Biosci. Biotechnol. Biochem., 75 (4), 669–673, 2011
Characterization of Chitosanase of a Deep Biosphere Bacillus Strain Tohru K OBAYASHI,y Osamu K OIDE, Shigeru D EGUCHI, and Koki H ORIKOSHI Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka 237-0031, Japan Received November 4, 2010; Accepted January 25, 2011; Online Publication, April 22, 2011 [doi:10.1271/bbb.100782]
A chitosanase of deep-biosphere Bacillus thuringiensis strain JAM-GG01 was purified. The optimal pH and temperature for the purified enzyme (Cho-GG) were about pH 6 and 60 C, but Cho-GG was unexpectedly unstable under incubation at over 40 C. This discrepancy between higher activity and lower stability in the same range of temperature was abolished by the addition of reaction products, chitotriose and chitotetraose. The Cho-GG gene was amplified by PCR and sequenced. The deduced amino acid sequence of Cho-GG showed more than 98% identity to those of other Bacillus enzymes belonging to GH family 8. Although Cho-GG did not show the definite characteristics of a sub-seafloor ectoenzyme, the thermal stability of many chitosanases of B. turingienesis and other related strains can be improved by adding chitotriose or chitotetraose. Key words:
chitosanase; chito-oligomer; deep biosphere; Bacillus; thermal stability
Chitosan, a D-glucosamine polymer, is a totally or partially deacetylated chitin (poly--1,4-D-N-acetylglucosamine) found in the cell walls of fungi of the class Zygomycetes1) and the exoskeletons of insects.2) Chitosan and chito-oligomers show several physiological activities, viz., antimicrobial, antitumoral, immunestimulating, and blood cholesterol-lowering actions.3–8) Thus they are used in the food, textile, and pharmaceutical industries.6,8) Chitosanase (EC 3.2.1.132), a chitosan-degrading enzyme, is produced by many organisms, including fungi, bacteria, and plants. Chitosanases are classified into glycoside hydrolase (GH) families 3, 5, 7, 8, 46, 75, and 80 according to the CAZy database (http://www.cazy.org/). Among them, many chitosanases are derived from the genus Bacillus, and belong to GH families 8 and 46.9–15) The reason is not clear, but one possibility is that chito-oligomers work as elicitors of phytoalexins.16) Most Bacillus strains are known to produce extracellular pectate lyases,17,18) and the products, oligogalacturonides, also show elicitor activity.19) Most Bacillus strains are not phytopathogenic, but there might be interrelationships between Bacillus strains and plants in nature. Recently, we isolated many aerobic, heterotrophic bacteria from deep sub-seafloor sediments off the Shimokita Peninsula of Japan and investigated the production of several enzymes.20) Under our test conditions, chitosanase activities were found only in cultures of Bacillus strains, and not in any other y
bacteria species from the deep sub-seafloor. We selected a representative chitosanase producer, Bacillus thuringiensis strain JAM-GG01. Here, we describe the purification of the chitosanase, its enzymatic properties, and the sequencing of its gene.
Materials and Methods Strains and propagation. Deep sub-seafloor sediments were obtained by the Shakedown Expedition CK06-06 of the deep-sea drilling vessel Chikyu. The water depth was 1,180 m, and 365 m of core sediments was recovered approximately 80 km from the eastern coast of the Shimokita Peninsula, Japan (41 10.63800 N, 142 12.081E). High numbers of aerobic, heterotrophic microorganisms were grown on several solid media. The production of various extracellular enzymes of the isolates was examined. Strain JAM-GG01 was selected as a chitosanase producer from sediments of 190.4 meters below the seafloor. The strain was cultured aerobically with shaking at 30 C for 24 h in a liquid medium (pH 6.5) that contained 0.1% casamino acids (Difco, Detroit, MI), 2% yeast extract (Difco), 0.1% chitosan 5 (Wako Pure Chemicals, Osaka, Japan; deacetylated value, 80%), and 2% NaCl. Purification of Cho-GG. Culture supernatant (1.3 L) was concentrated to 30 mL using an Amicon Ultra-15 (3 kDa Mr cut-off). The concentrate was diluted with distilled water, and then the solution was applied to a SuperQ column (2:5 10 cm; Tosoh, Tokyo, Japan) equilibrated with 25 mM borate buffer (pH 9.5). Chitosanase was passed through the column, and it was applied to a CM-Toyopearl column (2:5 6 cm; Tosoh) equilibrated with 25 mM borate buffer (pH 9.5). The proteins were eluted with a 300-mL linear gradient of 0 to 0.15 M NaCl in the buffer. Fractions containing enzyme activity, which were eluted at about 50 mM NaCl, were pooled and concentrated by ultrafiltration. The concentrate was applied to a CM-Toyopearl column (1:5 10 cm) equilibrated with 25 mM MOPS buffer (pH 6.0) plus 75 mM NaCl. Chitosanase was eluted with a 200-mL MOPS buffer with a linear gradient of 0.075 to 0.2 M NaCl. Fractions showing chitosanase activity were eluted at about 140 mM NaCl, and pooled and concentrated. The concentrate was stored at 20 C until use. Enzyme assay. Chitosanase activity was measured in 0.5 mL of reaction mixture that contained 100 mM acetate buffer (pH 6), 0.2% chitosan5, and suitably diluted enzyme. After incubation at 50 C for 30 min, 0.5 mL of dinitrosalicylic acid (DNS) reagent was added, and the reducing sugar formed was quantified. One unit (U) of chitosanase activity was defined as the amount of protein that released the reducing sugar equivalent to 1 mmol of glucosamine. Degradation of chitopentaose and chitohexaose was also measured using DNS reagent. All experiments to determine enzymatic properties were done at least in duplicate, and their mean values are given. The degradation pattern of chito-oligosaccharides was determined by thin-layer chromatography using a solvent system of n-propanol:ethyl acetate:ammonia: water ¼ 6/1/1/3 v/v. The chromatograms were developed by color reaction with ninhydrin reagent. Protein was measured using a DC-Protein Assay Kit (Bio-Rad, Hercules, CA) with bovine serum albumin as standard.
To whom correspondence should be addressed. Tel: +81-46-867-9718; Fax: +81-46-867-9719; E-mail:
[email protected]
670
T. KOBAYASHI et al.
Sequencing of the 16S rRNA gene of strain JAM-GG01. The 16S rRNA gene of strain JAM-GG01 was amplified by PCR using universal primers 27f and 1492r and a colony of strain JAM-GG01 as template in a DNA thermal cycler (Gene Amp PCR system 9700, Applied Biosystems, Foster City, CA) with an LA Taq DNA polymerase (Takara Bio, Ohtsu, Japan). After purification of the amplified fragment, sequencing was done with a DNA sequencer (Model 3100, Applied Biosystems) using an ABI Prism Big Dye Terminator sequencing kit (Applied Biosystems). Cloning and sequencing of the gene for Cho-GG. The gene for Cho-GG was amplified by PCR using primers 50 -AATCAACAAGTAGCTGCTGCAAAGG-30 and 50 -TTCTTCATCCAATCCCAACC30 , which were designed from the appropriately conserved regions of Bacillus thuringiensis family 8 chitosanases (EF143917–EF143925), with the genomic DNA of strain JAM-GG01 as template. The former primer sequence was 125 bp downstream of the ATG start codon, and the latter was 142 bp upstream of the TAA stop codon of the genes for family 8 chitosanases. The PCR conditions were 2 min at 94 C, followed by 30 cycles of 20 s at 94 C, 15 s at 57 C, and 90 s at 72 C, and extension of 5 min at 72 C. The entire gene for Cho-GG was amplified by PCR using primers 50 -ATCGTGATTGCTCATATACC30 (210 bp upstream of the ATG start codon) and 50 -GTAAGCCAAGTTTTATCACC-30 (581 bp upstream of the TAA stop codon), and 50 -TCATCAATACCTGGGTCTCC-30 (184 bp downstream of the TAA stop codon) and 50 -CAGAAGGTCAAGGGTATGGG-30 (361 bp downstream of the ATG start codon), which were designed from the complete genome sequence of Bacillus cereus G9842 (CP001186.1) and the genomic DNA of strain JAM-GG01 as template. The nucleotide sequences of both strands were determined. Electrophoresis and gel filtration chromatography. SDS-polyacrylamide gel electrophoresis (PAGE) was performed essentially by the method of Laemmli21) on a 12.5% acrylamide slab gel (Bio-Rad). A precision mass marker (Bio-Rad) was used as standard. Gel filtration chromatography on a Bio-Gel A-0.5 m column (0:8 50 cm; Bio-Rad) equilibrated with 10 mM MOPS buffer (pH 6) plus 0.1 M NaCl was also used to determine the molecular mass of the enzyme. Gel filtration calibration kit LMW (GE Healthcare, Fairfield, CT) was used as standard. Analysis of the N-terminal amino acid sequence of Cho-GG. After SDS–PAGE of the purified Cho-GG, it was blotted onto a methanoltreated PVDF membrane (Immobulon, Millipore, Billerica, MA). Small pieces of the membrane were subjected to amino acid sequencing (Model 476A, Applied Biosystems).
Results and Discussion Analysis of the 16S rRNA gene sequence of strain JAM-GG01 A 1,489-bp nucleotide sequence of the 16S rRNA gene of strain JAM-GG01 was analyzed and compared with those of other strains. The nucleotide sequence was entirely identical to those of Bacillus thuringiensis strain 4Q281 (AF155954.1) and B. cereus G9842 (CP001186.1). This clearly indicates that strain JAMGG01 belongs to the Bacillus cereus group.22) The nucleotide sequence of the 16S rRNA gene was submitted to the GenBank/EMBL/DDBJ databases under accession no. AB553285. Purification of Cho-GG Cho-GG was purified to homogeneity as judged by SDS–PAGE from culture broth of B. turingiensis strain JAM-GG01. The specific activity increased by 24 times during purification, with 9.8% recovery of initial activity. The purification process is summarized in Table 1. The molecular mass of the enzyme was determined at approximately 43 kDa by SDS–PAGE
Table 1. Summary of Purification of Cho-GG
Step Culture broth SuperQ Toyopearl CM Toyopearl (pH 9.5) CM Toyopearl (pH 6)
Total protein (mg)
Total activity (units)
Specific activity (units/mg)
Yield (%)
Fold
321 26.7 3.8
5478 2859 1506
17.1 107 396
100 52.2 27.5
1 6.3 23.2
1.3
536
9.8
24.1
A
B
412
kDa 200 150 100 75 50 37
25 20
Fig. 1. SDS–PAGE of Purified Cho-GG. Purified Cho-GG (4.1 mg, lane 1) and a molecular mass marker (lane 2) were electrophoresed in the presence of 0.1% SDS. Gels were stained with Coomassie Brilliant Blue R-250 and destained with 7% v/v acetic acid.
(Fig. 1) and Bio-Gel A-0.5 m chromatography, indicating that Cho-GG is a monomer. The N-terminal amino acid sequence of the purified enzyme was Ala-Ser-AlaX-Glu-Met-X-Pro-Phe-Pro-Gln-Gln-Val-Asn-Tyr, where X is an unidentified amino residue. Effects of pH on Cho-GG The optimal pH for Cho-GG activity was about pH 5.6 (actual pH values at 50 C) in 100 mM acetate buffer (Fig. 2A). Like many chitosanases, the enzyme was most active between pH 4.9 and 6.2, but not active below pH 3 and above pH 8. Cho-GG was very stable in the acidic pH region, pH 2 to 4, when incubated at 30 C for 30 min in various pH buffers, as shown in Fig. 2B. Effects of temperature on Cho-GG Cho-GG was most active at 60 C in acetate buffer (pH 5.6), and highly active between 50 C and 70 C (Fig. 3A). To assess the thermal stability of Cho-GG, it was incubated at 30 C to 70 C for 15 min in 100 mM acetate buffer (pH 6). Unexpectedly, it was gradually inactivated during incubation at temperatures above 45 C. It was completely inactivated at 55 C, which indicates a discrepancy between the higher optimal temperature and the lower thermal stability of Cho-GG. This phenomenon has also been observed in many other chitosanases.12–14,23–28) We noticed that chito-oligosac-
Chitosanase of a Deep Biosphere Bacillus
B
100 80
Residual activity (%)
Relative activity (%)
A
60 40 20
671
100 80 60 40 20
0
0 2
3
4
5
6
7
8
9
1 2
3 4
5 6
pH
7 8
9 10 11 12 13
pH
Fig. 2. Effects of pH on Cho-GG. A, Effect of pH on activity: Activity was measured at 50 C for 30 min in a total volume of 0.5 mL with 0.11 mg of purified Cho-GG. The following 100 mM buffers were used: glycine-HCl (pH 2.1–5.6) ( ), acetate (pH 4.0–5.5) ( ), MOPS (pH 5.9–7.5) ( ), phospate (pH 6.2–7.5) ( ), and Tris–HCl (pH 6.2–8.3) ( ). The pH values in the reaction mixture were measured at 50 C. Maximal activity is indicated as 100%. B, Effect of pH on stability: The enzyme (0.11 mg) was incubated at 30 C for 30 min in 20 mM buffers of various pH, as follows: glycine-HCl (pH 2.1–3.9) ( ), acetate (pH 4–6) ( ), MOPS (pH 6–8) ( ), Tris–HCl (pH 7–9) ( ), glycine-NaOH (pH 9.1–10.8) ( ), and KCl–NaOH (pH 11.6–12.4) (). Residual activity was measured under the standard assay conditions. Maximal residual activity was taken as 100%.
B
100
Residual activity (%)
Relative activity (%)
A
80
60
40
20
120 100 80 60 40 20 0
0 10
20
30
40
50
60
70
80
Temperature (ºC)
30
40
50
60
70
Temperature (ºC)
Fig. 3. Effects of Temperature on Cho-GG. A, Effect of temperature on activity: Activity was measured at the indicated temperatures for 30 min in 100 mM acetate buffer (pH 5.5) with 0.07 or 0.11 mg of purified Cho-GG. Maximal activity was taken as 100%. B, Effect of temperature on stability: Purified enzyme (0.54 mg) was incubated in 20 mM acetate buffer (pH 6) with and without 2.5 mM of each chito-oligomer at the indicated temperatures in a total volume of 0.05 mL. After incubation for 15 min, the solutions were diluted 10-fold with distilled water, and then residual activity was measured using 0.1 mL of dilute under the standard assay conditions. The residual activity at 30 C incubated without chito-oligosaccharide ( ) was taken as 100%. Glucosamine ( ), chitobiose ( ), chitotriose ( ), chitotetraose ( ).
charides prevent thermal inactivation of Cho-GG. As shown in Fig. 3B, the thermal stability of the enzyme in the presence of 2.5 mM chitotriose or chitotetraose was retained up to 55 C, whereas glucosamine and chitobiose did not affect thermal stability at all. Retention of thermostability depended on the concentration of chitotriose or chitotetraose. When the enzyme was incubated with 2.5, 5, 10, and 20 mM chitotriose or chitotetraose at 65 C for 15 min in acetate buffer (pH 6), the residual activity was 0%, 8.3%, 18.2%, and 21.5% in the presence of chitotriose, and 16.5%, 30.6%, 39.7%, and 31.4% respectively in the presence of chitotetraose. The residual activities of Cho-GG after 1 h of incubation at 60 C with 10 mM chitotriose or chitotetraose were 35.5% and 59.5% respectively, as shown in Fig. 4. Chitotetraose was more effective in preserving thermal stability than chitotriose.
Substrate specificity of Cho-GG Cho-GG (0.11 mg) hydrolyzed 0.2% chitosan5 (5– 10 cps at 0.5% solution), chitosan10 (5–20 cps at 0.5% solution), and chitosan100 (50–150 cps at 0.5% solution) in 100 mM acetate buffer (pH 5.5) at relative rates of 100%, 96%, and 90% respectively. This indicates that the degradation rates do not depend on the viscosity (molecular weight) of chitosan. The enzyme (0.28 mg) hydrolyzed neither glycolchitosan, chitin, carboxymethyl cellulose, barley -glucan, nor phosphoric-acid swollen cellulose (0.2% each). It hydrolyzed only lichenan at a relative rate of 18% of that of chitosan5. The hydrolysis patterns towards chitosan and chitooligosaccharides (chitobiose to chitohexaose) were analyzed by TLC, as shown in Fig. 5. Cho-GG hydrolyzed chitosan to chitobiose, chitotriose, and chitotetraose during a short incubation, and to chitobiose and
672
T. KOBAYASHI et al.
Residual activity (%)
100 50
1 0
10
20
30
40
50
60
Incubation time (min) Fig. 4. Time Course of the Thermal Stability of Cho-GG. Purified enzyme (2.2 mg) was incubated at 60 C in 100 mM acetate buffer (pH 6) with and without 10 mM chitotriose or chitotetraose in a total volume of 0.2 mL. Timed samples (30 mL) were withdrawn and diluted 10-fold with distilled water. Residual activities were measured under the standard assay conditions using 0.1 mL of dilute samples. The original activity without incubation was taken as the initial activity (100%). No addition ( ), chitotoriose ( ), chitotetraose ( ).
G1 G2 G3 G4 G5 G6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Fig. 5. Degradation of Chitosan and Chito-Oligomers by Cho-GG. The reaction mixture was composed of 0.3% chitosan or 7 mM chito-oligomers, 100 mM acetate buffer (pH 6), and 0.11 mg of purified enzyme in a total volume of 0.1 mL. The reaction was carried out at 50 C for 30 min to 18 h. Samples (20 mL each) were taken at several intervals and incubated at 100 C for 1 min to stop the reaction. The reaction products were analyzed by thin-layer chromatography, as described in ‘‘Materials and Methods.’’ Lanes 1 and 14, standards (n, number of glucosamine); lanes 2 and 3, products of chitobiose; lanes 4 and 5, products of chitotriose; lanes 6 and 7, products of chitotetraose; lanes 8 and 9, products of chitopentaose; lanes 10 and 11, products of chitohexaose; lanes 12 and 13, products of chitosan. Lanes 2, 4, 6, 8, 10, and 12, products after 30 min of incubation; lanes 3, 5, 7, 9, 11, and 13, products after 18 h of incubation.
chitotriose during a long incubation. It did not hydrolyze chitobiose or chitotriose. It hydrolyzed chito-oligomers longer than chitotetraose, which are barely degradable substrates. The final products of the reaction were chitotriose and chitobiose. Kinetics of chito-oligosaccharides The kinetic parameters of Cho-GG (0.11 mg) towards chitosan (0.05% to 0.4%), chitopentaose, and chitohexaose (0.3 to 2 mM) were determined using LineweaverBurk plots. The Km and Vmax values for chitosan were 0.42% and 833.3 mmol/min/mg of protein. The Km and kcat /Km values for chitopentaose and chitohexaose were 2.04 and 5.56 mM and 121.9 and 151.8 S1 respectively.
Effects of chemicals and metals on Cho-GG activity Cho-GG (0.54 mg) was incubated with several chemicals plus 20 mM acetate buffer (pH 6) in a total volume of 0.05 mL at 30 C for 30 min. After incubation, the treated solution was diluted 10-fold with distilled water, and enzyme activity was measured under the standard assay conditions. The enzyme was very stable under incubation with EDTA, EGTA, o-phenathroline, Nethylmaleimide, monoiodoacetate, and EDAC (5 mM each), 2-mercaptoethanoland dithiothreitol (2 mM each), N-bromosuccinimide (1 mM), and p-chlorolomercuribenzoate (0.1 mM). Only 2-hydoxy-5-nitrobenzyl bromide inhibited the enzyme activity, by 69% (5 mM) and 33% (1 mM). However, when the enzyme was incubated with the same chemicals in 20 mM acetate buffer (pH 4) at 30 C for 30 min, no inhibition was observed for any of the chemicals tested. No enzyme activity was activated at all when 100 mM of NaCl, KCl, or Na2 SO4 was added to the reaction mixture. When metal ions (1 mM each) were added to the reaction mixture, Hg2þ and Pd2þ strongly inhibited Cho-GG activity, by 98% and 96% respectively. Cu2þ , Co2þ , Fe3þ , and Ni2þ also moderately inhibited the enzyme activity, by 70%, 60%, 35%, and 25% respectively. Cho-GG was characteristically inhibited by transition elements belonging to families 8 to 11, which suggests that these metals easily coordinate with the enzyme molecule. Other metal ions, including Ca2þ , Sr2þ , Mn2þ , Fe2þ , Sn2þ , and Zn2þ , did not affect the enzyme activity at all. No metal ions tested activated it. Nucleotide and deduced amino acid sequences of Cho-GG The 1,828-bp nucleotide fragment amplified contained one open reading frame of 1,362 bp that started with an ATG codon and ended with a TAA codon. The deduced amino acid sequence was composed of 453 amino acids containing a putative signal sequence of 46 amino acids. The 15 N-terminal amino acid residues of the purified enzyme from B. thuringiensis strain JAMGG01 were found in the deduced amino acid sequence from Ala47 to Tyr61 . The molecular mass and pI value of the mature enzyme were calculated to be 45,910 Da and pH 8.3. The deduced amino acid sequence of Cho-GG (AB555586) showed the highest level of similarity to a chitosanase (YP 002446102.1) of B. cereus G9842, with 98.9% identity. The next best matches were also high levels of homology to family 8 chitosanases BTC31 (ABO61888.1), BCT30 (ABO61890.1) of B. thuringiensis serovar sandiego, BTC36 (ABO61891.1) of B. thuringiensis serovar darmstadiensis, at 98.7%, 98.5%, and 98.5% identity respectively. Thus, Cho-GG belongs to GH family 8 of chitosanases. The thermal stability of many chitosanases belonging to GH family 8 can be enhanced by adding chitotetraose or chitotriose. Furthermore, the thermal stability of chitosanases belonging to GH family 46 might also be retained by chitotetraose or chitotriose. Fukamizo et al.29) reported that the transition temperature of the thermal unfolding (Tm ) of B. circulans MH-K1 chitosanase, belonging to GH family 46, was elevated to 2.2 C (Tm ) when chitotriose was added. Recently, Roy et al.30) also reported that chitosan itself protects the thermal denaturation of family 46 chitosanase of
Chitosanase of a Deep Biosphere Bacillus
Streptomyces sp. N174. Though the Streptomyces enzyme degraded chitosan to 5 to 15 chito-oligomers during fluorometric analysis, chito-oligomers, including chitosan, securely protected the thermal unfolding of GH family 46 chitosanase. Although these experiments were performed to measure the thermal unfolding (Tm ), chito-oligomers might enhance the thermal stability of GH family 46 chitosanases as well as GH family 8 ones. B. thuringiensis JAM-GG01 appears to be originally dropped or co-sedimented with soil components, and then resides in the deep sub-seafloor. Hence Cho-GG did not show definite characteristics of a sub-seafloor ectoenzyme, but is the first finding that chito-oligomers, especially chitotriose and chitotetraose, enhance the thermal stability of chitosanase.
References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12)
Bartnicki-Garcia S, Ann. Rev. Microbiol., 22, 87–108 (1968). Hall SR, Proc. R. Soc. A, 465, 335–366 (2009). Sugano M, Fujikawa T, Hiratsuji Y, Nakashima K, Fukuda N, and Hasegawa Y, Am. J. Clin. Nutr., 33, 787–793 (1980). Suzuki K, Mikami T, Okawa Y, Tokoro A, Suzuki S, and Suzuki M, Carbohydr. Res., 151, 403–408 (1986). Roller S and Covill N, Int. J. Food Microbiol., 47, 67–77 (1999). Shahidi F, Arachchi JKV, and Jeon Y-J, Trends Food Sci. Technol., 10, 37–51 (1999). Gallaher CM, Munion J, Hesslink RJr, Wise J, and Gallaher DD, J. Nutr., 130, 2753–2759 (2000). Barreteau H, Delattre C, and Michaud P, Food Technol. Biotechnol., 44, 323–333 (2006). Tominaga Y and Tsujisaka Y, Biochim. Biophys. Acta, 410, 145–155 (1975). Pelletier A and Sygusch J, Appl. Environ. Microbiol., 56, 844– 848 (1990). Yoon H-G, Kim H-Y, Lim Y-H, Kim H-K, Shin D-H, Hong B-S, and Cho H-Y, Appl. Environ. Microbiol., 66, 3727–3734 (2000). Aktuganov GE, Shirokov AV, and Melent’ev AI, Appl. Biochem. Microbiol., 39, 536–541 (2003).
13) 14) 15) 16) 17)
18) 19) 20)
21) 22)
23) 24) 25) 26) 27) 28) 29)
30)
673
Camarillo RC, Pe´rez OS, Avelizapa NGR, Ramı´rez MG, and Avelizapa LIR, Folia Microbiol., 49, 94–96 (2004). Choi YJ, Kim EJ, Piao Z, Yun YC, and Shin YC, Appl. Environ. Microbiol., 70, 4522–4531 (2004). Lee HS, Jang JS, Choi SK, Lee DW, Kim EJ, Jung HC, and Pan JG, FEMS Microbiol. Lett., 277, 133–141 (2007). Kendra DF, Christian D, and Hadwiger LA, Physiol. Mol. Plant Pathol., 35, 215–230 (1989). Kobayashi T, Koike K, Yoshimatsu T, Higaki N, Suzumatsu A, Ozawa T, Hatada Y, and Ito S, Biosci. Biotechnol. Biochem., 63, 65–72 (1999). Payasi A, Sanwal R, and Sanwal GG, World J. Microbiol. Biotechnol., 25, 1–14 (2009). De Lorenzo G, Cervone F, Hahn MG, Darvill A, and Albersheim P, Physiol. Mol. Plant Pathol., 39, 335–344 (1991). Kobayashi T, Koide O, Mori K, Shimamura S, Matsuura T, Miura T, Takaki Y, Morono Y, Nunoura T, Imachi H, Inagaki F, Takai K, and Horikoshi K, Extremophiles, 12, 519–527 (2008). Laemmli UK, Nature, 227, 680–685 (1970). Guinebretie`re M-H, Thompson FL, Sorokin A, Normand P, Dawyndt P, Ehling-Schulz M, Svensson B, Sanchis V, NguyenThe C, Heyndrickx M, and De Vos P, Environ. Microbiol., 10, 851–865 (2008). Pedraza-Reyes M and Gutie´rrez-Corona F, Arch. Microbiol., 168, 321–327 (1997). Kurakake M, Yo-u S, Nakagawa K, Sugihara M, and Komaki T, Curr. Microbiol., 40, 6–9 (2000). Omumasaba CA, Yoshida N, Sekiguchi Y, Kariya K, and Ogawa K, J. Gen. Appl. Microbiol., 46, 19–27 (2000). Chiang C-L, Chang C-T, and Sung H-Y, Enzyme Microbial. Technol., 32, 260–267 (2003). Zhu X-F, Wu X-Y, and Dai Y, Biosci. Biotechnol. Biochem., 67, 284–290 (2003). Su C, Wang D, Yao L, and Yu Z, J. Agric. Food Chem., 54, 4208–4214 (2006). Fukamizo T, Amano S, Yamaguchi K, Yoshikawa T, Katsumi T, Saito J, Suzuki M, Miki K, Nagata Y, and Ando A, J. Biochem., 138, 563–569 (2005). Roy S, Fortin M, Gagnon J, Ghinet MG, LeHoux J-G, Dupuis G, and Brzezinski R, Biochim. Biophys. Acta, 1774, 975–984 (2007).
