Genetic and Structural Characterization of a Thermo-Tolerant, Cold

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Genetic and structural characterization of a thermotolerant, cold-active, and acidic endo-#-1,4-glucanase from Antarctic springtail, Cryptopygus antarcticus Jung Min Song, Seung Kon Hong, Young Jun An, MeeHye Kang, Kwon Ho Hong, Youn-Ho Lee, and Sun-Shin Cha J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05037 • Publication Date (Web): 03 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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Journal of Agricultural and Food Chemistry

Genetic and structural characterization of a thermo-tolerant, cold-active, and acidic endo-β-1,4-glucanase from Antarctic springtail, Cryptopygus antarcticus

Jung Min Song1¶ , Seung Kon Hong2¶, Young Jun An1, Mee Hye Kang1, Kwon Ho Hong3, Youn-Ho Lee1,4*, and Sun-Shin Cha2* 1

Korea Institute of Ocean Science and Technology,787 Haean-Ro, Sangnok-Gu, Ansan 426-

744, Republic of Korea 2

Department of Chemistry & Nano Science, Ewha Womans University, Seoul 03760,

Republic of Korea 3

Institute for Therapeutics Discovery and Development, University of Minnesota,

Minneapolis, 717 Delaware Street SE, Minneapolis, MN 55414, USA 4

University of Science and Technology, 217 Gajung-Ro Yuseong-Gu, Daejeon 305-333,

Republic of Korea

¶

These authors contributed equally to this work.

*

Corresponding author: Dr. Youn-Ho Lee & Prof. Sun-Shin Cha

E-mail address: [email protected] (Y.-H.L) & [email protected] (S.-S. C)

Running title: An acidic, cold-active, but thermo-tolerant endo-β-1,4-glucanase (cellulase) from C. antarcticus

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ACS Paragon Plus Environment


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ABSTRACT: The CaCel gene from Antarctic springtail Cryptopygus antarcticus codes for a

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cellulase belonging to the glycosyl hydrolase family 45 (GHF45). Phylogenetic, biochemical,

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and structural analyses revealed that the CaCel gene product (CaCel) is closely related to

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fungal GHF45 endo-β-1,4-glucanases. The organization of five introns within the open

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reading frame of the CaCel gene indicates its endogenous origin in the genome of the species,

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which suggests the horizontal transfer of the gene from fungi to the springtail. CaCel

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exhibited optimal activity at pH 3.5, retained 80% of its activity at 0~10°C, and maintained a

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half-life of 4 h at 70oC. Based on the structural comparison between CaCel and a fungal

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homolog, we deduced the structural basis for the unusual characteristics of CaCel. Under

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acidic conditions at 50°C, CaCel was effective to digest the green algae (Ulva pertusa),

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suggesting that it could be exploited for biofuel production from seaweeds.

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Keywords: cold-active cellulase; Cryptopygus antarcticus; endo-β-1,4-glucanase; horizontal

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gene transfer; biochemical and structural features; biofuel production from seaweeds

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INTRODUCTION

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Cellulose is the main component of the cell wall in terrestrial plants and marine algae.1 It is

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indispensable for many herbivores to produce cellulases in order to utilize cellulose as a

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major food source. Cellulase is an enzyme that hydrolyzes the 1,4-β-D-glycosidic linkages in

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cellulose polymer. They are commonly divided into two types in terms of their mode of

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cleavage: endo-β-1,4-glucanase (EC 3.2.1.4) and exo-β-1,4-glucanase (EC 3.2.1.91). The

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former cleaves the substrate in the middle of the cellulose polymer while the latter attacks

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cellulose from the ends of the molecule.2 Complete degradation of cellulose into glucose

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residues requires another enzyme, β-glucosidase (EC 3.2.1.21). These enzymes are attracting

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industrial interests since cellulose-derived glucose can be directly converted into biofuel by

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fermentation.3

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Cellulases have been isolated from various organisms, including bacteria, fungi, and some

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metazoans. Animal cellulases were once considered to be the products of the symbiotic

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microorganisms.4 According to recent studies, however, some metazoans including

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nematodes, mollusks, and arthropods are able to produce endogenous cellulases.5-10 Animal

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cellulases are classified into five glycosyl hydrolase families, GHF5, 6, 9, 10 and 45, on the

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basis of amino acid sequence similarities.11 GHF45 enzymes are endo-β-1,4-glucanases

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which have a relatively small molecular mass of 19~25 kDa. These enzymes have broad

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substrate specificities including β-1,3/1,4-glucans (lichenan and barley β-D-glucan) as well as 3



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cellulose.7 GHF45 enzymes from insects, nematodes, and some mollusks show high sequence

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similarity to fungal homologues, which suggests that the genes coding for animal GHF45

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enzymes are acquired by horizontal gene transfer from fungi.7, 10

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Collembola, commonly referred to as springtails, are cryptic arthropods which are usually

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found in top soils and decaying materials. They play an important role in decomposition of

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plant litters and formation of soil microstructures. These animals consume a wide variety of

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foods including decomposed plants, algae, pollens, fungi, diatoms, and bacteria.12 Based on

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food preferences, they are categorized into three feeding groups: (1) phycophages/herbivores

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feeding mainly on lichens, algae, and plant tissues, (2) primary decomposers feeding on

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litters and detritus, and (3) secondary decomposers feeding on fungi.13 Cellulase activities

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have been detected in all three feeding groups.14 However, biochemical and genetic

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characterization of collembolan cellulases has not been fully achieved.

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The Antarctic springtail, Cryptopygus antarcticus Willem (Collembola, Isotomidae) is the

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most abundant, widespread arthropod in the maritime Antarctic region. This organism feeds

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on decomposed plant, pollen, and unicellular algae,15 implying that C. antarcticus may

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contain various carbohydrolases to digest ingested food in the cold environment. We had

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reported two hemicellulose hydrolyzing enzymes, β-1,4-mannanase (CaMan) and endo-β-

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1,3-glucanase (laminarinase, CaLam) of the species which have the typical cold-adapted

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characteristics.16-18 We have also identified an endogenous gene (CaCel) coding for a GHF45 4


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endo-β-1,4-glucanase from the C. antarcticus EST library and characterized the recombinant

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protein (CaCel) produced in Bombyx mori-baculovirus expression systems.19 In this study,

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we analyzed the organization of introns in the CaCel gene and performed phylogenetic

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analyses to propose the horizontal transfer of the gene from a fungus to C. antarcticus.

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Furthermore, we revealed biochemical and structural properties of the recombinant CaCel

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protein produced in Escherichia coli at 10°C and examined the potential usage of CaCel for

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biofuel production through the saccharification of seaweeds.

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MATERIALS AND METHODS Construction of a cDNA Library and Screening of GHF45 Endo-β-1,4-Glucanase

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Gene (CaCel). Previously, we established 2,115 expressed sequence tags (ESTs) from

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Antarctic springtail, C. antarcticus.16-17 In this study, the sequences obtained from these ESTs

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were screened for a cellulase gene by the similarity search of the NCBI database

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(http://www.ncbi.nlm.nih.gov/), Pfam HMM database (http://pfam.sanger.ac.uk/), and the

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ExPASy proteomics database (http://www.expasy.ch/).

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Sequence Comparison and Phylogenetic Analysis. The amino acid sequence translated

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from the CaCel cDNA was compared with those of GHF45 cellulases from animals

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(mollusks, insects, and nematodes), fungi, and insect hindgut protists. The sequences were 5



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aligned using the computer program MUSCLE.20 Phylogenetic trees were reconstructed by

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the maximum likelihood method using PhyML3.021 with the WAG amino acid substitution

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model22 and also by the Bayesian method using Mrbayes23 with the WAG model. For the

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maximum likelihood method, the transition/transversion ratio, the proportion of invariable

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sites, and the gamma distribution parameter were optimized with four substitution categories.

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Bootstrapping was carried out with 1,000 replicates. For the Bayesian method, the number of

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generation was set to 1,000,000; the trees sampled every 100th generation and the posterior

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probability of each node estimated after burning the first 25% of the samples.

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Amplification of the CaCel gene from the genomic DNA. Genomic DNA was isolated

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from the individuals of C. antarcticus using UltraClean forensic DNA kit (MoBio, USA)

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according to the manufacturer’s instruction. The genomic region of CaCel was obtained by

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PCR amplification of the genomic DNA using a pair of primers designed in the signal peptide

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region and in the stop codon region of the cDNA so that the entire open reading frame can be

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amplified: the forward primer, Cant1,4gluF (5’-ATGAAGGTTTTCGTTTTGGCAGCTAT-3’)

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and the reverse primer, Cant1,4gluR (5’-TTAAGGTCCAGGAGTTCTGACGCATT-3’). The

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amplification was carried out with KOD Hot Start DNA polymerase (Novagen, USA) in the

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following reaction: an initial denaturation at 94°C for 3 min, 30 cycles of chain reaction

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(94°C for 40 sec, 50°C for 40 sec, and 72°C for 2 min), and a final extension at 72°C for 10 6


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min. The PCR product was cloned into the pCR2.1-TOPO vector (Invitrogen, USA) and

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sequenced.

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Expression, purification, crystallization, and structure determination. The mature

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protein coding region of the CaCel gene was cloned into pET-28a (+) and then transformed

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into E. coli Rosetta-gami2(DE3) (Novagen, USA). Detailed procedures for expression,

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purification, and crystallization of the recombinant CaCel produced in E. coli have been

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described elsewhere.24-25 In the previous report, the space group of CaCel crystals were

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determined to be P3121. However, during the molecular replacement (MR) with the crystal

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structure of Melanocarpus albomyces cellulase (MaCel) (PDB code: 1OA9) as a search

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model, we noticed that the correct space group was P31 with unit-cell parameters a=b=

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81.714, and c=89.352Å. The crystal volume per unit molecular weight (VM) was determined

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to 2.99Å3Da-1 with a solvent content of 58.84% by volume (Matthews, 1968) since the

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asymmetric unit contained three molecules. MR, model building, and refinement were

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performed by using MolRep,26 Coot,27 and Phenix,28 respectively. The Ramachandran plot of

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the final model with R and Rfree values of 21.9% and 28.9%, respectively, indicates that 95.31%

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of non-glycine residues are in the most favored regions and the remaining 4.69% residues are

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in the allowed region. The refinement statistics are summarized in Supporting Information

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(Table S1). 7



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Assessment of Endo-β-1,4-Glucanase Activity of the Recombinant CaCel.

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Endoglucanase activity of the recombinant CaCel was assessed by measuring the reducing

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sugars (D-glucose equivalents) liberated from the substrate, carboxymethyl cellulose (CMC),

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following the dinitrosalicylic acid (DNS) assay. An enzyme reaction mixture (500 µL) was

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made containing 1% CMC, 50 mM sodium citrate buffer (pH 3.5), and 1.75 µg of the purified

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recombinant CaCel. The mixture was incubated at 40°C for 30 min, and the reaction was

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stopped by addition of 1 mL of DNS reagent. Then, the reaction mixture was boiled for 5 min

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in a water bath and cooled quickly to room temperature. The extent of enzymatic hydrolysis

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of CMC was determined by measuring the absorbance at 540 nm. One unit of enzyme

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activity was defined as the amount of the enzyme liberating reducing sugars equivalent to 1

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µmole of glucose per minute. The enzyme assay was repeated three times each with three

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replicates.

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Characterization of CaCel. The substrate specificity of CaCel was evaluated with the

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following substrates: 1% lichenan (w/v), 1% barley β-1,4-D-glucan (w/v), 1% Avicel (w/v), 1%

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soluble starch (w/v) (Sigma-Aldrich, USA). Lichenan (P-LICHN) and barley β-1,4-D-glucan

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(P-BGBL) were purchased from Megazyme. Starch (S9765), Avicel (11365) and CMC

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(C5678) were obtained from Sigma-Aldrich. The enzyme activity was measured by the 8


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dinitrosalicylic acid (DNS) method as described in the previous paragraph. The optimum temperature was determined by measuring the enzyme activity at various

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temperatures from 0 to 80°C by the DNS method using CMC as the substrate. Thermal

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stability was assessed by the residual activity of the enzyme (17.5 µg/mL) at 40°C after

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incubation of CaCel at 50°C to 90°C in 50 mM sodium citrate buffer (pH 3.5) for 1-8 h. The

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optimum pH was determined by measuring the enzyme activity at pH ranges of 2.0 to 10.0

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using the following 50 mM buffers: sodium citrate (pH 2.0-4.0), sodium acetate (pH 4.0-5.0),

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sodium phosphate (pH 5.0-7.0), Tris-HCl (pH 7.0-9.0), and glycine-NaOH (pH 9.0-10.0).

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Data were obtained from three independent experiments each with triplicates.

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The reaction mechanism of CaCel was characterized by assessing the major digested

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products of cellooligosaccharides including cellobiose (G2), cellotriose (G3), cellotetraose

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(G4), and cellopentaose (G5) as well as carboxymethyl cellulose (CMC). The digestion

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reaction was carried out in the mixture of 0.1 mL containing 1.75 µg CaCel and 0.1 mg

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substrate in 50 mM sodium citrate buffer (pH 3.5). The reaction mixture was incubated at

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40oC for 5 min to 60 min. After incubation, 10 µL of the reaction mixture was applied to the

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TLC plate (silica gel-coated plate Type K6F, Whatman UK) and developed by a mixture of

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solvents (the volume ratios of n-butanol, acetic acid, and water; 2:2:1). The liberated sugars

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were visualized by putting the TLC plate in a staining solution containing 3 g 1-naphtyl-

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ethylendiamine, 50 mL sulfuric acid, and methanol to 1 L, followed by heating at 110°C for 9



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10 min

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Enzymatic Hydrolysis of the Green Algae, Ulva pertusa by CaCel. One gram of

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powdered Ulva pertusa was suspended in 100 mL distilled water in a 250 mL Erlenmeyer

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flask with adjustment of pH 3.5 by 1% sulfuric acid solution. After addition of 1 mg CaCel,

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the mixture was incubated at 50°C for 9 h with shaking at 100 rpm. An aliquot was taken

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during the reaction at regular intervals and the reducing sugars liberated from the seaweed

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were measured by the DNS assay. The digested products were visualized by the thin layer

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chromatography (TLC) method as described in the previous paragraph.

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RESULTS A collembolan endo-β β-1,4-glucanase gene from Antarctic springtail. Through the

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analysis of 2,115 ESTs obtained from Antarctic springtail, C. antarcticus, one clone was

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identified with high similarity to previously reported cellulase genes of the GHF45 family

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(endo-β-1,4-glucanase). The clone contained the full-length cDNA sequence which was

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subsequently designated as CaCel (GenBank accession number: FJ648735). It consists of 799

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nucleotides with an open reading frame of 678 nucleotides coding for a 225-residue protein

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(Figure 1). The amino acid sequence includes a hydrophobic signal peptide of 16 amino acids

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which was identified by the SignalP3.0 program.29 The sequences in the 5’-untranslated 10


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region (5’-UTR) and the 3’-untranslated region (3’-UTR) are 18 bp and 103 bp in length,

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respectively. The 3’-untranslated region includes a putative polyadenylation signal sequence

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(AAAATA) and a poly-A tail. A Blast search of homologous sequences in other collembolan

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species through the EST libraries in Collembase database

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(http://www.collembase.org/index.html) resulted in a partial fragment of cDNA from an arctic

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springtail, Onychiurus arcticus, which exhibits 65.8% amino acid sequence identity to CaCel

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(Figure 2).

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According to a multiple sequence alignment, CaCel is closely related with cellulases of

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fungi including Syncephalastrum racemosum (64% identity) and Humicola grisea (55%

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identity) which belong to GHF45 subfamily I with seven disulfide bonds (Figure 2). In

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contrast, CaCel shows only 27-30% sequence identities to mollusk cellulases which belong to

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GHF45 subfamily II with four disulfide bonds. Phylogenetic analysis confirmed that CaCel

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and its homologue in O. arcticus make a distinct clade in the tree of GHF45 enzymes which

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then clusters with cellulases of fungi such as S. racemosum and Rhizopus oryzae (Figure 3).

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Intron organization in the CaCel gene. To determine whether CaCel originated from

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the genome of C. antarcticus or from the genome of any symbiotic fungi, we examined the

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gene structure of CaCel. The genomic DNA was extracted from the individuals of C.

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antarcticus and used for PCR amplification of the CaCel gene. The PCR reaction resulted in 11



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a 952-nucleotide long DNA fragment (CaCel-gDNA, GenBank accession number: FJ648736)

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whose sequences were identical to the CaCel cDNA sequence with insertions of five introns

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(Figure 1A and 1B). The introns are 58, 52, 51, 58, and 55 nucleotides long, respectively, and

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use the canonical eukaryotic splicing sequences, GU at the 5’-splicing donor site and AG at

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the 3’ splicing acceptor site, except the fifth intron which uses GA instead of GU at the 5’-

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splicing donor site (Table 1). The intron positions are marked by arrows in the cDNA

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sequence in Figure 1A.

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Biochemical Characterization of the Recombinant CaCel Protein. CaCel was

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successfully expressed as a soluble protein maintaining the catalytic activity using the pET-

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28a (+) and E. coli Rosetta-gami2(DE3) expression system (Novagen, USA). A significant

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amount of active enzyme was obtained when the transformed cells were cultured for 7 days at

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the incubation temperature as low as 10°C.24 The recombinant enzyme hydrolyzed CMC with

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the specific activity, 35.8 U/mg at 40°C. It also digested lichenan (20.5 U/mg) and β-1,4-D-

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glucan (13.9 U/mg) while it did not show any enzymatic activity towards crystalline cellulose

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(Avicel), soluble starch, xylan, mannan, and laminarin. From CMC digestion, it produced

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cellobiose (G2), cellotriose (G3), and cellotetraose (G4) as the major products (Figure 4). To

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investigate the mode action of CaCel, the time-course hydrolysis pattern of

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cellooligosaccharides (G2 to G6) was monitored by using thin layer chromatography. 12


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Cellohexaose was completely hydrolyzed into cellotetraose and cellobiose (plus traces of

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cellotriose) within 5 min. Complete hydrolysis of cellopentaose to cellobiose and cellotriose

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was observed after 30 min and cellotetraose was partially degraded into cellobiose. CaCel

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displayed no hydrolytic activity against cellobiose and cellotriose (Figure 4).

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The optimum pH for the CaCel activity was measured to be 3.5 in the standard method

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using CMC as a substrate (Figure 5A). More than 60% of its activity was maintained at pH

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2.0~7.0. The optimum temperature for the CaCel activity was 40oC, and 60%~80% of the

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maximum activity was retained even at 0°C ~10°C (Figure 5B). The enzyme also displayed

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40% of its maximum activity at as high as 80°C. In a thermostability test, the enzyme

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remained active at 60°C for over 8 h without loss of any enzyme activity. Approximately 50%

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of the activity was still maintained 4 h after incubation at 70°C (Figure 5C). The enzyme

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activity was not affected by metal ions except Mn2+ which reduced the activity by 40%-50%

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(Figure 6). No significant change in the enzymatic activity was observed in response to 1 mM

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EDTA, suggesting that metal ions might not be involved in the catalytic reaction of CaCel.

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Structure of CaCel. The CaCel models of the three molecules (A, B, and C) in the

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asymmetric unit consist of residues 1-208 corresponding to the whole region of CaCel except

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the signal peptide. The three CaCel structures are nearly identical with root mean square

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deviations of ~0.296 Å for all Cα atoms. Therefore, molecule A is exploited for structural 13



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description in this study. Like other cellulases belonging to GHF45 subfamily I, the CaCel

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structure with seven disulfide bonds is built up on the basis of a six-stranded β-barrel domain

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and two connecting domains (CD-I and -II) located between strands in the barrel (Figure 7A).

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CD-I (residues 12-68) is located between strands 1 and 2, and this region is mostly composed

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of random coils except a short strand. This domain has two intradomain disulfide bonds

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(Cys15-Cys50 and Cys34-Cys58), and Cys14 and Cys19 also form additional disulfide bonds

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with Cys136 in CD-II and Cys87 in the connecting loop between strands 3 and 4, respectively

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(Figure 7A). It is highly likely that the four disulfide bonds contribute to the conformational

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stability of this random coil domain. CD-II (residues 125-184) is located between strands 5

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and 6 and has three helices that are involved in the formation of two disulfide bonds

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including the above-mentioned Cys14-Cys136 bond. The second disulfide bond in this

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domain is formed between Cys160 in helix 4 and Cys171 in helix 5 (Figure 7A). Cys193 and

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Cys203 in the C-terminal region of CaCel (residues 192-206) form two disulfide bonds with

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Cys88 and Cys90 in strand 4, respectively. The active site is situated at the interface of the

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barrel, CD-1, and CD-II domains, and harbors two catalytic aspartate residues, Asp13 and

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Asp122 (Figure 7A).

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Saccharification of the green algae, Ulva pertusa by CaCel. Potential usage as an endo-β-1,4-glucanase of CaCel for saccharification of seaweed was examined by measuring 14


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liberated sugars from the green algae Ulva pertusa when it was treated with the enzyme. After

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incubation of 1 gram powdered seaweed with 1mg CaCel for 9 h, approximately 80 mg of

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sugar was liberated (Figure 8A), and half of them were detected just after one hour of

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treatment. The digested products were visible in TLC assay of the reaction mixture (Figure

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8B).

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DISCUSSION Berg and coworkers reported that most of the collembolan species show cellulase

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activities digesting cellulosic materials regardless of their food preferences.14 However, there

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had been no report on isolation and characterization of cellulases from these species. We have

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identified an endogenous endo-β-1,4-glucanases gene (CaCel) through the analysis of EST

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library of C. antarcticus and succeeded in producing its recombinant protein using the E. coli

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expression system.24 The recombinant CaCel exhibited the highest hydrolytic activity (35.8

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U/mg) at 40°C toward CMC, a typical β-1,4-glucan substrate. It also hydrolyzed β-1,3/1,4-

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glucans such as lichenan and barley-β-D-glucan with specific activities of 20.5 U/mg and

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13.9 U/mg, respectively. The minimal length of digestible oligosaccharide substrates for

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CaCel was determined to be cellotetraose with the end product being cellobiose (Figure 4).

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The major hydrolysates of cellopentaose and cellohexaose were cellobiose and cellotriose,

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indicating that CaCel functions as an endo-β-1,4-glucanase (EC 3.2.1.4). 15



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Although CaCel originated from Antarctic springtail, phylogenetic analysis revealed its

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close relationship with fungal cellulases belonging to GHF45 subfamily I (Figure 3). CaCel

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shows 55–64% sequence identities to those of fungal cellulases whereas the sequence identity

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between CaCel and mollusk cellulases is only 27-30%. Structural homologue search using

269

the Dali server30 ascertained its close resemblance to fungal cellulases from Melanocarpus

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albomyces and Humicola insolens. According to the time course hydrolyzing experiments

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analyzed by TLC, CaCel exhibited higher activity toward cellohexaose than cellopentaose

272

and cellotetraose (Figure 4). Consistently, the catalytic efficiency (kcat/Km) of the homologous

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H. insolens cellulase towards cellohexaose is much higher than its kcat/Km values towards

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cellopentaose and cellotetraose.31

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The highest structural homologue of CaCel is the M. albomyces cellulase (MaCel)

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(PDB code: 1OA9, r.m.s.d. = 1.0 Å for all Cα atoms, and Z-score = 35.0). The sequence

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similarity and identity between CaCel and MaCel is 58.64 and 47.73%, respectively. The

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active site of MaCel has been proposed to consist of six subsites (+2, +1, -1, -2, -3, and -4

279

subsites) that interact with six glucose units.32 CaCel is virtually identical to MaCel in terms

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of the length, depth, and width of the active site (Figure 7B), strongly suggesting that its

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active site is also composed of six subsites. Since the cleavage occurs between -1 and +1

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subsites, the existence of six subsites in CaCel is in accord with the observation that the

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preferred substrate is cellohexaose and the final products are either cellobiose or cellotriose 16


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(Figure 4). It is notable that the -2 and -3 subsites accommodate cellobiose in the crystal

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structure of the MaCel/cellobiose complex,32 explaining why cellobiose cannot be substrates

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of enzymes with six subsites.

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The high sequence similarity between CaCel and fungal cellulases can be interpreted

288

that the CaCel gene comes from a symbiotic fungus associated with Antarctic springtail. In

289

fact, a parasitic fungus, Paecilomyces antarcticus, was isolated from the body of C.

290

antarcticus.33 However, our analysis of exon-intron organization in the CaCel gene supports

291

its endogenous origin from the Antarctic springtail genome. The CaCel gene has five introns

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within the coding region (Figure 1B) whereas the cellulase gene of the fungus M. albomyces

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contains only two introns at different positions (Table 1).34 Moreover, two genes isolated

294

from the fungus Rhizopus oryzae ,35 whose products make a close cluster with CaCel in the

295

phylogenetic tree (Figure 3), are known to have no intron. Recently, Palomares-Rius et al.

296

also revealed eleven intron positions from 289 fungal partial DNA fragments coding for

297

GHF45 cellulases. Most of the amplified fragments contained only one intron in their DNA

298

fragments. 43 fragments had one intron at position 6, and 15 amplified sequences had one

299

intron at position 11.36 The positions of the eleven introns found in fungal sequences are

300

different from intron positions of the CaCel gene (Figure 2). The disparity in intron

301

organization and the structural similarity between CaCel and fungal endo-β-1,4-glucanase

17



302

(cellulase) genes suggest that CaCel is currently an endogenous gene of C. antarcticus after

303

horizontal gene transfer from a fungus.

304

CaCel shows the highest sequence identity (65.8%) to the partial cellulase sequence of

305

an Arctic springtail O. arcticus identified from Collembase EST libraries (Figure 2). The two

306

collembolan cellulases make a distinct cluster in the molecular phylogeny of GHF45 family,

307

having fungal cellulases as neighboring clades (Figure 3). The Arctic springtail O. arcticus

308

and the Antarctic springtail C. antarcticus are distantly related species, belonging to the same

309

taxonomic group Arthropleona within Collembola. The close phylogenetic relationships of

310

cellulases between the two distantly-related collembolan species imply that the horizontal

311

gene transfer of the cellulase gene from fungi to collembolan species would have occurred

312

early in the evolution of the springtails. Another example of horizontal gene transfer of fungal

313

β-1,4-glucanase was reported from the pinewood nematode Bursaphelenchus xylophilus7 in

314

which three GHF45 endo-β-1,4-glucanase genes of B. xylophilus (BxENG 1 to 3) are known

315

to be closely related with a fungal homologue.

316

The recombinant CaCel is unique in that it is both cold-active and thermo-tolerant. In

317

general, cold-active enzymes show a high level of activity at low temperatures compared to

318

their mesophilic homologues and become thermo-labile at high temperatures.37 The

319

mannanase and laminarinase from C. antarcticus are also cold-active enzymes that are

320

thermolabile.16-17 Unlike other cold-active enzymes, however, CaCel is thermo-tolerant 18


Page 18 of 41

Page 19 of 41


321

(Figure 5C). An enzyme with a similar unusual property was reported from the blue mussel,

322

Mytilus edulis.10 The 19 kDa endo-β-1,4-glucanase of the mussel whose phylogenetic

323

relationship to CaCel is distant (Figure 3) is active at 0°C with 55-60% of its maximum

324

activity, but it withstands 100oC for 10 min without loss of enzymatic activity (Table 2).

325

Another interesting biochemical property of CaCel is its optimum pH 3.5. It has the lowest

326

optimum pH value among known GHF45 endo-β-1,4-glucanases (Table 2). Most fungal34-35,

327

38

328

CaCel maintained about 80% of its maximum activity at pH 2.5 and more than 60% even at

329

pH 2.0 (Figure 5A)

330

and animal cellulases7, 10, 39-40 have the optimum activity at pH 5.0~7.0. Furthermore,

Since MaCel is a thermostable enzyme, the high resemblance in both primary and

331

tertiary structures between CaCel and MaCel provides a rationale for the thermostability of

332

CaCel. In spite of the structural homology and the common thermostability, MaCel is not

333

active at low temperatures and its optimum pH value is 7.0. Therefore, MaCel is an ideal

334

target for structural comparison to reveal structural features of CaCel associated with its

335

activity at low temperatures and acidic pH. Superposed structures of CaCel and MaCel

336

revealed local conformational disparities in the context of overall resemblance (Figure 7B).

337

Among them, conformational differences observed in residues 119-122 containing β6 and the

338

glycine-rich loop (residues 113-118) are noteworthy. In CaCel, the side chains of His120 and

339

Asp122 face the glycine-rich loop while their equivalent histidine and aspartate residues in 19



340

MaCel have opposite side-chain orientations (Figure 7C). The glycine-rich loops also have

341

different conformations, which can be attributed to the fact that CaCel has a distinct amino

342

acid sequence with one less glycine residue compared to MaCel (Figure 7C). In CaCel, the

343

glycine-rich loop forms favorable contacts with the side chain of His120 (Figure 7D). In

344

particular, the side chain of His120 forms a hydrogen bond with the carbonyl oxygen of

345

Ala117. In contrast, the side chain of His120 in CaCel clashes with the glycine-rich loop of

346

MaCel in the superposed structures (Figure S1), which suggests that the side-chain

347

orientation of His120 is affected by the conformation of the glycine-rich loop.

348

Surface representation revealed the tunnel-like architecture of the active site of CaCel

349

(Figure 7E), for which the glycine-rich loop of CaCel bringing Ser114 close to Ile132 on the

350

opposite side of the active site cleft is responsible. In MaCel where the glycine-rich loop has

351

a different conformation and Ser114 is replaced by Gly112, such a close positioning cannot

352

be made (Figure 7E). In addition to the tunnel-like shape, the active site of CaCel is different

353

from that of MaCel in terms of its amino acid composition. Among residues lining the active

354

site, Gln47, Asn51, and Asn111 in CaCel are replaced by valine, glutamate, and serine,

355

respectively, in MaCel (Figure 7B). To be active at low temperatures, CaCel should lower the

356

activation energy through the efficient stabilization of the transition state. Consequently, it is

357

likely that the active site of CaCel is effective in stabilizing the transition state even at low

358

temperatures, which cannot be achieved by the active site of MaCel. 20


Page 20 of 41

Page 21 of 41

359


It has been proposed that Asp10 and Asp120 in MaCel corresponding to Asp13 and

360

Asp122 in CaCel (Figure 7B) function as the catalytic base and acid, respectively.32 The

361

appropriate protonation state of catalytic residues, which is determined by their pKa values at

362

a given pH, is critical for the general acid-base catalysis. Therefore, the disparity in optimum

363

pH between CaCel and MaCel is probably related to the disparity in the pKa values of the

364

catalytic aspartate residues. The local environment and the side-chain conformation of Asp13

365

in CaCel are identical to those of Asp10 in MaCel (Figure 7B). However, the side-chain

366

conformation and the local hydrogen-bonding network of Asp122 in CaCel are different from

367

those of Asp120 in MaCel (Figure 7C). The side chain conformation of Asp122 seems to be

368

affected by the side chain conformation of His120. In the superposed structures between

369

CaCel and MaCel, Asp122 of CaCel clashes with His118 of MaCel that corresponds to

370

His120 of CaCel (Figure 7C). As described above, the conformation of the glycine-rich loop

371

affects the side chain conformation of His120 (Figure 7D and S1). Consequently, the

372

conformations of the glycine-rich loop, His120, and Asp122 are correlated with each other.

373

Asp122 in CaCel forms a hydrogen bond with the side chain hydroxyl group of Thr6 while

374

the corresponding Asp120 in MaCel is connected to Thr6 and His118 by a hydrogen-bonding

375

network (Figure 7C). Therefore, the pKa values of Asp120 in MaCel and Asp122 in CaCel

376

might be different, conferring different pH optima on the two homologous enzymes.

377

Seaweeds are considered as a source of biomass for biofuel production.3 Procedure for 21



378

biofuel production from seaweeds contains the saccharification of seaweeds through

379

hydrolysis. In general, acid-hydrolysis is more efficient than enzyme hydrolysis in which

380

conversion yield becomes more efficient only when it is combined with acid hydrolysis.41 For

381

this reason, the acidic, cold-active, thermo-stable enzyme such as CaCel becomes useful in

382

the hydrolysis of seaweeds. Indeed, we found that CaCel could digest the green algae (Ulva

383

pertusa) at the acidic condition and produce small oligosaccharides (Figure 7A and 7B),

384

revealing the possibility of CaCel application to the saccharification reaction of seaweeds.

385

22


Page 22 of 41

Page 23 of 41


386

ASSOCIATED CONTENT

387

Supporting information

388

The Supporting information is available free of charge on the ACS Publication website.

389

Data collection and refinement statics and a figure showing the interaction between

390

His120 of CaCel and the glycine-rich loop of MaCel

391

Accession Codes

392

The atomic coordinates and structural factors of the final model have been deposited in the

393

Protein Data Bank with the accession code 5H4U.

394

395

AUTHOR INFORMATION

396

Funding

397

This study was supported by the KIOST in-house programs (PE99413, PE98933, and

398

PO00110), the National Research Foundation of Korea Grants (NRF-

399

2015R1A2A2A01004168 and NRF-2015M1A5A1037480) and a grant from the Marine

400

Biotechnology Program (PJT200620) funded by the Ministry of Oceans and Fisheries, Korea.

401

402

ACKNOWLEDGMENTS

403

We thank the beamline staffs at beamline 17A at the Photon Factory, Japan, for support with

404

the data collection. 23



405

REFERENCES

406

(1) Roberts, A. W.; Roberts, E. M.; Delmer, D. P. Cellulose synthase (CesA) genes in the

407

green alga Mesotaenium caldariorum. Eukaryotic Cell 2002, 1, 847-855. (2) Wilson, D. B.; Irwin, D. C., Genetics and Properties of Cellulases. In Recent Progress

408 409 410 411 412 413 414 415 416 417

in Bioconversion of Lignocellulosics, Springer Berlin Heidelberg: Berlin, Heidelberg, 1999, 1-21. (3) Taylor, G. Biofuels and the biorefinery concept. Energy Policy 2008, 36, 4406-4409. (4) Bayer, E. A.; Chanzy, H.; Lamed, R.; Shoham, Y. Cellulose, cellulases and cellulosomes. Curr. Opin. Struct. Biol. 1998, 8, 548-557. (5) Watanabe, H.; Noda, H.; Tokuda, G.; Lo, N. A cellulase gene of termite origin. Nature 1998, 394, 330-331. (6) Suzuki, K.; Ojima, T.; Nishita, K. Purification and cDNA cloning of a cellulase from

419

abalone Haliotis discus hannai. Eur. J. Biochem. 2003, 270, 771-778. (7) Kikuchi, T.; Jones, J. T.; Aikawa, T.; Kosaka, H.; Ogura, N. A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus.

420

FEBS Lett. 2004, 572, 201-205.

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(8) Linton, S. M.; Greenaway, P.; Towle, D. W. Endogenous production of endo-β-1,4glucanase by decapod crustaceans. J. Comp. Physiol. B 2006, 176, 339-348. (9) Nishida, Y.; Suzuki, K.; Kumagai, Y.; Tanaka, H.; Inoue, A.; Ojima, T. Isolation and

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primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus.

425

Biochimie 2007, 89, 1002-1011. (10) Xu, B.; Hellman, U.; Ersson, B.; Janson, J. C. Purification, characterization and

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426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442

amino-acid sequence analysis of a thermostable, low molecular mass endo-β-1,4-glucanase from blue mussel, Mytilus edulis. Eur. J. Biochem. 2000, 267, 4970-4977. (11) Cantarel, B. L.; Coutinho, P. M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat, B. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009, 37, D233-238. (12) Saur, É.; Ponge, J.-F. Alimentary studies on the Collembolan Paratullbergia callipygos using transmission electron microscopy. Pedobiologia 1988, 31, 355-379. (13) Chahartaghi, M.; Langel, R.; Scheu, S.; Ruess, L. Feeding guilds in Collembola based on nitrogen stable isotope ratios. Soil Biol. Biochem. 2005, 37, 1718-1725. (14) Berg, M. P.; Stoffer, M.; van den Heuvel, H. H. Feeding guilds in Collembola based on digestive enzymes. Pedobiologia 2004, 48, 589-601. (15) Broady, P. A. Feeding studies on the collembolan Cryptopygus antarcticus Willem at Signy Island, South Orkney Islands. Br. Antarct. Surv. Bull. 1979, 48, 37-46. (16) Song, J. M.; Nam, K. W.; Kang, S. G.; Kim, C. G.; Kwon, S. T.; Lee, Y. H. Molecular cloning and characterization of a novel cold-active β-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 24


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2008, 151, 32-40. (17) Song, J. M.; Nam, K.; Sun, Y. U.; Kang, M. H.; Kim, C. G.; Kwon, S. T.; Lee, J.; Lee, Y. H. Molecular and biochemical characterizations of a novel arthropod endo-β-1,3-glucanase from the Antarctic springtail, Cryptopygus antarcticus, horizontally acquired from bacteria.

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Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 2010, 155, 403-412. (18) Kim, M. K.; An, Y. J.; Song, J. M.; Jeong, C. S.; Kang, M. H.; Kwon, K. K.; Lee, Y.

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H.; Cha, S. S. Structure-based investigation into the functional roles of the extended loop and

450

substrate-recognition sites in an endo-β-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus. Proteins 2014, 82, 3217-3223. (19) Hong, S. M.; Sung, H. S.; Kang, M. H.; Kim, C. G.; Lee, Y. H.; Kim, D. J.; Lee, J. M.;

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Kusakabe, T. Characterization of Cryptopygus antarcticus endo-β-1,4-glucanase from Bombyx mori expression systems. Mol. Biotechnol. 2014, 56, 878-889. (20) Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792-1797. (21) Guindon, S.; Lethiec, F.; Duroux, P.; Gascuel, O. PHYML Online--a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res. 2005, 33, W557559. (22) Whelan, S.; Goldman, N. A general empirical model of protein evolution derived from

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multiple protein families using a maximum-likelihood approach. Mol. Biol. Evol. 2001, 18, 691-699. (23) Huelsenbeck, J. P.; Ronquist, F. MRBAYES: Bayesian inference of phylogenetic trees.

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Bioinformatics 2001, 17, 754-755.

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(24) Song, J. M.; An, Y. J.; Kang, M. H.; Lee, Y. H.; Cha, S. S. Cultivation at 6-10°C is an effective strategy to overcome the insolubility of recombinant proteins in Escherichia coli.

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Protein Expr. Purif. 2012, 82, 297-301. (25) An, Y. J.; Kim, M.-K.; Song, J. M.; Kang, M. H.; Lee, Y.-H.; Cha, S.-S. Vapor batch crystallization and preliminary X-ray crystallographic analysis of a cold-active endo-β-1,4glucanase that was produced through the cold temperature protein expression. Biodesign 2015, 3, 138-142. (26) Vagin, A.; Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 22-25. (27) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot.

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Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486-501. (28) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution.

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Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213-221.

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(29) Bendtsen, J. D.; Nielsen, H.; von Heijne, G.; Brunak, S. Improved prediction of signal

482

peptides: SignalP 3.0. J. Mol. Biol. 2004, 340, 783-795. (30) Holm, L.; Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res.

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2010, 38, W545-549. (31) Schou, C.; Rasmussen, G.; Kaltoft, M. B.; Henrissat, B.; Schulein, M. Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases. Eur. J. Biochem. 1993, 217, 947-953. (32) Hirvonen, M.; Papageorgiou, A. C. Crystal structure of a family 45 endoglucanase from Melanocarpus albomyces: mechanistic implications based on the free and cellobiosebound forms. J. Mol. Biol. 2003, 329, 403-410. (33) Bridge, P. D.; Clark, M. S.; Pearce, D. A. A new species of Paecilomyces isolated from the Antarctic springtail Cryptopygus antarcticus. Mycotaxon 2005, 92, 213-222. (34) Miettinen-Oinonen, A.; Londesborough, J.; Joutsjoki, V.; Lantto, R.; Vehmaanperä, J. Three cellulases from Melanocarpus albomyces for textile treatment at neutral pH. Enzyme Microb. Technol. 2004, 34, 332-341. (35) Murashima, K.; Nishimura, T.; Nakamura, Y.; Koga, J.; Moriya, T.; Sumida, N.; Yaguchi, T.; Kono, T. Purification and characterization of new endo-1,4-β-D-glucanases from Rhizopus oryzae. Enzyme Microb. Technol. 2002, 30, 319-326. (36) Palomares-Rius, J. E.; Hirooka, Y.; Tsai, I. J.; Masuya, H.; Hino, A.; Kanzaki, N.; Jones, J. T.; Kikuchi, T. Distribution and evolution of glycoside hydrolase family 45 cellulases in nematodes and fungi. BMC Evol. Biol. 2014, 14, 69. (37) Feller, G.; Gerday, C. Psychrophilic enzymes: hot topics in cold adaptation. Nat. Rev. Microbiol. 2003, 1, 200-208. (38) Wonganu, B.; Pootanakit, K.; Boonyapakron, K.; Champreda, V.; Tanapongpipat, S.; Eurwilaichitr, L. Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris. Protein Expr. Purif. 2008, 58, 78-86. (39) Lee, S. J.; Lee, K. S.; Kim, S. R.; Gui, Z. Z.; Kim, Y. S.; Yoon, H. J.; Kim, I.; Kang, P. D.; Sohn, H. D.; Jin, B. R. A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: gene structure, expression, and enzymatic activity. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 2005, 140, 551-560. (40) Li, Y. H.; Guo, R.; Yin, Q. Y.; Ding, M.; Zhang, S. L.; Xu, G. J.; Zhao, F. K.

515

Purification and characterization of two endo-β-1,4-glucanases from mollusca, Ampullaria crossean. Acta Biochim. Biophys. Sin. (Shanghai) 2005, 37, 702-708. (41) Choi, D.; Sim, H. S.; Piao, Y. L.; Ying, W.; Cho, H. Sugar production from raw

516

seaweed using the enzyme method. J. Ind. Eng. Chem. 2009, 15, 12-15.

514

517 26


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518


Figure Legends

519 520

Figure 1. Genetic information on an endo-β-1,4-glucanases gene (CaCel) of the Antarctic

521

springtail, C. antarcticus. (A) Full-length cDNA sequence and its deduced amino acid

522

sequence. The putative signal sequence for 16 residues is underlined. The stop codon is

523

indicated by a hyphen and the polyadenylation signal by a double underline. The canonical

524

endoglucanase catalytic sites are represented by rectangles. The positions of each intron are

525

indicated by arrows above the sequence. (B) Diagrams of the cDNA and genomic structure of

526

CaCel. Open and closed boxes represent exons and introns, respectively. The numbers

527

indicate sizes of cDNA, ORF, the exons, and introns.

528

529

Figure 2. Multiple alignments of the amino acid sequences of CaCel and GHF45 endo-β-1,4-

530

glucanases. Residues conserved between CaCel, and the other endo-β-1,4-glucanases are

531

marked in black. The numbers on the left side of the sequence indicate the amino acid

532

position of each protein. Cysteine residues involved in disulfide bond formation are

533

numbered in a pair. The two active site residues, aspartic acids (Asp29 and Asp138), are

534

marked with asterisks and the conserved catalytic motif with a bold line. Five amino acid

535

residues (Arg26, Lys32, Trp37, Asn127, and Asn199) and Asp131 important for the substrate

536

binding and catalysis are well conserved in GHF45 subfamily I glucanases, and they are

537

represented as capital “R, K, W, N, N, and D” above the amino acid sequence of CaCel. 27



538

Asn67 in CaCel and its corresponding residues in other GHF45 enzymes are shown by a gray

539

colored rectangle. The positions of each intron are indicated by arrows above the amino acid

540

sequence of CaCel. Eleven intron positions found in fungal sequences are shown by open

541

triangles under amino acid alignment of GHF45 proteins. The information on eleven intron

542

positions is obtained from the recently published paper.36

543

544

Figure 3. Phylogenetic relationship of CaCel with GHF45 endo-β-1,4-glucanases. The

545

unrooted tree was generated using the neighbor-joining method (NJ) and the maximum

546

likelihood method (ML). Values at nodes represent bootstrap support out of 1000 repetitions

547

in NJ (left) and ML (right). Branches with less than 50% support are collapsed.

548

549

Figure 4. Thin layer chromatography (TLC) analysis of the digested products of

550

cellooligosaccharides and carboxymethyl cellulose (CMC) after digestion with CaCel at 40°C,

551

pH 3.5 for different incubation time ranging from 5 min to 60 min. Left lane, standard marker:

552

G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose; G6,

553

cellohexaose.

554

555

Figure 5. Effects of pH and temperature on CaCel activity (A and B) and its thermostability

556

(C). (A) The effect of pH on cellulase activity was measured using the following 50 mM 28


Page 28 of 41

Page 29 of 41


557

buffers: sodium citrate (pH 2.0-4.0), sodium acetate (pH 4.5-5.0), sodium phosphate (pH 5.0-

558

7.0), Tris-HCl (pH 7.0-9.0), and Glycin-NaOH (pH 9.0-10.0). (B) The effect of temperature

559

on cellulase activity was measured in 50 mM sodium citrate buffer, pH 3.5, at various

560

temperatures. Each assay was performed for 30 min with 1.75 µg /ml of CaCel and CMC

561

substrate, and the activity was determined by the DNS method. (C) The thermostability of

562

CaCel was examined by treating the enzyme (17.5 µg /ml) at temperatures of 50°C (●),

563

70°C (○), 80°C (■), and 90°C (□) for 1-8 h in 50 mM sodium citrate buffer, pH 3.5 and by

564

measuring the residual activity at 40°C for 30 min. Data were obtained from three

565

independent experiments with triplicates.

566

567

Figure 6. Effects of metal treatment on the CaCel activity. Reaction conditions were identical

568

to those of Figure 5B. Data were generated from three independent experiments.

569

570

Figure 7. (A) A ribbon diagram of CaCel is shown with the secondary structures labeled. The

571

six-stranded β-barrel, CD-I, and CD-II domains are coloured in orange, magenta, and cyan,

572

respectively. The glycine-rich loop and the C-terminal region are shown in light blue and blue,

573

respectively. Fourteen cysteine residues forming seven disulfide bonds and two catalytic

574

aspartate residues (Asp13 and Asp122) are shown in the sticks. (B) Stereo view of the

575

superimposed Cα tracing of CaCel (cyan) and MaCel (chocolate: PDB code, 1OA9). Active 29



576

site residues of CaCel (cyan) and MaCel (chocolate, italic) are shown in the sticks. Black and

577

green dotted lines represent hydrogen bonds in MaCel and CaCel, respectively. The circle

578

indicates residues 119-122 containing β6 and the glycine-rich loop. (C) Structural and

579

sequential comparison of the circled region in B between CaCel (cyan) and MaCel

580

(chocolate). The positions of glycine residues are indicated, and black dotted lines represent

581

hydrogen bonds. Overlapped cyan and pink dots are van der Waals radii of an oxygen atom in

582

Asp122 of CaCel and a carbon atom in His118 of MaCel. (D) The interaction between

583

His120 and the glycine-rich loop in CaCel. Magenta and green dots represent the van der

584

Waals surfaces of His120 and the glycine-rich loop, respectively. (E) Surface representation

585

of CaCel and MaCel.

586

587

Figure 8. Saccharification of the green algae, Ulva pertusa by the recombinant CaCel as an

588

endo-β-1,4-glucanase (A) a number of liberated sugars through incubation time. One gram of

589

powdered U. pertusa was loaded with 1 mg of CaCel and incubated at 50°C, pH 3.5 for 9 h.

590

(B) TLC analysis is showing the digested products of the U. pertusa powder by treatment

591

with CaCel.

30


Page 30 of 41

Page 31 of 41


Table 1. Comparison of CaCel cDNA and genomic structures with other GHF45 endo-β-1,4-glucanases Phylum

Species of origin

Arthropoda

Cryptopygus antarcticus

Apriona germari

GenBank Accession Number For cDNA (size) FJ648735 (799 bp)

GenBank Accession number For gDNA (size) FJ648736 (952 bp)

Intron Number (Intron size) 5 (58,52,51,58,55)

n.a

AY451326 (1036 bp)

2 (217, 99)

Position of Intron and their sequences 209 266 AATGGAGGCT gtac ------ ttag CTTCATACGTC 458 509 GACTTGAAT gtag---- ttag GGTAACCAT 550 600 GGTGGAGTAG gtac ---- ttag GTATCTTTAAC 655 712 GGATGGGGTC gtaa ---- aaag AACGTTACGGT 777 831 TTCGAGCCGG gaaa ---- ccag ATGCAATTGG 41 257 CTCTGCACAT gtaa ---- tcag TTGAAGCATC 626

724

GGCAAACAG gtgc---- ccag ATGATTGTC

Fungi

Syncephalastrum racemosum Rhizopus oryzae Rhizopus oryzae Melanocarpus albomyces

EU057152 (1023 bp)

n.a

n.a

n.a

AB056667 (1083 bp) AB047927 (1017 bp) AJ515703 (936 bp)

0 0 2 (71,70)

n.a

n.a

116 186 CAGTCCACGAG gtaa ---- ccag ATACTGGGAC 436 505 GCCTGCTACGC gtga ---- ccag TCTGACCTTT

Nematode

Bursaphelenchus xylophilus

AB047927 AB179542 (1302 bp) AB179543

Mollusca

Mytilus edulis

AB179544 AJ271364 (3304 bp)

0 1 (98) 1 (132) 0 2 (2171,497)

n.a: information not available. 31


933 1030 GATTTGGCT gtaa ----- tcag ATTCCCGGT 1194 1325 GATTTGGCT gtaa ----- ttag ATTCCTGGT 35 2205 CTCGTTCTTG gtaa ---- tcag TATACAGTGTT 2458 2954 ACAACAACAG gtat ---- gtag TTATAAAGATT


Page 32 of 41

Table 2. Comparison of biochemical properties of CaCel with those of other GHF45 endo-β-1,4-glucanases Phylum

Species of origin

Optimum pH

% remaining activity (Thermostability)

% activity at the lowest temperature

specific activity (U/mg)

Reference

3.5

Optimum temperature (°C ) 40

Arthropoda

Cryptopygus antarcticus

50-60% at 70°C for 4 h

60-70% at 0-10°C

this study

6

50

Loss activity at 80°C 10 min

30% of the maximum at 30°C

35.6 (CMC) 20.5 (Lichenan) 13.9 (β-glucan) 812

Apriona germari

3842424242424 24242424139383 83533303029272 52220161312111 010221

Fungi

Syncephalastrum racemosum Rhizopus oryzae

5~6

70

55-60% at 70°C for 4 h

30-40% of the maximum at 30°C

56.9

37

5

55

n.a

20% of the the maximum at

110

3443434343434

30°C

34343434240393 93634313130282 62321171413121 111332

Melanocarpus albomyces

6-7

70

52% at 100°C for 30 min

70% at 50°C

Nematode

Bursaphelenchus xylophilus

5.8

60

less 50% at 70°C for 10 min

n.a

Mollusca

Mytilus edulis Ampullaria crossean

5.5 5.5

30-50 50

100% at boiling water for 10 min less 10% at 50°C for 24 h

55-60% at 0°C 10-15% at 25°C

n.a: information not available

32


270 (hydroxyethylcell ulose) 203 (β-glucan) 85.2 (CMC) 367 (Lichenan) 10.4 146.5

33

7

10 39

Page 33 of 41


FIGURES Figure 1.

33



Figure 2.

34


Page 34 of 41

Page 35 of 41


Figure 3.

35



Figure 4.

36


Page 36 of 41

Page 37 of 41


Figure 5.

37



Page 38 of 41

Figure 6. MnCl2 + EDTA

MnCl2

100

50

150

100

% of Activity

% of Activity

% of Activity

EDTA

150

150

50

100

50

0

0

TA

10

0

ED

nC

5

10

+

5

M

0

l2

0

0

(mM)

M

nC

l2

(mM)

MgCl2

CaCl2

50

150

% of Activity

% of Activity

% of Activity

100

100

50

0

0 0

5

FeSO 4

5

10

0

ZnSO4

50

0

NaCl

100

50

10

100

50

0

0 5

10

150

% of Activity

% of Activity

100

5

(mM)

150

(mM)

50

(mM)

150

0

100

0 0

10

(mM)

% of Activity

CuCl2

150

150

0

5

10

(mM)

38


0

5

(mM)

10

Page 39 of 41


Figure 7.

39



Figure 8.

40


Page 40 of 41

Page 41 of 41


A TOC graphic 82x44mm (300 x 300 DPI)


Genetic and Structural Characterization of a Thermo-Tolerant, Cold

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