Ann Microbiol (2016) 66:907–919 DOI 10.1007/s13213-015-1170-6

ORIGINAL ARTICLE

First report of a tropical enzymogenes producing bifunctional endoglucanase activity towards carboxymethylcellulose and chitosan

Siraprapa Saraihom 1,2 & Donald Y. Kobayashi3 & Pongtharin Lotrakul2 & Sehanat Prasongsuk2 & Douglas E. Eveleigh4 & Hunsa Punnapayak2

Received: 25 June 2015 /Accepted: 15 October 2015 /Published online: 14 November 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015

Abstract In the past, Lysobacter spp. were found only in amino acid sequences of the endoglucanase derived were de- temperate and sub-tropical zones. In order to assess the exis- duced from draft genome sequencing. Phylogenetic analysis of tence of Lysobacter spp. in tropical environments, samples the endoglucanase gene and multiple alignment of the deduced were collected from grass-covered soil in Thailand. Among amino acid sequence illustrated that the endoglucanase (Cel8A) several obtained, isolate 521 (NCCB 100553) was from L. enzymogenes 521 displayed homology to family 8 gly- identified as Lysobacter enzymogenes based on morphology cosidehydrolases(GHF-8),andwasfoundtobesimilartothe and physiological tests along with 16S rRNA gene sequence bifunctional endoglucanase (Cel8A) from Lysobacter sp. IB- analysis. Phenotypic characteristic variations of 9374. Bifunctional CMCase and chitosanase activities of a pu- L. enzymogenes 521 with respect to the most studied strain from rified endoglucanase with a mass of 41 kDa from temperate regions, L. enzymogenes C3, were observed. The L. enzymogenes 521 were revealed by zymogram analysis. growth rate, carboxymethylcellulase (CMCase) and This is the first report on the phenotypical characteristics, chitosanase activity of L. enzymogenes 521 were clearly higher the endoglucanase with bifunctional CMCase and chitosanase than those of L. enzymogenes C3. Nucleotide and predicted activities, and the endoglucanase gene, from L. enzymogenes from a tropical environment. Electronic supplementary material The online version of this article (doi:10.1007/s13213-015-1170-6) contains supplementary material, which is available to authorized users. Keywords Bifunctional endoglucanase . Carboxymethyl-cellulase . Chitosanase . Tropical Lysobacter * Donald Y. Kobayashi enzymogenes . Glycoside hydrolase family 8 [email protected] * Pongtharin Lotrakul [email protected] Introduction * Hunsa Punnapayak [email protected] Lysobacter enzymogenes () represents the most studied member of the genus Lysobacter and, as such, is of 1 Biological Sciences Program, Faculty of Science, Chulalongkorn considerable ecological and biotechnological interest. It is a pro- University, Bangkok 10330, Thailand ducer of novel antibiotics and extracellular lytic enzymes that are 2 Plant Biomass Utilization Research Unit, Department of Botany, believed to function as mechanisms of antagonism against fungi Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand and other microbes. Moreover, L. enzymogenes species have 3 attracted considerable attention because of their rich stock of lytic Department of Plant Biology and Pathology, School of β Environmental and Biological Sciences, Rutgers, The State enzymes (protease, chitinase, -1,3-glucanase and lipase), which University of New Jersey, New Brunswick, NJ 08854, USA have been investigated intensively for their application in biolog- 4 Department of Biochemistry and Microbiology, School of ical control (Kobayashi and Yuen 2007). Besides, it has been Environmental and Biological Sciences, Rutgers, The State reported that Lysobacter species produce high activity of University of New Jersey, New Brunswick, NJ 08854, USA endoglucanases that exhibit bifunctional activity towards 908 Ann Microbiol (2016) 66:907–919 carboxymethylcellulose (CMC) and chitosan (Hedges and Wolfe In this study, we report the existence of a tropical 1974; Ogura et al. 2006). endoglucanase-producing L. enzymogenes isolated from a Cellulase is one of the most useful enzymes for industrial grass-covered soil collected from natural grassland nearby applications (Kuhad et al. 2011). Both fungi and bacteria have the Mekong River, the boundary river between Thailand and been investigated intensively because of their ability to produce Laos. Isolate 521 was identified by observing its morpholog- a wide variety of cellulases. In particular, bacterial cellulase- ical and physiological characteristics, and its identity was con- producing strains have been increasingly isolated and investi- firmed by molecular biological analysis. The effect of incuba- gated as a potential producer because they generally have tion temperature on growth, bifunctional CMCase and higher growth rates than those of fungi. Furthermore, bacteria chitosanase activities of L. enzymogenes 521 was investigated. are resistant to environmental stresses, and are often able to Moreover, the dual mode of endoglucanase activities on CMC produce enzymes that are stable even under extreme conditions. and colloidal chitosan was displayed by zymogram analysis. Theycanbemodifiedeasilybygenetic engineering in order to The deduced amino acid sequence of the endoglucanase enhance their cellulase production. Moreover, bacterial cellu- (Cel8A) of L. enzymogenes 521 was compared with that of lases are less inhibited by end-product (feedback inhibition), endoglucanases from other Lysobacter strains. and often have multiple activity modes (Maki et al. 2009). A bifunctional endoglucanase with hydrolytic activity on both CMC and chitosan has been reported in several bacterial Materials and methods species, including Bacillus cereus S1 (Kurakake et al. 2000), Bacillus circulans WL-12 (Mitsutomi et al. 1998), Bacillus Sample collection and isolation of Lysobacter spp. megaterium P1 (Pelletier and Sygusch 1990), Paenibacillus fukuinensis D2 (Kimoto et al. 2002), Streptomyces griseus Grass-covered soil samples were collected in Nong Kai prov- HUT 6037 (Tanabe et al. 2003), Lysobacter sp. IB-9374 ince (17°52′36.84″N102°42′47.52″E) located along the (Ogura et al. 2006)andL. enzymogenes (Myxobacter AL-1) Mekong River, Northeastern Thailand in May 2013. The soil (Hedges and Wolfe 1974). Interestingly, endoglucanase pro- samples were used as an inoculum for bacterial isolation. The duced from Lysobacter species exhibited higher activity on isolation method was modified from the enrichment culture CMCthanonchitosanevenwhenculturedinmediawithout method of Kobayashi and El-Barrad (1996). Cultures were added CMC or chitosan as a carbon substrate. At present, grown in 100 mL M9 mineral salt medium (Sambrook et al. endoglucanase from Lysobacter spp. has still not been well stud- 1989) supplemented with 0.1 % (w/v) colloidal shrimp chitin ied, and the ecological role of the bifunctional endoglucanase in (Kobayashi and El-Barrad 1996) in 250-mL Erlenmeyer these bacteria remains unclear. The fact that the bifunctional flasks. To aid isolation of the bacterial group of interest, the endoglucanase is an extracellular enzyme suggests that it may following antimicrobials were added to the enrichment medi- have an advantage in biotechnological applications over the usu- um: 50 mg/L kanamycin, 50 mg/L penicillin and 100 mg/L al intracellular and periplasmic enzymes. Although bifunctional Rose Bengal. Enrichment cultures were initiated by adding 1 g endoglucanases have been used as endoglucanase in the soil sample to 100 mL medium followed by shaking at room cellulose-based textile industry (Kuhad et al. 2011), and as temperature (28±2 °C), 150 rpm for 7 days. The cultured chitosanase in the production of chitooligosaccharides (Xia medium solutions were then serially diluted and spread on a et al. 2008), they have not been used with their dual mode of yeast cell agar (YCA) consisting of 0.5 % (w/v) autoclaved activity in any application. Baker’s yeast cell wall and 1.5 % (w/v) agar (Kobayashi and So far, the genus Lysobacter has been isolated mainly from El-Barrad 1996). Colonies capable of lysing autoclaved yeast habitats in temperate areas; especially the Americas, Europe, cell wall, as depicted by clearing zones around colony edges, Japan, and Korea and some sub-tropical areas in China were selected and further purified by repeatedly streaking to (Reichenbach 2006; Wei et al. 2012). The bacteria live in soil yield a single colony on fresh YCA. and fresh water under diverse environments in those temperate regions. However, the existence of this bacterial species in Identification of Lysobacter spp. tropical countries has not been reported. As shown in a study of hydrolytic enzyme degradation in grassland soils (Trasar- Morphological characterization Cepeda et al. 2007), increasing the incubation temperature generally increases the rate of enzyme catalysis. Therefore, Lysobacter enzymogenes strain C3 (Sullivan et al. 2003), pro- extending the search for these bacteria to tropical habitats vided by the Department of Plant Biology and Pathology, would be of interest, as it would open up the opportunity to Rutgers, The State University of New Jersey, United States, discover new potential lytic-enzyme producers with interest- wasusedasareferencestrain.Gram’s staining of bacteria was ing properties, such as thermophilic and thermotolerant conducted by first growing cells on skim milk acetate agar [0.5 % strains. (w/v) skim milk powder, 0.05 % (w/v) yeast extract, 0.02 % Ann Microbiol (2016) 66:907–919 909

(w/v) sodium acetate, 1.5 % (w/v) agar] at 25 °C for 3 days. emulsified in 1 % (w/v) Na2HPO4, respectively. The cultures Gliding motility and colonial characteristics were observed by were incubated aerobically for 48 h under constant shaking at growing cells on Cook’s Cytophaga agar (CCA) and casein yeast 200 rpm at 30 °C, and were collected and centrifuged at 4 °C (10, extract agar (CYA) at 20 °C for 5 days (Reichenbach 2006). 000 g, for 5 min) for further enzymatic assays. Experiments were performedintriplicate(N=3). Physiological and biochemical characterization CMCase, chitosanase and chitinase activities were assayed using the DNS method (Miller et al. 1960). The reaction mixture Biochemical tests were performed using API 20NE kit included 0.45 mL of either 1.0 % (w/v) CMC, 1.0 % (w/v) (bioMérieux, Marcy l’Etoile, France). Bacterial growth were colloidal chitosan or 1.0 % (w/v) colloidal chitin in 50 mM so- observed at various temperatures (20 °C, 25 °C, 30 °C, 35 °C, dium acetate buffer, pH 5.0 and 50 μL crude enzyme. The mix- 37 °C and 40 °C) in LB broth on a shaking incubator at tures were incubated at 40 °C for 15 min and the reducing sugars

200 rpm, for 24 h. The optical density (OD600) of the culture released in the reaction mixtures were determined using 0– was measured and the growth determined by comparison to 1500 μg/mL glucose (CMCase), glucosamine (chitosanase) the McFarland standard scale (bioMérieux). Solid phase and N-acetylglucosamine (chitinase) as standards. The reaction microextraction (SPME) GC-MS analysis was performed at mixtures were added to 1.5 mL DNS reagent and subsequently Central Instrument Facility, Mahidol University, Bangkok, boiled for 5 min. The absorbance of the mixture was measured at Thailand in order to provide a volatile compound profile of 540 nm in order to quantify the amount of reducing sugar re- the bacterial culture odors. A brief description of the SPME leased in the reaction. Note that one unit of enzyme activity was process can be found in Online Resource 1. defined as the amount of enzyme that releases 1 μmol glucose/ glucosamine/N-acetylglucosamine equivalent per minute under Expression of secreted lytic enzymes on agar plates constant conditions. CMCase, chitosanase, chitinase, protease and lipase were pro- Protease activity was assayed following the procedure de- duced following the methods described by Kobayashi and El- scribed by Cupp-Enyard (2008). The reaction mixture (in trip- Barrad (1996) and Reichenbach (2006). Colloidal chitosan was licate) included 0.45 mL 0.65 % (w/v) casein in 50 mM potas- prepared from 75–85 % deacetylated chitosan (Sigma-Aldrich, sium phosphate buffer, pH 7.5 and 50 μL crude enzyme. After St. Louis, MO) as previously described (Yabuki et al. 1988). incubation at 40 °C for 15 min, the reaction was stopped by Secreted CMCase and chitosanase were detected by decolori- adding 0.5 mL 110 mM trichloroacetic acid (TCA). The amino zation of degraded CMC (dp: 400, ds: 0.65–0.90, Sigma- acids released in the reaction mixture were determined using 0– Aldrich) or colloidal chitosan after having grown the culture 100 μg/mL L-tyrosine as a standard. A 0.4 mL aliquot of reac- at 30 °C for 3 days on M813 (Palumbo et al. 2003)agarsup- tion mixture and the standard were mixed with 1 mL 0.5 M plementedwith1%(w/v)ofthesubstrates and then stained sodium carbonate and 0.2 mL 0.5 mM Folin-Ciocalteu’s with 0.5 % (w/v) Congo Red. The production of chitinase was Phenol Reagent, and incubated at 40 °C for 30 min. The absor- indicated by the appearance of clear zones after having cultured bance of the mixture was measured at 660 nm. One unit of the bacteria at 30 °C for 5 days on M813 agar containing 1 % enzyme activity was defined as the amount of enzyme that (w/v) colloidal shrimp chitin as a substrate. The production of releases 1 μmol tyrosine per minute under constant conditions. protease was detected by the appearance of clear zones after Lipase activity was assayed by the colorimetric method having cultured the bacteria on skim milk agar medium at 30 °C (Glogauer et al. 2011)usingp-nitrophenylpalmitate (pNPP) as for 24 h. The production of lipase was detected by observing a substrate. Substrate solutionwaspreparedbymixing50mM the color change of an indicator around bacterial colony gener- Tris–HCl buffer, pH 7.5, 1 mM CaCl2, 0.3 % (v/v) Triton ated by free fatty acid after culturing at 30 °C for 5 days on X-100, 4 % (v/v) isopropanol, 1 % (v/v) acetonitrile and M813 supplemented with 1 % (v/v) olive oil emulsified in 1 % 1mMpNPP, in a shaking water bath at 60 °C. The reaction

(w/v) Na2HPO4, and 0.01 % (w/v) phenol red. mixture included 0.45 mL substrate solution and 50 μLcrude enzyme. The mixtures were incubated at 40 °C for 15 min, and Enzyme activity assay Seed cultures of L. enzymogenes 521 the absorbance of the mixture was measured at 410 nm by using and L. enzymogenes C3 were prepared by growing them in LB at 0–500 μg/mL p-nitrophenol as a standard. One unit of enzyme 30 °C for 18–20 h in order to reach exponential growth phase. activity was defined as the amount of enzyme that releases

The OD600 of the seed cultures was adjusted to 0.1 in 0.85 % 1 μmol p-nitrophenol per minute under constant conditions. (w/v) NaCl. The adjusted cell suspensions at 10 % (v/v) inocu- lum size were subsequently added to 100 mL of each enzymatic Protein determination Protein concentration was determined production medium. Production of CMCase, chitosanase, by the Lowry method (Lowry et al. 1951)using0–300 μg/mL chitinase, protease and lipase was carried out in M813 supple- bovine serum albumin as a standard. The reaction including mentedwith1%(w/v)CMC,1%(w/v) colloidal chitosan, 1 % 0.5 mL either crude enzyme or appropriately diluted purified (w/v) colloidal chitin, 0.5 % (w/v) casein and 1 % (v/v) olive oil enzyme and 2.5 mL Lowry reagent was incubated for 10 min at 910 Ann Microbiol (2016) 66:907–919 room temperature. After that, 0.25 mL Folin-Ciocalteu’s at different temperatures between 30 °C and 39 °C. The cultures Phenol reagent was added to the reaction mixture and were sampled every 24 h and centrifuged at 4 °C (10,000 g,for incubated for 30 min at room temperature. Absorbance 5 min). The supernatants were assayed for CMCase and of the reaction was then measured at 750 nm in order to chitosanase activity using the DNS method (Miller et al. 1960) calculate the protein concentration by comparison with as described previously. Experiments were performed in tripli- the standard protein. cate (N=3). Bacterial cell density was measured using a standard direct Effect of temperature on growth rate, CMCase and plate count method. Cell suspensions were sampled from cul- chitosanase activities Seed cultures of L. enzymogenes 521 tures grown at different incubation times (24 h–96 h) and and L. enzymogenes C3 were prepared by growing them in mod- temperatures (33 °C–39 °C), and subsequently serially diluted ified M813 supplemented with 0.75 % (w/v) glucose, 0.45 % with 0.85 % NaCl to reach the appropriate proportions. Each (w/v) casein and 0.2 % (w/v) CMC at 30 °C for 18–20 h. The diluted mixture (10 μL) was dropped on an LB agar plate and

OD600 of the seed cultures was adjusted to 0.1 and added at 10 % incubated for 48 h. Experiments were performed in triplicate (v/v) inoculum size to 100 mL fresh M813. The cultures were (N=3). The original cell density and bacterial growth rate incubated aerobically for 96 h under constant shaking at 200 rpm were calculated using the following equation:

Number of colony x Dilution factor Colony−forming units per milliliterðÞ¼ CFU=ml ÀÁ Diluted mixture volume Bacterial growth rate; k h−1 ¼ lnðÞ N2=N1 =ðÞt2−t1 where k is the growth constant, N1 is the initial cell number, sequencing and genome assembly were conducted at N2 is the final cell number, t1 is the initial time and t2 is the Rutgers, The State University of New Jersey using the final time. Illumina’s desktop sequencing system and de novo assembly with default assembly parameters, respectively. The annotated data were searched for endoglucanases, and those sequences Molecular characterization were compared with the endoglucanase gene sequences found in the GenBank database using the BLAST algorithm (http:// 16S rRNA gene analysis blast.ncbi.nlm.nih.gov/Blast.cgi). Genomic DNA was isolated following the protocol described by Sambrook et al. (1989). The 16S rRNA gene was amplified Phylogenetic analysis using the universal primer set 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′ (M=C: A) and 1525R (5′-AAG GAG GTG Phylogenetic trees of the 16S rRNA sequence and the cel8A WTC CAR CC-3′ (W=A: T; R=A: G) (Lane 1991). DNA gene sequence were constructed using maximum-likelihood amplification was conducted by predenaturation at 94 °C for analysis by Bayesian phylogenetic inference analysis using 3min,andwasfollowedbyameltingstepat94°Cfor1min, the program MrBayes version 3.2.2 (Ronquist et al. 2012). In an annealing step at 52 °C for 1 min, and an extension step at order to construct the tree of the 16S rRNA gene, the almost- 72 °C for 2 min. The reaction was repeated for 30 cycles and complete DNA sequence (1418 bp) of L. enzymogenes 521 was followed by a final extension for 15 min. The PCR product aligned with sequences in the GenBank database and the am- was purified using the QIAquick PCR Purification kit biguous regions were subsequently excluded. The evolutionary (Qiagen, Valencia, CA), and subsequently sequenced by distances were calculated using the Kimura two-parameter Macrogen, Seoul, Korea. The full sequences so obtained were model. Analysis of 310,000 generations was performed with compared with the sequences in the GenBank database using tree diagnosing every 500 generations. A final 129 likely trees the Basic Local Alignment Search Tool (BLAST) algorithm. were obtained and the most likely consensus tree is presented The pairwise alignment was conducted with closely related bac- with a 103 bootstraps data set. The cel8A gene sequence terial 16S rRNA gene sequences in the GenBank using Clustal (1169 bp) of L. enzymogenes 521 was aligned with the X program (ftp://ftp.ebi.ac.uk/pub/software/clustalw2/2.0.11/). GenBank database sequences and the ambiguous regions were subsequently excluded. The general time reversible (GTR) Analysis of the full-length endoglucanase gene model was used to calculate evolutionary distances. A final 79 likely trees were obtained from analysis of 380,000 genera- The isolation of chromosomal DNA was modified as previ- tions. The most likely consensus tree is shown with a 103 boot- ously described (Staskawicz et al. 1987). The genome straps data set at the branch points. Ann Microbiol (2016) 66:907–919 911

Nucleotide sequence accession numbers (10 μg protein) mixed with sample buffer [2 % (w/v) SDS, 5 % (v/v) β-mercaptoethanol] was applied to each native gel. The DNA sequence of the 16S rRNA gene and the DNA Electrophoresis was conducted on ice at constant current of sequence encoding the Cel8A enzyme of L. enzymogenes 30 mA for 2 h, and enzyme activity bands were detected in the 521 were deposited with the GenBank database under acces- gels. CMCase and chitosanase zymograms were modified sion numbers KR445659 and KR445660, respectively. from a method described previously by Bischoff et al. (2006). After electrophoresis, 20 % (v/v) isopropanol in Purification of endoglucanase 50 mM sodium acetate buffer (assay buffer) was used to wash the gel for 20 min. The gel was then washed three times Endoglucanase preparation was carried out at 4 °C, and all (20 min each) in assay buffer. The gel was incubated at reagents and chemicals were of analytical grade. After incu- 40°C in assay buffer for 1 h, stained with 0.1 % (w/v) bating the bacterium in M813 for 48 h, the cultured broth (3 Congo Red for 30 min, and destained with 1 M NaCl for L) was centrifuged at 10,000 g for 20 min. Supernatants were 30 min. The CMCase and chitosanase activities were visual- concentrated by ultrafiltration using 10 kDa MW membrane ized on the gel as a yellow halo resulting from the hydrolysis cut-off Vivaflow 50 (Sartorius, Goettingen, Germany) and of the CMC and colloidal chitosan, respectively. subsequently precipitated using ammonium sulfate (50 %– 80 % saturation). The precipitate was collected and dissolved in 50 mM sodium acetate buffer, pH 5.0 and filtrated using Results and discussion Vivaflow 50 to remove salt. The filtrate was then exchanged to dissolve into the start buffer (1 M ammonium sulfate in Isolation, identification and characterization of Lysobacter 50 mM sodium phosphate, pH 7.0) of HiTrap Phenyl HP spp. (Amersham Pharmacia Biotech, GE Healthcare, Uppsala, Sweden), a hydrophobic interaction column, and subsequent- Lysobacter-like bacteria were isolated successfully from ly loaded into the column. The bound proteins were eluted grassland soils in Thailand using an enrichment procedure. with a linear gradient from 1 M to 0 M ammonium sulfate at Since chitinolysis is a distinguishable character of a flow rate of 1.0 mL/min. Fractions were collected and Lysobacter spp. (Christensen and Cook 1978), in this work assayed for CMCase and chitosanase activities as described we used an enrichment procedure consisting of colloidal previously. Protein content in each fraction was determined shrimp chitin supplemented with selective antimicrobials. using the Lowry method as described previously. Fractions containing both enzyme activities were pooled, salt was re- Morphological characterization moved and buffer was changed using vivaflow 50 into the start buffer (1 M sodium chloride in 50 mM sodium acetate In order to distinguish the Lysobacter-like bacteria from other buffer, pH 4.0) of Hitrap SP HP (Amersham Pharmacia taxonomic groups, single colony-pure bacterial isolates were Biotech), for cation exchange chromatography. The fractioned investigated first by comparing morphological and physiolog- proteins were subsequently loaded onto the column. Fractions ical traits with the reference strain. Among several chitinolytic were collected at a flow rate of 5.0 mL/min and assayed for bacteria isolated from the soil samples, isolate 521 (NCCB CMCase and chitosanase activities and protein content as de- 100553) was the only isolate belonging to the genus scribed previously. Purified protein fractions were pooled, salt Lysobacter, and was further identified as Lysobacter was removed and the purified protein was concentrated into enzymogenes based on morphological and physiological char- 50 mM sodium acetate buffer, pH 5.0 and kept at 4 °C until it acteristics and 16S rRNA gene sequence analysis. Typical of was used. L. enzymogenes, the tropical isolate 521 was a Gram-negative, rod-shaped bacterium (Fig. 1a) displaying gliding motility on Zymographic analysis CCA and CYA (Fig. 1b, c). Colony morphology was a creamy-yellow, with irregular, flat to convex form with undu- SDS-PAGE was performed in 5 % and 10 % acrylamide stack- lated edges. Cells were morphologically diverse in population, ing and resolving gels, respectively. Proteins were detected by which is a distinguishing characteristic of Lysobacter strains Coomassie Brilliant Blue G250 staining (Neuhoff et al. 1985). (Reichenbach 2006). Standard molecular weight marker, BLUeye Prestained Protein Ladder (MW from 10 kDa to 245 kDa) (GeneDireX, Physiological and biochemical characterization Las Vegas, NV), was used. CMC and colloidal chitosan were separately added at 0.2 % (w/v) of final concentration into a The main distinguishable characteristics of L. enzymogenes native-PAGE with 5 % and 10 % stacking and resolving gel, 521 with respect to the reference strain L. enzymogenes C3 respectively (Sambrook et al. 1989). Purified endoglucanase (Sullivan et al. 2003) and the other representatives of the 912 Ann Microbiol (2016) 66:907–919

Fig. 1a–c Morphological characters of Lysobacter enzymogenes 521 (CYA). The media (skim milk acetate agar, CCA, and CYA) were (left) compared with the reference strain, L. enzymogenes C3 (right). a prepared following Christensen and Cook (1978) and Reichenbach Cell morphology on skim milk acetate agar. b Gliding motility on Cook’s (2006) Cytophaga agar (CCA). c Gliding motility on casein yeast extract Agar

Gram-negative genus Lysobacter are shown in Table 1.Most A summary of differential phenotypic characteristics of the Lysobacter strains display gliding motility. All strains produce tropical L. enzymogenes 521 in comparison with the temperate proteolytic activity—a trait typical of Lysobacter strains that L. enzymogenes C3 is shown in Table 2. Both strains shared have been previously characterized for their antagonistic ac- many similar biochemical traits. They showed positive cata- tivity against nematodes and bacteria (Kobayashi and Yuen lase, oxidase, β-glucosidase, getalinase and β-galactosidase 2007). Some of the strains also produce lipase. We observed activities, while they were negative for nitrate reduction, in- that the enzymatic activity seemed to depend on the isolation dole production, glucose fermentation, arginine dihydrolase procedure. Note that the Lysobacter strains of Table 1 not and urease activities. The strains were able to utilize D-glu- displaying chitinolytic activity were isolated using non- cose, D-mannose, D-maltose and N-acetylglucosamine. On the selective media, while the strains of L. enzymogenes, other hand, none of them was able to utilize L-arabinose, D- Lysobacter antibioticus,andLysobacter gummosus producing mannitol, capric acid, adipic acid, phenylacetic acid and po- both chitinase and CMCase activities were isolated using tassium gluconate. chitin-amended enrichment culture. The use of other enrich- Slight characteristic variations were observed between the ment substrates such as yeast cell wall, which is a rich nutrient tropical L. enzymogenes 521 and the temperate L. enzymogenes compared to chitin, might yield more diverse species of C3 in gliding features on CCA and CYA (Fig. 1b–c), cell size, Lysobacter by differential enhancement of growth. colony color and shape (Fig. 1a,Table2). The two strains had

Table 1 Gliding motility and enzyme activities of Lysobacter enzymogenes 521 compared with L. enzymogenes C3 and other species in the genus Lysobacter

Species Gliding motility Enzyme activity Chitosanase Reference

Chitinase Protease Lipase CMCase

L. enzymogenes 521 +a +++++ Thisstudy L. enzymogenes C3 + + + + + NR Sullivan et al. 2003 Lysobacter antibioticus ATCC 29479 + + + + + NR Christensen and Cook 1978 Lysobacter brunescens ATCC 29482 + + + −− NR Christensen and Cook 1978 Lysobacter gummosus ATCC 29489 + + + + + NR Christensen and Cook 1978 Lysobacter yangpyeongensis GH 19-3 + − + −− NR Weon et al. 2006 Lysobacter ginsengisoli Gsoil 357 + − + −− NR Jung et al. 2008 Lysobacter daecheongensis Dae08 + − + −− NR Ten et al. 2008 Lysobacter oryzae YC6269 + − + −− NR Aslam et al. 2009 Lysobacter panaciterrae Gsoil 068 + − + −− NR Ten et al. 2009 Lysobacter ximonensis XM415 + − ++− NR Wang et al. 2009 Lysobacter soli DCY21 + + + + − NR Srinivasan et al. 2010 Lysobacter korlensis ZLD-17 + − + −− NR Zhang et al. 2011 Lysobacter bugurensis ZLD-29 + − + −− NR Zhang et al. 2011 Lysobacter xinjiangensis RCML-52 −−++− NR Liu et al. 2011 Lysobacter thermophilus YIM 77875 −−+ −− NR Weietal.2012 a +Positive,− negative, NR not reported Ann Microbiol (2016) 66:907–919 913 very different odors generated by different volatile compounds lytic enzymes expressed on agar media. Secretion levels of produced in the culture medium (Online Resource 1). Based on CMCase, chitosanase, chitinase, protease and lipase were GC-MS analysis, the major volatile compounds produced by slightly higher for isolate 521 with respect to strain C3 strain C3, including 2-ethyl-1-hexanol, nonanoic acid and 4- (Fig. 2). Quantitative assay of the lytic enzymes clearly morpholineethanamine, were not detected in strain 521. The showed that strain 521 produced higher specific activities in optimal growth temperature of both strains was between 20°C all type of lytic enzymes under constant conditions. It is un- and 40°C. The tropical L. enzymogenes 521 grew much better clear whether the variation in morphological and physiologi- than the temperate L. enzymogenes C3 when cultured at high cal features between strains was related to the geographical temperature (37°C and 40°C). Strain C3 could utilize malic acid locations where the stains were isolated. weakly but could not utilize trisodium citrate, while isolate 521 could utilize both substrates (Table 2). Effect of temperature on growth rate, CMCase and chitosanase activities In order to investigate the effect of Expression of secreted lytic enzymes on agar plates and temperature on growth rate, CMCase and chitosanase activi- enzyme activities Both strains showed positive results for ties of the tropical L. enzymogenes 521 in comparison with the

Table 2 Differential phenotypic characteristics of L. enzymogenes 521 and L. enzymogenes C3

Characteristic L. enzymogenes 521 L. enzymogenes C3

Cell size (μm) 0.5−1 × 3.5-5 0.5–1×.2-8.4 Colony color Creamy yellow Pale yellow Odorous compounds 2-Methylbutan-1-ol 2-Methylbutan-1-ol, 2-Ethyl-1-hexanol, monanoic acid, 4-morpholine-ethanamine Growth at 20 °C +a + 25 °C + + 30 °C ++ ++ 35 °C + + 37 °C + w 40 °C + w Catalase test + + Oxidase test + +

Reduction of NO3 to NO2/ Reduction of NO2 to N2 −/−−/− Indole production −− Glucose fermentation −− Arginine dihydrolase −− Urease −− β-Glucosidase + + Gelatinase + + β-Galactosidase + + Assimilation:

D-Glucose ++ −− L-Arabinose D-Mannose ++ D-Maltose ++ −− D-Mannitol N-Acetyl-glucosamine + + Capric acid −− Adipic acid −− Malic acid + w Phenylacetic acid −− Potassium gluconate −− Trisodium citrate + − a ++ Strongly positive, + positive, w weakly positive, − negative 914 Ann Microbiol (2016) 66:907–919

Fig. 2 Specific lytic enzyme activity of L. enzymogenes 521 and M813 agar supplemented with 1 % CMC (CMCase), 1 % colloidal L. enzymogenes C3 cultures grown at 30°C for 48 h. Data are the chitosan (chitosanase), 2 % colloidal chitin (chitinase), 0.5 % casein means and standard deviations of three replicates. Below the graphs (protease), or 1 % olive oil (lipase) enzyme activities are indicated by zones around colonies grown on temperate L. enzymogenes C3, both cultures were grown in temperature for growth of several L. enzymogenes strains of modified M813 for 96 h at different temperatures. Profiles of between 25°C and 35°C. Interestingly, our results showed that growth rate, CMCase and chitosanase production of L. enzymogenes 521 had higher growth rate, CMCase and L. enzymogenes 521 and L. enzymogenes C3 are illustrated chitosanase activities than those of L. enzymogenes C3 at all in Fig. 3. The bifunctional endoglucanase was produced dur- incubation temperatures, and especially at temperatures be- ing growth phase to stationary phase (Fig. 3a). The enzyme tween 33°C and 39°C. The maximum CMCase and produced from both strains had maximum activity towards chitosanase activity of L. enzymogenes 521 was about 1.5 CMC after incubating for 48 h at 30°C, while both enzymes times higher than that obtained from L. enzymogenes C3 showed maximum activity towards chitosan after incubating (Fig. 3a) at the optimal incubation temperature. The for 24 h at the same temperature. At temperatures higher than CMCase activity produced by several aerobic bacteria was 30°C, both the growth rate and the CMCase and chitosanase reported to be between 0.43 U/mL and 2.00 U/mL (Da activities decreased substantially (Fig. 3a, b). These results are Vinha et al. 2011;Dekaetal.2011;Macedoetal.2013; consistent with those of Christensen and Cook (1978) and Shankar and Isaiarasu 2011). The maximum CMCase activity Reichenbach (2006),whoreportedthatanoptimal obtained from the bifunctional endoglucanase of

Fig. 3 Growth and lytic enzyme activity profiles of L. enzymogenes 521 (■) CMCase and ( ) chitosanase activity of L. enzymogenes 521; and L. enzymogenes C3 (a), and growth rate of L. enzymogenes 521 and (−○−) cell number and growth rate of L. enzymogenes C3; ( ) L. enzymogenes C3 (b) cultured in modified M813. The cultures were CMCase and ( ) chitosanase activity of L. enzymogenes C3. incubated aerobically for 96 h, at 30 °C, 33 °C, 36 °C, and 39 °C. Growth Experiments were performed in triplicate (N=3). Error bars Standard rate, CMCase and chitosanase activities were determined every 24 h. deviation Symbols: (−●−) cell number and growth rate of L. enzymogenes 521; Ann Microbiol (2016) 66:907–919 915

Fig. 4 Phylogenetic relationship of the L. enzymogenes 521 with Lysobacter spp. and other species (GenBank accession numbers in parentheses) of the family Xanthomonadaceae. The tree was constructed employing the maximum-likelihood (ML) analysis on the basis of 16S rRNA gene sequences using the program MrBayes version 3.2.2. The Kimura two-parameter model was used to calculate the evolutionary distances and bootstrap values (expressed as percentagesof103 replications) greater than 50 % are shown at the branch points

L. enzymogenes 521 was 2.46±0.19 U/mL, which is higher Molecular characterization than activities seen in previous reports. On the other hand, chitosanase activity produced by several aerobic bacteria Phylogenetic analysis of 16S rRNA gene sequence was reported at between 1.0 U/ml and 1.76 U/mL (Mitsutomi et al. 1998; Pelletier and Sygusch 1990;Kimoto Phylogenetic analysis on the basis of the 16S rRNA et al. 2002; Tanabe et al. 2003). The maximum gene sequence employing maximum-likelihood (ML) chitosanase activity of the isolate 521 bifunctional analysis showed that isolate 521 belongs to Lysobacter endoglucanase was 1.3±0.18 U/mL, which fits within species lineage with high bootstrap value (100 %). the range seen in most reports. Based on percent nucleotide sequence identity, isolate

Fig. 5 Maximum likelihood tree of the cel8A gene sequence of hydrolase 8 superfamily protein from Jeongeupia naejangsanensis L. enzymogenes 521 and other related family 8 glycoside hydrolase Njc02; endoglucanase (Egl257) from Bacillus circulans N257; β- proteins found in the GenBank database (GenBank accession numbers glucanase from Lysobacter capsici AZ78; Cel8A from Lysobacter in parentheses). The tree was constructed employing ML analysis using enzymogenes 521; endoglucanase (Cel8A) from Lysobacter sp. IB- the program MrBayes version 3.2.2. Evolutionary distances were 9374; endoglucanase Y (Cel8A) from vesicatoria ATCC calculated using the general time reversible (GTR) model. Bootstrap 35937; cellulase (Celf0376) from Cellulomonas fimi ATCC 484; cellulase values (expressed as percentages of 103 replications) greater than 50 % (CelB) from Streptomyces coelicolor A3(2); endoglucanase Y (Cel8A) are shown at the branch points. Sequences aligned: endoglucanase from from 91–118; endo-1,4-beta-glucanase (EglS) Janthinobacterium agaricidamnosum NBRC 102515; glycosyl from Bacillus subtilis subsp. subtilis 168 916 Ann Microbiol (2016) 66:907–919

521 was most similar to L. enzymogenes strain 495 Phylogenetic analysis of the endoglucanase gene (99.3 %) and L. enzymogenes C3(99.3%),andmore and deduced amino acid sequence analysis distantly related to Lysobacter gummosus KCTC 12132 (97.2 %), Lysobacter antibioticus USAM 3C (97.1 %), Sequence relatedness of the bifunctional endoglucanase en- Lysobacter koreensis dae16 (95.9 %), Lysobacter zyme (Cel8A homologue) from L. enzymogenes 521 was daejeonensis GH1-9 (92.8 %), Lysobacter concretionis compared phylogenetically along with similar glycoside hy- Ko07 (90.6 %) and Lysobacter defluvii IMMIB APB-9 drolase protein sequences. The results showed that the gene (88.1 %) (Fig. 4). Phylogenetic analysis was consistent sequence derived from the draft genome sequencing of with the morphological and physiological characteristics L. enzymogenes 521 was very similar to that of endoglucanase and further supported identification of isolate 521 as (Cel8A, bifunctional enzyme) from Lysobacter sp. IB-9374 L. enzymogenes. (97.0 %), β-1,4-glucanase from L. capsici AZ78 (82.5 %)

Fig. 6 Multiple deduced amino acid sequence alignment of Cel8A and 27405 (A3DC29), chitosanase (ChoK-BK17) from Bacillus sp. K17 related cellulases. Sequences aligned: Cel8A (Cel8A-LE521) from (1V5C_A). Identical and similar (>60 %) amino acid residues among Lysobacter enzymogenes 521 (GenBank accession number: all enzymes are shaded in black and gray, respectively. The conserved KR445660); endoglucanase (Cel8A-LEIB) from Lysobacter sp. IB- amino acid residues are marked by a black line below the alignment. The 9374 (AB244037); β-glucanase (Bglu-LC) from Lysobacter capsici asterisks indicate the conserved catalytic residues in GHF-8 proteins and AZ78 (EYR68409); endoglucanase (Egl257-BC) from Bacillus the hash tags indicate the substrate recognition sites in Cel8A from circulans N257 (AB059267); endoglucanase Y (Cel8A-XV) from C. thermocellum ATCC 27405. @ and + indicate conserved substrate Xanthomonas vesicatoria ATCC 35937 (EGD1117); endoglucanase Y binding and substrate recognition sites, respectively, in chitosanase from (Cel8A-XP) from Xanthomonas perforans 91–118 (EGD14463); Bacillus sp. K17. Gaps in the sequence alignment are indicated by dashes. endoglucanase A (Cel8A-CT) from Clostridium thermocellum ATCC The alignment was performed using the program Clustal X Ann Microbiol (2016) 66:907–919 917 and other related proteins (56.1 %–46.6 %) as shown in Figure 5. All the related proteins showed homology to family 8 glycoside hydrolases (GHF-8), which include endoglucanase, lichenase, chitosanase and endoxylanase. The multiple sequence alignment of the deduced amino acid sequence of the Cel8A from L. enzymogenes 521 was analyzed Yield (%) 73 75 with selected GHF-8 proteins (Fig. 6). The enzyme showed 100 100 per minute under the assay homology to endoglucanase (Cel8A, bifunctional enzyme) from Lysobacter sp. IB-9374 (98.3 %), β-1,4-glucanase from L. capsici AZ78 (78.1 %), and had less similarity (43 %–22 %) to endoglucanase from B. circulans N257, Clostridium thermocellum ATCC 27405, Xanthomonas vesicatoria ATCC 35937, X. perforans 91–118, and chitosanase from Bacillus sp. K17. Alignment analysis showed that the Cel8A contained the

GHF-8 catalytic module region (Ogura et al. 2006) from mol glucosamine equivalent μ 2.5 2.6 1.0 1.0 Ala158 to Gly180. Based on analysis of the crystal structure of Cel8A from C. thermocellum ATCC 27405 (Alzari and Dominguez 1996) and chitosanase from Bacillus sp. K17 (Adachietal.2004), it has been reported that several aromatic and acidic residues act as substrate binding sites and substrate recognition sites (Fig. 6). Note that a few residues acting as substrate binding sites or substrate recognition sites reported in Cel8A and chitosanase are present also in the Cel8A from L. enzymogenes 521. These data suggested that substrate rec- ognition and binding of the bifunctional CMCase-chitosanase Specific activity (U/mg) Purification (fold) from L. enzymogenes 521 might differ from those of C. thermocellum ATCC 27405 and Bacillus sp. K17. ount of chitosanase required to release 1

Purification of endoglucanase in L. enzymogenes 521 a and zymographic analysis 521

Lysobacter enzymogenes has been reported previously to ex- 00 1333.70 2.64 1.43 press the dual endoglucanase activities of CMCase and CMCase Chitosanase CMCase Chitosanase CMCase Chitosanase CMCase Chitosanase 2454. chitosanase (Hedges and Wolfe 1974; Ogura et al. 2006). In 3348.00 1780.85 1.05 0.56 order to investigate the dual mode of activities of the L. enzymogenes endoglucanase from L. enzymogenes 521, culture filtrate was purified and zymographic analysis was performed. Table 3 shows the purification summary of the endoglucanase. The mol glucose equivalent per minute or the am μ purified enzyme yielded specific activities of CMCase at al protein (mg) Total activity (U) 930.75 41.4 U/mg protein and chitosanase at 23.38 U/mg protein, 3177.00 which is about a 41-fold enrichment. High protein purity with asinglebandof∼41 kDa was observed on Coomassie-stained SDS-PAGE. The result was similar to those previously report- ed in Lysobacter sp. IB-9374 (Ogura et al. 2006)andBacillus 750 (Kurakake et al. 2000; Mitsutomi et al. 1998; Pelletier and 110 122.43 712.47 391.47 5.82 3.20 5.5 5.7 21 22 Sygusch 1990) species, and close to the predicted values ob- tained from the deduced amino acid sequence analysis (44 kDa, data not shown). A purified protein containing endoglucanase activity from L. enzymogenes 521 exhibited a precipitation clear activity band on both the CMC-zymogram gel and the Purification of bifunctional endoglucanase produced from 4 80 % saturation) chitosan-zymogram gel, both corresponding to a single pro- SO 2 – ) tein band observed on the Coomassie-stained SDS-PAGE gel 4 (50 1U=the amount of CMCase required to release 1 (NH Ultrafiltration Culture supernatant 3000 HiTrap Phenyl HPHitrap SP HP 30 10 18.99 1.70 371.61 194.56 73.53 19.57 39.75 10.25 43.25 23.38 18.6 18.3 41.0 41.7 11 11 2.2 2.2 Purification step Volume (mL) Tot Table 3 a (Fig. 7). This result demonstrated that the endoglucanase from conditions 918 Ann Microbiol (2016) 66:907–919

living in hot, humid tropical environments will be necessary in order to explain the relationship between high temperatures and bacterial physiology, such as growth and lytic enzyme production. Moreover, it would be very interesting to study on endoglucanase production by L. enzymogenes 521 further in order to understand its function at the molecular level and to explore its potential as a useful bifunctional CMCase- chitosanase producer.

Acknowledgments The authors wish to thank Chulalongkorn Univer- sity Dutsadi Phiphat Scholarship, Ratchadaphiseksomphot Endowment Fund 2014 of Chulalongkorn University (CU-57-043-EN) and Eveleigh- Fenton Fund of Rutgers, the State University of New Jersey for financial support. The authors are grateful to Assistant Professor Tosak Seelanan, PhD (Chulalongkorn University) for molecular technical support. Assis- tance from members of the Plant Biomass Utilization Research Unit (PBURU), Department of Botany, Faculty of Science, Chulalongkorn University is also acknowledged. Fig. 7 SDS-PAGE (a) and zymogram (b, c) analysis of the purified endoglucanase from L. enzymogenes 521. Protein mass and purity were determined by SDS-PAGE stained with Coomassie Brilliant Blue (a). CMCase activity of the protein was determined in a native PAGE References containing 0.2 % (w/v) CMC (b) and chitosanase in a native PAGE containing 0.2 % (w/v) colloidal chitosan (c). The native-PAGE gels Adachi W, Sakihama Y, Shimizu S, Sunami T, Fukazawa T, Suzuki M, were stained with 0.1 % (w/v) Congo Red. Lanes: 1 Molecular weight Yatsunami R, Nakamura S, Takénaka A (2004) Crystal structure of markers (BLUeye Prestained Protein Ladder, molecular size markers in family GH-8 chitosanase with subclass II specificity from Bacillus kDa), 2–4 purified endoglucanase (10 μg) sp. K17. J Mol Biol 343:785–795 Alzari PM, Dominguez R (1996) The crystal structure of endoglucanase L. enzymogenes 521 also exhibits CMCase-chitosanase bi- CelA, a family 8 glycosyl hydrolase from Clostridium – functional activity. thermocellum. Structure 4:265 275 Aslam Z, Yasir M, Jeon CO, Chung YR (2009) Lysobacter oryzae sp. nov., isolated from the rhizosphere of rice (Oryza sativa L.). Int J Syst Evol Microbiol 59:675–680 Conclusions Bischoff KM, Rooney AP, Li XL, Liu S, Hughes SR (2006) Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis.BiotechnolLett28: Bacterial isolate 521 expressing endoglucanase activity, iden- 1761–1765 tified as Lysobacter enzymogenes based on morphological and Christensen P, Cook FD (1978) Lysobacter, a new genus of nonfruiting, physiological characteristics combined with 16S rRNA gene gliding bacteria with a high base ratio. Int J Syst Bacteriol 28:367– sequence analysis, was isolated successfully from a soil sam- 393 ’ ple in Thailand. Lysobacter enzymogenes 521 isolated from a Cupp-Enyard C (2008) Sigma s non-specific protease activity assay- casein as a substrate. J Vis Exp 19:e899. doi: 10.3791/899 tropical environment had several characteristic variations with Da Vinha FNM, Gravina-Oliveira MP, Franco MN, Macrae A, Da Silva respect to its counterpart of temperate strain, L. enzymogenes Bon EP, Nascimento RP, Coelho RRR (2011) Cellulase production C3. In particular, growth, CMCase and chitosanase activity of by Streptomyces viridobrunneus SCPE-09 using lignocellulosic bio- L. enzymogenes 521 were less affected by high temperature mass as inducer substrate. Appl Biochem Biotechnol 164:256–267 than those of L. enzymogenes C3. A homologue of Cel8A, Deka D, Bhargavi P, Sharma A, Goyal D, Jawed M, Goyal A (2011) Enhancement of cellulase activity from a new strain of Bacillus originally characterized from a gene identified within subtilis by medium optimization and analysis with various cellulosic L. enzymogenes 521, showed homology to family 8 glycoside substrates. Enzyme Res 2011:1–8 hydrolases (GHF-8). The deduced amino acid sequence of the Glogauer A, Martini VP, Faoro H, Couto GH, Müller-Santos M, gene was very similar to that of bifunctional endoglucanase Monteiro RA, Mitchell DA, Souza EM, Pedrosa F, Krieger N (2011) Identification and characterization of a new true lipase iso- (Cel8A) from Lysobacter sp. IB-9374, and is predicted to lated through metagenomic approach. Microb Cell Fact 10:54 encode CMCase activity in L. enzymogenes 521. Zymogram Hedges A, Wolfe RS (1974) Extracellular enzyme from Myxobacter AL- analysis confirmed that the purified endoglucanase (41 kDa) 1 that exhibits both β-1, 4-glucanase and chitosanase activities. J produced by L. enzymogenes 521 exhibited both CMCase and Bacteriol 120:844–853 chitosanase activities as a single band. Thus, the present report Jung HM, Ten LN, Im WT, Yoo SA, Lee ST (2008) Lysobacter ginsengisoli sp. nov., a novel species isolated from soil in Pocheon of L. enzymogenes 521 extends our knowledge about the geo- Province, South Korea. J Microbiol Biotechnol 18:1496–1499 graphical range of Lysobacter species to tropical climates. Kimoto H, Kusaoke H, Yamamoto I, Fujii Y,Onodera T, Taketo A (2002) Further isolation and characterization of Lysobacter strains Biochemical and genetic properties of Paenibacillus glycosyl Ann Microbiol (2016) 66:907–919 919

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