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Table 1 Compared characteristics of the gene candidates (Halo-cellulase 1 and 2) and
the profile halophilic cellulase gene, Hu-CBH1.
After using our HMM to search against our metagenomic data, we found two
candidates that have the potential to function in ionic liquids to break down
cellulose. The first gene, Halo-cellulase 1, matched the HMM the closest. It
contains a cellulase and fibronectin type III-like domain similar to the profile
gene, Hu-CBH1. The second candidate, Halo-cellulase 2, shares homology
to archaeal genes and contains only a cellulase domain.
In this study, we ran a Hidden Markov Model (HMM) against
metagenomic libraries collected in salt flats to find novel halo-cellulase
candidates.
Methods
Results
IntroductionBiofuel, as an alternative fuel source, can be produced by the
enzymatic breakdown of cellulose through ionic liquids and cellulases.
• Substrate: Cellulose
• the most abundant organic compound
• composed of metabolically versatile glucose
• Pre-Treatment: Ionic liquids
• high salt solutions used as an alternative to heat pretreatment in
order to solubilize cellulose for efficient digestion.
• Enzyme: Halophilic Cellulases
• Salt-tolerant enzymes that hasten the digestion of cellulose
• Normal cellulases are unable to work in the ionic liquid’s high
salt environment
Our research seeks to:
• Identify candidate halophilic cellulase (“halo-cellulases”) genes from
metagenomic data libraries
• Insert two strong candidates into the amplification/overexpression
vector, pTA963 (see Fig. 4) via Gibson assembly
• Amplify the expression vectors in E. coli
• Express the novel halo-cellulase candidates in the halophilic
archaeon Haloferax volcanii
• Characterize the halo-cellulase activity with a reducing sugar assay
References and Acknowledgments1. Finn RD, Clements J, Eddy SR (2011) HMMER web server: interactive
sequence similarity searching. Nucleic Acids Research 39:W29 - W37.
2. Eddy SR. (2004) What Is a Hidden Markov Model? Nature Biotechnology
22.10: 1315-316.
3. Allers T, Barak S, Liddell S, Wardell K, Mevarech M (2010) Improved strains
and plasmid vectors for conditional overexpression of his- tagged proteins
in Haloferax volcanii. Appl Environ Microbiol 76:1759–1769.
Special Thanks To: Dr. David Bernick, Dr. Hugh Olsen, Dr. Mark Akeson, Jessica
Kost, Charles Paine, College 9, Crown College, Merrill College
Identification of Novel Halo-Cellulases for Applications in Biofuel
Shadie W. Nimri, Tyler D. Ortega, Christian P. Pettet, Arjun S. Sandhu,
Kaylee C. Walker, David L. BernickUniversity of California, Santa Cruz
Biomolecular Engineering and Bioinformatics
Fig. 1 A wooden post
from metagenomic
sample site A23 in
Fremont, CA,
showing degradation
via ionic liquids
(photo credit: Wade
Dugdale).
Fig. 2 Proposed
future system for the
production of
cellulose-based
biofuel (butanol) in an
ionic-liquid system.1
Fig. 3 Hidden Markov Model
searches utilize already
known genes to create a
profile against which novel
genomic data is statistically
compared.2
Halo-cellulase 1 and 2 will each be
cloned into a modified expression
vector for Haloferax volcanii, pTA963:
• The histidine tag allows proteins
expressed on this vector to be
purified on a cobalt column.
• The E. coli origin of replication
allows the plasmids to be amplified
in the bacterium.
For our following experiment we used PCR
assembly to assemble two gene-block
fragments for Halo-cellulase 2, represented in
lanes 5 & 6. The band lengths for our product
were measured to be about 1200 bp ,as
expected, compared to the DNA Ladder
standard in lane 1.
Fig. 4 Graphical
map of the shuttle
vector, pTA963.3
• Halo-cellulase 1 and 2 will be annealed to the pTA963 expression vector
through Gibson assembly.
• The construct will be transformed into Haloferax volcanii for protein
expression.
• The protein can be purified by binding the histidine tag on the
recombinant protein onto a Ni2+ column.
• The protein will be characterized with a 3,5-dinitrosalicylic acid (DNS)
assay to detect simple sugars (cellulase product).
Future Work
Fig. 5 The assembled Halo-
Cellulase 2 gene quantified
and characterized on an
agarose gel.
Results (cont’d)
The candidates were further analyzed with several bioinformatic tools
including BLAST, Pfam, and EMBOSS tools. Through metagenomic
analysis, potential genes were filtered to result in two strong candidates
for cellulosic enzymatic activity and halotolerance.