Synthesis of Vitamin A Esters by Immobilized Candida sp. Lipase in Organic Media

Vitamin A ester was synthesized in organic solvents with immobilized lipase from Candida sp. The types of lipases, influences of solvent, the molar ratio of substrates, the reaction temperature and the water activity in the reaction were studied in detail in order to obtain the optimum conditions for Vitamin A palmitate synthesis. In a system of hexane, 100mg immobilized Candida sp. lipase was used in the presence of 1.2mmol vitamin A acetate and 3.6mmol palmitic acid. The yield of vitamin A palmitate reached 81% in 12h at 25℃. The immobilized Candida sp. lipase was prepared by adsorbing Cand/da sp. fermentation broth on pretreated textile and could be reused for at least six batches.

Zwitterionic polymer-coated porous poly(vinyl acetate–divinyl benzene)microsphere: A new support for enhanced performance of immobilized lipase

Enzyme immobilization has attracted great attention for improving the performance of enzymes in industrial applications.This work was designed to create a new support for Candida rugosa lipase(CRL)immobilization.A porous poly(vinyl acetate–divinyl benzene)microsphere coated by a zwitterionic polymer,poly(maleic anhydride-alt-1-octadecene)and N,N-dimethylethylenediamine derivative,was developed for CRL immobilization via hydrophobic binding.The catalytic activity,reaction kinetics,stabilities and reusability of the immobilized CRL were investigated.It demonstrated the success of the zwitterionic polymer coating and subsequent CRL immobilization on the porous microsphere.The immobilized lipase(p2-MS-CRL)reached27.6 mg·g^-1 dry carrier and displayed a specific activity 1.5 times higher than free CRL.The increase of Vmax and decrease of Kmwere also observed,indicating the improvement of catalytic activity and enzyme-substrate affinity of the immobilized lipase.Besides,p2-MS-CRL exhibited significantly enhanced thermal stability and pH tolerance.The improved performance was considered due to the interfacial activation regulated by the hydrophobic interaction and stabilization effect arisen by the zwitterionic polymer coating.This study has thus proved the advantages of the zwitterionic polymer-coated porous carrier for lipase immobilization and its potential for further development in various enzyme immobilizations.

Lipase immobilized on HOOC-MCF:A highly enantioselective catalyst for transesterification resolution of (R,S)-1-phenylethanol

Pseudomonas cepacia lipase （PSL） immobilized on the carboxyl-functionalized meso-cellular foams （HOOC-MCF） was used for the transesterification resolution of （R,S）-l-phenylethanol in organic solvent. The results showed that the ee value of （S）-1- phenylethanol and （R）-1-phenylethyl acetate reached 99% with 50% conversion of 1-phenylethanol using toluene as solvent. Furthermore, it was found that PSL/HOOC-MCF exhibited high enantioselectivity in organic solvent with log P ≤ 2 such as toluene and hexane.

Study on Immobilized Lipase Catalyzed Transesterification Reaction of Tung Oil

The transesterification reaction conditions of tung oil with methanol have been studied in this article, with immobilized lipase NOVO435 as catalyst. The response surface methodology was used to optimize the transesterification reaction of tung oil in a nonsolvent system. The optimal conditions were rotation rate 200 r/min, molar ratio of methanol to oil 2.2： l, reaction temperature 43℃, and the catalyst amount 14% （based on the weight of oil）. After reacting for 18 h, 67.5% of the oil was converted to its corresponding methyl esters （the theoretical ester conversion was 73.3%）. The lipase was washed by organic solvents after each reaction and was reused again. The esters conversion of tung oil was decreased by 6% after the lipase was reused for 120 h. The theoretical amount of methanol was added in two steps, 85% ester conversion was obtained after 36 h of reaction （theoretical ester conversion was 100%）. The molar ratio of methanol to oil, the catalyst amount, the reaction temperature, and reaction time were all highly significant factors, and there was a relative significant interaction between every two factors.

Activity and Enantioselectivity of Lipase Immobilized in CTAB Microemulsion-based Gels

Introduction The formation of gelatin-containing mieroemulsionbased gels（MBGs） was first described in 1986 and the physical/structural characterization was carried out by a number of groups with a variety of techniques including tracer diffusion, electrical conductivity, NMR, X-ray and small angle neutron scattering. The MBGs were proposed to comprise an extensive, rigid, interconnected network of gelatin/water rods stabilized by a monolayer of surfactant, in coexistence with a po- pulation of conventional W/O microemulsion droplets.

Corrosion Inhibition by Co-Immobilized Lysozyme and Lipase in Circulating Cooling Water System

The corrosion inhibition performance of co-immobilized lysozyme and lipase was investigated in a recirculating cooling water system. Four methods were carried out in co-immobilization, and the operating parameters were optimized by using the respond surface methodology(RSM). The corrosion inhibition performance of co-immobilized lipase and lysozyme was evaluated by weight loss measurements and electrochemical measurements. The results revealed that the optimal co-immobilization method should be the sequential immobilization of lysozyme and then lipase. The inhibition efficiency was 86.10% under the optimal co-immobilized conditions. Electrochemical data showed that co-immobilized lysozyme and lipase was a mixed-type inhibitor and the corrosion inhibition efficiency was 81%.

Hydrophobic surface modification of chitosan gels by stearyl for improving the activity of immobilized lipase

The hydrophobic surface modification of chitosan gels was carried out using the amidating reaction of amido groups on a gel surface with stearic acid activated by 1-ethyl-3-（3-dimethylaminopropyl）-carbodiimide hydrochloride （EDC） and N-hydroxysuccinimide （NHS）. Lipases from Candida rugosa were adsorbed on the nascent chitosan gels （CS） and stearyl-modified gels （SCS） with different degrees of amidation. The increased surface hydrophobicity of chitosan gels improved the adsorption capacity and activity of the immobilized lipase. SCS with 31.46% amidation showed the maximum activity retention （83.43%）. The experimental results suggest that the moderate surface hydrophilicity/hydrophobicity of chitosan gels is necessary for the improvement of the activity of immobilized lipase. ？2009 Hong Tao Deng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Combined Use of Co-immobilized Lysozyme and Lipase and Chemical Inhibitors in Circulating Cooling Water System

In order to improve the stability and corrosion inhibition performance of bioenzyme, lipase and lsozyme were co-immobilized on the mesoporous molecular sieve MCM-41 by the adsorption method. Then the immobilized enzymes were combined with amino trimethylene phosphonic acid and polyaspartic acid to prevent corrosion caused by circulating cooling water. The weight-loss method and electrochemical techniques were used to evaluate the performance of composite inhibitors. The co-immobilized lysozyme and lipase achieved good inhibition effects. After they were combined with amino trimethylene phosphonic acid and polyaspartic acid, the corrosion inhibition properties were further improved. The inhibition efficiency was promoted to 94.4%. During the corrosion inhibition process, the immobilized enzymes played an important role. The addition of corrosion inhibitor could inhibit the anodic dissolution and cathodic hydrogen evolution process of carbon steel at the same time. The adsorption of co-immobilized lysozyme and lipase composite inhibitor on the steel surface was a joint action involving physical adsorption and chemical adsorption.

Synthesis of Wax Esters by Lipase-catalyzed Esterification with Immobilized Lipase from Candida sp. 99-125

Wax esters were synthesized in a solvent free system catalyzed by immobilized lipase from Candida sp. 99-125, with oleic acid and cetyl alcohol. The effects of substrate molar ratio, lipase dosage and water removal were investigated in a 50 ml flask incubated in a thermostatic cultivation cabinet. The optimized conditions were: temperature 40 ℃, shaking at 170 r·min-1, acid/alcohol molar ratio 1:0.9, lipase dosage in 10% (by mass) of oleic acid, and open reaction for water removal. As a result, the conversion rate reached 98% for reaction of 8 h. The volume of reactor was scaled up to 1 L three-neck flask. The optimized parameters were: 200 r·min-1 agitation speed, 2.5% (by mass) lipase dosage, others were the same as the parameters described above. The conversion rate reached 95% for reaction of 24 h. The lipase retained 46% conversion rate after reuse for 6, 7 batches. The products were purified by removing remained cetyl alcohol and fatty acids with ethanol and saturated sodium carbonate so-lution, respectively. The purity of the wax ester, cetyl oleate, was 96%. The physical and chemical properties of cetyl oleate were tested and compared with those of jojoba oil. The results show that the product cetyl oleate has great potential to use as the substitute of natural jojoba oil.

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.

Catalytic Characteristics of Lipase Immobilized in CTAB/TX-100 MBG

CTAB/TX-100 microemtdsion-based gel（MBG）, which could be soaked in aqueous solution for a long time without mechanical strength and shape changes, was successfully prepared. Lipase immobilized in this gel had a higher activity than that in CTAB MBG when catalyzed the esterification reaction between n-hexanoic acid and n-octanol and its enantioselectivity was slightly higher when catalyzed the stereoselective esterification reaction between racemic ibuprofen and n-octanol. In aqueous solution, lipase in CTAB/TX-100 MBG could catalyze smoothly the hydrolysis reaction of olive oil and could be reused many times without the evident change of MBG mechanical strength although its activity decreased 41.8% after nine-time recycles.

Lipase Immobilized on Magnetic Nanoparticles: A New Tool for Synthesis of Disulphide Compounds

The study describes chemo-enzymatic synthesis of organic disulphide compounds. The reaction was initiated by hydrolysis of thiol acetates using hydrolytic enzyme lipase (PPL) immobilized on to magnetic nanoparticles and subsequent formation of organic disulphide compounds. Lipase was immobilized on to the magnetic nanoparticles by co-precipitation method via epichlorohydrin chitosan cross-linking, under mild and eco-friendly conditions. The immobilized lipase enzyme exhibited broad range of substrate specificity in synthesizing disulphide compounds, which involves both intra and inter-molecular disulphide bond formation under anaerobic conditions. The disulphide compounds synthesized also show a promising antimicrobial activity.

STUDY ON IMMOBILIZED PORCINE PANCREATIC LIPASE CATALYZING TRANSESTERIFICATION BETWEEN METHYL－BUTYRATE AND 1－BUTANOL IN NONAQUEOUS SYSTEMS

Transesterification between methyl-butyrate and 1-butanol in nonaqueous systems was catalyzed by porcine pancreatic lipase which was immobilized on cross- linked polystyrene. Organic solvents, substrate concentration, contents of water and other parameters which affect the immobilized enzyme activity were studied. Lipase immobilized on hydrophobic crosslinked polystyrene can reduce its diffusion limit in the reaction. It was found that the activity of immobilized lipase in organic systems was two times as high as that of free lipase.

Determination of External Mass Transfer Model for Hydrolysis of Jatropha Oil Using Immobilized Lipase in Recirculated Packed-Bed Reactor

In this study, a simple and effective technique for establishing an external mass transfer model in a recirculated packed-bed batch reactor (RPBBR) with an immobilized lipase enzyme and Jatropha oil system is presented. The external mass transfer effect can be represented with a model in the form of Colburn factor JD = K Re-(1–n). The value of K and n were derived from experimental data at different mass flow rates.The experiment shows an average increment of 1.51% FFA for calcium alginate and 1.62% FFA for carrageenan after the hydrolysis took place. Based on different biopolymer material used in immobilized beads, JD = 1.674 Re-0.4 for calcium alginate and JD = 1.881 Re-0.3 for k-carrageenan were found to be adequate to predict the experimental data for external mass transfer in the reactor in the Reynolds number range of 0.2 to 1.2. The purposed model can be used for the design of industrial bioreactor and scale up. Besides, the external mass transfer coefficients for the hydrolysis of Jatropha oil reaction and the entrapment efficiency for the two biopolymer materials used were also investigated.

Non-Alcohol Route of Biodiesel Synthesis from Fried Palm Oil using Immobilized Candida rugosa Lipase

Synthesis biodiesel using biocatalyst is an emerging and attracting alternative process to replace the conventional process. However, biocatalyst is easy to be deactivated by alcohol, which is a reactant in biodiesel synthesis reaction. Therefore, it is needed to develop new method to maintain the activity and stability of the biocatalyst during reaction. New method to be developed is by changing the reaction route which is using alcohol to the reaction route which is not using alcohol. Route reaction of non alcohol can be done by changing the alkyl alcohol with alkyl acetate. Both have the same function as alkyl supplier during the reaction. In this research, methyl acetate was reacted with triglyceride from fried palm oil using Candida rugosa lipase in batch reactor. The reactants and products were analyzed using HPLC. The effect of operating factors such as enzyme concentration, substrates ratio, operating temperature and addition of inhibitor using free and immobilized enzyme were investigated. The experimental results showed that 89.6% of triglyceride from fried palm oil was converted to its corresponding methyl esters under the condition of 4% wt lipase based on substrate weight, 1/12 mol rasio of oil/methyl acetate after 50 hours reaction using immobilized lipase. Stability test indicated that the activity of the immobilized biocatalyst was still remained after three reaction cycles.

Hydrolysis of Olive Oil with Immobilized Lipase in a Tapered Column Reactor

Lipase was immobilized in ion exchange resin and then used in the hydrolysis of olive oil to produce fatty acids and glycerol. The time course of hydrolysis of olive oil was investigated in a stirred tank reactor using both of the free and immobilized lipases to find the yield of activity of immobilized enzyme. Continuous hydrolysis of olive oil was also carried out in a tapered column reactor and a cylindrical column reactor with a bottom ID of 10 mm at different upward flow rates. It can be known from experimental results that the degree of hydrolysis of olive oil in the tapered column reactor is moderately better than that in the cylindrical column reactor, the pressure drop in the tapered column reactor is much smaller than that in the cylindrical column reactor.

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.

Carboxyl Ester Lipase Protects Against Metabolic Dysfunction-Associated Steatohepatitis by Binding to Fatty Acid Synthase

Carboxyl ester lipase(CEL),a pivotal enzyme involved in lipid metabolism,is recurrently mutated in obese mice.Here,we aimed to elucidate the functional significance,molecular mechanism,and therapeutic potential of CEL in metabolic dysfunction-associated steatohepatitis(MASH).Hepatocyte-specific carboxyl ester lipase gene(Cel)knockout(Cel^(DHEP))and wildtype(WT)littermates were fed with cholinedeficient high-fat diet(CD-HFD)for 16 weeks,or methionine-and choline-deficient diet(MCD)for three weeks to induce MASH.Liquid chromatography–mass spectrometry and co-immunoprecipitation were employed to identify the downstream targets of CEL.CD-HFD/MCD-fed WT mice received intravenous injections of CEL-adeno-associated viral,serotype 8(AAV8)to induce specific overexpression of CEL in the liver.We observed a decrease in CEL protein levels in MASH induced by CD-HFD or MCD in mice.Cel^(DHEP) mice fed with CD-HFD or MCD exhibited pronounced hepatic steatosis,inflammation,lipid peroxidation,and liver injury compared to WT littermates,accompanied by increased hepatic nuclear factor kappa-light-chain-enhancer of activated B cell(NF-jB)activation.Consistently,Cel knockdown in mouse primary hepatocytes and AML12 cells aggravated lipid accumulation and inflammation,whereas CEL overexpression exerted the opposite effect.Mechanistically,CEL directly bound to fatty acid synthase(FASN),resulting in reduced FASN SUMOylation,which in turn promoted FASN degradation through the proteasome pathway.Furthermore,inhibition of FASN ameliorated hepatocyte lipid accumulation and inflammation induced by Cel knockdown in vivo and in vitro.Hepatocyte-specific CEL overexpression using AAV8-Cel significantly mitigated steatohepatitis in mice fed with CD-HFD or MCD.CEL protects against steatohepatitis development by directly interacting with FASN and suppressing its expression for de novo lipogenesis.CEL overexpression confers a therapeutic benefit in steatohepatitis.

Prokaryotic Expression of Pseudomonas Aeruginosa Lipase Gene

[Objective] The aim of this study was to investigate the prokaryotic expression of pseudomonas aeruginosa Lipase gene.[Method]Lipase gene was amplified by PCR from the genome DNA of pseudomonas aeruginosa,and its nucleotide sequence was determined.The prokaryotic expression vector of Lipase gene was constructed by the gene recombination technique.The protein expression was induced for 4 hours by IPTG with the final concentration of 1.0 mmol/L,and then SDS-PAGE electrophoresis was analyzed.[Result]The sequence of mature peptides in Lipase gene cloned from pseudomonas aeruginosa had a 99.36% homology with that of pseudomonas aeruginosa lipase submitted in NCBI,so the prokaryotic expression vector of Lipase gene pET32a-Lip was successfully constructed.Furthermore,the results of SDS-PAGE electrophoresis showed that the target gene was expressed highly and effectively.[Conclusion]The cloned pseudomonas aeruginosa lipase with its signal peptide could be normally expressed in E.coli and also used for further study.

Recent advances in immobilized enzymes on nanocarriers

Recent progress in nanotechnology has provided high-performance nanomaterials for enzyme immobilization.Nanobiocatalysts combining enzymes and nanocarriers are drawing increasing attention because of their high catalytic performance,enhanced stabilities,improved enzyme-substrate affinities,and reusabilities.Many studies have been performed to investigate the efficient use of cellulose nanocrystals,polydopamine-based nanomaterials,and synthetic polymer nanogels for enzyme immobilization.Various nanobiocatalysts are highlighted in this review,with the emphasis on the design,preparation,properties,and potential applications of nanoscale enzyme carriers and nanobiocatalysts.