodiesel yield was as high as 98.3% and the reaction time was successfully shortened from 60 h to 21 h. Moreover, the combined lipases retained a yield of about 80% after 20 cycles in a solvent-free system. In addition to ROL, the performance of other lipases whose regioselectivity were similar to ROL was improved when they were combined with Novozym 435. In conclusion, synergic catalysis is a promising strategy for enhancing biodiesel yield and minimizing reaction time for ROL.