- Open Access
Identification and immobilization of a novel cold-adapted esterase, and its potential for bioremediation of pyrethroid-contaminated vegetables
- Xinjiong Fan†1,
- Weiqu Liang†2,
- Yanfang Li2,
- He Li3 and
- Xiaolong Liu1Email authorView ORCID ID profile
© The Author(s) 2017
- Received: 6 July 2017
- Accepted: 7 September 2017
- Published: 11 September 2017
Pyrethroids are potentially harmful to living organisms and ecosystems. Thus, concerns have been raised about pyrethroid residues and their persistence in agricultural products. To date, although several pyrethroid-hydrolyzing enzymes have been cloned, very few reports are available on pyrethroid-hydrolyzing enzymes with cold adaptation, high hydrolytic activity and good reusability, indispensable properties in practical bioremediation of pyrethroid-contaminated vegetables.
Here, a novel gene (est684) encoding pyrethroid-hydrolyzing esterase was isolated from the Mao-tofu metagenome for the first time. Est684 encoded a protein of 227 amino acids and was expressed in Escherichia coli BL21 (DE3) in soluble form. The optimum temperature was 18 °C. It maintained 46.1% of activity at 0 °C and over 50% of its maximal activity at 4–35 °C. With the goal of enhancing stability and recycling biocatalysts, we used mesoporous silica SBA-15 as a nanometer carrier for the efficient immobilization of Est684 by the absorption method. The best conditions were an esterase-to-silica ratio of 0.96 mg/g (w/w) and an adsorption time of 30 min at 10 °C. Under these conditions, the recovery of enzyme activity was 81.3%. A large improvement in the thermostability of Est684 was achieved. The half-life (T1/2) of the immobilized enzyme at 35 °C was 6 h, 4 times longer than the soluble enzyme. Interestingly, the immobilized Est684 had less loss in enzyme activity up to 12 consecutive cycles, and it retained nearly 54% of its activity after 28 cycles, indicating excellent operational stability. Another noteworthy characteristic was its high catalytic activity. It efficiently hydrolyzed cyhalothrin, cypermethrin, and fenvalreate in pyrethroid-contaminated cucumber within 5 min, reaching over 85% degradation efficiency after four cycles.
A novel cold-adapted pyrethroid-hydrolyzing esterase was screened from the Mao-tofu metagenome. This report is the first on immobilizing pyrethroid-hydrolyzing enzyme on mesoporous silica. The immobilized enzyme with high hydrolytic activity and outstanding reusability has a remarkable potential for bioremediation of pyrethroid-contaminated vegetables, and it is proposed as an industrial enzyme.
Pyrethroids are one of the most important insecticides globally. They already have high usage, and the total demand continues to grow. Pyrethroid residues are toxic, which may presents a risk to human health and poses a significant threat to ecosystems [1, 2]. Enzyme biotechnology offers an attractive solution for the removal of highly toxic pollutants such as pyrethroid residues. The main metabolic pathway of pyrethroids includes oxidation and ester-bond hydrolysis, which produces nontoxic acid and alcohol compounds [3, 4]. Native enzymes, such as PytY, EstP, PytH, Pye3, PytZ, EstSt7, and CMO, have been explored for the biodegradation of pyrethroids in the past 10 years [5–10]. Very few reports are available on pyrethroid-hydrolyzing enzyme with novel characteristics, such as cold adaptation and high hydrolytic activity, because such enzymes are rare. Cold-adapted enzymes exhibit high catalytic efficiency at moderate and low temperatures and are thus versatile biocatalysts in many applications , especially for bioremediation of pyrethroid-contaminated vegetables.
However, the cost of the enzyme is a key determinant of the economic feasibility of biotransformations. The ability to fully recycle biocatalysts would help to minimize waste disposal and optimize economic benefits. Enzyme immobilization might be the best method for reducing the cost of the process because it would permit reuse of the enzymes . Enzymes immobilized on the surface of functional materials have many advantages over soluble enzymes, including the possibility for recovery and reuse, enhanced stability and catalytic activity, and easy operation [13–16]. Several methods are available for immobilizing proteins onto solid supports, and they are divided into four main categories: adsorption, entrapment, cross-linking, and covalent binding using commercial materials . Each method has advantages and disadvantages. In physical adsorption, enzyme conformation is largely protected because the adsorption is mainly achieved by either electrostatic interaction or van der Waals’ force . However, such bonding is relatively weak, and enzyme leakage from carrier can occur during operational process. Sol–gel entrapment is widely used because of its mild reaction conditions. Various methods for sol–gel formation have been proposed for building nanostructures and immobilizing enzymes. However, some limitations exist, such as enzyme leakage from the support. Cross-linking and covalent binding have the advantage of strong irreversible binding of the enzyme to the supporting agent, which can protect against enzyme leakage. A drawback of this strong binding is the risk of a reduction in enzyme activity .
In addition to the different immobilization approaches, a variety of carriers have been used for enzyme immobilization. Mesoporous silica materials have attracted significant interest because of their stable structures, larger surface areas, good biocompatibility, and tunable pore sizes and volumes [20–22]. As immobilizing carriers, mesoporous materials can incorporate proteins or enzymes through physical or chemical reactions with good adsorption because of their large specific surface area. The available size range, about 3–10 nm, is comparable with the hydrodynamic radius of most enzymes being used. Although many reports exist on the properties of enzymes immobilized on mesoporous silica, the effect of mesoporous silica on the properties of pyrethroid-hydrolyzing esterase has not yet been investigated.
In this study, we reported the cloning, identification and biochemical characterization of one novel cold-adapted pyrethroid-hydrolyzing esterase derived from the Mao-tofu metagenome. With the goal of enhancing stability and recycling biocatalysts, we immobilized this esterase on a matrix of mesoporous silica SBA-15. The stability and reusability of the immobilized enzyme were estimated and compared with the soluble enzyme. In addition, the potential use of the immobilized pyrethroid-hydrolyzing esterase for removal of pyrethroids on vegetables was investigated.
Screening for pyrethroid-hydrolyzing esterase from a metagenomic library
Mao-tofu, is a type of fermented tofu, is primarily fermented by Mucor spp., which can degrade the starches, proteins, and lipids of soybean . Presumably, there are a variety of hydrolytic enzymes excreted by Mao-tofu microbes. The metagenomic library-based technique has been successfully applied to screen novel genes . Here, the total DNA was extracted from Mao-tofu microbes and used to construct a metagenomic library containing approximately 13,200 clones. Several transformants with hydrolysis zones were obtained by functional expression screening of plasmid clones with esterase/lipase activity. One of them harboring the pUC118No.2 showed pyrethroid-hydrolyzing activity.
Heterologous expression and purification of recombinant Est684
Est684 was cloned and expressed in Escherichia coli BL21 (DE3), and then purified by a Ni–NTA His·Bind column and a Superdex 200 (16/60) size-exclusion column. SDS-PAGE analysis showed that Est684 migrates as a single band with a molecular mass lower than 35 kDa (Additional file 1: Figure S1), in accordance with its predicted molecular mass of 30.58 kDa, containing the 227 amino acids and a fusion of 51 amino acids corresponding to polyhistidine tag (His-tag), a unique thrombin cleavage site (Thrombin).
Substrate specificity of Est684
Effect of temperature on the activity and stability of recombinant Est684
Thermostability was determined by analysis of the residual activity toward p-nitrophenyl butyrate at regular intervals after preincubation for durations up to 24 h, at temperatures ranging from 18 to 35 °C. Est684 was very stable at 18 °C, with 52% residual activity after incubation for 18 h (Fig. 3b). However, the activity decreased dramatically at 35 °C, and remained 28% activity for 2 h, much worse than Sys410 and its mutants [34, 35], which proved that thermostability and high activity at low temperatures are apparently incompatible in natural enzymes.
Immobilization of purified esterase onto mesoporous silica SBA-15
The remaining activity of the immobilizing Est684 on different carriers
The remaining activity
10.1 ± 0.2
12.7 ± 2.1
Mesoporous silica SBA-15
66.3 ± 0.9
Kinetic parameters determination
p-Nitrophenyl butyrate was used to test the activity and kinetic parameters of the soluble and immobilized enzyme. The Km and kcat values were calculated by fitting the data to the Michaelis–Menten equation. The Km and kcat values of the soluble enzyme were 6.01 ± 2.74 μM and 417.15 ± 1.51 s−1, respectively. The Km and kcat values of the immobilized enzyme were 7.92 ± 2.10 μM and 403.47 ± 2.71 s−1, respectively. The Km value was much lower than the pyrethroid-hydrolyzing esterase Sys410 and its mutants [34, 35], which demonstrated that both soluble and immobilized Est684 had better affinity for the substrate. Compared to the soluble enzyme, Km of the immobilized Est684 showed a little increase upon immobilization, which reflected a slightly decreased affinity for the substrate and the presence of partitioning and diffusional effects in the pores of the SBA-15 matrix. The slight change in the affinity for p-nitrophenyl acetate may have been caused by structural changes in the enzyme introduced by the immobilization procedure and lower accessibility of the active site of the immobilized enzyme for the substrate [38, 39]. Nevertheless, the immobilized enzyme had high catalytic activity and a high kcat value. Efficient catalytic activity is a valuable enzymatic property for practical applications.
Comparison of catalytic properties of soluble and immobilized Est684
Operational stability of the immobilized Est684
The outstanding operational stability could be attributed in part to the suitable pore size of SBA-15. Supports with small pore diameters would impede the exit of enzyme, while large pore diameters offer less resistance to enzyme release . The good thermostability of Est684 itself may contribute to the stability. Xu et al.  reported that the relative activity of NH-SBA-15-PPL remains at 49.2% after the 5th reuse. Cai et al.  reported that 92.5% of the initial glycerolysis activity of SBA-15(6.6)-CALB remained after five cycles, each lasting 12 h. SBA-15 clearly serves as an excellent support for immobilizing enzymes, which enhances operational stability and provides a highly biocompatible environment for enzymes. These results suggest that the immobilized Est684 possesses high catalytic activity, outstanding operational stability, and good adaptability at low and moderate temperatures, which may be useful in the bioremediation of pyrethroids. However, further experiments are needed to ensure the suitability of the procedure for removal of pesticides from vegetables.
Gas chromatography analysis of the degradation of pyrethroids in cucumbers
In this study, we identified a novel pyrethroid-hydrolyzing esterase Est684 from the Mao-tofu metagenome for the first time. The recombinant enzyme displayed high activity but weak stability. With the aim of improving stability and recycling enzymes, Est684 was immobilized on mesoporous silica SBA-15 using absorption method. The results showed that immobilization could improve the properties of the enzyme. Thermostability and operational stability were notably enhanced, and the immobilized enzyme retained broad substrate specificity and catalytic activity. The enzyme efficiently hydrolyzed several pyrethroids on contaminated cucumbers within a very short time, reaching over 90% degradation efficiency after two cycles. These favorable features make the process ideal for biodegradation of pyrethroids on contaminated vegetables.
Chemicals and materials
All p-nitrophenyl esters were purchased from Sigma. Mesoporous silica SBA-15 (<150-μm particle size, 8-nm pore size) was purchased from Rusology Technology Co. LTD (Guangxi, China). The chitosan beads, Chitopearl BCW-3010 (BCW), were purchased from Wako Chemicals GmbH (Neuss, Germany). T4 DNA ligase, restriction endonuclease, and DNA polymerase were purchased from TaKaRa (Dalian, China) and used according to manufacturer recommendations. E.Z.N.A. Plasmid Mini Kit and E.Z.N.A. Gel Extraction Kit were purchased from OMEGA (Norcross, USA). All chemicals were of analytical or electrophoresis grade, and unless stated otherwise, they were purchased from Sigma Aldrich (St. Louis, MO, USA).
Bacterial strains and plasmids
Escherichia coli DH5α and E. coli BL21 (DE3) (Novagen, Madison, WI, USA) was used as the host for gene cloning and protein expression. pUC118 (TaKaRa) and pET28a (+) (Novagen) was used to construct metagenomic library and express the target gene.
Metagenomic library construction and pyrethroid-hydrolyzing gene screening
Microbes from Mao-tofu were used to construct one metagenomic library. The total DNA was extracted on the base of the method described before [26, 43]. The purified DNA was digested into 3.0–10 kb fragments by incomplete digestion of EcoRI. The metagenomic library was constructed based on the previous method . Initially, the transformants were replicated on LB agar plates supplemented with 0.1% (v/v) tributyrin. Transparent zone due to tributyrin hydrolysis appeared after cultivation at 37 °C for 1–2 days. The positive clones with hydrolysis zones were selected, and further tested for the ability to hydrolyze pyrethroids confirmed by gas chromatography (GC) analysis. One clone with pyrethroid-hydrolyzing activity was screened. The recombinant plasmid (pUC118No.2) was sequenced on ABI 377 DNA sequencer.
Cloning, expression, and purification of pyrethroid-hydrolyzing esterase
The putative pyrethroid-hydrolyzing gene est684 was amplified from the pUC118No.2 plasmid by using the primers. The following primers were used: fw (5′- CGCGGATCCATGCCGTCTGGAGAAAATCAGTTTT-3′; the BamHI cutting site is underlined) and rv (5′- CCCAAGCTTCTACTTCTTCAGCGTTTCCTCGAAC-3′; the HindIII cutting site is underlined). Amplified DNA was digested by BamHI/HindIII, ligated into the vector pET-28a (+) which was linearized by BamHI/EcoRI, then transformed into E. coli BL21 (DE3) cells. The procedure was carried out according to the method of Fan et al. . Recombinant cells were cultivated in a 250-mL flask containing 50 mL of Luria–Bertani (LB) (50 μg/mL kanamycin) at 37 °C until the cell concentration reached an OD600 of 1.0, then induced with 0.6 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37 °C for 8 h with shaking at 220 rpm. The protein of interest was purified with a Ni–NTA His·Bind column according to the method of Fan et al. . The sample was loaded onto a Ni–NTA His·Bind column pre-equilibrated with binding buffer. Then the column was washed with binding buffer and washing buffer (0.5 M NaCl, 60 mM imidazole, 20 mM Tris–HCl, pH 7.9). Finally, the bound protein was eluted with eluting buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris–HCl, pH 7.9). The eluted samples were further purified using a Superdex 200 (16/60) size-exclusion column (GE Healthcare) with 20 mM Tris–HCl, 200 mM NaCl, pH 7.5 . The purified protein corresponding to Est684 was collected and stored at −80 °C. The molecular mass of the denatured protein was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). A 12% SDS-PAGE was prepared by the previous method . Proteins were stained with Coomassie brilliant blue G-250.
Determination of substrate specificity
p-nitrophenyl esters, as general substrates of esterases/lipases, were used to determine substrate specificity of est684 according to the method of Fan et al. , with slight modification. The reaction was performed at pH 6.5 and 18 °C for 5 min. The production of ρ-nitrophenol was measured at 405 nm by Labsystems Dragon Wellscan MK3. One unit of enzyme activity was defined as the rate of the reaction that the enzyme produced 1 μmol of ρ-nitrophenol per minute under the conditions. In each measurement, the effect of nonenzymatic hydrolysis of substrates was considered and subtracted from the value measured when the enzyme was added.
Effect of temperature on activity and stability of recombinant enzyme
The optimum temperature was evaluated by testing esterases/lipases activity at pH 6.5 in temperature ranges of 0–50 °C based on the previous method , with slight modification. Thermostability was measured by preincubation of the purified enzyme in 50 mM potassium phosphate buffer (pH 6.5) at different temperatures for intervals. Then, the residual activity was measured using p-nitrophenyl butyrate as a substrate at pH 6.5 and 18 °C based on the method described previously .
Investigation of immobilization conditions
Immobilization of the pyrethroid-hydrolyzing esterase on chitosan beads was performed using glutaraldehyde as the crosslinker . The immobilization on alginate was performed as previously described , with some modifications as follows: 4% sodium alginate, 1 (mg/g) enzyme/sodium alginate ratio, 4% CaCl2, and 4 h of crosslinking time at 4 °C.
Immobilization of the enzyme on mesoporous silica SBA-15 was performed as follows: the enzyme solution was diluted by potassium phosphate buffer (pH 7.5) to a specific concentration (728 U/mL, 0.32 mg/mL). One hundred milligrams of SBA-15 was dispersed in appropriate amounts of Est684. The volumes varied from 100 to 2000 μL. After 4 h of incubation at 4 °C with shaking at 100 rpm, the suspension was centrifuged (6000×g, 2 min) to precipitate the bound enzymes, which were then resuspended in potassium phosphate buffer. The process was repeated 3 times to ensure removal of the unbound enzyme.
The effects of varying immobilizing time and temperature for the immobilization of the esterase onto mesoporous silica SBA-15 were investigated separately. One hundred milligrams of carrier was dispersed in 300 μL of Est684 (728 U/mL). After incubation at 4 °C with shaking at 100 rpm for various intervals, the unbound enzyme was washed out and the immobilized enzyme was obtained. Immobilizing intervals were 10, 20, 30 min, 1, 2, 3, 4, and 5 h. For experiments to test the effect of the immobilizing temperature, 100 mg of carrier was dispersed in 300 μL of Est684 (728 U/mL, 0.32 mg/mL). After incubation at 0, 5, 10, 15, 20, 25, 30, 35, or 40 °C at 100 rpm for 30 min, the unbound enzyme was washed out and immobilized enzyme was obtained.
The activity of the immobilized enzyme and the supernatant was measured using p-nitrophenyl butyrate as a substrate at pH 6.5 and 18 °C. The immobilized enzyme was dried at room temperature and stored at 4 °C for further study. The activity recovery was defined as the activity of the immobilized enzyme compared to the total starting activity of the free enzyme: the activity recovery (%) = (Immobilized enzyme activity/total enzymes activity) × 100%.
The samples were coated with platinum and analyzed using Scanning Electron Microscopy (SEM) (Hitachi SU3500, Krefeld, Germany) using the secondary electron emission mode at 1 kV. Images were collected at 100,000× magnifications.
Determination of kinetic parameters
Enzyme activity was tested under conditions of pH 6.5 and 18 °C by using p-nitrophenyl butyrate as a substrate, according to the method of Fan et al. . The free and immobilized enzymes were incubated with various concentrations of p-nitrophenyl butyrate. The final concentrations ranged from 1.0 to 10.0 mM in potassium phosphate buffer (pH 6.5). The kinetic parameters were calculated by fitting the initial rate data into the Michaelis–Menten equation using GraFit software version 6 (Erithacus Software Ltd., Horley, UK).
Comparison of catalytic properties of free and immobilized enzyme
Effects of temperature and pH on the reaction rates of free and immobilized enzyme were determined by using p-nitrophenyl butyrate as a substrate. The optimum pH was measured at 18 °C. The pH buffers included citric acid-NaOH buffer (pH 3.5–5.5), potassium phosphate buffer (pH 5.0–7.0), and Tris–HCl buffer (pH 6.5–9.0). The optimum temperature and thermostability were determined according to the method 2.6.
Operational stability of the immobilized enzyme
Gas chromatography analysis of the degradation of different pyrethroids in cucumbers
Cucumbers are one of the most common consumed vegetables. In this study, organic cucumbers without pesticide application during growth were collected from an experimental plot of Dongguan Agriculture Research Center. Fresh cucumbers were immersed in pyrethroid solutions consisting of 0.2 mg/L cyhalothrin, 0.2 mg/L cypermethrin, and 0.2 mg/L fenvalreate for 5 min and then air-dried in a fume hood. The pyrethroid-contaminated cucumbers were randomly divided into three groups, including a blank group, control group, and experimental group. For each group, approximately 1 kg of pyrethroid-contaminated cucumbers were weighed into a plastic pot. Control samples were immersed in 1.0 L of tap water at 30 °C for 5 min with slow stirring and then air-dried in fume hood. For the experimental group, 1 kg of cucumbers were immersed in 1.0 L of tap water containing 10.0 mg of immobilized enzyme (1.5 U/mg) at 18 °C for 5 min with slow stirring and then air-dried in fume hood. The immobilized enzyme was then collected by natural precipitation and filtration, and then used for the next four reactions. Blank samples without any further processing, control samples, and experimental samples were separately blended in a variable speed laboratory blender until homogenized. Ten grams of each comminuted homogenous sample was placed a 50-mL centrifuge tube. Next, 20 mL of acetonitrile and 4.0 g of sodium chloride were added, and the samples were immediately vortexed for 20 min and then centrifuged for 5 min at 6000×g. A 4.0-mL aliquot of each extract was transferred into a 20-mL glass cuvette and dried under nitrogen, after which the extract was redissolved in 2 mL of n-hexane. The residues detected by the gas chromatography analysis were based on the method described previously . All experiments were carried out in triplicate.
XJF performed construction of expression, purification and immobilization, and revised the manuscript. WQL performed enzyme characterization. YFL grew and prepared organic cucumber samples. HL performed construction of metagenomic library and gas chromatography analysis. XLL designed and supervised the experiments and wrote the manuscript. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analysed during this study are included in this published article.
Consent for publication
Ethics approval and consent to participate
This work was supported by the National Natural Science Foundation of China (31400680), School Research Fund (2017xkj007) and Early Research Training Program (2013-ZQKY-09) from Anhui Medical University, Open Project from Anhui Province Biomass Clean Energy Key Laboratory, Science and Technology Plan Project of Guangdong Province (2013B010404044, 2017A010105011), Education Project of Guangdong Province (2013KJCX0107, 2016KTSCX067), and Guangdong Climbing Plans (pdjh2017b0262).
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- Li QY, Li Y, Zhu XK, Cai BL. Isolation and characterization of atrazine-degrading Arthrobacter sp. AD26 and use of this strain in bioremediation of contaminated soil. J Environ Sci. 2008;20:1226–30.View ArticleGoogle Scholar
- Saxena P, Saxena AK. Cypermethrin induced biochemical alterations in the blood of albino rats. Jordan J Biol Sci. 2010;3:111–4.Google Scholar
- Kasai S. Role of cytochrome P450 in mechanism of pyrethroid resistance. J Pestic Sci. 2004;29:220–1.View ArticleGoogle Scholar
- Sogorb MA, Vilanova E. Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis. Toxicol Lett. 2002;128:215–28.View ArticleGoogle Scholar
- Ruan ZY, Zhai Y, Song JL, Shi YH, Li K, Zhao B, Yan YC. Molecular cloning and characterization of a newly isolated pyrethroid-degrading esterase gene from a genomic library of Ochrobactrum anthropi YZ-1. PLoS ONE. 2013;8:e77329.View ArticleGoogle Scholar
- Wang BZ, Guo P, Hang BJ, Li L, He J, Li SP. Cloning of a novel pyrethroid-hydrolyzing carboxylesterase gene from Sphingobium sp. JZ-1 and characterization of the gene product. Appl Environ Microbiol. 2009;75:5496–500.View ArticleGoogle Scholar
- Li G, Wang K, Liu YH. Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the metagenome. Microb Cell Fact. 2008;7:38.View ArticleGoogle Scholar
- Zhai Y, Li K, Song JL, Shi YH, Yan YC. Molecular cloning, purification and biochemical characterization of a novel pyrethroid-hydrolyzing carboxylesterase gene from Ochrobactrum anthropi YZ-1. J Hazard Mater. 2012;221:206–12.View ArticleGoogle Scholar
- Wei T, Feng SX, Shen YL, He PX, Ma GL, Yu X, Zhang F, Mao DB. Characterization of a novel thermophilic pyrethroid-hydrolyzing carboxylesterase from Sulfolobus tokodaii into a new family. J Mol Catal B Enzym. 2013;97:225–32.View ArticleGoogle Scholar
- Chen SH, Lin QS, Xiao Y, Deng YY, Chang CQ, Zhong GH, Hu MY, Zhang LH. Monooxygenase, a novel beta-cypermethrin degrading enzyme from Streptomyces sp. PLoS ONE. 2013;8:e75450.View ArticleGoogle Scholar
- Rahman MA, Culsum U, Tang W, Zhang SW, Wu GB, Liu ZD. Characterization of a novel cold active and salt tolerant esterase from Zunongwangia profunda. Enzyme Microb Technol. 2016;85:1–11.View ArticleGoogle Scholar
- Chaubey A, Parshad R, Koul S, Taneja S, Qazi G. Arthrobacter sp. lipase immobilization for improvement in stability and enantioselectivity. Appl Microbiol Biotechnol. 2006;73:598–606.View ArticleGoogle Scholar
- Villeneuve P, Muderhwa JM, Graille J, Haas MJ. Customizing lipases for bio-catalysis: a survey of chemical, physical and molecular biological approaches. J Mol Catal B Enzym. 2000;9:113–48.View ArticleGoogle Scholar
- Shimada Y, Watanabe Y, Sugihara A, Tominaga Y. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J Mol Catal B Enzym. 2002;37:133–42.View ArticleGoogle Scholar
- Almeida RV, Branco RV, Peixoto B, Lima C, Alqueres S, Martins OB, Antunes OAC, Freire DMG. Immobilization of a recombinant thermostable esterase (Pf2001) from Pyrococcus furiosus on microporous polypropylene: isotherms, hyperactivation and purification. Biochem Eng J. 2008;39:531–7.View ArticleGoogle Scholar
- Branco RV, Gutarra MLE, Guisan JM, Freire DMG, Almeida RV, Palomo JM. Improving the thermostability and optimal temperature of a lipase from the hyperthermophilic archaeon Pyrococcus furiosus by covalent immobilization. Biomed Res Int. 2015;2015:250532.View ArticleGoogle Scholar
- Rodrigues DS, Mendes AA, Adriano WS, Goncalves LRB, Giordano RLC. Multipoint covalent immobilization of microbial lipase on chitosan and agarose activated by different methods. J Mol Catal B Enzym. 2008;51:100–9.View ArticleGoogle Scholar
- Sassolas A, Blum LJ, Leca-Bouvier BD. Immobilization strategies to develop enzymatic biosensors. Biotechnol Adv. 2012;30:489–511.View ArticleGoogle Scholar
- Kloskowski A, Pilarczyk M, Chrzanowski W, Namie´snik J. Sol-gel technique—a versatile tool for adsorbent preparation. Crit Rev Anal Chem. 2010;40:172–86.View ArticleGoogle Scholar
- Bernardos A, Mondragon L, Aznar E, Marcos MD, Martinez-Manez R, Sancenon F, Soto J, Barat JM, Pérez-Payá E, Guillem C, Amorós P. Enzyme-responsive intracellular controlled release using nano-metric silica mesoporous support capped with saccharides. ACS Nano. 2010;4:6353–68.View ArticleGoogle Scholar
- Popat A, Hartono SB, Stahr F, Liu J, Qiao SZ, Lu GQ. Mesoporoussilica nanoparticles for bioadsorption, enzyme immobilisation, and delivery carriers. Nanoscale. 2011;3:2801–18.View ArticleGoogle Scholar
- Hartmann M, Kostrov X. Immobilization of enzymes on porous silicas–benefits and challenges. Chem Soc Rev. 2013;42:6277–89.View ArticleGoogle Scholar
- Zhao XH, Zheng XT. A primary study on texture modification and proteolysis of mao-tofu during fermentation. Afr J Biotechnol. 2009;8:2294–300.Google Scholar
- Handelsman J. Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev. 2004;68:669–85.View ArticleGoogle Scholar
- Piper KR, von Bodman SB, Farrand SK. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature. 1993;362:448–50.View ArticleGoogle Scholar
- Fan XJ, Liu XL, Liu YH. The cloning and characterization of one novel metagenome-derived thermostable esterase acting on N-acylhomoserine lactones. J Mol Catal B Enzym. 2012;83:29–37.View ArticleGoogle Scholar
- Zhou XW, Liu HF, Zhao XH. The potencies of three microorganisms to dissipate four organophosphorus pesticides in three food materials during traditional fermentation. J Food Sci Technol. 2015;52:7353–60.View ArticleGoogle Scholar
- Esteban-Torres M, Mancheño JM, de las Rivas B, Muñoz R. Characterization of a cold-active esterase from Lactobacillus plantarum suitable for food fermentations. J Agric Food Chem. 2014;62:5126–32.View ArticleGoogle Scholar
- Yu EY, Kwon MA, Lee M, Oh JY, Choi JE, Lee JY, Song BK, Hahm DH, Song JK. Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome. Appl Microbiol Biotechnol. 2011;90:573–81.View ArticleGoogle Scholar
- Mander P, Cho SS, Simkhada JR, Choi YH, Park DJ, Ha JW, Yoo JC. An organic solvent-tolerant alkaline lipase from Streptomyces sp. CS268 and its application in biodiesel production. Biotechnol Bioprocess Eng. 2012;17:67–75.View ArticleGoogle Scholar
- Wu G, Zhang S, Zhang H, Zhang S, Liu Z. A novel esterase from a psychrotrophic bacterium Psychrobacter celer 3Pb1 showed cold-adaptation and salt-tolerance. J Mol Catal B Enzym. 2013;98:119–26.View ArticleGoogle Scholar
- Wu G, Wu G, Zhan T, Shao Z, Liu Z. Characterization of a cold-adapted and salt-tolerant esterase from a psychrotrophic bacterium Psychrobacter pacificensis. Extremophiles. 2013;17:809–19.View ArticleGoogle Scholar
- Novototskaya-Vlasova K, Petrovskaya L, Yakimov S, Gilichinsky D. Cloning, purification, and characterization of a cold-adapted esterase produced by Psychrobacter cryohalolentis K5T from Siberian cryopeg. FEMS Microbiol Ecol. 2012;82:367–75.View ArticleGoogle Scholar
- Fan XJ, Liu XL, Huang R, Liu YH. Identification and characterization of a novel thermostable pyrethroid-hydrolyzing enzyme isolated through metagenomic approach. Microb Cell Fact. 2012;11:33–43.View ArticleGoogle Scholar
- Liu XL, Liang MJ, Liu YH, Fan XJ. Directed evolution and secretory expression of a pyrethroid-hydrolyzing esterase with enhanced catalytic activity and thermostability. Microb Cell Fact. 2017;16:81.View ArticleGoogle Scholar
- Kato K, Seelan S. Enhancing activity and stability of Burkholderia cepacia lipase by immobilization on surface-functionalized mesoporous silicates. J Biosci Bioeng. 2010;109:615–7.View ArticleGoogle Scholar
- Kumari A, Kaur B, Srivastava R, Sangwan RS. Isolation and immobilization of alkaline protease on mesoporous silica and mesoporous ZSM-5 zeolite materials for improved catalytic properties. Biochem Biophys Rep. 2015;2:108–14.Google Scholar
- Bayramogri G, Altınok H, Bulut A, Denizli A, Arıca MY. Preparation and application of spacer-arm-attached poly (hydroxyethyl methacrylate-co-glycidyl methacrylate) films for urease immobilization. React Funct Polym. 2003;56:111–21.View ArticleGoogle Scholar
- Kumar D, Muthukumar M, Garg N. Kinetics of fungal extracellular α-amylase from Fusarium solani immobilized in calcium alginate beads. J Environ Biol. 2012;33:1021–5.Google Scholar
- Serra E, Mayoral Á, Sakamoto Y, Blanco RM, Díaz I. Immobilization of lipase in ordered mesoporous materials: effect of textural and structural parameters. Microporous Mesoporous Mater. 2008;114:201–13.View ArticleGoogle Scholar
- Xu YQ, Zhou GW, Wu CC, Li TD, Song HB. Improving adsorption and activation of the lipase immobilized in amino-functionalized ordered mesoporous SBA-15. Solid State Sci. 2011;13:867–74.View ArticleGoogle Scholar
- Cai CH, Gao YQ, Liu Y, Zhong NJ, Liu N. Immobilization of Candida antarctica lipase B onto SBA-15 and their application in glycerolysis for diacylglycerols synthesis. Food Chem. 2016;212:205–12.View ArticleGoogle Scholar
- Zhou J, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. Appl Environ Microbiol. 1996;62:316–22.Google Scholar
- Zhang WC, Wang CL, Song Y, Shao C, Zhang X, Zang JY. Structural insights into the mechanism of Escherichia coli Ymdb: a 2′-O-acetyl-ADP-ribose deacetylase. J Struct Biol. 2015;192:478–86.View ArticleGoogle Scholar
- Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–5.View ArticleGoogle Scholar
- Liburdi K, Benucci I, Palumbo F, Esti M. Lysozyme immobilized on chitosan beads: kinetic characterization and antimicrobial activity in white wines. Food Control. 2016;63:46–52.View ArticleGoogle Scholar
- Liu JX, Liu J, Xu DF, Ling WT, Li SY, Chen MD. Isolation, immobilization, and degradation performance of the 17β-estradiol-degrading bacterium Rhodococcus sp. JX-2. Water Air Soil Pollut. 2016;227:422.View ArticleGoogle Scholar