Identification of a Novel Esterase from Marine Environmental Genomic DNA Libraries and Its Application in Production of Free All- trans -Astaxanthin

Article abstract of DOI:10.1021/acs.jafc.7b06062

Astaxanthin is a pigment with various functions. Free astaxanthin is obtained mainly through saponification methods, which could result in many byproducts. Enzymatic methods using lipases have been used in a few cases, while there are no reports on the us

Full text of DOI:10.1021/acs.jafc.7b06062

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Article
Identification of a novel esterase from marine environmental genomic
DNA libraries and its application in production of free all-trans-astaxanthin
Ping Lu, Xinwei Gao, Hao Dong, Zhen Liu, Francesco Secundo, Changhu Xue, and Xiangzhao Mao
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06062 • Publication Date (Web): 22 Feb 2018
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Identification of a novel esterase from marine environmental genomic DNA
libraries and its application in production of free allꢀtransꢀastaxanthin
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1#
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Ping Lu , Xinwei Gao , Hao Dong , Zhen Liu , Francesco Secundo , Changhu
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1,2 *
Xue , Xiangzhao Mao
1 College of Food Science and Engineering, Ocean University of China, Qingdao
266003, China
2 Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for
Marine Science and Technology, Qingdao 266237, China
3 Istituto di Chimica del Riconoscimento Molecolare, CNR, v. Mario Bianco 9, Milan
20131, Italy
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* Corresponding author: Professor Xiangzhao Mao
Address: College of Food Science and Engineering, Ocean University of China,
Qingdao 266003, China
Tel.: +86ꢀ532ꢀ82032660
Fax: +86ꢀ532ꢀ82032272
Eꢀmail: xzhmao@ouc.edu.cn
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Abstract
Astaxanthin is a pigment with various functions. Free astaxanthin is obtained
mainly through saponification methods, which could result in many byꢀproducts.
Enzymatic methods using lipases have been used in few cases, while there are no
reports on the use of esterases for the production of free astaxanthin. Herein we
present the screening and identification of a novel esterase (Est3ꢀ14) from a marine
mud metagenomic library. Est3ꢀ14 is pHꢀsensitive and keeps good stability in akaline
buffers (residual activity 94%, pH 8.0, 4 °C and 36 h). Meanwhile, Est3ꢀ14 keeps a
good stability in the medium temperature condition (residual activity 56.7%, pH 8.0,
40 °C and 84 h). Est3ꢀ14 displayed high hydrolysis activity to prepare free
allꢀtransꢀastaxanthin in biphasic systems. Furthermore, under optimal conditions (0.5
mL ethanol, 6 mL 0.1 M TrisꢀHCl buffer, pH 8.0, 0.5% (w/v) H. pluvialis oil, 40 °C),
the hydrolytic conversion ratio was 99.3% after 36 h.
Key words: Metagenomic Library, Alkaline Esterase, Free Ax, Enzymatic Methods,
High Hydrolytic Conversion Ratio
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■ INTRODUCTION
Astaxanthin (3,3’ꢀdihydroxyꢀβ,βꢀcaroteneꢀ4,4’ꢀdione) (Ax) is a terpenoid with
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various functions, such as antioxidant activity, antitumor activity, immune response
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enhancement, antiꢀnerve injury, inhibition of Helicobacter pylori infection, and
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improvement of memory. Thanks to these properties, it has been widely applied to
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food, cosmetic, aquaculture and poultry.
Ax is widely distributed in aquatic organisms, such as the shrimp, crab shells and
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microalgae.
In particular, the green alga Haematococcus pluvialis (H. pluvialis) is
considered as the most suitable source for natural Ax being the content of this
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carotenoid about 4% (w/w) in the dry biomass.
However, 95% of Ax in H.
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pluvialis is esterified with long chain (C16ꢀC20) fatty acids. Ax has several isomers,
such as three geometrical isomers (9Zꢀ, allꢀEꢀ and 13Zꢀ) and three optical isomers
((3S,3’S) (allꢀtrans), (3R,3’S) and (3R, 3’R)). Under natural conditions, allꢀtrans
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configuration is the most stable structure (Figure S1, Supporting Information). The
configuration of Ax in H. pluvialis is consistent with the Ax in wild salmon, which is
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allꢀtrans.
It has been demonstrated that Ax esters may have different chemical
functionalities that confer them a different physiological role, which may limit the
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wide application of Ax from H. pluvial.
Free allꢀtransꢀAx has been served as a
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standard to compare the different functions of Ax esters. Ax also has wide
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application in aquaculture field. Choubert et al. found that the inclusion of Ax in the
diet of rainbow trout had a great influence on the muscle pigmentation of this fish.
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Therefore, the preparation of free allꢀtransꢀAx from its esters produced in H. pluvialis
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is a biotechnological field of major interest.
Numerous attempts have been made to hydrolyze Ax esters for obtaining free Ax.
Saponification is an efficient method to cleave esters, however, alkaline conditions
can cause the formation of byꢀproducts (astacene) that may have biological functions
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different from Ax.
Enzymeꢀcatalyzed hydrolysis is expected to be an
environmentally friendly promising alternative to saponification, thanks to the mild
hydrolysis conditions, especially if carried out under a nitrogen atmosphere to inhibit
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the formation of byꢀproducts.
In particular, lipases (EC 3.1.1.3) and esterases
(EC 3.1.1.1) are a family of hydrolases that catalyze the cleavage of ester bonds and
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are widespread in nature.
mainly focused on lipaseꢀmediated catalysis.
could hydrolyze Ax esters from H. pluvialis oil, which could hydrolyze 63.2% Ax
Up to now, some studies about preparing the free Ax are
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Zhao et al. found five lipases
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esters. Nagao et al. attempted to obtain purified Ax by twoꢀstep process, they got
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63.9% Ax of the initial content in the cell extract finally. However, this group never
used this enzyme for the recovery of free Ax from a natural source. Lipases are
important industrial biocatalysts and have been widely used for the preparation of free
Ax, however, the shortcomings of above researches are the long reaction time and the
low conversion efficiency. Indeed, a long reaction time is not suitable for storing Ax,
which could cause the degradation of Ax. Besides lipases also esterases could be
suitable to improve the catalytic efficiency. It is well known that esterases (EC3.1.1.1)
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could hydrolyze esters with shortꢀchain fatty acids (less than 10 carbon atoms).
Instead, lipases (EC3.1.1.3) prefer hydrolyzing esters with long chain fatty acids
(≥10). Nevertheless, only in a few cases lipases have been used for the cleavage of Ax
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esters. This lack is due to the absence of known esterases active on these substrates.
Screening of novel biocatalysts is the subject of investigation of many scientists.
These studies have facilitated the progress of new methods to separate novel genes.
Nevertheless, though the biocatalysts obtained from new isolated genes are abundant,
most of the microorganisms from where the isolated genes originated are not
cultivable. It is estimated that less than 1% of microorganisms in nature have ever
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been cultured by traditional methods in lab. Thanks to the development of
metagenomics, the loss of diverse biocatalysts can be avoided. Isolation of
metagenomic DNA from the environmental samples and cloning it into a special
vector can be constructed a metagenomic library. The desired genes can be isolated
through functional screening from this metagenomic library. Up to now, this approach
is one of the most powerful methods to screen novel biocatalysts without the need for
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culturing.
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Within this work, we constructed a fosmid metagenomic library and screened the
lipolytic genes on large scale. A novel esterase (Est3ꢀ14) was identified and purified
for further characterization. The Est3ꢀ14 also displayed high hydrolytic activity in
aqueous biphasic systems to prepare the free allꢀtransꢀAx from Ax esters.
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■ MATERIALS AND METHODS
Materials. DNA amplification used the DNA polymerase from Vazyme (Nanjing,
China). Restriction enzymes (NotI，BamHI, HindIII, Sau3AI and Calfꢀintestinal
alkaline phosphatase (CIP)) were bought from NEB (New England Biolabs) (MA,
USA). The pꢀNitrophenol (pNP) and its esters were bought from Sigma (MO, USA).
H. pluvialis oil (0.5% (w/w) Ax esters) was bought from Yunnan Alphy Biotech
(Yunnan, China). Preꢀprepared plates of silica (GF254) were supplied by Qingdao
Shenghai silica Chemical Co. Ltd. (Qingdao, China). Methanol, Methyl
tertꢀbutylether (MTBE) was purchased from EMD Millipore Corporation (Billerica,
MA) and other chemicals and organic solvents were analytical reagents.
The construction of fosmid library and screening. The metagenomic DNA was
extracted from marine mud. DNA fragments from 15 to 45 kb were gained by partial
digestion with the restriction enzyme NotI and used to build a fosmid library by
protocol of CopyControl pCC1FOS Fosmid Library Production Kit (Epicentre
Biotechnologies). Transformants, with recombinant fosmids, were cultured on
LuriaꢀBertani (LB) agar (containing chloramphenicol (Chl)) at 37 °C for 20 h. To
identify the effectiveness of library, 5 clones were stochastically chosen and digested
with the restriction enzyme NotI. The method of construction of metagenomic library
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is similar to that reported by Gao et al.
Construction of subꢀcloning genomic library and sequence analysis of
lipolytic genes. For screening the colonies exhibiting lipolytic genes, the positive
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pools from fosmid library were diluted with sterile saline solution and cultivated at
37 °C for 48ꢀ72 hours on LB agar (containing Chl and emulsified tributyrin). The
positive pools from above LB agar were partially digested with restriction enzyme
Sau3AI to gather 3ꢀ9 kb DNA fragments. Prepared DNA fragments were ligated into
pBluescript II SK (+) vector. The products of ligation were introduced into E. coli
DH5α cells. The transformants were cultivated on LB agar plates (containing
ampicillin and tributyrin) at 37 °C for 48 h. Then, positive colonies with clear halos
were cultured in LB medium and sent to be sequenced.
NCBI open reading frame (ORF) finder (https://www.ncbi.nlm.nih.gov/orffinder/)
was used to identify the ORFs in the nucleotide sequences. The functions of ORFs
were predicted using BLASTP search (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Multiple sequence alignments were analyzed by Clustal X and ESPript 3.0
(http://espript.ibcp.fr/ESPript/ESPript/). The neighborꢀjoining phylogenetic tree
among hydrolases was analyzed by MAGA 6.0 software.
Heterologous expression and purification of esterase Est3ꢀ14. According to
the one positive clone with predicted lipolytic gene (est3-14), the gene was ligated to
pETꢀ28a (+) vector and introduced into the expression host E. coli BL21 (DE3).
Correct recombinant strain was cultivated at 37 °C until the value of OD600 reached
0.6ꢀ0.8. Then, after adding isopropylꢀβꢀDꢀ1ꢀthiogalactopyranoside (IPTG), it was
further cultivated at 20 °C for 20 h. Centrifugation was used to collect cells and the
cells were washed with sterile NaCl solution. The collected cells were ruptured by
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sonication in 0.1 M, TrisꢀHCl buffer, pH 8.0 (Buffer A). After centrifugation (10,000
× g) for 30 min at 4 °C, the supernatant of Est3ꢀ14 was loaded onto a NiꢀNTA column
(1 mL, Qiagen, Hilden, Germany). The target protein was purified by elution with 0.2
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M imidazole in Buffer A and dialyzed against the same buffer to remove imidazole.
Finally, the expression level and the purity of Est3ꢀ14 was measured by SDSꢀPAGE.
The esterase activity was determined by using pꢀnitro phenyl butyrate (pNPB) (0.02
M dissolved in buffer of isopropanol and dimethyl sulfoxide) as substrate and
following the formation of pNP at 405 nm according to the method reported by
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Margesin with minor modification. One unit of esterase activity was defined as the
amount of esterase needed to form 1 ꢁmol pNP per minute. Soluble protein content
was estimated by the method of Coomassie brilliant blue.
Enzyme characterization of esterase Est3ꢀ14. Esterase activity and soluble
protein content were estimated as described in the section heterologous expression
and purification of esterase Est3ꢀ14.
Substrate specificity of Est3ꢀ14 was tested by the same method as described
above but adopting pNP esters with variable chain length: pNP acetate (C2), pNP
butyrate (C4), pNP caprylate (C6), pNP decanoate (C8), pNP caprate (C10), pNP
laurate (C12), pNP myristate (C14), and pNP palmitate (C16).
To identify the optimal pH of hydrolysis, the esterase activity of Est3ꢀ14 was
determined at pH values from 4.0 to 10.0 in four different buffers. In particular
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sodium citrate, sodium phosphate, TrisꢀHCl and Na CO ꢀNaHCO were used (0.1 M)
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for the pH range of 4.0ꢀ6.0, 6.0ꢀ8.0, 8.0ꢀ9.0 and 9.0ꢀ10.0, respectively.
To identify the pH stability of Est3ꢀ14, the purified enzyme was incubated in
different buffers with pH ranging from 5.0 to 10.0 for 36 h. The residual activity of
Est3ꢀ14 was determined by the method mentioned above.
The determination of the optimal temperature was conducted at temperatures
from 30 °C to 80 °C using the pNP butyrate as substrate. Meanwhile, the thermal
stability of Est3ꢀ14 was detected by putting the purified enzyme into different
temperature conditions (40 °C, 45 °C, 50 °C, 55 °C, 60 °C) for up to 84 h, and then
the remaining activity was measured.
Effects of surfactants, metal ions and organic solvents on Est3ꢀ14 activity.
The influence of surfactants on the esterase activity was determined in Buffer A
including 0.5% (w/v) variable surfactants (Triton Xꢀ100, Tween 80, Tween 60, SDS,
Tween 20). The activity measured without adding surfactants was used as the
negative control. The effect of metal ions (CoCl , FeCl
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, KCl, LiCl, FeSO
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, MnCl ,
CaCl , MgCl , ZnCl and NiCl ) and chelating agent Na ꢀEDTA on Est3ꢀ14 activity
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were analyzed at the concentration of 1 mM and 10 mM in Buffer A.
To estimate the tolerance of Est3ꢀ14 to organic solvents (methanol, ethanol,
acetonitrile, nꢀhexane, chloroform, dimethylsulfoxide (DMSO), acetone, nꢀpropanol,
isopropanol, isooctane, cyclohexane), the catalytic activity of Est3ꢀ14 was determined
after placing the enzyme solution at 30 °C for 3 h in organic solvent/water mixture
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(25%, 50% (v/v), respectively). To reduce the influence of the organic solvents
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during the activity assay, the hydrophobic organic solvents were removed by
centrifugation or, in the case of hydrophilic organic solvents, diluted to 5%.
The identification of pure organic solvents effect on Est3ꢀ14 was tested according
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to the method described by Li et al. Est3ꢀ14 powder was added to organic solvents
and placed at 30 °C while shaking for 3 h. Centrifugation was taken to remove
organic solvents. The remaining esterase activity was measured under standard
conditions.
Hydrolysis of Ax esters from H. pluvialis oil. In order to test the hydrolysis
ability of Est3ꢀ14 towards Ax esters, the supernatants of E. coli cell lysate of Est3ꢀ14
was lyophilized by vacuum freezing, and the dried crude powder was used to catalyze
the hydrolysis reaction. Esterase activity per milligram of dried cell powder was
determined as above. The hydrolysis reaction was carried out adding the supernatant
of Est3ꢀ14 lysate to a biphasic system (0.5 mL ethanol; 6 mL Buffer A; 0.5% (w/v) H.
pluvialis oil) at 40 °C and shaking for 48 h in a thermostatic bath. Free allꢀtransꢀAx
was extracted from the reaction medium by 1/1 (v/v) isopropanol/dichloromethane
mixture for several times until the aqueous phase was colorless. After extraction, the
method of TLC was used to analyze whether Est3ꢀ14 could hydrolyze Ax esters. The
mobile phase was the mixture of nꢀhexane and acetone and the ratio was 4:1 (v/v).
The standard of Ax was used as a positive control.
Identification of free allꢀtransꢀAx in the hydrolysis medium. The reaction
samples were analyzed by using a highꢀperformance liquid chromatograph (HPLC)
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(Agilent) equipped with Cꢀ30 column (YMC, 250 × 4.6 mm; 5 ꢁm). The detector
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wavelength was set to 476 nm and the temperature of column was kept at 35 °C.
The elution conditions were as follows: 0ꢀ15 min, isocratic conditions 90% A and 10%
B, then gradient to 40% A and 60% B until t = 25 min, 25ꢀ35 min, the gradients came
to initial concentration. Eluent A consisted of methanol, Eluent B consisted of MTBE.
Analysis was conducted in triplicate.
The standard curve of the standard of free allꢀtransꢀAx was used to quantify the
production of free allꢀtransꢀAx. (Figure S5, Supporting Information) The Ax ester
hydrolysis rate was equal to decrease rate of Ax esters. The equation was as follows:
Conversion=Area of residual Ax esters after hydrolysis/ Area of initial Ax esters in
the reaction medium
HPLCꢀMS/APCI was adopted to analyze free allꢀtransꢀAx in the hydrolysis
medium. The gradient elution program used in HPLCꢀMS/APCI was consistent with
the method mentioned in HPLC determination. The MS instrument was Bruker maXis
II (Bruker, Germany) equipped with a quadrupole mass spectrometer system
Navigator. The instrument was fitted with APCI with the positive ionization mode.
The voltage of the fragmentation was set to 35 eV and the flow rate of nitrogen was 5
L/min. The range of scanning was set from 200 to 650 (m/z) with the scan time of 0.5
s.
The reaction curve of preparing free allꢀtransꢀAx under different reaction
times and enzyme loading. Under the initial reaction conditions, the hydrolysis curve
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was drawn by sampling the reaction mixture at different times (12 h, 24 h, 36 h, 48 h,
72 h and 84 h) and enzyme loading (180 U, 300 U, 900 U, 1800 U, 2700 U, 3600 U)
to identify the efficiency of hydrolysis.
■ RESULTS AND DISCUSSION
The construction of fosmid library and functional screening. According to the
results of identification of the marine mud metagenomic library (average insert size
40 kb, 40,000 clones), it was observed that the constructed fosmid metagenomic
library is various. The results of digestion with NotI of 5 randomly chosen clones
suggested that 90% of clones included different DNA fragments. It is suggested that
this metagenomic library is of high efficiency. The functional screening was based on
the ability of producing transparent circle on the plates of LB agar (containing
tributyrin and Chl). After incubation at 37 °C for 48 h, the positive fosmids (40 kb)
(Figure S2, Supporting Information) were extracted and used for the construction of
subꢀclones.
To identify the effective fragments among the positive fosmids, the fosmids were
digested by restriction enzyme Sau3AI to gather 3.0ꢀ9.0 kb fragments (Figure S3,
Supporting Inforamtion). Then, the fragments were ligated to pBluescript II SK (+)
vector and introduced into E. coli. The subꢀclones that showed lipolytic activity were
sent to be sequenced. After sequenced by Sangon Biotech (Shanghai, China) and on
the basis of the ORF finder and BLASTP search, the inserted DNA fragment was
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identified.
The sequence analysis and phylogenetic tree construction of esterase gene
est3-14. The putative lipolytic gene was named est3-14 and had sequence identities of
51% with the esterase gene (KRO42937.1) from Acidimicrobium sp., which has not
been expressed and characterized. The low similarity with other esterase genes shows
that est3-14 is a new esterase gene. The nucleotide sequence of esterase Est3ꢀ14 was
uploaded to Genebank database with the accession number MF568505.
Phylogenetic analysis of est3-14 was carried out according to the classification
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reported by Arpigny and Jaeger. On the basis of this classification and on the amino
acid sequences of different esterase genes belonging to the Families IꢀVIII, esterase
Est3ꢀ14 belongs to family V (Figure 1a). Furthermore, the multiple sequence
alignment (Figure 1b) revealed that Est3ꢀ14 has the typical catalytic triad (Ser, 115;
Asp, 237; His, 266), confirming, Est3ꢀ14 was a novel esterase.
Heterologous expression and purification of Est3ꢀ14. The est3ꢀ14 gene
(BamHI/HindIII restriction site) was ligated to pETꢀ28a (+) and transformed into
BL21 (DE3) to express active protein. The Est3ꢀ14 was expressed successfully in E.
coli and the expression level was estimated by SDSꢀPAGE (Figure S4, Supporting
Inforamtion). The esterase activity determination showed that this vector could be
used to express active esterase. Based on the existence of the terminal 6×His affinity
tag, esterase Est3ꢀ14 was purified successfully. The purified Est3ꢀ14 was separated as
a single protein and consistent with the predicted molecular weight (32.8 kDa), which
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was shown in Figure 2.
Enzyme characterization of purified esterase Est3ꢀ14. The catalytic activity of
Est3ꢀ14, measured by using pNP esters with different acyl chain lengths, showed that
this enzyme preferentially hydrolyzed short chain fatty acids (C<10) and showed the
highest activity towards pNP butyrate (292 U/mg, at pH 8.0 and 37 °C), and the
lowest towards pNP palmitate (20.9% of the pNP butyrate) (pH 8.0 and 37 °C)
(Figure 3a). These results indicated that Est3ꢀ14 is a “true” esterase that preferentially
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hydrolyzed the substrate with short acyl chain length.
The effects of pH on Est3ꢀ14 activity were tested using pNPB as substrate.
Est3ꢀ14 was very pHꢀsensitive and showed relatively higher activities at alkaline
conditions, with optimal pH at 9.0 in phosphate buffer and inactivation at pH < 6.0
(<10% maximum activity in optimal pH) (Figure 3b). The pH stability of Est3ꢀ14 was
analyzed by testing the residual activity at different time intervals after pretreatment
(4 °C, up to 36 h) in different pH value buffers (pH 5.0ꢀ10.0). Data in Figure 3c
showed that at pH 8.0 even after 36 h, the residual activity of Est3ꢀ14 only slightly
decreased (residual activity was 94%). Instead, after incubation for 6 hours at pH 6.0
the enzyme kept about 62% of the original activity. However, the residual activity of
Est3ꢀ14 decreased sharply at pH < 6.0 buffer, and it plunged to 23.7% of the original
activity at pH 5.0. According to these results, Est3ꢀ14 displayed high activity in
alkaline buffers. Therefore, we can conclude that Est3ꢀ14 is a member of alkaline
esterases.
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The influence of temperature on Est3ꢀ14 catalytic activity was tested in the
30ꢀ80 °C range, and it was found that it increased quickly with the temperature to
60 °C and then declined at 70 °C and 80 °C (Figure 3d). In a recent report, EstK1
2
9
displayed highest activity at 50 °C (Est3ꢀ14, 60 °C), which indicated that Est3ꢀ14
has a promising application in the high temperature condition. Thermal stability of
Est3ꢀ14 was identified by measuring the remaining activity at various time intervals
after pretreatment of the purified enzyme for 84 h at different temperatures (40 °C,
45 °C, 50 °C, 55 °C, 60 °C). Est3ꢀ14 preserved over 60% of its original activity after
incubation at 40 °C for 78 h. At 55 °C, when the enzyme was pretreated for 24 h, the
residual activity of Est3ꢀ14 decreased to 50%. However, at 60 °C, the residual activity
decreased to 35% of original activity within 6 h (Figure 4e). Based on these data,
Est3ꢀ14 appeared stable at medium temperature (e.g., 40 °C), which was consistent
38
with esterase EST4 (40 °C) reported by Gao et al. It is a beneficial feature in the
perspective of applying this enzyme in the hydrolysis of Ax esters.
The various surfactants (0.5%) could cause different effects on esterase activity.
As shown in Figure 4f, the activity of Est3ꢀ14 was slightly stimulated by Tween 20,
Tween 60, Tween 80, Triton Xꢀ100. SDS, an anionic surfactant, had a strong
inhibitory effect on Est3ꢀ14 activity, which is similar to that observed with the
43
esterase Est_p6 reported by Peng et al. and with the esterase Est4 reported by Gao et
38
al.
2
Concerning the influences of the metal ions and Na ꢀEDTA on the esterase
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activity, Est3ꢀ14 was slightly stimulated by adding Co , K at the low concentration
(1 mM) (Table 1). While when the addition of metal ions and Na ꢀEDTA was
ꢀEDTA caused a
2
2
+
2+
2+
2+
2+
3+
2+
increased to 10 mM, Ca , Mn , Mg , Co ，Ni , Fe , Zn and Na
2
2
+
decrease of Est3ꢀ14 activity by 20ꢀ80%, and the Zn displayed highest inhibiting
effect to activity of Est 3ꢀ14 (decreased by 77%).
The effect of organic solvents on Est3ꢀ14 was studied at various concentrations of
different organic solvents (Table 2) with different Log P values from ꢀ1.3 to 4.5. Log
P is an indicator of solvent polarity, and the log P value of hydrophobic organic
39
solvents was ≥ 2. Detailed assay method was carried out according to previous
40
report. Methanol, chloroform, acetonitrile and isopropanol had slight inhibitory
effects on Est3ꢀ14 activity at a concentration of 25% (v/v). However, high organic
solvent concentration (50% v/v) decreased the activity sharply by 25.7ꢀ81.5%.
Furthermore, the purified enzyme was highly denatured by the hydrophilic organic
solvents (log P < 2). Generally, Est3ꢀ14 was more stable in hydrophobic organic
41
solvents, which was similar to lipase SML reported by Li et al. For example, the
residual activity of Est3ꢀ14 in absolute nꢀhexane was 88.2% of original activity, which
indicates that Est3ꢀ14 is stable in organic solvents. Compared with other reports,
4
4
45
Est3ꢀ14 was much more stable than rEstSL3 and LipBA in organic solvents. It was
worth pointing out that Est3ꢀ14 displayed high enzyme activity (91.2%) in low
concentration of ethanol (25% v/v), which could guarantee the hydrolysis reaction of
Ax esters in the biphasic system.
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Application of Est3ꢀ14 in hydrolysis of Ax esters from H. pluvialis oil and the
identification of free allꢀtransꢀAx. In order to test the ability of Est3ꢀ14 of
hydrolyzing Ax esters (Scheme 1), 1200 U crude powder was added to start the
reaction (0.5 mL ethanol; 6 mL Buffer A; 0.5% (w/v) H. pluvialis oil). The TLC result
of the hydrolysis of Ax esters was shown in Figure 4. It was observed that Est3ꢀ14
can be used to remove the ester bonds of Ax esters to prepare free allꢀtransꢀAx. After
analyzing by HPLC, the result is consistent with result of TLC. We can see from the
Figure 6, the peak of retention time of 7.7 min was the free allꢀtransꢀAx. In order to
identify the product, we used MS/MS to do further verification. In Figure 5a, the
retention time of free allꢀtransꢀAx (retention time 7.7 min) in the mixture is consistent
with the standard. The MS result of peak (7.7 min) from Figure 5a was expressed in
Figure 5c, a strong protonated quasimolecule ion was seen at m/z 597.3934, which
+
13
corresponded to the [M+H] of free allꢀtransꢀAx reported by Zhao et al. In the
Figure 5d, the peaks at m/z 147.1484 and 173.1588 were characteristic fragments of
free allꢀtransꢀAx, which were consistent with the standard of free allꢀtransꢀAx (Figure
5b). The characteristic fragments of free allꢀtransꢀAx corresponded to the results
2
3
reported by Dong et al. Therefore, the peak (retention time 7.7 min) was eventually
identified as allꢀtransꢀAx.
The reaction curve of preparing allꢀtransꢀAx under different reaction times
and enzyme loading. At industrial level, reaction time and enzyme loading play
important roles on the final cost of the product. Thus, the time of reaction and the
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enzyme loading to complete conversion of substrate and the spaceꢀyield are crucial
parameters to be evaluated.
We studied the relationship among reaction time, conversion ratio of Ax esters
hydrolysis and spaceꢀtimeꢀyield. As shown in Figure 6a, with the loading of 1200 U
Est3ꢀ14 crude powder and the reaction time changing, the tendency of hydrolysis
could be detected by HPLC; the conversion of Ax esters reached (88.9%) at 36 h and
96.7% at 48 h (Figure 6b); the spaceꢀtimeꢀyield was shown in Figure 6c, at last we got
ꢀ1
200 ꢁg•mL free allꢀtransꢀAx.
Under the optimal reaction time, we also studied the relationship between
enzyme loading and conversion ratio, in Figure 7a, we could find that the content of
free allꢀtransꢀAx with different enzyme loading and at 1800 U (allꢀtransꢀAx: 195
ꢀ1
ꢁg•mL , the reaction came to balance and the final conversion reached 99.3% (Figure
7b).
It is important to emphasize that the conversion obtained in the present study is
higher than that reported in previous studies. After the reaction, 99.3% Ax esters were
ꢀ1
hydrolyzed and the production was about 195 ꢁg•mL . According to previous studies,
the conversion is higher than them. In a recent study, the hydrolysis of Ax esters was
carried out using an alkaline lipase from Penicillium cyclopium and the final recovery
13
is about 63.2%. In another report, they used two steps to get the free Ax, first step
was used to remove FFA, the second step was used to get free Ax. It is worth pointing
that the second hydrolysis step needed 68 h and the total reaction time was 110 h
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(final recovery, 63.9%). However, our reaction time is less than 110 h, which is
benefit to application. Fermentation also was used to prepare Ax, the conversion of
23
Ax esters was 98.08% at 60 h. Saponification is a traditional method to prepare the
24, 42
free Ax,
low temperature (5 °C) was chosen to minimize the degradation of Ax
during saponification, and 94.4% Ax esters was transformed into free Ax after
4
2
saponification for 12 h. However, the low temperature control needs more cost and
the hydrolysis of astaxanthin esters by chemical methods from both H. pluvialis rarely
produced Ax but always astacene and was therefore not considered applicable for
industrial use. Therefore, Est3ꢀ14 is a promising alternative biocatalyst in preparation
of free allꢀtransꢀAx.
This is the first report to describe the esterase to prepare the allꢀtransꢀAx. In this
study, we identified a novel esterase (est3ꢀ14) gene (51% similarity) from a marine
mud metagenomic library. Est3ꢀ14 is pHꢀsensitive and keeps good stability in alkaline
buffers, especially in Buffer A (residual activity 94%, 36 h, 4 °C). At the same time,
Est3ꢀ14 keeps a good stability in the medium temperature condition (residual activity
56.7%, Buffer A, 40 °C, 84 h). Under optimal conditions, the final hydrolysis rate was
ꢀ1
99.3% and the yield of free allꢀtransꢀAx was 200 ꢁg•mL . According to these results,
we found that the hydrolysis rate was high and the byꢀproducts of enzymatic method
24
were less than using saponification, which is a preꢀrequisite for the development of a
green chemistry process. The whole reaction system is mild (moderate temperature
23
and less chemical solvents) and the less byꢀproducts. Thus, it can be concluded that
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Est3ꢀ14 is an efficient biocatalyst exploitable for the development of a competitive
industrial process for obtaining free allꢀtransꢀAx. Certainly, the reaction system needs
further optimization to improve the volume of production of prepared free
allꢀtransꢀAx. Furthermore, by the means of enzyme engineering for improving the
enzymatic properties, including substrate specificity, new applications for Est3ꢀ14 can
also be envisaged.
■ ABBREVIATIONS
Ax: astaxanthin; LB: LuriaꢀBertani; CIP: alkaline phosphatase calf intestinal;
pNP: pꢀnitrophenol; pNP acetate (C2): pꢀnitrophenyl acetate; pNP butyrate (C4
pNPB): pꢀnitrophenyl butyrate; pNP caprylate (C6): pꢀnitrophenyl caprylate; pNP
decanoate (C8): pNP decanoate, pNP caprate (C10): pꢀnitrophenyl caprate; pNP
laurate (C12): pꢀnitrophenyl laurate; pNP myristate (C14): pꢀnitrophenyl myristate;
pNP palmitate (C16): pꢀnitrophenyl palmitate; ORF: open reading frame; MTBE:
methyl tertꢀbutyl ether; HPLC: highꢀperformance liquid chromatography; IPTG:
isopropylꢀβꢀDꢀ1ꢀthiogalactopyranoside; TLC: thin layer Chromatography; LB:
LuriaꢀBertani; EDTA: ethylene diamine tetraacetic; SDS: sodium dodecyl sulfate;
PAGE: polyacrylamide gel electrophoresis;
■ FUNDING
419
This work was supported by the China Agriculture Research System (CARSꢀ48),
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Major Special Science and Technology Projects in Shandong Province
(2016YYSP016), Applied Basic Research Program of Qingdao (16ꢀ5ꢀ1ꢀ18ꢀjch),
Natural Science Foundation of Shandong
Province
(ZR2017MC024),
China Postdoctoral Science Foundation (2017M612354) and the Laboratory for
Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science
and Technology (LMDBKF201705).
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Notes
The authors declare that they have no competing interests.
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Supporting Information
Figure S1 The structures of Ax. allꢀtransꢀAx, 9ꢀcisꢀAx, 13ꢀcisꢀAx, respectively.
Figure S2 The functional screening of positive strains from marine mud metagenomic
library. M1, M2: with the clear hydrolysis halos on the LB agar with
tributyrin (0.5%, v/v).
Figure S3 Partial digestion of positive strains with Sau3AI. The digestion time was:
lane 1, M1ꢀ5 min; lane 2, M1ꢀ10 min; lane 3, M2ꢀ5 min; lane 4, M2ꢀ10
min.
Figure S4 Analysis of the expression level and purification of Est3ꢀ14 by SDSꢀPAGE.
lane1, supernatant of pETꢀ28a (+) cell lysate; lane 2, supernatant of
est3-14ꢀpETꢀ28a (+) cell lysate; lane3, precipitation of est3ꢀ14ꢀpETꢀ28a
(+) cell lysate; lane 4, 0.02 M imidazole with buffer A; lane 5, 0.05 M
imidazole with buffer A; lane 6, 0.1 M imidazole with buffer A; lane 7,
0.2 M imidazole with buffer A; lane 8, 0.5 M imidazole with buffer A;
Lane M, marker.
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