Encapsulation of lipases on coordination polymers and their catalytic performance in glycerolysis and esterification

In this study,lipases of CALB(Candida antarctica lipase B),TLL(Thermomyces lanuginosa lipase),RML(Rhizomucor miehei lipase),CALA(Candida antarctica lipase A)and LU(Lecitase?Ultra)were encapsulated into the nucleotidehybrid metal coordination polymers(CPs)for diacylglyerols(DAG)preparation.Guanosine 5'-monophosphate(GMP)and adenosine 5'-monophosphate(AMP)were used as coordinating molecules,and metal ions of Fe^(3+),Ba^(2+),Mn^(2+),Ni^(2+)and Cr^(3+)were applied to prepare matrix.Results indicated that,besides Ba^(2+)with AMP,all other metal ions can coordinate with AMP and GMP to generate CPs.In addition,the AMP/Ni was amorphous when standing temperature was 4℃,while it was crystalline when standing temperature was from 30 to 180℃.DAG content from 47.55%to 64.99%was obtained from glycerolysis by CALB@GMP/Ba,RML@GMP/Ba,TLL@GMP/Ba,RML@GMP/Mn and TLL@GMP/Mn.Additionally,CALB@GMP/Fe showed selectivity towards DAG formation in the esterification and DAG content up to 61.88%was obtained.

Lipases in the castor bean seed of Chinese varieties:Activity comparison,purification and characterization

The lipase activities in the three main Chinese castor bean varieties [ZiBi 05 （ZB 5）,TongBi 06 （TB 6）,JinBi 02 （JB 2）] were investigated.Two lipases were found in extracts from the endosperms of all the three varieties.One lipase is located in the lipid bodies,showing higher activity at pH 4.5 and exhibiting the highest activity at day 1 of germination.The other lipase is located in the particulates,showing higher activity at pH 9.5 and particularly active at day 4 of germination.The lipid bodies from ZB 5 show the highest lipase activity at day 1 and pH 4.5 among the three varieties during the 8 d of germination.The acid lipase located in the lipid bodies of ZB 5 of germination 1 d was purified to homogeneity by DEAE-sepharose CL-6B and butyl-sepharose CL-4B chromatography after isolation,delipidation and solubilization,with a purification factor of 16.1 and overall activity recovery of 17.7%.The characterizations （such as optimum pH,pH stability,optimum temperature,thermal stability,effect of metal ions,organic solvents stability,substrate specificity） of the purified acid lipase were conducted in detail.A combination of the characterized properties may make this enzyme a useful biocatalyst for industrial application.

Performance Comparison of Commercial and Home-Made Lipases for Synthesis of Poly(δ-Valerolactone)Homopolymers

Novozyme 435,which is the commercially available immobilized form of Candida antarctica lipase B,has been successfully conducted ring opening polymerization of lactones in organic solvents.In this paper,it was aimed to introduce an alternative biocatalyst for Novozyme 435.Candida antarctica lipase B immobilized onto rice husk ashes via physical adsorption(with a specific activity of 4.4 U/mg)was prepared in previous studies and used as a biocatalyst for poly(δ-valerolactone)synthesis in the present work.Polymerization reactions were proceeded at various reaction temperatures and periods via both two immobilized enzyme preparations.The resulting products were characterized spectroscopically and thermally.The highest molecular weight(Mn=9010 g/mol)was obtained via Novozyme 435 catalysis at 40℃and 24 hours.The performance of home-made lipase,which resulted in a molecular weight of 8040 g/mol,was close to commercial one.

New Colorimetric Method for Lipases Activity Assay in Microbial Media

A simple, rapid and precise method has been developed for determination of lipase activity in microbial media. The method is based on using phenyl acetate as substrate for lipase and determination of liberated phenol by Folin Ciocalteu reagent. Reaction mixture containing substrate 2.4 ml of phenyl acetate 165 μM in Tris HCl buffer, 0.1 M and pH 7, with 1% (v/v) Triton X-100) and 0.1 ml lipase is incubated at 40?C during 10 minutes and the absorbance was measured at 750 nm. Linearity was observed in the concentration range 0-0.8 g/L lipase.

Characterization of Lipases from Geobacillus stearothermophilus and Anoxybacillus flavithermuscell Lysates

The aim of this study was to characterize lipases from two thermophilic bacteria, Geobacillus stearothermophilus (GS) and Anoxybacillus flavithermus (AF) in heat treated cell lysates. The pH optimum, pH stability, temperature stability and substrate kinetics and specificity of the lipases were determined. Optimum activity of the lipase from GS (LGS) was observed at pH 7.5, and the optimum activity of the lipase from AF (LAF) was at pH 8.0. LGS was stable up to 70°C after 12 hrs while LAF was stable up to 90°C after 12 hrs. Both enzymes were stable at a pH range of 6 to 8 over 12 h at 4°C. LGS had a highest Vmax value of 22 mM·min-1·mg-1 with p-nitrophenyl acetate while the lowest Km was 0.8 mM with p-nitrophenyl laurate. The highest Vmax of LAF was 2.5 mM·min-1·mg-1 with p-nitrophenyl myristate, and the lowest Km was 0.4 mM with p-nitrophenyl octanoate. LGS preferentially hydrolyzed p-nitrophenyl acetate and p-nitrophenyl octanoate while LAF preferentially hydrolyzed p-nitrophenyl myristate and p-nitrophenyldodecanoate. Lipases from both GS and AF showed characteristics that would be beneficial in food processing.

Characterization of neutral lipases revealed the tissue-specific triacylglyceroi hydrolytic activity in Nilaparvata lugens

The lipid metabolism plays an essential role in the development andreproduction of insects, and lipases are important enzymes in lipid metabolism. In Nilaparvatalugens, an important insect pest on rice, triacylglycerol hydrolytic activities weredifferent among tissues, with high activity in integument, ovary, and fat body, but low activityin intestine. To figure out the tissue-specific triacylglycerol hydrolytic activity, weidentified 43 lipases in N. lugens. Of these 43 lipases, 23 belonged to neutral lipases, sothis group was selected to perform further experiments on triacylglycerol hydrolysis. Thecomplete motifs of catalytic triads, β9 loop, and lid motif, are required for the triacylglycerolhydrolytic activity in neutral lipases, which were found in some neutral lipases withhigh gene expression levels in integument and ovary, but not in intestine. The recombinantproteins of 3 neutral lipases with or without 3 complete motifs were obtained, and the activitydetermination confirmed the importance of 3 motifs. Silencing XM_022331066.1,which is highly expressed in ovary and with 3 complete motifs, significantly decreasedthe egg production and hatchability of N. lugens, partially through decline of the lipidmetabolism. In summary, at least one-third of important motifs were incomplete in allneutral lipases with high gene expression in intestine, which could partially explain whythe lipase activity in intestine was much lower than that in other tissues. The low activityto hydrolyze triacylglycerol in N. lugens intestine might be associated with its food resourceand nutrient components, and the ovary-specific neutral lipases were important forN. lugens reproduction.

Gastric and small intestinal lipid digestion kinetics as affected by the gradual addition of lipases and bile salts

We studied the effect of evolving from static in vitro digestion conditions towards the gradual addition of essential digestive compounds for lipid digestion.Two oil-in-water emulsions were considered:a low(5%w/w)and high(20%w/w)triolein-based emulsion with identical surfactant-to-oil ratio(0.2).Emulsions were subjected to in vitro static digestion conditions or gradual gastric lipase addition,gradual pancreatic lipase addition,and/or gradual bile salt addition.For these three latter cases,similar amounts of gastric/pancreatic lipase and/or bile salts were provided as in the static case,however divided over 4 doses added during the first 30 min of each digestive phase.For the low-lipid emulsion,gradually adding lipases and bile salts did not significantly affect lipolysis kinetics.This can be related to the sufficient amounts of digestive compounds present even in the smallest initial dose.For the high-lipid emulsion,the gradual addition of bile salts significantly reduced the lipolysis rate.Bile salts are essential to remove lipid digestion products from the interface and thus allow the continuation of the lipid digestion process at the interface.Oppositely,the lipolysis extent after 2 h of small intestinal phase was not significantly influenced by the digestion approach.This is again explained by the simple nature of the emulsions studied and the excess of lipase even in the smallest initial dose.Overall,this work showed that evolving towards more(semi-)dynamic digestion conditions can impact(lipid)digestion kinetics,even for relatively simple food compositions,and is of interest to obtain more physiological relevant digestion kinetics.

Microbial lipases:Propitious biocatalysts for the food industry

Lipases(triacylglycerol acylhydrolases,EC 3.1.1.3)are one of the largest groups of enzymes and are used in various industrial processes.Lipases of microbial origin are currently receiving increased attention for industrial application as microorganisms grow quickly and are easily genetically manipulated.Furthermore,they offer several advantages,such as catalysis of diverse reactions,high specificity,high yields,low energy consumption and reduced processing time and production costs.There is a relentless ongoing effort to optimise the production of microbial lipases for potential application in the food industry.In this context,this review highlights the most promising techniques for producing microbial lipases and the recent applications of these lipases in dairy,oils and fats,bakery and confectionery,meat,flavours and aromas and other food industries.Microbial lipases are normally obtained by fermentation,but the high costs of carbon and nitrogen sources limit the process.To overcome this problem,low-cost agro-industrial residues in the lipase production process are explored.To obtain lipases with high yields and improved characteristics,the technique of protein engineering is described as promising,and the immobilization method that allows the recycling of lipases to improve their catalytic performance is focused.Due to their catalytic properties and versatility,lipases of microbial origin are considered extremely important catalysts in the food industry,meeting the demand for tastier foods with pleasant aromas and textures.Therefore,microbial lipases are considered safe and sustainable biocatalysts.

Lipase and photodecarboxylase coexpression: A potential strategy for alkane-based biodiesel production from natural triglycerides

Alkane-based biodiesel is considered the next generation of biodiesel owing to its potential environmental benefits and the fact that it exhibits much higher specific caloric values than traditional biodiesel.However,the formidable obstacle impeding the commercialization of this cutting-edge fuel alternative lies in the cost associated with its production.In this study,an engineered strain Escherichia coli(E.coli)showcasing harmonized coexpression of a lipase(from Thermomyces lanuginosus lipase,TLL)and a fatty acid photodecarboxylase(from Chlorella variabilis,CvFAP)was first constructed to transform triglycerides into alkanes.The potential of E.coli BL21(DE3)/pRSFDuet-1-TLL-CvFAP for alkane synthesis was evaluated with tripalmitin as a model substrate under various process conditions.Following a comprehensive examination of the reaction parameters,the scope of the biotransformation was expanded to‘real’substrates(vegetable oils).The results showed that bioderived oils can be transformed into alkanes with high yields(0.80-10.20 mmol·L^(-1))under mild conditions(35℃,pH 8.0,and 36 h)and blue light illumination.The selected processes were performed on an increased lab scale(up to 100 ml)with up to 24.77 mmol·L^(-1) tripalmitin,leading to a yield of 18.89 mmol·L^(-1) pentadecane.With the employment of a method for efficiently producing alkanes under mild conditions and a simple procedure to isolate alkanes from the reaction system,the utilization of sustainable biomass as a fundamental feedstock emerges as the primary solution to lower the cost of alkane-based biodiesel.Thus,this study proposes a readily implementable and highly effective approach for alkane-based biodiesel production.

Research on the quality deterioration of UHT milk products during shelf life:core microorganisms and related characteristics

Commercial sterility does not guarantee the sustained stability of ultrahigh temperature(UHT)milk over 6 months shelf life.We explore the microbiota presented in normal(SZ)and quality deteriorated UHT milk(QY and WY)products from the same brand.Based on high-throughput sequencing research results,11 phyla and 54 genera were identified as dominant microbiota.Pseudomonas,Streptococcus,and Acinetobacter as core functional microbiota significantly influenced the UHT milk quality properties.Moreover,principal component analysis(PCA)and multivariate analyses were used to examine the quality characteristics,including 11 physicochemical parameters,10 fatty acids,and 2 enzyme activities,in normal and quality deteriorated UHT milk.We found that the abundance of Pseudomonas increased in quality deteriorated milk(WY)and showed a significant positive correlation with heat-resistant protease content.Acinetobacter in quality deteriorated milk(QY)also considerably contributed to the content of heat-resistant lipase,which resulted in spoilage deterioration of UHT milk.

Host-Induced Gene Silencing of Effector AGLIP1 Enhanced Resistance of Rice to Rhizoctonia solani AG1-IA

Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing(TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into si RNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.

Physicochemical and Bacteriological Profile of Bilanko and Ngamakala Peat Bog Soils (Republic of Congo)

Peatlands are unique and complex natural ecosystems that are part of the most important carbon reservoirs on our planet, home to a diversity of microorganisms responsible for fermentation, humification or peat. The aim is to understand chemical and biological indicators of peatland soils. This work aims to determine the physicochemical and bacteriological profile and lipolytic activity of soil bacteria in Bilanko peatlands. The bacterial profile with the production of lipases is carried out by classical microbiology techniques. The results show that the soils are moderately acidic with temperatures of 27.8˚C ± 0.01˚C for Bilanko and 27.1˚C ± 0.57˚C for Ngamakala. The electroconductivity (EC) varies from (9.52 ± 0.002) μS/cm to (39.01 ± 1.4) μS/cm with low turbidity of (2.04 ± 0.66) mg/L to (31.02 ± 0.84) mg/L and low ion concentrations with, however, a richness in phenolic compounds for Bilanko compared to Ngamakala. FMAT diversity ranged from (1.71 ± 0.88)∙104 UFC/g to (2.92 ± 0.07)∙105 UFC/g for Bilanko and (1.30 ± 0.73)∙104 UFC/g to (2.89 ± 0.06)∙104 UFC/g for Ngamakala. Bacillus loads ranged from (5.20 ± 1.40)∙103 CFU/g to (1.22 ± 0.13)∙104 CFU/g and from (1.11 ± 0.13)∙104 CFU/g to (9.20 ± 2.05)∙103 CFU/g;enterobacteria loads from (1.40 ± 0.76)∙103 CFU/g to (8.80 ± 1.73)∙103 CFU/g and from (1.01 ± 0.02)∙103 CFU/g to (9.20 ± 2.05)∙103 CFU/g;in Pseudomonas from 0 to (2.30 ± 0.53)∙102 CFU/g and from 0 to (8.90 ± 2.35)∙102 CFU/g for Bilanko and Ngamakala respectively. These results reveal a variation in bacterial similarity and distribution in the Bilanko and Ngamakala peat bogs.

Lipases-Catalyzed Alcoholysis for the Preparation of Chiral 1- or 2-Hydroxyalkanephosphonates

Enzymatic alcoholysis has been developed for the preparation of some chiral 1- and 2-hydroxyalkanephosphonates with high optical purity. This method ensures the convenient access to the optically pure phosphocarnitine, phosphogabob and phosphomycin.

Triacylglycerol lipases of the yeast

All eukaryotes including the yeast contain a lipid storage compartment which is named lipid particle,lipid droplet or oil body.Lipids accumulating in this subcellular fraction serve as a depot of energy and building blocks for membrane lipid synthesis.In the yeast,the major storage lipids are triacylglycerols(TGs)and steryl esters(SEs).An important step in the life cycle of these non-polar lipids is their mobilization from their site of storage and channeling of their degradation components to the appropriate metabolic pathways.A key step in this mobilization process is hydrolysis of TG and SE which is accomplished by lipases and hydrolases.In this review,we describe our recent knowledge of TG lipases from the yeast based on biochemical,molecular biological and cell biological information.We report about recent findings addressing the versatile role of TG lipases in lipid metabolism,and discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.

Lipases catalyzed acetylation of meso-2,4-dimethyl-1,5-pentanediol and the synthesis of Prelog-Djerassi lactone

Lipase catalyzed acetylation of 2 afforded 3 with the e.e. up to 98%. Utility of 3 was illustrated by the preparation of Prelog-Djerassi lactones 7a and 7b.

Effect of sodium replacement on the quality characteristics of pastırma(a dry-cured meat product)

This study aims to investigate the effect of sodium replacement on the quality characteristics of pastırma.For this purpose,pastırma production with four different salt mixtures(Ⅰ.100%NaCl;Ⅱ.50%NaCl+50%KCl;Ⅲ.40%NaCl+40%KCl+20%CaCl_(2);Ⅳ.30%NaCl+40%KCl+20%CaCl_(2)+10%MgCl_(2))were carried out using traditional method.The use of different salt mixtures for pastırma had no statistically significant effect on the microbial counts and residual nitrite of the final product.The a*and b*values were affected by this treatment.The salt mixture containing CaCl_(2) or CaCl_(2)+MgCl_(2) significantly decreased the pH values and sensory scores.The eighteen volatile compounds were affected by the salt mixtures.Na^(+),K^(+),Ca^(2+),and Mg^(2+)had a certain increase depending on their ratio in the salt mixtures.The highest Cathepsin activity in all pastırma samples was observed in Cathepsin B+L.The salt mixture with NaCl+KCl+CaCl_(2)+MgCl_(2) increased acid lipase activity.However,this mixture had no significant effect on neutral lipase activity.

Bound lipase:an important form of lipase in rice bran (Oryza sativa)

Rice bran residue possessed a steady lipase activity((26.68 ± 3.69)%)after its endogenous lipase was extracted continuously by phosphate buffer solution(PBS)for 24 h. T herefore, the aim of this research was to explore whether there exist any bound lipases in rice bran(Oryza sativa). Three physical treatments(grinding, homogenizing and ultrasound crush)and 6 enzymatic treatments(cellulase, hemicellulase, pectinase, complex cellulase, glucoamylase and α-amylase)were applied to rice bran in order to investigate this bound lipase. The relative catalytic activities of extraction supernatant and residue for pectinase group were(437.63 ± 22.54)% and(159.26 ± 2.12)%, respectively, which were significantly higher(P < 0.05)than other groups. This phenomenon demonstrated that lipase was the most likely to combine with pectin. Molecular simulation proved that pectin could combine with two rice bran lipases(lipase 315 and lipase 308)and cover the catalytic centers so as to prevent the lipases from encountering the substrate and inhibiting their catalytic activities. During combination, pectin could make the lipases more compact and reduce the solvent accessible surface area of lipases, which would make the lipases inactive to molecular interaction. In summary, part of rice bran lipase was proved to exist in bound form and combined with the pectin.

Facile Preparation of Dopamine-Modified Magnetic Zinc Ferrite Immobilized Lipase for Highly Efficient Synthesis of OPO Functional Lipid

1,3-Dioleoyl-2-palmitoylglycerol(OPO)has been a hotspot of functional oils research in recent years,but due to the high cost of sn-1,3 specific lipase in enzymatic synthesis and the lack of biocatalyst stability,large-scale industrial application is difficult.In this study,the prepared magnetic ZnFe_(2)O_(4) was functionalized with dopamine to obtain ZnFe_(2)O_(4)@PDA,and the nano-biocatalyst ZnFe_(2)O_(4)@PDA@RML was prepared by immobilizing sn-1,3 specific lipase of Rhizomucor miehei lipase(RML)via a cross-linking method.The existence of RML on ZnFe_(2)O_(4)@PDA was confirmed by XRD,FTIR,SEM,and TEM.This strategy proved to be simple and effective because the lipase immobilized on magnetic nanoparticles could be quickly recovered using external magnets,enabling reuse of the lipase.The activity,adaptability to a high temperature,pH value,and operational stability of immobilized RML were superior to those of free RML.After optimizing the synthesis conditions,the OPO yield was 42.78%,and the proportion of PA at the sn-2 position(PA-Sn2)was 54.63%.After the first four cycles,the activity of ZnFe_(2)O_(4)@PDA@RML was not significantly affected.The magnetically immobilized lipase has good thermal stability,long-term storage stability,reusability,and high catalytic activity.It can be used as a green and efficient biocatalyst to synthesize the OPO functional lipid.

Conjugation of Candida rugosa lipase with hydrophobic polymer improves esterification activity of vitamin E in nonaqueous solvent

We described a novel polymer-lipase conjugate for high-efficient esterification of vitamin E using vitamin E and succinic anhydride as the substrates in nonaqueous media.In this work,the monomer,N-isopropylacrylamide(NIPAM),was grafted onto Candida rugosa lipase(CRL)to synthesize poly(NIPAM)(pNIPAM)-CRL conjugate by atom transfer radical polymerization via the initiator coupled on the surface of CRL.The result showed that the catalytic efficiencies of pNIPAM-CRL conjugates(19.5-30.3 L·s^(-1)·mmol^(-1))were at least 7 times higher than that of free CRL(2.36 L·s^(-1)·mmol^(-1))in DMSO.It was attributed to a significant increase in Kcat of the conjugates in nonaqueous media.The synthesis catalyzed by pNIPAM-CRL co njugates was influenced by the length and density of the grafted polymer,water content,solvent polarity and molar ratio of the substrates.In the optimal synthesis,the reaction time was shortened at least 7 times,and yields of vitamin E succinate by pNIPAM-g-CRL and free CRL were obtained to be 75.4%and 6.6%at 55℃after the reaction for 1.5 h.The result argued that conjugation with pNIPAM induced conformational change of the lid on CRL based on hydrophobic interaction,thus providing a higher possibility of catalysis-favorable conformation on CRL in nonaqueous media.Moreover,pNIPAM conjugation improved the thermal stability of CRL greatly,and the stability improved further with an increase of chain length of pNIPAM.At the optimal reaction conditions(55℃and 1.5 h),pNIPAM-g-CRL also exhibited good reusability in the enzymatic synthesis of vitamin E succinate and kept~70%of its catalytic activity after ten consecutive cycles.The research demonstrated that pNIPAM-g-CRL was a more competitive biocatalyst in the enzymatic synthesis of vitamin E succinate and exhibited good application potential under harsh industrial conditions.

Novel Lipase from Golden Pompano(Trachinotus ovatus)Viscera:Purification,Characterization,and Application in the Concentrating of n-3 Polyunsaturated Fatty Acids

Lipases have been widely applied in a variety of industrial fields,such as food,pharmaceuticals,biofuels,and biotechnology.Recent years have witnessed a great interest in modifying lipids for the production of triacylglycerols enriched with n-3 polyunsaturated fatty acids(PUFAs).Here,a novel salt-tolerant,organic solvent-stable,and bile salt-activated lipase was purified from golden pompano(Trachinotus ovatus)viscera,which was named as golden pompano lipase(GPL).GPL had a specific activity of 57.2U mg^(-1)with an estimated molecular weight of 14 k Da,exhibited optimal activity at 40℃a nd pH 8.0,and showed K_(m)and V_(max)of 40.16μmol L^(-1)and 769.23μmol L^(-1)min^(-1),respectively.GPL activity was enhanced by Mn^(2+)and sodium deoxycholate.It was active in organic solvents,including methanol,ethanol,chloroform,and hexane.GPL also showed a good salinity tolerance of up to 1 mol L^(-1).n-3PUFA enrichment in the glyceride fraction of golden pompano oil was performed by GPL-catalyzed hydrolysis and yielded a total PUFA concentration of 56.99%.EPA,DHA,and DPA were enriched by 10.4-,3.2-,and 1.8-fold of their initial levels,respectively.This study recognized the industrial applicability of GPL to prepare enriched C_(20-22)n-3 PUFA.