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ADVISORY BOARDS
KEN BUCKLE
University of New South Wales, Australia
MARY ELLEN CAMIRE

University of Maine, USA
ROGER CLEMENS
University of Southern California, USA
HILDEGARDE HEYMANN
University of California, Davis, USA
ROBERT HUTKINS
University of Nebraska, USA
RONALD JACKSON
Brock University, Canada
HUUB LELIEVELD
Global Harmonization Initiative, The Netherlands
DARYL B. LUND
University of Wisconsin, USA
CONNIE WEAVER
Purdue University, USA
RONALD WROLSTAD
Oregon State University, USA
SERIES EDITORS
GEORGE F. STEWART (1948–1982)
EMIL M. MRAK (1948–1987)
C. O. CHICHESTER (1959–1988)
BERNARD S. SCHWEIGERT (1984–1988)
JOHN E. KINSELLA (1989–1993)
STEVE L. TAYLOR (1995–2011)
JEYAKUMAR HENRY (2011–2016)
FIDEL TOLDRÁ (2016– )
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Notices
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Practitioners and researchers must always rely on their own experience and knowledge in
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CONTRIBUTORS
L.Y. Chen
School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China
S. Das
Indian Institute of Technology Kharagpur, Kharagpur, India
K. Doriya
Indian Institute of Technology, Hyderabad, Telangana, India
L. Gopalakrishnan
PES University, Bangalore, India
M. Gowda
NITK-Surathkal, Bangalore, India
N. Jose
U. Kabilan
School of Bioengineering, SRM University, Kattankulattur, Tamil Nadu, India
D. Kezia
Center for Biotechnology, Andhra University, Visakhapatnam, India
D.S. Kumar
R.N. Lima
Laboratório de Quı́mica Org^anica e Biocatálise, Instituto de Quı́mica de São Carlos,
Universidade de São Paulo, São Carlos, São Paulo, Brazil
H.S. Patnala
A.L.M. Porto
Laboratório de Quı́mica Org^anica e Biocatálise, Instituto de Quı́mica de São Carlos,
Universidade de São Paulo, São Carlos, São Paulo, Brazil
R.M.D. Rao
D. Roy
T. Sathish
Bioengineering and Environmental Centre, Indian Institute of Chemical Technology,
Hyderabad, India
R. Sen
ix
x Contributors
K.B. Uppuluri
Bioprospecting Laboratory, School of Chemical and Biotechnology, SASTRA University,
Thanjavur, India
A.R. Uria
Research and Development Center for Marine and Fisheries Product Processing and
Biotechnology, Central Jakarta, Indonesia
P. Veera Bramha Chari
Krishna University, Machilipatnam, India
V. Venugopal
Seafood Technology Section, Bhabha Atomic Research Centre, Mumbai, India
X.N. Xu
School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China
X.Q. Zhao
State Key Laboratory of Microbial Metabolism and School of Life Science and
Biotechnology, Shanghai Jiao Tong University, Shanghai, China
D.S. Zilda
Research and Development Center for Marine and Fisheries Product Processing and
Biotechnology, Central Jakarta, Indonesia
PREFACE
In the last decades, progress on the knowledge of marine enzymes has

advanced exponentially. The growing interest on marine enzymes is related
to their relevant properties that make them quite attractive because some-
how they are different from the well-known terrestrial enzymes. Marine
organisms may have to face extreme environmental conditions and this
makes that most of their enzymes be active and stable under extreme con-
ditions like very high or very low temperatures, high pressure, tolerance to
high salt concentration, stability to acid or basic pH, easy adaptation to cold
conditions, etc. All these properties make marine enzymes very attractive for
new catalytic reactions and, of course, new applications in food and nutri-
tion. In view of this increased interest, Advances in Food and Nutrition Research
is publishing three consecutive volumes focused on the topic Marine
Enzymes Biotechnology: Production and Industrial Application. This volume
78 corresponds to Part I that is mainly dealing with the production of
enzymes from marine sources, next volume 79 will correspond to Part II
dealing with the marine organisms producing enzymes, and volume 80 will
correspond to Part III dealing with the applications of marine enzymes.
This volume brings a variety of chapters reporting the production of
enzymes from marine sources. So, this includes the metagenomics approach
for discovering novel marine enzymes and the production of enzymes from
different marine sources like fishery wastes, algae, sponge, fungi, and bacteria.
Reported enzymes include proteases, lipases, amylases, cellulases, xylanases,
chitinase, laccase, alkaline phosphatase, transglutaminase, hyaluronidase,
acetyl glycosaminidase, glutaminase, asparaginase, and carrageenase, among
others. The use of solid-state fermentation vs submerged fermentation for
the production of marine enzymes is also discussed in several chapters. This
volume presents the combined effort of more than 28 professionals with
diverse expertise and background. The Guest Editors wish to thank the
production staff and all the contributors for sharing their experience and
for making this book possible.
SE-KWON KIM
FIDEL TOLDRÁ
Guest Editors
xi
CHAPTER ONE
Metagenomics-Guided Mining of
Commercially Useful Biocatalysts
from Marine Microorganisms
A.R. Uria1, D.S. Zilda
Research and Development Center for Marine and Fisheries Product Processing and Biotechnology,
Central Jakarta, Indonesia
1
Corresponding author: e-mail address: agustinus.uria@gmail.com
Contents
1. Introduction 1
2. Metagenome Isolation 7
3. Metagenomic Library Construction 9
4. Functional Screening 12
5. Homology Screening 16
6. Conclusions 19
References 21
Abstract
Marine microorganisms are a rich reservoir of highly diverse and unique biocatalysts
that offer potential applications in food, pharmaceutical, fuel, and cosmetic industries.
The fact that only less than 1% of microbes in any marine habitats can be cultured under
standard laboratory conditions has hampered access to their extraordinary biocatalytic
potential. Metagenomics has recently emerged as a powerful and well-established tool
to investigate the vast majority of hidden uncultured microbial diversity for the discov-
ery of novel industrially relevant enzymes from different types of environmental sam-
ples, such as seawater, marine sediment, and symbiotic microbial consortia. We discuss
here in this review about approaches and methods in metagenomics that have been
used and can potentially be used to mine commercially useful biocatalysts from
uncultured marine microbes.
1. INTRODUCTION
Marine microorganisms play various ecological roles in the oceans,
including their involvement in nearly all primary and secondary production
as well as in all biogeochemical cycles (Strom, 2008). The Census of Marine
Advances in Food and Nutrition Research, Volume 78 # 2016 Elsevier Inc. 1

ISSN 1043-4526 All rights reserved.
http://dx.doi.org/10.1016/bs.afnr.2016.05.001
2 A.R. Uria and D.S. Zilda
Life program between 2000 and 2010 estimated that the diversity of marine
microbes accounts for 1 billion with 38,000 species and 5000–9000 species
of microbial bacteria in a liter of seawater and a gram of sand, respectively
(Costello et al., 2010; www.coml.org, 2010). The great microbial diversity
and vital ecological roles suggest that marine microorganisms are a rich res-
ervoir of highly diverse and unique biocatalysts, either as novel enzymes
encoded on discrete single genes or as modular enzymes encoded on gene
clusters (Ferrer, Golyshina, et al., 2005; Ferrer, Martinez-Abarca, &
Golyshin, 2005).
Biocatalysts have attracted much attention for various applications in
food, pharmaceutical, fuel, and cosmetic industries, because they are consid-
ered as environmental friendly, economical, and clean catalysts that can pro-
duce a wide variety of chemical substances (Ferrer, Golyshina, et al., 2005;
Ferrer, Martinez-Abarca, et al., 2005). The high efficiency and stereo-
selectivity of enzyme-mediating reactions make biocatalysts suitable for
being applied in various industrial processes, ranging from food-related con-
version, laundry detergent, and paper processing to the production of useful
fine-chemicals and research reagents (Arnold, 2001; Wahler & Reymond,
2001). For such reasons, it is not surprising that the industrial demand for
novel enzymes to improve existing or to establish novel processes has
increased dramatically in the past few years (Schmid et al., 2001). Recent
particular industrial interests have been focused on novel enzymes capable
of mediating new chemical reactions that are difficult or expensive to be
achieved by chemical catalysis or traditional organic synthesis (Ferrer,
Beloqui, Timmis, & Golyshin, 2009).
The general approach of obtaining appropriate microbial biocatalysts
with desired specific properties is screening of natural diversity for hitherto
unknown enzymes based on traditional cultivation-dependent microbiolog-
ical methods. This classical approach includes enrichment of microorgan-
isms from environmental samples, isolation of pure cultures, screening of
microbial isolates expressing desired enzymatic activity, characterization
of the isolated enzymes of interest, and optimizing the enzyme production
and recovery (L€ammle et al., 2007). Recent advances in DNA sequencing
technologies have contributed to the huge accumulation of new fully
sequenced genomes from bacteria and archaea available in the public
databases. Examining genome database, popularly called genome mining,
provides exciting opportunities of discovering genes encoding novel
commercially valuable enzymes with interesting properties (Lee et al.,
2010; Lόpes-Lόpes, Cerdán, Siso, 2014). However, this cultivation-based
Metagenomics-Guided Mining 3
discovery of novel enzymes has so far been limited to only a tiny fraction of
the existing microbial diversity in any given habitat, because less than 1% of
microorganisms in most environments can be cultivated by conventional
methods, as laboratory cultivation conditions do not reflect natural living
conditions (Amann, Ludwig, & Schleifer, 1995).
The limitation to cultivate most microorganisms under standard labora-
tory conditions can be overcome by metagenomics, a powerful cultivation-
independent tool for analysing the genome sequences of an uncultured
microbial community in any given environment (Hugenholtz & Tyson,
2008). In recent years, metagenomics has emerged as a well-established
approach to exploit the biocatalytic potential of the vast majority (>99%)
of hidden uncultured microbial diversity, leading to the discovery of novel
proteins (Lorenz & Eck, 2005). This approach, also called metagenome min-
ing, has been developed to examining metagenome (the collective genomes
of all microorganisms present in a given habitat) for the discovery of novel
biocatalysts without cultivating any particular microbes (Ferrer et al., 2009;
Handelsman, Rondon, Brady, & Clardy, 1998; Steele, Jaeger, Daniel, &
Streit, 2009). Metagenomics-guided searching for enzymes from uncultured
marine microbes involves direct isolation of total DNA from an environ-
mental sample followed with cloning of the DNA obtained into an easily
culturable microbial host. The resulting clones collectively termed as a meta-
genomic library can be screened to obtain genes of interest (Handelsman,
2004; Handelsman et al., 1998). Further gene sequence analysis, over-
expression and characterization of the expression product can provide useful
information on the enzyme properties.
Approaches for metagenomic library screening can be divided into two
general categories, namely, functional screening and homology screening.
Functional screening relies on the heterologous expression of the cloned
genes in a cultured host to generate phenotypes of interest. Homology
screening relies on the use of DNA sequence similarity to identify clones
harboring genes of interest in a metagenomic library (Iqbal, Feng, &
Brady, 2012). Development of metagenomics techniques has allowed the
discovery of an unlimited pool of new potential biocatalysts, genes, and bio-
synthetic pathways from uncultivable marine microorganisms (Arnold,
2001; Daniel, 2001). Some examples of industrially relevant enzymes dis-
covered from marine microorganisms using metagenomics strategies were
listed in Table 1. We describe here common steps in metagenomics that
can be used for discovery of marine biocatalysts (Fig. 1), which include
metagenome isolation, metagenomic library construction, library screening
Table 1 Examples of Biocatalysts Discovered from Uncultured Marine Microorganisms Through Metagenomics
Marine Screening Insert
Biocatalyst Source Vector Approach Size (kp) Σ Clone + Clone References
Chitinases Coastal seawater Phagemid Homology 1.8–4.2 75,000 14 Cottrell, Moore, and Kirchman
(1999)
Coastal and Plasmid Homology 0.9 850,000 1 Cottrell, Wood, Yu, and
estuarine water Kirchman (2000)
Sargasso Sea Plasmid PCR 0.9 – 108 LeCleir, Buchan, and Hollibaugh
Homology (2004)
Amylase Marine sediment Fosmid – 30–40 20,000 1 Liu et al. (2012)
Laccase Surface seawater BAC Homology 50–150 20,000 1 Fang et al. (2011)
Esterases Deep-sea Λ-Phagemid Functional ND ND 5 Ferrer, Golyshina, et al. (2005)
hypersaline basin and Ferrer, Martinez-Abarca,
et al. (2005)
Deep-sea sediment Fosmid Functional 40 ND 1 Park et al. (2007)
Surface seawater BAC Functional 70 20,000 4 Chu, He, Guo, and Sun (2008)
Arctic sediment Fosmid Functional – – 2 Jeon, Kim, Kang, Lee, and Kim
(2009)
Sea sediment Fosmid Functional 36 – 1 Fu et al. (2011)
Deep-sea sediment Fosmid Functional 36 20,000 12 Jiang et al. (2012)
Marine sediment Plasmid Functional 1–8.5 118,000 15 Peng et al. (2011)
Lipases Tidal flat sediment Fosmid Functional 35 386,400 4 Lee, Lee, Oh, Song, and Yoon
(2006)
Deep-sea sediment Fosmid Functional 21–40 8823 1 Jeon et al. (2009)
Baltic sea sediment Fosmid Functional 24–39 >7000 1% of Hårdeman and Sj€
oling (2007)
library
Deep-sea sediment Fosmid Functional 15–33 81,100 6 Jeon et al. (2011)
Marine sediment Plasmid Functional 1–8.5 118,000 1 Peng et al. (2014)
Proteases Antarctic seawater – Genome – – 1 Acevedo et al. (2008)
walking
Deep-sea sediment Fosmid Functional ND ND 1 Lee et al. (2007)
Amidases Marine sediments Plasmid Functional 5.2 25,000 6 Gabor, de Vries, and Janssen
(2004)
Cellulase Sargasso Sea Fosmid Homology – – 1 Cottrell, Yu, and Kirchman
(WGS) (2005)
β-Glucosidase Hydrothermal Plasmid Functional 4–10 ND 1 Schr€
oder, Elleuche, Blank, and
spring Antranikian (2014)
β-1,4- Seaweed- Plasmid Functional 1.5–7 40,000 11 Martin et al. (2014)
Endoglucanase associated
microbiota
Fumarase Seawater Plasmid Homology 1–15 50,000 1 Jiang et al. (2010)
Note: WGS, whole-genome sequencing; ND, not determined.
Direct extraction Indirect extraction
Seawater
Marine sponge
Small and middle-
sized DNA fragments
(1–50 kb)
Single cells
Metagenome Marine sediment Large DNA

cloning fragments (> 50 kb)
Metagenome
cloning
MDA
Agar plate format Microplate format 3D format
Metagenome
sequencing
Indicator media Hybridization Pooling-PCR-dilution
Single-cell
Clone sequencing sequencing
Metagenomic datasets
Bioinformatic analysis, gene overexpression-

and enzyme characterization
Fig. 1 A general scheme of metagenomics for gaining access to commercially useful

enzymes from uncultured marine microorganisms. Metagenome can be extracted
directly or indirectly from an environmental sample, either seawater, marine sediment,
or microbial consortia of marine invertebrates exemplified by sponge. The isolated
metagenome was cloned into a heterologous host, usually E. coli, to create a meta-
genomic library. The vector used for cloning depends on the DNA fragment length.
DNA fragments of less than 8 kb are usually cloned using a plasmid, thereby creating
a small insert library. Cosmid or fosmid is used to clone fragments of 25–50 kb, gener-
ating a middle-size insert library. DNA fragments of >50 kb can be cloned using bac-
terial artificial chromosome (BAC) to create a large-insert metagenomic library. MDA,
multiple displacement amplification.
by function, homology-driven screening, and metagenome sequencing.

Some approaches described here were applied for terrestrial samples, but
they can potentially be used or developed for marine samples.
2. METAGENOME ISOLATION
In metagenome preparation, three things should be taken into consid-
eration depending on the requirement for downstream steps: DNA yield,
length/size, and purity. Metagenomic DNA from an environmental sample
can directly be isolated without prior cultivation of microorganisms or cell
isolation. This direct metagenome isolation approach usually relies on deter-
gents and enzymes to break and process samples, followed with phenol/
chloroform extraction and isopropanol/ethanol precipitation. This
approach results in relatively high DNA yield, but it usually generates small
DNA fragments (1–50 kb) due to the shearing or mechanical forces during
the extraction process (Desai & Madamwar, 2007; Xing, Zhang, & Huang,
2012). The DNA obtained using this direct approach can be used for the
construction of plasmid-based small insert libraries and cosmid/fosmid-
based intermediate insert libraries. An example of direct DNA isolation is
based on a lysis buffer containing lauryl sarcosyl and urea at high concentra-
tion to break microbial cell walls, as reported by Piel et al. (2004). This
method is applicable not only for a marine sponge known to harbor the asso-
ciated complex symbiotic microbes, but also for fish gut sample and marine
sediment sample. This method may involve grinding a sample using liquid
nitrogen followed with phenol and chloroform extractions. If a sample con-
tains huge amount of polysaccharides as found in many sponges, the
cetyltrimethylammonium bromide (CTAB) treatment can be included for
polysaccharide removal prior to isopropanol and ethanol precipitation
(Gurgui & Piel, 2010; Hrvatin & Piel, 2007).
In metagenomics, it is sometimes very important to obtain larger DNA
fragments of more than 50 kb, especially for the purpose of generating large
insert libraries based on bacterial artificial chromosome (BAC) vector.
Avoiding intensive DNA shearing to get larger DNA fragments can be car-
ried out through mechanical separation of prokaryotic or bacterial cells prior
to cell lysis and DNA preparation. The drawback of these indirect isolation
approach is the relatively low yield, which is 10–100 times lower compared
with the direct approach (Parachin, Schelin, Norling, Radstrom, & Gorwa-
Grauslund, 2010). A simple way to separate prokaryotic cells from a marine
sediment sample is based on low-speed centrifugation as reported by Bakken

and Lindahl (1995) and Hårdeman and Sj€ oling (2007). Briefly, a sediment
sample is suspended in TE-pyrophosphate buffer and gently agitated,
followed with blending in a CTAB buffer and low-speed centrifugation
to recover prokaryotic cells (Hårdeman & Sj€ oling, 2007). Nycodenz gradi-
ent separation method used by Bertrand et al. (2005) to extract bacterial cells
from soil samples might be applicable for any marine sediment sample. For a
seawater sample, bacterial and archaeal cells can simply be collected by sea-
water filtration on a 0.22-μm pore size membrane and subsequent membrane
washing to release the cells, as reported by Stein, Marsh, Wu, Shizuya,
and DeLong (1996) and Chu et al. (2008). To obtain symbiotic microbial
symbionts of marine invertebrates, cell separation from the hosts can
be conducted, either by simple squeezing as reported for tunicates by
Schmidt et al. (2005) or by homogenization/blending and subsequent
differential centrifugation as reported for sponges by Bewley, Holland, and
Faulkner (1996).
The microbial cells obtained from the environmental sample can subse-
quently be lysed to recover the DNA through several ways. Simple heating
reported by Syn and Swarup (2000) was found to be useful in preparing
HMW DNA from uncultivated bacterial cells. This protocol involves treat-
ment of the bacterial cells with NE (NaCl and EDTA) buffer to remove
extracellular exopolysaccharides followed with heating the cell suspension
at 75°C to allow cell lysis. Further phenol/chloroform extraction, iso-
propanol precipitation, and ethanol washing steps allow the recovery of
HMW DNA (Syn & Swarup, 2000). Enzymatic treatment using combina-
tion of lysozyme and achromopeptidase allows efficient cell lysis (Bertrand
et al., 2005; Courtois et al., 2003). The enzymatically lysed cells are subse-
quently treated with proteinase K and N-lauroyl sarcosine, followed with
applying a cesium chloride density gradient to purify the DNA (Bertrand
et al., 2005). Another method to recover HMW from the isolated bacterial
cells is the gel-embedding method, as reported by Liles et al. (2008) for soil
microorganisms and Quyang et al. (2009) for bacterial cells from diverse
sponges. This method starts with embedding the isolated cell pellet in aga-
rose plugs. This is followed with treatment of the plugs with a buffer con-
taining lysozyme to break the embedded cells and then with a buffer
containing proteinase K for protein degradation. Further plug electropho-
resis allows DNA fractionation and recovery of over 400-kb fragments
(Quyang et al., 2009). The HMW DNA fragments of >30 kb length, either
as the cell-free form or embedded in low-melting point (LMP) agarose plug

can be purified by enzymatic gel extraction using GELase commercially pro-
vided by Epicentre or by column-based gel extraction. GELase (Epicentre)
contains β-agarose-digesting enzyme, enabling breaking down the carbohy-
drate backbone of LMP gel into small, soluble oligosaccharides (http://
www.epibio.com). The DNA fragments are then recovered by isopropanol
or ethanol precipitation (Gurgui & Piel, 2010).
Ion exchange chromatography based on resin, commercially provided
by QIAGEN (Hilden, Germany, www.qiagen.com), is designed for the
recovery of chromosomal DNA of 20–150 kb in size and has been proven
to be useful in obtaining pure HMW metagenomic DNA from the uncul-
tured bacterial cell fraction dominated by Entotheonella, filamentous symbi-
onts of marine sponges (Wilson et al., 2014). This method involves careful
cell lysis with lysozyme and binding of all the negatively charged molecules
(DNA, RNA, and protein) to the resin containing very little salt. When the
salt concentration is gradually increased using a medium-salt buffer, traces of
RNA and protein become unbound and nonspecific interaction disrupts,
thereby leaving DNA on the chromatography column. By passing a higher-
salt buffer, the DNA is efficiently eluted from the column, which can be con-
centrated using isopropanol/ethanol precipitation (Qiagen; Brown, 2010).
3. METAGENOMIC LIBRARY CONSTRUCTION

For metagenomic library construction, metagenome obtained from an
environmental sample needs to be cloned into a cultured bacterial host, usu-
ally Escherichia coli, using an appropriate vector. The type of a vector chosen
depends on the length of foreign DNA fragments to be cloned. Cloning of
DNA fragments of up to 8 kb is usually based high-copy plasmid and
phagemid (Brown, 2010). A wide variety of plasmid vectors are available
commercially from suppliers. Small-insert libraries based on plasmid are
often sufficient to screen a discrete enzyme-encoding genes (average size
of 1 kb). Small insert libraries offers an advantage that most genes are present
in the appropriate orientation under the influence of the very strong vector
expression signals, suggesting a good chance of being expressed and
detected by activity screens (Ferrer et al., 2009). Knietsch, Waschkowitz,
Bowien, Henne, and Daniel (2003) reported construction of libraries with
small inserts to find a clone expressing alcohol dehydrogenase activity.
Total DNA retrieved from an environmental sample was partially digested

using the restriction enzyme, Bsp1431. The resulting small DNA fragments
are ligated with pBluescript SK+, tranformed into E. coli DH5α for clone
amplification, and transferred to E. coli ECL707 for insert expression. The
expression product was then prepared from the host cells at the stationary
phase and tested for alcohol dehydrogenase activity (Knietsch et al., 2003).
In a similar way, Tirawongsaroj et al. (2008) constructed a small-insert
metagenomic library from Thailand hot spring sediments to screen
clones expressing lipolytic activity. The metagenomic DNA partially digested
with Sau3AI was subjected to size fractionation. Fractions containing
DNA fragments of 1–10 kb were selected and cloned into E. coli
using a BamHI-linearized zero-background cloning vector (Tirawongsaroj
et al., 2008).
Creating large-insert libraries increases the chance of finding genes of
interest, because less clone numbers need to be screened. The high-capacity
vectors such as cosmid can manage DNA fragments of 30–50 kb. An exam-
ple of a large metagenomic library based on cosmid was reported by
Courtois et al. (2003). To increase the ligation process, the DNA fragments
of approximately 50 kb was added with polynucleotide (poly-dT) tails prior
to ligation with the cosmid previously linearized enzymatically and tailed
with poly-dA. The ligation product was packaged in λ phage particles,
which were subsequently used to infect E. coli. The resulting clones were
arranged and grown in 96-well microtiter plates, allowing long-time storage
of the library in glycerol at 80°C (Courtois et al., 2003). Fragments up to
400 kb can be handled using BAC (Brown, 2010; Kim, Shizuya, de Jong,
Birren, & Simon, 1992; Shizuya et al., 1992). Interestingly, using BAC,
Rondon et al. (2000) constructed two large metagenomic libraries of >1
Gb-inserts and identified genes for lipase, amylase, and nuclease in the
libraries.
The main problem in cloning of large fragments is the instability and high
potential undesired modifications such as rearrangements and deletions as
has been shown by Kim et al. (1992) for complex mammalian genomic
DNA inserts. To overcome this problem, it requires a high-capacity vector
present in low copy number, which can subsequently be induced to high
copy number. Obtaining high yields of the cloned DNA is important
for downstream applications such as subcloning, enzymatic restriction,
sequencing, and recombination. Fosmid is designed to meet this require-
ment related to the instability and modification of larger inserts. This is best
exemplified by the fosmid-based vector pCC1FOS (Epicentre) coupled

with E. coli host harboring trfA gene that has been proven to be useful
in maintaining the stability of large inserts and reducing potential
rearrangements. The F factor system present in pCC1FOS controls the
low copy number (1–2 copies per cell). If L-arabinose is added into the
growth media, this sugar induces the araC-PBAD promoter/regulator sys-
tem controling trfA transcription, leading the increased copy number to
about 10–50 copies per cell in the presence of an oriV replication origin
(Westenberg, Bamps, Soedling, Hope, & Dolphin, 2010; Epicentre;
Wild, Hradecna, & Szybalski, 2002). Fosmid pCC1FOS is provided by
Epicentre in the dephosphorylated linear form to avoid self-ligation.
Prior to ligation with this blunt-ended fosmid, DNA fragments should
be repaired at the both termini, because the DNA fragment ends can
be fray without phosphate residues during extraction and purification
processes. This end repairing step relies on T4 DNA polymerase to
blunt the frayed ends by filling the excessed 30 -termini and removing
the protruding 30 -termini and terminal phosphotransferase to convert
the hydroxyl group at the 30 -termini into a phosphate group (Brown,
2010). In a metagenomic library construction based on this fosmid vector,
the repaired blunt DNA fragments of approximately 40 kb are ligated
with fosmid and packaged in bacteriophage viral particles that are subse-
quently used to infect E. coli, thereby generating a complex fosmid library
as has been reported for sponge samples (Ueoka et al., 2015; Uria,
2012) (Fig. 2).
Metagenomic libraries can be generated in different formats, usually agar
plates and microplates, depending on the screening approach to be used.
Because library in agar plate format cannot be kept alive for a long time,
it can be combined into pools, mixed with glycerol and stored at 80°C
for a long time. A library in the microplate format can be added with glycerol
and stored in the freezer for being used in later screening. However, con-
structing a very large metagenomic library (up to 1 millions) either in agar
plates or microplates is inefficient in term of labor, space, and consumables
because it requires a lot of plates or microtiters, a lot of colony picking and
larger space for storage. Hrvatin and Piel (2007) have recently developed an
efficient 3D library format containing a very large number of clones (up to 1
million clones) with the clone density of 1000 cfu/mL. This library format
allowed its long-time storage in smaller space at the freezer and further faster
whole-cell screening.
Cell separation
Sponge
sample Bacterial fraction
DNA DNA
isolation isolation
HMW metagenomic cos camR

DNA
Fragment selection
Selected metagenomic Ligation

fragment (~ 40 kb) Fosmid molecules
Metagenomic fragment
camR
cos Catenane
Packaging
Catenane
Packaging
Clones harboring Clones harboring

circular recombinant circular recombinant
molecules molecules
Transfection Transfection
into E. coli into E. coli
Chloramphenicol
Chloramphenicol
medium
medium
Fig. 2 Construction of a metagenomic library using pCC1FOS fosmid vector. Meta-

genomic DNA obtained from either a sponge sample or bacterial cells associated with
sponge is separated on low-melting gel (LMP) via electrophoresis. Subsequently, DNA
fragments of approximately 40 kb are ligated with the fosmid pCC1FOS. The ligation
product is packaged in bacteriophage viral particles and used to infect E. coli, thereby
resulting in recombinant clones, collectively called a metagenomic fosmid library.
Redrawn from Uria, A. R. (2012). Investigating natural product biosynthesis in uncultivated
symbiotic bacteria of the marine sponge, Theonella swinhoei. PhD dissertation, University
of Bonn, Germany. 218 p.
4. FUNCTIONAL SCREENING
Functional screening is generally based on detection of the expression
products encoded on the individual genes of interest, and therefore it relies
on the ability of transformants to express genes of interest (Wahler &

Reymond, 2001). The success in this functional screening depends on
the compatibility of the cloned genes with the expression machinery of
the heterologous host, usually E. coli. The most simple functional screen-
ing is the use of indicator media containing a substrate for an enzyme of
interest. Positive clones expressing the desired enzyme activities can be
identified based on the halo formation or color change on agar plate assays.
For example, the presence of one of hydrolase members is visualized
with a clearing zone around the positive transformants (Rondon et al.,
2000). Using indicator media or agar plate assays offer a major advantage
that it is highly scalable, which can be integrated with robotics enabling
screening of many thousands clones per day (Suenaga, Ohnuki, &
Miyazaki, 2007).
Functional screening based on indicator media has been developed to
gain access to novel esterases from seawater and marine sediment, as recently
reported by Chu et al. (2008), Fu et al. (2011), and Jiang et al. (2012). Chu
et al. (2008) prepared HMW DNA from bacterial cells collected by filtration
of surface seawater. The DNA fragments of >50 kb was cloned into E. coli
EPI300 using a BAC. Functional screening of the resulting BAC library
consisting of approximately 20,000 clones led to the isolation of four
BAC clones with lipolytic activity. Subcloning of two BAC inserts enabled
identification of two ORFs, designated as estA and estB, responsible for lipo-
lytic activity. Overexpression and biochemical characterization of them rev-
ealed the remarkable activity of EstB toward p-nitrophenyl esters, suggesting
its potential industrial applications (Chu et al., 2008). Jiang et al. (2012) have
discovered nine genes coding for novel lipolytic enzymes that shared
56–84% identity at the amino acid sequence level to other lipolytic enzymes
in the public database. Briefly, they initially prepared DNA from the deep-
sediments obtained at the depth of 5886 m with the temperature of 1.5°C.
A metagenomic library with the average clone insert size of 36 kb was con-
structed in E. coli using a fosmid, and functionally screened on Luria–Bertani
(LB) agar medium containing 1% tributyrin. They found 12 clones
exhibiting lipolytic activity based on the clearing zone formation around
colonies. Expression and characterization of one of the discovered genes,
designated as Est6, revealed that its expression product was an esterase with
the preference for mid-length acylglycerols, suggesting its potential indus-
trial application (Jiang et al., 2012).
In a separate study by Fu et al. (2011), a metagenomic library constructed
from a South China Sea Sediment core was screened for clones exhibiting
lipolytic activity using the tributyrin agar diffusion assay. This led to the iso-
lation of a positive clone harboring a 36-kb insert fragment. Subcloning of
the insert and subsequent sequencing of the positive subclones enabled iden-
tification of an esterase gene, designated as EstF. Further phylogenetic anal-
ysis, overexpression, and characterization revealed that EstF was active at
very low temperatures with the preference for the short acyl chain substrate,
suggesting it was a cold-active “true” esterase (Fu et al., 2011). A lipase gene,
designated as Est_p6, was isolated by Peng et al. (2014) through functional
screening of a marine sediment metagenomic library. Subsequent gene over-
expression and protein characterization revealed that this lipase showed a
high hydrolytic specificity toward myristate and palmitate as well as a distinc-
tive and desirable flavor and odor in milkfat flavor production, suggesting its
potential application for commercial lipolyzed milkfat flavor production and
manufacturing processes (Peng et al., 2014). Lipases have been applied in
various industries for food, cosmetics, pharmaceutical, chemical, and deter-
gent productions (Kim et al., 2007). Lee et al. (2006) constructed a meta-
genomic fosmid library from tidal flat sediment and screened for lipolytic
activity using LB media containing emulsified tricaprylin. This led to the
identification of four clone exhibiting lipolytic activity as shown by a halo
surrounding the colonies. Phylogenetic analysis indicated that enzymes
expressed by the clones belong to a new family of bacterial lipases (Lee
et al., 2006).
Functional screening of a metagenomic library for clones capable of
secreting cellulase can be based on a colorimetric assay. This assay involves
the use of carboxymethylcellulose in the growing media and staining with
the dye congo red (Kasana, Salwan, Dhar, Dutt, & Gulati, 2008). The dye
interacts with β-D-glucans resulting in the dye–glucan complex indicated
with the formation of a yellow halo surrounding of cellulase is the clones
expressing cellulase activity (Teather & Wood, 1982). Cellulase has attracted
increasing attention in recent years due its great future potential in biofuel
production through biodegradation of plant biomass containing lignocellu-
lose (Maki, Leung, & Qin, 2009). Functional screening based on chromo-
genic or fluorogenic substrates has been used to detect clones expressing
certain enzymes that convert the substrates selectively into products which
can visually be detected in vivo (Wahler & Reymond, 2001). This screening
approach has successfully been developed to identify oxidoreductases,
which is applicable for marine metagenomic libraries. For example, clones
expressing dioxygenase activity can be screened using horseradish peroxi-
dase (HRP). In this screening, a fluorogenic aromatic substrate is hydrolyzed
by oxygenases to form a phenol or catechol that is further polymerized by

HRP, thereby producing a fluorescent dimer/polymer readily detected
spectroscopically using a digital imaging system or plate readers (Joo,
Arisawa, Lin, & Arnold, 1999). Another example is the identification of
multicopper oxidase in a metagenomic library based on the formation of
a catechol (reddish brown) resulted from the guaiacol hydrolysis catalyzed
by this oxidase (Ype, Li, Liang, & Liu, 2010).
The major disadvantage of indicator media approach is that it relies on
transport of either substrate or enzyme of interest to detect activity, and
many potential enzymes might not be simply detected based on colony
appearance or color changes in the reaction product (Suenaga et al.,
2007). To overcome this limitation, some methods have recently been
developed to detect the enzyme-catalyzed reaction products. For example,
Rabausch et al. (2013) developed functional screening based on a
semiautomated thin-layer chromatography (TLC) to screen clone pools
in a metagenomic library, allowing the rapid identification of clones
expressing flavonoid-modifying enzymes. Clone pools were transferred to
a biotransformation medium containing flavonoid, and the reaction prod-
ucts were subjected to TLC analysis. Positive pools were downsized until
the responsible single clones were detected (Rabausch et al., 2013).
Flavonoid-modifying enzymes can mediate the regio- and stereochemical
modification of flavonoids, and therefore they have gained increasing
demand for producing specific flavonoids required in the cosmetic,
pharma-, and nutraceutical industries (Das & Rosazza, 2006). Another
method to overcome such problem is the use of a reporter gene assay
called SIGEX (substrate-induced gene expression screening) designed by
Uchiyama, Abe, Ikemura, and Watanabe (2005) to screen clones expressing
enzymes involved in catabolism. In this method, green fluorescent protein
(GFP) was used as reporter to detect metagenomic clones expressing desired
catabolic enzymes induced by suitable substrates. SIGEX has allowed dis-
covery of genes involved in benzoate and catechol degradation in a ground-
water metagenomic library (Uchiyama et al., 2005). Subsequently, PIGEX
(product-induced gene expression) was developed by Uchiyama and
Miyasaki (2010) to screen amidase activity in a wastewater sludge meta-
genomic library. In this method, GFP gene was placed under the control
of benzoate-sensitive transcription factor. Amidases expressed by positive
clones convert benzamine into benzoate. The resulting benzoate induces
the activation of the transcription factor leading to the expression of GFP
(Uchiyama & Miyasaki, 2010). In more recently, Hollfelder and his
colleagues reported an impressive ultrahigh-throughput functional screen-

ing for new hydrolases from various metagenomic sources (Colin et al.,
2015). This approach was based on the use of microfluidic droplets, in which
E. coli cells transformed with environmental DNA using a plasmid were
individually encapsulated into single water-in-oil droplets containing sub-
strate and lysis agents. Subsequently, emulsion droplets were incubate off-
chip and assayed for catalytic activities of cytoplasmically expressed hydro-
lases after cell lysis (Colin et al., 2015).
5. HOMOLOGY SCREENING
Homology-driven screening can be based on PCR using degenerate
primers designed specifically to target genes coding for new members of a
known enzyme family in an environmental sample. Cloning and sequencing
of the PCR product resulted from the degenerate primers can provide
insight into the sequence diversity of partial genes with the same function.
The sequence information of an amplified partial gene can then be used as
the basis for designing new specific primers to recover an entire gene from a
metagenomic library. Subsequent heterologous expression of the entire
gene may lead to the discovery and overproduction of the encoded biocat-
alyst. This PCR-based screening is best exemplified by identification of a
laccase from the surface water of the South China Sea, as has recently been
reported by Fang et al. (2011). The DNA fragments of 50–150 kb prepared
from the surface water were cloned into E. coli using a BAC. The resulting
clones placed in 385-well plates were screened using degenerate primers
designed based on the conserved region of laccases in copper-binding sites.
Subsequently, the full-length laccase gene obtained was expressed using
the pET expression system. Interestingly, the resulting recombinant laccase
(439 amino acids) harboring three conserved copper-binding domains shares
less than 40% of sequence identities with known bacterial multicopper
oxidases. The unusual properties possessed by this new laccase, such as
alkalescence-dependent activity, high chloride tolerance, and dye decolor-
ization ability, makes it an alternative for specific industrial applications
(Fang et al., 2011).
PCR-based identification of chitinase genes in a seawater sample
reported by Cottrell et al. (2000) involved the use of the degenerate
primers designed based on deduced amino acid sequences of chitinases in
four γ-proteobacteria (Alteromonas sp., Aeromonas caviae, Serratia marcescens,
Enterobacter agglomerans). First, bacterial cells were collected from seawater
samples using 0.22-μm filtration cartridge. Subsequently, PCR amplifica-

tion was performed using the degenerate primers with total DNA from such
bacteria cells as the template. The resulting PCR products were cloned into
E. coli, and the resulting clones were selectively sequenced (Cottrell et al.,
2000). This PCR-based screening has also been used to identify alkaline
proteases as reported by Niehaus et al. (2011).
Homology screening based on in situ hybridization can also be used to
identify biocatalyst-encoding genes of interest, as shown by Ginolhac et al.
(2004) for identifying polyketide synthase in a metagenomic library. This
begins with designing degenerate primers to isolate partial genes of interest.
The obtained gene fragments are labeled with [α-33P]dCTP and used
to screen a metagenomic library. In library screening, colonies on plates
are initially transferred on to nylon membranes and lysed to expose the
DNA. The DNA on membranes is immobilized and treated with probe
to allow visualization of hybridization signals as an indicator of positive
clones on a master plate (Ginolhac et al., 2004). Homology-based screening
of a metagenomic library constructed from Pacific deep-sea sediment
led to the identification of two novel alkane hydroxylases (Xu, Xiao, &
Wang, 2008).
A very large metagenomic library can effectively be screened using pool
dilution and whole-cell PCR method if the library is generated in 3D format
(Hrvatin & Piel, 2007) as mentioned in Section 3. In this screening method
(Fig. 3), a library is initially grouped into pools with the clone density of
1000 cfus/mL. Then, a small aliquot of several pools is combined as a super-
pools. Superpools are screened by whole-cel PCR, and the pools from a
PCR-positive superpool are subsequently screened. Positive clone pools
are diluted at lower cell density, followed by PCR-rescreening. This cell dilu-
tion coupled with PCR screening is carried out until the clone density of
approximately 20–40 cfus/mL. Cell aliquot with such clone density is then
plated at around 200 cfus/plate. Some colonies can be checked by colony
PCR to isolate a single positive clone (Hrvatin & Piel, 2007; Uria, 2012).
Recent advances in DNA sequencing technologies marked with
the development of next-generation sequencing (NGS) has contributed
significantly to a great accumulation of metagenomic sequences from various
environmental samples. Subsequent bioinformatic analysis of metagenomic
datasets allows identifying genes encoding commercially useful enzymes
(CUEs). Obtaining the complete sequence of a genome from complex
metagenomic DNA is very challenging to accomplish since the meta-
genomic data come from heterogenous microbial communities, often
Library screening
Production of
A positive pool
recombinant
(~4000 cfus/mL)
biocatalyst
+
4 x dilution PCR
Gene cloning and
overexpression
Subpools x 10
(~1000 cfus/mL)
+ PCR-amplification
5 x dilution PCR of a target gene
Sub-subpools
(~200 cfus/mL) x 10
Subcloning and
10 x dilution + Sequencing
PCR
Sub-sub-subpools x 10
(~20 cfus/mL)
+
PCR
Construct
Plating
preparation
Fig. 3 An example of mathematical calculations for the high-throughput screening of a

fosmid-based complex 3D metagenomic library. In an attempt to screen a complex
library at high clone density (for instance 4000 cfus/mL), a small aliquot of a positive
pool is initially screened by PCR, followed by the combination of pool dilution and
whole-cell PCR analysis until the clone density of 20 cfus/mL. Cell suspension is then
plated at the clone density of 200 cfus/plate, followed with colony PCR to isolate a
single positive clone.
containing more than 10,000 species (Wooley, Godzik, & Friedberg,

2010). In NGS technology, metagenome is usually fragmented into small
fragments, generating millions of short reads, commonly in the range
of 75–1000 bp depending on the sequencing platform used. The short
sequence reads must be assembled to generate contigs, usually up to
5000 bp. Due to the small reads and low depth of coverage, generating accu-
rate and high-quality genome assembly becomes difficult. Although recon-
struction of an entire genome present in a complex metagenomic sequences
is generally not possible (Riesenfeld, Schloss, & Handelsman, 2004; Wooley
et al., 2010), an incomplete metagenomic dataset consisting of contigs
assembled from short sequence reads can still offer great opportunities of
mining novel functional genes, new activities and products (Ferrer et al.,
2009). For example, using the Sargasso Sea whole-genome sequence
(WGS) data set prepared by Venter et al. (2004), Cottrell et al. (2005) iden-
tified genes coding for hydrolases potentially used by marine Cytophage-like
bacteria to degrade biopolymers in HMW dissolved organic matter (DOM).

PCR primers were then designed to amplify an entire hydrolase gene, des-
ignated celM, from a fosmid library constructed with prokaryotic DNA of
western Arctic Ocean. Subsequent subcloning of the gene into E. coli and
expression product analysis revealed that it has peptidase activity (Cottrell
et al., 2005).
The existing metagenomic datasets available in public databases can be
examined and used as the basis for the discovery of novel industrially relevant
biocatalysts. Although the DNA samples are not available, discrete genes that
are relatively small in size can be chemically synthesized and heterogously
expressed to allow functional analysis, as shown by Bayer et al. (2009) for
methyl halide transferase. Samar, Kumar, Prakash, and Taylor (2010) devel-
oped MetaBioMe, a database to explore CUEs in metagenomic datasets.
Using homology-based approach, they identified several novel homologues
for known CUEs, providing exciting opportunities for further experimental
verification. Recently single-cell genomics offers great potential to be devel-
oped for enzyme discovery. This technique involves isolation of single cells
from complex microbial consortia, either by a fluorescence-activated cell
sorter as has been shown for bacterial symbionts of a sponge (Unson,
Holland, & Faulkner, 1994), microfluidic chip (Marcy et al., 2007), or
micromanipulation as reported by Grindberg et al. (2011) for single cells
of Lyngbya bouillonii. Subsequent cell lysis and multiple displacement ampli-
fication (MDA) to amplify whole genome (Dean et al., 2002; Grindberg
et al., 2011) enable obtaining sufficient genomic DNA of an uncultured bac-
terial species for sequencing. The MDA method relies on the φ29 DNA
polymerase coupled with random primers to amplify the entire genome.
This polymerase has special characteristics, such as its extremely high
processivity and its strong strand displacing activity, which allow copy over
the sample template multiple times (Lasken, 2009). Bioinformatic analysis of
the MDA-amplified datasets opens exciting chances to identify potential
functional genes in an uncultured bacterial species.
6. CONCLUSIONS
The enormous abundance and diversity of marine microorganisms
offer exciting opportunities and challenges in discovery of highly diverse
and unique novel biocatalysts that are commercially useful or industrially rel-
evant. The fact that less than 1% of microorganisms in any given environ-
ment cannot be cultivated under traditional laboratory conditions has
hampered exploring and exploiting the vast majority of marine microorgan-

isms. Metagenomics has recently emerged as powerful and well-established
tool to access the biocatalytic potential of uncultured microbial communities
living in marine habitats, leading to the discovery of novel enzymes with
industrial potential applications. Metagenomics-guided mining of marine
microorganisms for CUEs involves direct or indirect extraction of total
DNA from an environmental sample (called metagenome) followed with
metagenome cloning, thereby resulting in a metagenomic library. Libraries
can be generated in different formats, either agar plates, microplates, or 3D
semiliquid, depending on the library size and the screening approach to be
used. Subsequent library screening, gene analysis, and overexpression may
lead to the discovery of novel recombinant enzymes that can be produced
at the large scale in sustainable way. Further enzyme characterization pro-
vides useful information on the enzyme properties that are important for
industrial applications.
Approaches for metagenomic library screening can be divided into two
general categories, namely, functional screening and homology screening.
Functional screening relies on the heterologous expression of the cloned
genes in a cultured host to generate phenotypes of interest. This screening
approach includes the use of indicator media containing a substrate for an
enzyme of interest to identify clones expressing the desired enzyme activities
based on the halo formation or color change on agar plate assays. Another
functional screening is based on the use of chromogenic/fluorogenic sub-
strates to identify clones expressing certain enzymes capable of converting
the substrates into in vivo-detectable products. Many potential enzymes
might not be simply detected based on indicator media or chromogenic/
fluorogenic substrates. To overcome such limitation, other methods have
developed to detect the reaction product catalyzed by the enzymes of inter-
est, exemplified by automated TLC and using the reporter gene encoding
GFP placed under the transcription factor. Homology-driven screening is
usually based on PCR or hybridization. Especially, the pool dilution and
whole-cell PCR method developed by Hrvatin and Piel (2007) allows
faster screening of a highly complex metagenomic library. Recent advances
in DNA sequences technologies have enabled fast sequencing of the
metagenome come from a uncultured bacterial community, leading to the
accumulation of metagenomics datasets. Single-cell genomics has recently
emerged as a powerful tool to examine a genome from an uncultured single
bacterial species. Bioinformatic analyses of metagenomic datasets open
exciting chances to identify potential novel genes encoding CUEs.
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[120] Gesta Regum, i. 4.
[121] Haddan and Stubbs, Councils, iii. 483. The names stand
as follows: “+ Ego Offa Rex Dei dono propriam donationis
libertatem signo sanctæ crucis confirmo. + Ego Ecgferth, filius
Regis, consensi. + Signum Hygeberhti Archiepiscopi. + Signum
Ceolulfi Episcopi. + Signum Æthelheardi Archiepiscopi.” Followed
by eight bishops and three abbats.
[122] It has already been noted that Alcuin found it very difficult
to shed tears.
[123] “Ceolmund the duke,” “Ceolmund the minister,” often
appears in the Mercian documents of the time.
[124] Simeon of Durham, under the year 779, has the entry,
Duke Aldred, the slayer of King Ethelred, was slain by Duke
Thorhtmund in revenge for his lord.
[125] This amounts to an official representation of the three
great powers, the West Saxons, the Mercians, and the
Northumbrians.
[126] Haddan and Stubbs, iii. 486.
[127] An Irishman.
[128] From 784 to 819.
[129] Haddan and Stubbs, iii. 487.
[130] We know nothing certain of this person.
[131] We cannot trace his pedigree.
[132] Simeon of Durham says that he committed suicide.
[133] In theory, at least, we know better now.
[134] a.d. 779 to 788.
[135] James ii. 13.
[136] Pet. iv. 17.
[137] He died in 703.
[138] He resigned in 716, and took from the library of
Wearmouth the Codex Amiatinus as a present to the Pope. This
huge and noble codex is now in the Laurenziana, in Florence.
See my Lessons from Early English Church History, pp. 72-75.
[139] See my Theodore and Wilfrith, pp. 106, 124, and for
Acca’s Cross, pp. 257-61.
[140] Bishop of Whithern (Candentis-Casae, Ep. 20, usually
Candidae Casae), 777-789; of Hexham, 789-797.
[141] Writing to an Englishman, Alcuin gives his Anglian name
in its Anglian spelling and without a Latin termination.
[142] See p. 123. The full story is given by Simeon of Durham
under the year 790, meaning 791: “In the second year of Ethelred
(i. e. of his restored sovereignty) Duke Eardulf was captured and
taken to Ripon, and was ordered by the said king to be put to
death outside the gate of the monastery. The brethren carried the
body to the church with Gregorian chants, and placed it in a shed
outside the door. He was found after midnight in the church,
alive.”
[143] In April, 796, the Patrician Osbald was made king by
certain leading men of the nation. But after twenty-seven days he
was deserted by the whole of the royal family and the chief men,
and was put to flight and banished from the kingdom. He escaped
with a few followers to the Isle of Lindisfarne, and thence went by
sea with some of the brethren to the king of the Picts. Sim. Dur.
795.
[144] Slain at Cobre (Corbridge has been suggested), April 18,
796.
[145] The Picts of the east of Scotland.
[146] Matt. xviii. 15, “Go and tell him his fault between thee and
him alone.”
[147] John viii. 34.
[148] Cant. viii. 7.
[149] 1 Tim. v. 20.
[150] Matt. xii. 50. It will be seen that Alcuin does not quote
exactly. The Vulgate has frater et soror et mater.
[151] Insula Sanctorum et Doctorum, p. 272.
[152] No doubt oil specially pure, and vegetable; we may safely
say olive oil, for purposes of chrism. Theodore of Canterbury
informs us (Theodore and Wilfrith, S.P.C.K. p. 180) that
“according to the Greeks a presbyter can ... make the oil for
exorcism and the chrism for the sick, if necessary; but according
to the Romans only a bishop can do so”. Hence the mention of
bishops in the letter of Alcuin. See also page 245, note 2.
[153] In this case Alcuin writes Karli regis; in other cases he
uses the full form Carolus, which comes from rolling the r in
Karlus.
[154] Shekels. On the argument that the didrachma was the
shekel in the New Testament the sicle may be put at 1s. 7½d., but
that gives no idea of its purchasing power then, which was
probably nearer £1. It will be seen that in a later sentence sicles
of pure gold are specified.
[155] See p. 79.
[156] As in year the Anglo-Saxon g was pronounced as y,
hence the name Mayo. In east Yorkshire a gate is still called a
yet.
[157] See Appendix B.
[158] The passage is incomplete, but this is the sense of it.
[159] This is not Lull of Malmesbury, who was so great a help to
Boniface; he died an archbishop in 787.
[160] A presbyter, who succeeded his namesake in the
archbishopric.
[161] We cannot imagine another dignity open to an aged
Archbishop of York to be preferred to that which he already held.
But it is evident that Alcuin referred to his retirement upon an
abbacy, which would set him comparatively free from calls for
exertion.
[162] Eph. v. 23.
[163] It has been supposed that Alcuin refers to some purpose
of bequeathing the library of York to Eanbald II.
[164] Ecclus. vi. 6.
[165] Ethelred of Northumbria was killed and Offa of Mercia
died in this year 796.
[166] James v. 11. Our version would have suited the occasion
better than the Vulgate, “Ye have heard of the patience of Job.”
[167] In the older MSS. in Deo, which has a subtle unintentional
bearing on the controversy with which we are dealing;
unintentional if, as seems certain, we possess MSS. of the
Athanasian symbol of a date earlier than the beginning of the
heresy of Felix.
[168] The punctuation is that of Wattenbach and Dümmler.
Migne puts a full stop after the Pope and another after the
Patriarch: this would seem to make singuli refer to two persons
only, the two bishops. The Roman controversialist makes a
different punctuation, putting a full stop after the Pope and
running the three others together. The whole passage ought to be
read in the Latin without any punctuation. See Appendix C, p.
319.
[169] Ep. 30, a.d. 793.
[170] But see p. 283.
[171] Bede i. 25, “Imaginem Domini salvatoris in tabula
depictam.”
[172] The historian-monk of St. Gallen says that his new eyes
were better than his old ones, both for use and to look at.
[173] Ep. 120, to Arno.
[174] The account which follows is taken from the
contemporary annals of Eginhart.
[175] Under the year 800.
[176] The actual words are given by Baronius, but with a vague
reference to his authority. They are given at length by Milman,
Hist. of Lat. Christianity, ii. 205.
[177] The ordinary word for the crypt or other receptacle of the
body of a saint.
[178] Stephen I was Pope 252 to 257. Another Stephen was
elected on March 14, 752, but died before his consecration. On
March 26, 752, the Stephen here spoken of was elected. He is
thus more properly called Stephen II than Stephen III; and
Stephen IV, who appears in Karl’s time, should be called Stephen
III. Many writers, however, call them Stephen III and Stephen IV.
[179] Labbe, Concil. xii. 539.
[180] Labbe, Concil. xii. 543.
[181] See p. 26.
[182] The district was rich in wine, fruit, flowers, and honey.
[183] Archbishop Albert of York; see p. 84.
[184] Solomon’s Song, iv. 12—v. 2.
[185] Isaiah, lv. 1.
[186] But see p. 209.
[187] There are great difficulties in the way of accepting this
statement of a mission by Karl in 773. The passage calls Albinus
deliciosus ipsius regis, and is quoted by Ducange as an evidence
of the use of the word. It appears to imply a more intimate
acquaintance than at that early date there can have been.
[188] In modern times, better wine is grown near Tours than
near Orleans. The wines of Vouvray, for example, beyond
Marmoutier, are much esteemed. A waiter at Tours concedes that
wine is still grown at Orleans, mais pas de spécialité comme ici.
[189] The spellings of ordinary names are varied in those times
almost at will, and it is interesting to note how often the letter h
plays a part in the variation.
[190] 1 Chron. xxvii. 27.
[191] Song of Songs, ii. 4. Alcuin takes on the whole the
Vulgate version. It will be seen by reference to the text and
margin of the Authorised and the Revised Versions that there is
much variety in the rendering of the Hebrew, especially as
regards the word here rendered “flowers”. The Septuagint gives a
sixth meaning, “perfumes” or “unguents”.
[192] 1 Chron. xxvii. 32. Alcuin makes here an unusually bold
use of Scripture, first in taking to himself the description of David’s
uncle, Jonathan, and then in putting into his mouth a cento of
phrases from Judges xvi. 4, Jer. xlviii. 33, Prov. v. 16.
[193] This song is built up from Song of Solomon vii. 12, v. 1, 2,
vii. 9, vi. 3, and Isa lv. 1.
[194] Song of Songs v. 3.
[195] Luke xi. 5, 7.
[196] 2 Sam. xxi. 1, 2.
[197] This appears to be going beyond a joke.
[198] Prov. xxv. 24.
[199] This is of course not the usually assigned derivation; but it
sounds the more reasonable of the two.
[200] Plate II.
[201] Plate III.
[202] Plate IV.
[203] Multitudo paganorum idolatriis dedita. Per cryptas et
latibula cum paucis Christianis per eumdem conversis, mysterium
solemnitatis diei Dominici clanculo celebrabat.
[204] See p. 221.
[205] For further extracts from Hadrian’s decree, see p. 228.
[206] His last testament is printed by Migne in the Appendix to
the works of Gregory of Tours, columns 1148-51. “Simul et omnes
libros meos praeter Evangeliorum librum quem scripsit Hilarius
quondam Pictavensis sacerdos quem tibi Eufronio fratri et
consacerdoti dilectissimo cum prefata theca do lego volo statuo.”
This theca was one of silver, containing relics of saints, which he
used to carry about with him. Another theca, gilt, was in his chest,
with two chalices of gold and a gold cross made by Mabuin; these
he left to his church.
[207] Gesta Regum, i. 3.
[208] See p. 203.
[209] But see p. 50.
[210] See p. 217.
[211] Printed in Gallia Christiana under Tours. See p. 228.
[212] See p. 217.
[213] It may be helpful to remember that the abbey was
originally outside the ancient Roman city, and its district was
called Martinopolis. The ancient Gallican bishoprics were
bishoprics of cities rather than of dioceses in our wide sense of
the word. This may conceivably have a bearing on the curious
question raised by Hadrian.
[214] See my Constitution of French Chapters, Proceedings of
St. Paul’s Ecclesiological Society, Vol. III, 1895.
[215] Micah v. 5, 6.
[216] James ii. 13.
[217] We know from other sources that this “&c.” meant Most
Serene Augustus, crowned by God, great peace-making
Emperor, Governor of the Roman Empire, by the mercy of God
King of the Franks and of the Lombards.
[218] The emperor irresistibly reminds us of the Eton master
and the boy who complained that his name was not that called for
punishment:—
Sive tu mavis Bōsănquet vocari

Sive Bōsănquet,
Te vapulabo.
[219] That is, Theodulfus, the Bishop of Orleans.

[220] Romans xiv. 4.
[221] 1 Kings xx. 42.
[222] This refers, no doubt, to the immunity of St. Martin’s from
the intervention of the Archbishop.
[223] Eulogias. Wattenbach and Dümmler gloss this cibos.
From its original meaning of the consecrated wafer it came to
mean the pain benit, then any present, and then a salutation.
There is no clue to its special meaning here.
[224] The character of the Latin verse may be gathered from
the closing words of this hexameter, est non laudabile cui nil.
[225] In another poem Theodulf begs Queen Luitgard to send
him some oil of balsam, to enable him to compose and
consecrate cream for chrism. We must suppose that Luitgard had
some special connexion with ports to which balsams were
brought.
Balsameum regina mihi transmitte liquorem,

Quo bene per populos chrismatis unguen eat.
Inde seges crescet tibimet mercedis opimae
Christicolum nomen cum dabit unguen idem.
[226] See p. 33.

[227] That is, a summary, epitome; not as yet a service-book.
[228] Ps. lxx. 14. The Vulgate, which Alcuin quotes, has more
point for his present purpose, adiiciam super omnem laudem
tuam, “I will add Thy praise above all praise.”
[229] Exod. xxiii. 8. Alcuin reads corda sapientium where the
Vulgate has prudentes.
[230] The letter was written in Lent. Easter day in 800 was April
19.
[231] These were Gisla, Charlemagne’s sister, and Rodtruda,
his daughter; see also p. 253.
[232] Adapted from chapters i and ii of Solomon’s Song.
[233]
Nomine pandecten proprio vocitare memento

Hoc corpus sacrum, lector, in ore tuo.
Quid nunc a multis constat bibliotheca dictum
Nomine non proprio, ut lingua pelasga probat.
A pandect was the whole Bible, Old and New Testament, as its
name, “containing everything,” implies. A bibliotheca, like our
word “library,” meant both a room or case where books were
stored, and also the collection of books in the place; hence it
might be used for the pandect, on the ground that it was a
collection of all the books of the Bible.
[234] Wattenbach and Dümmler, 223-4.
[235] See on this point pp. 86-9.
[236] See my Anglo-Saxon Coronation Forms, and the use of
the word Protestant in the Coronation Oath, S. P. C. K.
[237] That is, if the Pope has recovered from the attempt to
blind him and cut out his tongue.
[238] Presumably, if new charges are made against the Pope.
[239] A reference to Pliny’s Natural History, where wolves are
credited with this power; see also Virgil, Ecl. ix. 53, 54.
[240] A reference to Leo’s denial of the charges against him at
Paderborn, and also to St. Peter’s denial. We must credit Alcuin
with having seen that he would be taken to mean that one was as
true as the other. The denial was renewed at Rome, see p. 189.
[241] See p. 208.
[242] St. Martin’s at Tours.
[243] His pupils.
[244] See p. 72.
[245] It is a curious coincidence that the ivory comb found in St.
Cuthbert’s coffin, provided by Westone after the Norman
Conquest, had—as nearly as we can count—sixty teeth, sixteen
large and forty-four small. Alcuin’s comb may have had the same
double row of teeth, with a knob in the shape of a lion’s head
projecting from the ends of the central ivory.
[246] Monumenta Alcuiniana, Wattenbach and Dümmler, p. 63.
[247] Italian Alps, Longmans, 1875, Appendix D, pp. 371-3.
[248] Kemble, Cod. Dipl. ii. 208-62. Coolidge, Swiss Travel,
160. “Perpessus sit gelidis glacierum (and glaciarum) flatibus, et
pennino exercitu malignorum spirituum.”
[249] Gesta Pontificum, Rolls series, pp. 25, 26, 265.
[250] See my Lessons from Early English Church History, pp.
45, 46.
[251] Written from Rome; not preserved.
[252] Leo III.
[253] See p. 281 note.
[254] Leo III, see p. 188.
[255] There were two monasteries with this dedication. One of
these, Iuvavense, was at Salzburg, and probably it is the one to
which reference is made.
[256] See p. 168.
[257] It is probable that he was called Cuckoo from the refrain
of some favourite song of his. The Teutonic name for the “bird of
spring” was not a likely personal name, any more than cuckoo is
with us.
[258] See also Epistle 186 in Appendix A.
[259] Here, and in Ep. 108, to Arno, Alcuin combines two
phrases from the Song of Solomon, v. 7 and 8: “The watchmen
have wounded me,” “I am sick of love.” In the letter to Arno he
appears to quote the actual words of a text in his possession:
vulnerata karitate ego sum; in the present letter he writes caritatis
calamo vulneratus sum. The Vulgate has vulneraverunt me—
amore langueo. See p. 275.
[260] Eginhart in his life of Karl (ch. 25) states that the king
studied grammar under Peter of Pisa, an aged deacon.
[261] This was Angilbertus.
[262] That is, Eginhart, the man skilled in many arts, as was
Bezaleel, the chief architect of the Tabernacle.
[263] See p. 33.
[264] The Wends.
[265] Eginhard tells us under this year 789 that Karl crossed the
Rhine at Cologne with a great army, pushed through Saxony as
far as the Elbe, and brought the Wiltzi to terms. That, he says, is
their name in the Frank tongue. In their own tongue they are
Welatabi.
[266] The Huns, or Avars, had in the previous year invaded Italy
and Bavaria.
[267] See p. 151.
[268] “Amice carissime.”
[269] See my Aldhelm, S.P.C.K., p. 129.
[270] Mansi, Concilia, xiii. 937.
[271] Vienna, 1904.
[272] Cummings, History of Architecture in Italy, ii. 71.
[273] Pertz, Monumenta (Scriptores), ii. 665, 6.
[274] de ista die.
[275] savoir et pouvoir me donne.
[276] chacune.
[277] comme homme.
[278] droit.
[279] faciet.
[280] secundum meum velle.
[281] Concilium Liptinense.
[282] A photograph of this inscription is reproduced at p. 209 of
my Conversion of the Heptarchy.
[283] This must have come very near to being an umbrella.
[284] Dan. xiv. 35, Vulgate.
[285] Bonefatii. This was, of course, the great English
missionary Archbishop of Maintz, martyred at Dorkum in 755.
[286] 1 Cor. xv. 58.
[287] Rom. xii. 2.
[288] Based on 1 Pet. ii. 1.
[289] He was Abbat of Fulda from 780 to 802, when he
resigned the office.
[290] This, no doubt, is the origin of the tradition that Alcuin
wrote the Office for Trinity Sunday. See pp. 20, 173.
[291] Rom. xiv. 5.
[292] It will be observed that no mention is made of a king of
Kent. See p. 91.
[293] See the list on the next pages.
[294] This would indicate that the aula at which they had met
the king and held the council was one of Offa’s outlying manors,
and not his central royal residence.
[295] Supposed, on slight reasoning, to have been held at
Corbridge, see p. 216.
[296] Besides those in the Pope’s list.
[297] Sacerdos. It is uncertain to how late a date sacerdos is to
be rendered bishop.
[298] Wattenbach and Dümmler give only the headings of the
chapters, as here. The chapters themselves will be found in
Haddan and Stubbs, iii. 448-58.
[299] There are many injunctions that priests and others
serving at the altar must wear drawers. There is quite a large
literature on the subject of these garments (femoralia), in which
such of the early fathers as are given to symbolism find symbolic
meanings. They were an essential part of the dress of the
Levitical priesthood (Exod. xxviii. 42, 43).
[300] Probably referring to the practice of tattooing.
[301] Prudentius (Dipt. i. 3) has “Adam” not “humum”.
[302] This was Tilbert, Bishop of Hexham (Augustald) 781-789.
There is no reason of seniority or priority that should make him
sign above the Archbishop. If, as is probable, the Council was
held at Corbridge, in his diocese, he might sign first as bishop of
the place.
[303] Praesul. In the other signatures episcopus is used.
[304] Candens-casa, usually Candida-casa, so named from its
being the first church built of white stone in that region.
[305] Myensis. see p. 156. Aldulf was consecrated in 786, the
year of this Council, by Eanbald, Tilberht, and Hygbald, at
Corbridge. It is on this account that the Germans think the Council
was held at Corbridge. Hexham would equally meet the case, and
better meets the suggestion of a previous note.
[306] Not as yet identified.
[307] It is rather quaint that Sigha should have chosen placido
mente as the phrase to describe his manner of assent to No. 12
above, for two years later he killed King Aelfwald, and he
eventually died by his own hand.
[308] Of Ripon, 786-787.
[309] Some read Alquinum here, and make Alcuin one of the
two lectores.
[310] The text has two forms of this variously spelled name.
[311] Higbert of Lichfield 779-802.
[312] Lindsey 767-796. The Lindisfaras had nothing to do with
Lindisfarne.
[313] Leicester 781-802.
[314] Elmham 786-811, see p. 159.
[315] London 794-801.
[316] Kinbert of Winchester 785-801.
[317] Hendred of Dunwich, 781-789.
[318] Esne of Hereford 781-789.
[319] Tolta of Selsey 781-789.
[320] Rochester 785-803.
[321] Sherborn 766-793.
[322] Worcester 781-798.
[323] “Aimoini monachi, qui antea Annonii nomine editus est,
Historiae Francorum” Lib. V. Parisiis. 1567.
[324] Omitted in the quotation.
INDEX
A
Abrenuntiatio diaboli, 295.
Abulabaz, 289, 324.
Acca, 137.
Adalbert, 2, 27.
Adoptionism, 24, 174.
Adoration of Charlemagne, 190, 191.
Aigulf, 32.
Aimoin, 322.
Albert (York), 16, 53, 80.
Albinus (Alcuin), 15.
Alchfrith, 8.
Alchred, 122.
Alcuin, called Flaccus, 1;
Albinus, 15;
studies Virgil, 2, 11;
conversion, 11;
trained by Ecgbert, 12,
by Albert, 16;
a vision, 18;
ordained deacon, 19;
master of the School of York, 20;
joins Karl, 22;
revisits England, 24;
returns to France, 24;
refutes Felix, 25, 176, 299;
wishes to retire to Fulda, 26;
manner of life, 26;
knowledge of secrets, 2, 29, 30, 33, 34, 223;
extinguishes fire, 37;
writings, 42, 51-3,
poem on York, ch. iv,
lesser poems, 268, 277, 298,
a riddle, 268;
drinks wine, 45,
beer (not English, 267), 45 n.;
interview with the devil, 43;
death, 46, 303;
miracles, 49;
alms for his soul, 224;
his chief dates, 52, 85, 172;
the pallium, 77;
inherits the library of York, 84;
a fisherman, 268;
liturgical work, 260, 308;
singing, 260;
interest in missions, 285-9;
Bibles, 257;
advises reference to Hadrian, 177-9;
settles at Tours, 202;
his styles, 264-9;
mentions Eginhart, 283;
is mentioned by Eginhart, 284;
praised by William of Malmesbury, 52;
described by Theodulf, 45 n., 245.
Alcuin’s letters to:—
Abbat, an, 285.
Adalhard, 264.
Arno, 170, 235, 268, 270, 271, 273-6, 287, 289, 298, 299,
300.
Athilhard, 94, 115, 116, 117.
Beornwin, 98.
Bishop, a, (sanctuary), 234.
Britain, the pontiffs of, 157.
Candida Casa, 301.
Candidus and Nathanael, 231.
Charles (Karl’s son), 248, 250.
Colcu, 150, 286.
Cuckoo, the, 168, 169.
Dunwich and Elmham, 159, 301.
Eanbald I, 161.
Eanbald II, 164, 166, 167.
Eardulf, 141.
Elmham and Dunwich, 159, 301.
Etheldryth, 146, 148.
Fulda, 305.
Gisla and Rotruda, 253.
Hexham, 137.
Hibernia, 153.
Higbald, 132, 170, 287.
Jarrow, 135.
Joseph, 267.
Karl, 117, 191, 202, 208, 238, 247 (?), 254, 287, 300, 301,
302.
Kenulf, 109.
Leo III, 77.
Lindisfarne, 132, 135.
Mayo, 154.
Megenfrid, 288.
Mercian, a, 107.
Offa, 93, 103.
Osbald, 143.
Paulinus, 270.
Pepin, 252.
Peter of Milan, 270.
Pontiffs of Britain, 157.
Remedius, 270.
Rotruda, 253.
Theodulf, 206.
Uulfhard, 205.
Wearmouth, 135.
York, 162.
Aldhelm, 52, 107.
Aldric, 2.
Alfwold, 123.
Alps, 269-72.
Amalgarius, 242.
Anglo-Saxon, Coronation Forms, 261-3.
Anglo-Saxon, Earliest Examples of, 296, 297.
Archpresbyters, 230.
Areida, 151.
Arno, 47, 276.
Aust, 114.
B
Baldhuninga, 151.
Balther, 79.
Bede, letter to Ecgbert, ch. iii.
Bede, Story of, 70.
Beer, 45 n.
Beer, acid in Northumbria, 267.
Benedict of Aniane, 30.
Beornrad, 7.
Bewcastle Cross, 9, 57, 296.
Bibles, Alcuin’s, 257.
Billfrith, 125.
Biscop, 127.
Bishops, their conduct, 55.
Bishops, dioceses too large, 57.
Bishops, election of, 163.
Boniface, 26, 285, 305.
Boniface, his abrenuntiatio diaboli, 295.
Bouulf, 307.
Bremen, 285.
British Museum, Alcuin’s Bible, 257.
C
Ceolfrith, 78.
Charlemagne, see Karl.
Chur, 269.
Coire, 269.
Colcu, 150-3.
Cold in the Alps, 271-3.
Columba, see Rotruda.
Comb, riddle of, 269.
Constantine Copronymus, 201.
Constantine the Great, Donation by, 195, 320.
Constantine VI, 193.
Cormery, 31, 223-8.
Coronation Forms, Anglo-Saxon, 261-3.
Cuckoo, the (Cuculus), Arno’s letter to, 276.
Cuckoo, Alcuin’s lament on, 277.
D
Danes, 126.
Devil, interview of Alcuin with, 43;
of St. Martin, 44.
Dictated letters, &c., 7.
Donation of Constantine, 195, 320, 321.
Drithelme, vision of, 273.
Dunwich, 159, 301.
E
Eadbert, 76, 122.
Eadfrith, 125.
Eanbald I (York), 21, 161.
Eanbald II, 163-9.
Eanred, 124.
Eardulf, 123.
Eata, 79.
Ecgbert (Ireland), 8.
Ecgbert (York), 12, 13, 53, 54, 76.
Ecgfrith, 106.
Eginhard (Bezaleel), 33, 283, 284.
Elephants, 289-92.
Elfwald, 124.
Elmham, 159, 301.
Epternach, 6.
Ethelred I, 123.
Ethelred II, 124.
Ethelwald, 122, 125.
F
Felix, 174-9, 298.
Ferrières, 1, 28.
Frankfort, Council of, 183.
Fredegisus (Fridugisus), 27, 226, 227, 256.
Fulda, 26, Appendix A.
G
George, legate, 310.
Gisla (Lucia), 253;
letter to Alcuin, 254, 256.
Graduale, 260.
Gregory, Pope, the Pastoral Care, 169-71.
H
Hadrian I, 21;
raises Lichfield to an Archbishopric, ch. v;
fears Offa, 92;
to be consulted on a treatise of Felix, 177;
letter to Karl, 323.
Harun al Raschid, 324.
Hereditary descent, 5-7.
Hexham, 137.
Higbald, 132.
Huguenots, 210.
I
Image-worship, 181-3.
Irene, 193.
Itherius, 217, 224-6.
J
Jaenbert of Canterbury, despoiled, ch. v.
Jarrow, 127, 135.
Joseph, Archbishop of Tours, ch. xiv.
K
Karl, 21, 22;
visits Alcuin at Tours, 31;
loved foreigners, 33;
invites Alcuin, 54;
quarrels with Offa, 98;
letters to Offa, 99, 119;
letter to Athelhard, 120;
his visits to Rome, 186-91;
grants to Cormery, 225;
blames the brethren of St. Martin’s, ch. xiv;
letter to Alcuin (sanctuary), 235;
described by Theodulf, 245;
questions to Alcuin, 280-4;
described by Eginhart, 284.
Kenulf, 93, 111.
L
Languages of Carolingian times, 292-6.
Lectores, 317.

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Marine Enzymes Biotechnology

Production and Industrial Applications

Marine Enzymes Biotechnology Production and Industrial

Marine Enzymes Biotechnology Production and Industrial

Seafood Science Advances in Chemistry Technology and

Current Developments in Biotechnology and

Molybdenum and Tungsten Enzymes - Biochemistry 1st

Enzymes in Food Technology Improvements and Innovations

Microbes and Enzymes in Soil Health and Bioremediation

Molybdenum and Tungsten Enzymes - Bioinorganic

MARY ELLEN CAMIRE

First edition 2016

Copyright © 2016 Elsevier Inc. All Rights Reserved

For information on all Academic Press publications

Publisher: Zoe Kruze

In the last decades, progress on the knowledge of marine enzymes has

Advances in Food and Nutrition Research, Volume 78 # 2016 Elsevier Inc. 1

Direct extraction Indirect extraction

Metagenome Marine sediment Large DNA

Bioinformatic analysis, gene overexpression-

Fig. 1 A general scheme of metagenomics for gaining access to commercially useful

by function, homology-driven screening, and metagenome sequencing.

sediment sample is based on low-speed centrifugation as reported by Bakken

as the cell-free form or embedded in low-melting point (LMP) agarose plug

3. METAGENOMIC LIBRARY CONSTRUCTION

Total DNA retrieved from an environmental sample was partially digested

exemplified by the fosmid-based vector pCC1FOS (Epicentre) coupled

HMW metagenomic cos camR

Selected metagenomic Ligation

Clones harboring Clones harboring

Fig. 2 Construction of a metagenomic library using pCC1FOS fosmid vector. Meta-

on the ability of transformants to express genes of interest (Wahler &

by oxygenases to form a phenol or catechol that is further polymerized by

colleagues reported an impressive ultrahigh-throughput functional screen-

samples using 0.22-μm filtration cartridge. Subsequently, PCR amplifica-

Fig. 3 An example of mathematical calculations for the high-throughput screening of a

containing more than 10,000 species (Wooley, Godzik, & Friedberg,

bacteria to degrade biopolymers in HMW dissolved organic matter (DOM).

hampered exploring and exploiting the vast majority of marine microorgan-

Evidence-Based Complementary and Alternative Medicine. 2011, http://dx.doi.org/

Sive tu mavis Bōsănquet vocari

[219] That is, Theodulfus, the Bishop of Orleans.

Balsameum regina mihi transmitte liquorem,

[226] See p. 33.

Nomine pandecten proprio vocitare memento

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