SHORT COMMUNICATION

Identification and Phylogenetic Characterizationof a New Subfamily of α-Amylase Enzymesfrom Marine Microorganisms

Yang Liu & Yin Lei & Xuecheng Zhang & Yi Gao &

Yazhong Xiao & Hui Peng

Received: 17 June 2011 /Accepted: 2 October 2011 /Published online: 11 November 2011# Springer Science+Business Media, LLC 2011

Abstract A gene encoding a starch-hydrolyzing enzyme wasisolated from a marine metagenomic library and overex-pressed in Escherichia coli. The enzyme, designated AmyP,shows very low similarity to full-length sequences of knownα-amylases, although a catalytic domain correlated with theα-amylase superfamily was identified. Based on the range ofsubstrate hydrolysis and the product profile, the protein wasclearly defined as a saccharifying-type α-amylase. Sequencecomparison indicated that AmyP was related to four putativeglycosidases previously identified only in bacterial genomesequences. They were all from marine bacteria and formed anew subfamily of glycoside hydrolase GH13. Moreover, thissubfamily was closely related to the probable genuinebacterial α-amylases (GH13_19). The results suggested thatthe subfamily may be an independent clade of ancestralmarine bacterial α-amylases.

Keywords Metagenomic cloning .α-Amylase . Glycosidehydrolase family GH13 . Subfamily classification

Introduction

α-Amylases (EC 3.2.1.1) are widely occurring enzymes,which hydrolyze the α-1,4-glycosidic linkages in starch,

glycogen, and related oligosaccharides with a random endo-mechanism. They are among the most important commercialenzymes, having wide applications in starch-processing,textile treatment, fuel ethanol, and other industries. Based onsequence similarity, the vast majority of α-amylases areclassified in glycoside hydrolase family 13 (GH13) [see theCarbohydrate-Active Enzyme (CAZy) database at www.cazy.org] (Cantarel et al. 2009). The GH13 family that constitutesalmost 30 different reactions and product specificities is thelargest family of glycoside hydrolases. All enzymes of thisfamily share a (β/α)8-barrel domain as the common super-secondary structure, but the overall sequence similaritysometimes can be extremely low (about 10%) (Machovičand Janeček 2003). Recently, the family has been subdividedinto 35 subfamilies on the basis of sequence similarity andphylogenetic reconstruction criteria (Stam et al. 2006). α-Amylases are scattered over 11 subfamilies, including twoanimal subfamilies (GH13_24 and GH13_15), one plantsubfamily (GH13_6), one fungi and yeast subfamily(GH13_1), one archaea subfamily (GH13_7), and sixbacterial subfamilies (Stam et al. 2006; Da Lage et al.2004). The phylogeny of α-amylases is generally inagreement with their origin, except bacterial α-amylases.Five out of the six bacterial subfamilies are group withanimal, plant, or fungal α-amylases, which can be explainedas the results of horizontal gene transfer from Eukarya toBacteria (Da Lage et al. 2004). Only the subfamilyGH13_19 containing no eukaryotic enzymes forms aseparate branch, and seems to be a genuine bacterial α-amylases type (Stam et al. 2006; Da Lage et al. 2004).

Marine environments possess extremely abundant micro-organisms and a unique microbial diversity, providing us avast resource for mining novel genes and biocatalysts.Several microorganisms from marine habitats producing α-amylases have been described (Chakraborty et al. 2009;

Y. Liu :Y. Lei :X. Zhang :Y. Xiao :H. Peng (*)Engineering Technology Research Center of Microorganismsand Biocatalysis, Anhui Province, School of Life Sciences,Anhui University,Hefei 230039 Anhui, People’s Republic of Chinae-mail: pph0259@126.com

Y. GaoSchool of Resources and Environmental Engineering,Anhui University,Hefei 230039 Anhui, People’s Republic of China

Mar Biotechnol (2012) 14:253–260DOI 10.1007/s10126-011-9414-3

Najafi and Kembhavi 2005; Zhang and Zeng 2008).However, these enzymes have all been isolated fromthe cultivable microorganisms. As we know <0.1% ofmicrobes in seawater can be cultured by conventionmethods (Amann et al. 1995). Hence, metagenomic basedstrategies have began to employ as powerful tools toprovide new insights and understanding of enzymes frommarine environments (Kennedy et al. 2008). To extend ourknowledge on marine amylases, we screened a previouslyconstructed marine metagenomic library by an activity-based approach. One novel gene, AmyP, was isolated andidentified as an α-amylase by catalytic characterization,and the discovery led to the identification of a newbacterial subfamily of GH13.

Materials and Methods

Functional Screening for Amylolytic Activity

A marine metagenomic library in 96-well plates was a giftfrom Dr. Tong Zhang (Sun Yat-sen University, China).Samples for DNA extraction were collected from foursubsurface sediments in South China Sea. This library thatconsisted of about 20,000 colonies was created using theCopycontrol Fosmid Library Production kit (Epicentre) andEscherichia coli EPI300™ (Epicentre). Screening of posi-tive clones with amylolytic activity was performed aspreviously described (Ballschmiter et al. 2006). Briefly,Luria–Bertani (LB) agar plates supplemented with 1%soluble starch were incubated for 24 h at 37°C and thenincubated for 30 min at 65°C. This excessive incubationallowed detection of intracellular enzymes. Subsequently, the

plates were flooded with Lugol solution (0.3% I2, 0.6% KI inH2O). Positive clones were detected by the formation ofclear halos against a dark violet background. The plasmids ofthe positive clones were isolated and retransformed into E.coli to confirm the hydrolytic activity.

Subcloning of Amylolytic Positive Clones

DNA inserts of positive clones were partially digested withSau3A1. The product of 2–5 kb in length was recoveredand ligated into the BamHI digested and dephosphorylatedpUC19 vector. The ligation product was transformed into E.coli DH5α competent cells (TransGen Biotech, China),producing a library of 1,100 clones. The transforments wereplated onto screening plates containing starch and grown at37°C for detection of amylolytic activity. Positive cloneswere sequenced, and the open reading frames (ORFs) wereidentified via ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).

Sequence Analysis and Evolutionary Tree

Homologues of AmyP and conserved domains wereobtained by searching the NCBI protein database usingthe PSI-BLASTP program (Altschul et al. 1997) associatedwith the Conserved Domain Database search (Marchler-Bauer et al. 2005). Signal sequence was predicted using theSignalP 3.0 server (www.cbs.dtu.dk/services/SignalP/). Allsequence alignments were performed using the ClustalX(Thompson et al. 1997) and then manually tuned whererequired. Published three-dimensional structures of rep-resentatives of GH13 were used as templates that servedas definition criteria for a conserved catalytic module

Fig. 1 Partial alignment of the amino acid sequences of AmyP, thefour putative glycosidases, and the clan GH13 representativemembers. The regions I, II, III, IV, and VI correspond to the strands,β3, β4, β5, β7, and β2, respectively, of the catalytic (β/α)8-barrel

domain (Machovič and Janeček 2003). The clan GH13 representativemembers were referred to published data (Machovič and Janeček2003; MacGregor et al. 2001). Asterisks Four invariant residues, graycatalytic aspartates and glutamate, black functional histidines

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(Machovič and Janeček 2003; Da Lage et al. 2004;MacGregor et al. 2001). The alignment of α-amylases wasperformed for the sequence segments (approximately 300residues) spanning the region from β1 to β8 strands of thecatalytic (β/α)8-barrel domain (Hostinová et al. 2010; vander Kaaij et al. 2007). A total of 54 sequence segmentswere extracted and used for phylogenetic tree construc-tion. Most of the sequences were referred to publisheddata (Da Lage et al. 2004; Hostinová et al. 2010; Stam etal. 2006; van der Kaaij et al. 2007). The evolutionary treewas calculated with the neighbor-joining method (Saitouand Nei 1987) implemented in the ClustalX package usingthe final alignment including the gaps (van der Kaaij et al.2007). The tree was displayed with the program TreeView(Page 1996).

Enzyme Overexpression and Purification

AmyP gene was amplified by using the primers 5′-CCCGGATCCATGTGCGATAGCGCTTTGA-3′ with a

BamHI site and 5′-CCCGTCGACTTACGGACACTTAGAGACC-3′ with a SalI site. The PCR was carried outwith Pyrobest DNA polymerase (Takara). The product wasdigested with BamHI and SalI and ligated into pET32avector (Novagen). The construct (pET32a-AmyP) wastransformed into E. coli BL21 (DE3) cells. The transformedcells were grown with agitation at 37°C until the opticaldensity at 600 nm of 0.5–0.7 was reached. At this point,isopropyl-β-D-thiogalactopyranoside (IPTG) was added tothe final concentration of 1 mM, and the cells wereincubated at 16°C for 12 h to induce AmyP. The cells wereharvested and lysed by ultrasonication. AmyP protein waspurified with one-step purification procedure using Ni2+

affinity chromatography (Invitrogen).

Activity Assay and Protein Analysis

The activity of α-amylase was determined by measuringthe amount of reducing sugar using 3,5-dinitrosalicylic acidas described by Miller (1959). A standard reaction wasdetermined at 50°C for 3 min. The reaction mixturecontained 100 μl of 1% (w/w) soluble starch (or otherpolysaccharides as specified in the text), 190 μl of 50 mMsodium citrate buffer, and 10 μl of purified AmyP. One unitof activity was defined as the amount of enzyme thatliberated 1 μmol of reducing groups as glucose per minute(Dong et al. 1997).

The optimum pH was measured at 50°C in 50 mMsodium citrate (pH 4.0–6.5), 50 mM sodium/potassiumphosphate (pH 7.0–8.0) and 50 mM glycine–NaOH(pH 8.0–9.0), adjusted at this temperature to various pHvalues, using the standard assay described above. Theoptimum temperature was determined at pH 6.5 using thestandard assay reaction mixtures incubated at temperaturesranging from 0°C to 80°C. Thermostability of AmyP wasdetermined by pre-incubating the enzyme at differenttemperature ranging from 0°C to 50°C during 20–120 min, followed by residual activity determination withadded substrate at 50°C.

Protein concentration was determined using Bradfordmethod with bovine serum albumin as a standard. Proteinsamples were analyzed by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE).

Fig. 2 SDS-PAGE (12 %) analysis of recombinant AmyP. Lane MSize marker proteins; lane 1 cell extract of E. coli harboring pET32a(64 μg); lane 2 cell extract of E. coli harboring pET32a- AmyP(63 μg); lane 3 the purified AmyP protein (11 μg)

Fig. 3 Effects of temperatureand pH on the activity of AmyP.a Temperature; b pH; c thermo-stability: 0°C ( filled square),10°C (empty square), 20°C( filled triangle), 30°C (emptytriangle), 40°C ( filled circle)and 50°C (empty circle)

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Analysis of Sugars

Thin-layer chromatography (TLC) of mono- and oligosac-charides was done on 0.2-mm silica gel plates (G60, QingdaoHaiyang Chemical Co. Ltd., China) with a solvent systemcomposed of isopropanol/ethyl acetate/H2O at a volume ratioof 3:1:1. The plate was dried completely and dipped rapidlyinto a methanol solution containing 3 g of N-(1-naphthyl)-ethylenediamine and 50 ml of concentrated H2SO4 solutionper liter (Yun et al. 2004). The plate was dried and placed at110°C for 10 min for visualization.

Results and Discussion

Isolation of a Novel Gene by Functional Screening

Amylase screening resulted in only one amylolytic clonefrom among 20,000 clones on LB agar plates containing

soluble starch and its activity was reconfirmed afterretransformation. In order to identify the hydrolyticgene, the insert was subjected to further subcloning.Two clones with amylolytic activity were obtained fromthe subclone library, among which one clone showedsuperior activity and was thus sequenced. We designatedthis gene as AmyP. The nucleotide sequence wasdeposited in the GenBank database with accession numberof HM572234.

Sequence Analysis

AmyP gene was 1,917 bp long, and the molecular mass ofthe translated protein was estimated to be 70 kDa. AmyPapparently was an intracellular enzyme. No signal peptidecan be found in the amino acid sequence. The PSI-BLASTP search results for the amino acid sequence ofAmyP showed that AmyP had similarity to many putativeglycosidases. AmyP exhibited the highest identity (72%over 463 amino acids) to the putative glycosidase(CAG22972) of the deep-sea bacterium Photobacteriumprofundum, followed by a 67% (over 430 amino acids)identity to the putative glycosidase (CAV27328) of themarine microorganism Vibrio splendidus, a 53% (over 275amino acids) identity to the putative glycosidase(YP_437477) of the marine microbe Hahella chejuensis,and a 50% (over 248 amino acids) identity to the putativeglycoside hydrolase family protein (YP_001143410) of themarine fish pathogen Aeromonas salmonicida ssp. salmo-nicida. Many of these putative glycosidases were revealedby whole genome sequencing, but none of them has beenbiochemically characterized. Most importantly, the glyco-sidase (EC 3.2.1.–) is a group of enzymes, which have beenclassified into more than 100 different families (the CAZydatabase). Therefore, the results of sequence homologysearch did not reveal the valuable information for classifi-cation and function.

However, a significant but undefined relatedness wasindicated by results achieved with the Conserved DomainDatabase search. AmyP possessed a conserved catalyticdomain of α-amylase superfamily members that includingthe GH families 13, 70, and 77 (MacGregor et al. 2001).Figure 1 shows the comparisons of the five conservedregions with other enzymes belong to the GH13. These

Table 1 Hydrolysis activities of AmyP on various substrates

Substrate Activity (U/mg) Relative activity (%)

Soluble starch 476±14.5 100

Amylose 143±5.37 30

Amylopectin 73.4±5.52 15

Pullulan 49.7±4.79 10

Glycogen 15.2±3.46 3

α-Cyclodextrin 0 0

β-Cyclodextrin 0 0

Trehalose 0 0

Xylan (birch wood) 0 0

α-PNPG 0 0

Fig. 4 TLC analysis of product formation during degradation ofsoluble starch. Soluble starch (0.5%) was digested with 50 μg AmyPat 30°C in 50 mM sodium citrate buffer, pH 6.5. Lane StdMaltooligosaccharide standards. G1 glucose, G2 maltose, G3 malto-triose, G4 maltotetraose, G5 maltopentaose

Fig. 5 Amino acid sequence alignment of α-amylases from differentGH13 subfamilies. Subfamilies and GenBank accession numbers areindicated. Special symbols: boxed the saccharifying-type α-amylases;underlined the GenBank accession numbers for the marine bacterialproteins; two asterisks the three invariant residues of the α-amylasesuperfamily; one asterisk the conserved residues characteristic ofGH13; arrows aromatic residues specifically conserved within themarine bacterial proteins; black the specifically conserved residues ofthe marine bacterial proteins; various shades of gray the specificallyconserved residues of other α-amylases

b

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regions, especially four amino acid residues necessary foractivity, were highly conserved throughout the GH13

(Machovič and Janeček 2003). The corresponding regionsand residues can also be identified in the four putative

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glycosidases from marine bacteria. The results suggestedAmyP and these homologues were classified into the GH13family.

Physicochemical Properties of AmyP

AmyP from the marine metagenome was overexpressedin soluble form in E. coli and purified over Ni2+ affinitychromatography. The apparent molecular weight ofAmyP was about 90 kDa shown by SDS-PAGE (Fig. 2),which was larger than the molecular calculated from theprimary structure. This increase was due to the N-terminal Trx-tag™ and His-tag® fused expression ofAmyP.

With a assay using soluble starch as the substrate, thepurified recombinant AmyP displayed the maximumactivity at 50°C and pH 6.5 (Fig. 3a, b). The optimal pHis in agreement with most pH values previously reported(neutral pH or below) for intracellular amylases (Pandey etal. 2000; Gupta et al. 2003). AmyP retained most of theactivity after incubation at 0°C for 120 min, while stabilityof the enzyme at increased temperatures was rather poor(Fig. 3c). Under its optimum temperature AmyP lost half ofits activity after 12 min of incubation. The activitydecreased to 60% after 90 min treatment at 10°C, reflectingthe thermosensitivity of AmyP. Moreover, addition of 1 or5 mM CaCl2 did not have a significant effect on heatresistance. Many α-amylases, especially from GH13, areknown to require Ca2+ to stabilize protein structures andincrease thermostability (Pandey et al. 2000; Gupta et al.

2003). There were only a few reports where Ca2+ did nothave any effect on the enzyme, e.g., the α-amylases fromPyrococcus furiosus (Dong et al. 1997) and Bacillusthermooleovorans (Malhotra et al. 2000).

Hydrolysis Properties of AmyP

At optimal temperature and pH, the hydrolysis activity ofAmyP was highest toward soluble starch rather than towardamylose, amylopectin, pullulan or glycogen (Table 1). Cyclo-dextrins, trehalose, xylan, and methyl-α-D-glucopyranoside(α-PNPG) were not hydrolyzed. The hydrolysis pattern ofsoluble starch digested with purified AmyP was analyzed byTLC with samples drawn at different time points duringdegradation (Fig. 4). Glucose, maltose, and maltotriose wereformed in great amounts at the early stage of hydrolysis. Onfurther incubation, maltotriose increased, while no newoligosaccharide was observed. Therefore, AmyP is asaccharifying-type enzyme, which produces predominantlyglucose (G1), maltose (G2), or maltotriose (G3) duringstarch hydrolysis (Emori et al. 1990; Ohdan et al. 1999;Sabathé et al. 2002). Additionally, the enzyme hydrolyzedmaltotetraose to produce maltose and hydrolyzed maltopen-taose to maltose and maltotriose (data not shown). However,no activity was observed on maltose or maltotriose. On thebasis of its substrate specificity and characteristics of productformation by cleavage of glycosidic bonds in polysacchar-ides, AmyP was classified as an α-amylase. To ourknowledge, it was the first α-amylase isolated from a marinemetagenomic library.

Fig. 6 Phylogenetic tree of α-amylases from different living king-doms. Species and GenBank accession numbers are indicated. Thetree is based on the alignment made in ClustalX of the part of the

sequences encoding the (β/α)8-barrel domain. The separate group ofα-amylases, marine bacterial subfamily, is clearly distinguishable. Thescale bar indicates 0.1 amino acid replacements per site

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To evaluate whether sequence features exist betweenAmyP and other typical saccharifying-type α-amylases(Emori et al. 1990; Sabathé et al. 2002), a comparisonwas performed with their (β/α)8-barrel domains (Fig. 5).No significant residue was observed.

Evolutionary Analysis

Based on the previously reported classification of α-amylases in GH13 (Da Lage et al. 2004; Hostinová et al.2010; Stam et al. 2006; van der Kaaij et al. 2007), we chose54 amino acid sequences of known α-amylases to representthe 10 established subfamilies. These proteins together withAmyP and its four homologues were used for analyzing thesequence features (Fig. 5) and calculating the phylogenetictree (Fig. 6).

Different types of (β/α)8-barrel domain define groups ofα-amylase family members (Janeček et al. 1997). AmyPand its homologues share several distinctive sequencefeatures (Fig. 5). These features are as follows. (1)phenylalanine (Phe122, AmyP numbering), glycine(Gly128), tryptophan (Trp175), glutamine (Gln179), tyro-sine (Tyr181), and valine (Val299) are specifically con-served among the AmyP group. Especially, the other α-amylases studied here contain the asparagine preceding thecommon conserved histidine (His129) in the strand β3,while the AmyP group members have the glycine substi-tuted by asparagine. (2) It can be seen that the AmyP groupmembers possess more aromatic residues (highlighted byarrows in Fig. 5) within the very short, well-defined regionsthan that of the other α-amylases. The aromatic residues areconcentrated within the (β/α)8-barrel domains representingthe remote homologies of α-amylase superfamily members,supporting the idea that the original domains shared acommon ancestor (Janeček et al. 2007).

The phylogenetic tree clearly shows AmyP, and itshomologues could not be assigned to any previouslyidentified α-amylase grouped in GH13 and are most closelyrelated to the probable genuine bacterial α-amylase typerecently grouped in subfamily GH13_19. Therefore, wepostulated that they comprised a new subfamily of GH13.

It is also interesting to note that the members of the newsubfamily are from marine environments. The ocean hasbeen regarded as the origin of life on earth, and the bacterialα-amylases from ancestral marine Alteromonadaceae havebeen described as the ancestor of α-amylase (Da Lage et al.2004). The new subfamily may be an independent clade ofancestral marine bacterial α-amylases, not only since itsphylogenetic position but also its ecological conditions arequite different from those mentioned terrestrial species. It isalso worth mentioning that the new subfamily containsmore aromatic residues, implying the hypothesis that thesubfamily members retain more primordial sequence

features of the ancestor. The results provide the new cluesof the biodiversity and distribution of α-amylases in themarine environment. In nutrient-limited marine environ-ments, these previously unknowable α-amylases may playan important role in carbon access and ecological systemsustenance.

To conclude, AmyP is a novel saccharifying-type α-amylase from a marine metagenomic library and has verylow similarity to full-length sequences of known α-amylases. AmyP and four putative glycosidases formedthe new subfamily that may be an independent clade ofancestral bacterial α-amylases.

Acknowledgments We thank Tong Zhang for providing the marinemetagenomic library. This work was supported by the Natural ScienceFoundation of Anhui Province, China (11040606M65) and theFoundation for Key Program of Ministry of Education, China(211073).

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