Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis

International Biodeterioration & Biodegradation 96 (2014) 26e32

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Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis Qingyang Liu 1, Tiehan Zhang 1, Nan Song, Qian Li, Zhi Wang, Xuewen Zhang, Xiangyang Lu, Jun Fang, Jinjun Chen* College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 April 2014 Received in revised form 15 July 2014 Accepted 18 August 2014 Available online

A feather-degrading bacterium was isolated from the gut of the tarantula Chilobrachys guangxiensis, and was classified as Bacillus subtilis (named Bacillus subtilis CH-1) according to both the phenotypic characteristics and 16S rRNA profile. The improved culture conditions for feather-degrading were 10.0 g l
1 mannitol, 10.0 g l
1 tryptone, 0.1 g l
1 MgCl2, 0.4 g l
1 KH2PO4, 0.3 g l
1 K2HPO4, 0.5 g l
1 NaCl, and 2.0 g l
1 intact feather, with pH 8.5 and 37  C. In the optimized medium, the intact black feather was completely degraded by Bacillus subtilis CH-1 in 24 h. Furthermore, four kinds of enzymes which include extracellular protease Vpr, peptidase T, g-glutamyl transpeptidase and glyoxalmethylglyoxal reductase were identified as having principal roles. Simultaneously, the relationship between the disulfide bond reducing activity (DRT) and the keratinase activity (KT) in B. subtilis CH-1 fermentation system was discussed. This is the first report for a feather-degrading enteric bacterium from tarantula. The identification of the enzymes shines a light on further understanding the molecular mechanism of featherdegrading by microbes. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Bacillus subtilis Keratinase Enteric bacteria Enzyme system

Introduction Fibrous proteins are widely distributed in most animal tissues and play an important role in conferring strength and rigidity. Currently, millions of tons of fibrous proteins are discarded annually worldwide, which are largely comprised of “keratin”, such as feather (b-keratin), wool (a-keratin) and human hair (Brandelli et al., 2010). Keratins are recalcitrant to the common known proteolytic enzymes such as trypsin, pepsin and papain, since they consist of highly rigid and strongly cross-linked structures (Gupta and Ramnani, 2006). In particular, feather is the largest source of keratin, and used as organic fertilizers and feed supplements since it is rich in proteinaceous compounds that contain high levels of nitrogen and important amino acids like cystine, threonine and arginine (CAaC, 2008). Keratinases [EC 3.4.21/24/99.11] were

* Corresponding author. Tel./fax: þ86 731 84673602. E-mail addresses: [email protected] (Q. Liu), [email protected] (T. Zhang), [email protected] (N. Song), [email protected] (Q. Li), [email protected] (Z. Wang), [email protected] (X. Zhang), chhncjj@126. com (J. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ibiod.2014.08.008 0964-8305/© 2014 Elsevier Ltd. All rights reserved.

extensively sought from diverse microorganisms involving different bacteria (Cedrola et al., 2011; Mazotto et al., 2011; Sahoo et al., 2012), actinomycetes (Chitte RR, 1999; Gousterova et al., 2005) and fungi (el-Naghy et al., 1998; Kim, 2007; Rodrigues Marcondes et al., 2007) to degrade a variety of substrates, such as chicken feather (Yamamura et al., 2002), human hair (Mazotto et al., 2010) and wool (Cai et al., 2011). The commercial preparations (i.e. Versazyme, Valkerase and Prionzyme) have already been produced based on keratinase following the recent research progresses (Odetallah et al., 2005; Rutala et al., 2010). Meanwhile, keratinase researches are being further conducted, from basic enzymology metabolism to potential environmental and biotechnological applications. As we know, the tarantulas regurgitate digestive juice onto the immobilized prey through the mouth to break down the prey body until the body is completely liquefied, and suck up the pre-digested prey. The digestive juice of tarantula has high digestive ability to hydrolyze most internal structure of the prey within a short time. Of particular note, the usual preys of tarantula are orthopteran and blattaria whose internal structures are mainly made of firm protein and cellulose (Gerholdt, 1995). We speculate that some bacterial gut symbionts which produce high-efficiency enzymes help the

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host's digestive fluid to degrade firm fibrin. In this study, we isolate B. subtilis CH-1 showing high keratinolytic activity from the gut of tarantula. In addition, four key enzymes including two kinds of keratinases and two kinds of resductases of B. subtilis CH-1 are isolated and analyzed, which develops an understanding of featherdegradation mechanism. Materials and methods Collection of spider digestive fluid and native feather The tarantula C. guangxiensis was collected in Hainan Province of China and fed with sterile pig's liver as food in a germfree chamber. The digestive fluid was collected from the enteric canal of mature individuals, which surface disinfected with 70% ethyl alcohol, in a laminar flow cabinet. The fresh feather mainly composed of keratin (91.90% of dry matter), water (8.0%) and a low lipid content (1.3%) (Lederer, 2005) was collected from a local poultry-processing store, and then soaked with tap water 3 times each for 15 min and finally with double-distilled water for 3 times, followed by drying at 60  C for 12 h. Isolation and screening of feather-degrading bacteria The digestive fluid was coated on agar plates with a basal medium (0.4 g l
1 KH2PO4, 0.3 g l
1 K2HPO4, 0.5 g l
1 NaCl) with 2.0 g l
1 feather-powder at pH 8.5 and 37  C for 48 h. The feather, only source of carbon and nitrogen in the medium, limits the growth of microorganism which is incapable of degrading feather. The growth of isolates on the plates was checked, and each of wellgrown isolate was picked and inoculated individually in a basal whole-feather liquid medium (the basal medium added with 2.0 g l
1 whole black feather) and incubated at 37  C, 160 rpm for 48 h to confirm the capability of feather-degrading. The effective colonies which degraded whole-feather within two days were selected for further investigation. The purity of the isolate was confirmed with single colony through repeated streaking. Identification of feather-degrading strain Isolated strains were identified by sequence analysis of 16S rRNA genes. Genomic DNA was extracted as follows. Briefly, the strain was inoculated into an LB medium and cultured at 37  C for 16 h. Cultures (1 ml) were centrifuged at 4000 g for 5 min. Supernatant was removed, and the pellet was resuspended in 250 ml of precooled sucrose-N-[Tris (hydroxymethyl) metyl]-2aminopropanesulfonic acid and 40 ml of lysozyme (10.0 g l
1, pH 8.0) at 37  C for 15 min, followed by mixing with a DNA extraction buffer (60 ml of 10% SDS, 250 ml of 0.25 mM EDTA, pH 8.0) at 37  C for 30 min. The suspension was extracted successively by a mixture of phenol and chloroforms (1:1 volume) and by pure chloroform. DNA was precipitated by adding two volumes of 100% pre-cooled ethanol. The pellet was then dissolved into 50 ml of distilled water and quantified using a spectrophotometer. The 16S rRNA genes of the isolated microorganisms were amplified by PCR using both a forward primer (27F, 50 -AGAGTTTGATCMTGGCTCAG, M ¼ C: A) and reverse primer (1492R, 50 -TACGGYTACCTTGTTACGACTT, Y ¼ C: T). PCR products were purified using the columns from Wizard PCR preps DNA purification systems (Promega, USA). PCR product with the expected insert size was cloned into pUM-T (BioTeke, China) as recommended by the manufacturer. The recombinant plasmids were constructed on Shanghai Major Pharmaceutical Technology Co., Ltd (Shanghai, China). The BLAST algorithm was used to search homologous sequences in GenBank (http://blast.ncbi.nlm.nih.gov/

27

Blast.cgi). The nucleotide sequences reported here have been submitted to the GenBank database and an assigned accession number KC167327.1 for 16S rRNA of strain B. subtilis CH-1. Feather degradation The percentage of feather degradation was measured by weight loss. The feather still present in medium after the cultivation was filtered through Whatman Grade No. 1 filter paper. Then feather pellet was thoroughly washed with 70% alcohol and oven dried at 60  C for 48 h, and finally weighed to determine weight loss. The percentage of residual feather lower 5% meaning that feather was completely degraded (Mazotto et al., 2011). Assay of disulfide bond reducing activity (DRT) The DRT was measured by modified Pathange Prakash method (Prakash et al., 2010). The culture supernatant (500 ml) was incubated with 0.02 g of feather powder at 37  C for 1 h. The reaction was terminated by adding 4.0 ml of 10.0 g l
1 trichloroacetic acid (TCA). The resulting solution was mixed with 1.0 ml of 10.0 mM 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) for staining. The disulfide bond reducing activity was determined spectrophotometrically at 412 nm. One unit of DRT (IU ml
1) was defined as the amount of enzyme needed to catalyze 1 mM of disulfide bonds per minute. TCA was added to the control group before enzymatic reaction. Assay of keratinase activity (KT) The KT was measured by the modified Helena method using soluble keratin (0.5%, w/v) as substrate. Soluble keratin was prepared according to method of Wawrzkiewicz et al. (1987). Native chicken feathers (10 g) were soaked in 500 ml of dimethyl sulfoxide and heated in a hot air oven at 100  C for 2 h, and then precipitated by addition of 1000 ml pre-cold acetone at
70  C for 2 h, followed by centrifugation at 10,000 g for 10 min. The supernatant was removed immediately. Precipitate was washed twice with distilled water and dried at 40  C in a vacuum dryer. Then, 1 g of the precipitate was dissolved in 20 ml of 0.05 M NaOH, with pH adjusted to 8.0 via 0.1 M HCl, finally diluted to 200 ml soluble keratin solution by using phosphate buffer (0.05 M, pH 8.0). The cultures solution was centrifuged at 4000 g for 10 min, and the supernatant was used as a crude enzyme preparation. The reaction consisting 1.0 ml of culture supernatant and 1.0 ml of keratin solution was incubated at 37  C water bath for 1 h, then terminated by adding 3.0 ml 0.4 M TCA. After centrifugation at 1500 g for 30 min at 4  C, the supernatant was collected and used for assaying the absorbance at 280 nm against a control, which was prepared by adding with 3.0 ml 0.4 M TCA before enzymatic reaction. One unit (IU ml
1) of KT was defined as an increase of corrected absorbance of 280 nm with the control for 0.01 per minute (Gradisar et al., 2005). Effect of culture conditions Culture conditions were optimized by testing a range of initial pH from 6.0 to 9.0 with increases of 0.5 while keeping temperature at 37  C, and by varying temperature (25, 32, 37, 42e50  C) while keeping initial pH at 8.5. 500 mL erlenmeyer flasks contained 100 ml of the basal whole-feather liquid medium were incubated with 1 ml of 106 colony-forming units (CFU) ml seed culture, and shaken at 160 rpm for 24 h. The medium components were investigated in the basal wholefeather liquid medium at pH 8.5 and 37  C, and supplemented with various carbon sources, nitrogen sources and metal ions separately.

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Q. Liu et al. / International Biodeterioration & Biodegradation 96 (2014) 26e32

The additional carbon sources (10.0 g l
1) included glucose, maltose, sucrose, citric acid, mannitol and inositol respectively (Conville and Witebsky, 2001). Then, additional nitrogen sources (10.0 g l
1), including beef extract, carbamide, tryptone, yeast extract, yeast nitrogen base and NH4NO3, were selected individually. Additional metal ion compounds (0.1 g l
1), including MgCl2, NaCl, CaCl2, CuSO4, ZnSO4 and KCl, were also added separately (Alvarado-Cuevas et al., 2013). Measurement of cell growth and soluble protein In order to evaluate the effects of different culture conditions on B. subtilis CH-1 feather-degrading capability, we measured the three factors including cell growth, soluble protein concentration and DRT of culture solution at 18 h of cultivation (Jeong et al., 2010). First, cell growth was estimated by the measuring the absorbance at 600 nm with UVmini-1240 (SHIMADZU, Japan). An uninoculated basal feather medium was used as the control. And then, the culture solution was centrifuged at 4000 g for 10 min, and the supernatants were used for soluble protein concentration and DRT assays. The soluble protein concentration was determined by using a bicinchoninic acid (BCA) assay kit (Sigma, USA) according to the manufacturer's instructions with bovine serum albumin as the standard. Statistical analysis All experimental results were conducted independently in quadruplicate each time and repeated 3 times by different operators at different times. The results presented as mean value ± standard deviations (SD). Analysis of variance (ANOVA) with repeated measures was carried out using GLM command of software SPSS ver18.0.

In-gel digestion and LC-MS/MS analysis All in-gel digestions of proteins were performed in a laminar flow hood all the time. After SDS-PAGE, the gel pieces were cut into 5-mm-thick pieces and washed twice with Milli-Q water (Millipore Corporation, USA) and then 50% acetonitrile/25 mM ammonium bicarbonate in order. Then, the gel fragments were soaked in a solution containing 10 mM DTT and 55 mM iodoacetamide in 25 mM ammonium bicarbonate at 56  C for 45 min in the dark. The sample was incubated in 10 ml of modified trypsin digestion buffer (0.1 mg ml
1) with 25 mM ammonium bicarbonate at 37  C overnight. After the supernatant was transferred to an Eppendorf tube, 20 ml of 50% acetonitrile/5% formic acid was added, and the peptides were sonicated for 10 min to aid extraction. After centrifuging 2000 g for 10 min, this supernatant was combined with the previous supernatant and then dried down. Finally, each peptide sample in 20 ml of 5% acetonitrile/0.1% formic acid was subjected to LC-MS/MS analysis. Peptides were separated by reverse-phase chromatography on 20-cm columns (inner diameter 75 mm, 1.8 mm ReproSil-Pur C18-AQ media). The nanoACQUITY UPLC (Waters Corporation, USA) was coupled to a Finnigan LTQ-Orbitrap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). Peptides were loaded onto the column with buffer A (0.5% acetic acid) and eluted with a 150 min linear gradient from 2 to 35% buffer B (80% acetonitrile, 1.0% formic acid). After the gradient the column was washed with 90% buffer B and re-equilibrated with buffer A. MS spectra were acquired in the Orbitrap analyzer, with a mass range of 400e2000 Da and a target value of 106 ions. Peptide fragmentation was performed with the HCD method (Olsen et al., 2007) and MS/MS spectra were acquired in the Orbitrap analyzer and a target value of 40,000 ions. Ion selection threshold was set to 5000 counts. In silico analysis

Purification of enzymes All operations were done below 10  C. The supernatant was filtered through a 0.22-mm cellulose membrane (Millipore, Bredford, USA) and precipitated by adding solid ammonium sulphate (0e80% saturation). The protein precipitate obtained was collected by centrifugation at 10,000 g for 20 min at 4  C and then dissolved in phosphate buffer (50 mM, pH 8.0), and then dialyzed (Cellophane membrane, Sigma) with three changes at 8 h intervals. After dialysis, the enzyme example was adjusted to pH 4.2 with acetic acid and directly applied to a CM32-cellulose cation-exchange column, which was equilibrated with 0.1 M sodium acetate at a flow rate of 4 ml min
1. Upon reaching a steady base line, the column was washed by stepwise elution with 0.1, 0.3, 0.5, and 1.0 M NaCl equilibration buffer. These elution solutions were desalinated and concentrated by using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-3 membrane (EMD Millipore, USA) and preserved at
70  C for further study. Analysis on sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) Samples were prepared by mixing with 5 X electrophoresis buffer (10 mmol l
1 TriseHCl, pH 8.0; 2.5% SDS, w/v; 10% glycerol, v/v; 5% b-mercaptoethanol, v/v; 0.002% bromophenol blue, w/v), then heated at 98  C for 5 min. Then, Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was performed with 12% polyacrylamide resolving gels and 5% stacking gels (Laemmli, 1970). The relative molecular mass of the protease was determined using a prestained protein marker (GEN-VIEW SCIENTIFIC INC, USA). After electrophoresis, the gels were stained with Coomassie Brilliant Blue G-250.

Database searches for MS/MS spectra were conducted using MASCOT search engine against the complete proteome NCBInr database for known proteins from Bacillus subtilis. The gene ontology (GO) annotation of results were performed using Blast2GO version 2.7.0 following settings: a pre-E-value-Hit-Filter of 1.0E-6, a GO weight of 5, and the annotation cut-off of 55 (Neilson et al., 2011). The pathways of partial enzymes were predicted using BIOCYC software (http://www.biocyc.org/). Results Isolation and identification of the feather-degrading bacterium B. subtilis CH-1 A total of 7 high hydrolysis activity bacterial stains were obtained and these strains belonged to three different genre including Bacillus subtilis, Pseudomonas aeruginosa and Xanthomonas maltophilia (data not shown). Since bacillus subtilis is globally known Gram-positive bacterial strain categorized as totally harmless eubacteria. B. subtilis CH-1, the most efficient Bacillus subtilis, was chosen to be investigated further. The degrading processes of whole intact feathers incubated with B. subtilis CH-1 were observed. At first, the feather barbules were digested into pieces after 16 h of incubation in basal feather medium. After 24 h, feather powder appeared in the medium, and feather was fractured to shorter pieces, but not separated from rachis. After 36 h, feather pieces were degraded thoroughly (Fig. 1). The 16S rRNA sequence (Table A1) of B. subtilis CH-1 was submitted to the GenBank database of NCBI (accession No. KC167327). The sequence showed 99% homology with Bacillus subtilis DSM 10

Q. Liu et al. / International Biodeterioration & Biodegradation 96 (2014) 26e32

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low at the very beginning 14 h of the fermentation. There has been a significant increase performance of KT in the next 7 h. The highest peak (3.8 IU ml
1) of the value of KT exists with the same time point as the lowest peak of DRT. Effect of culture conditions

Fig. 1. The degradation of feather in a mineral basal medium for 36 h fermentation at 37  C (on the right); The control is on the left.

(Genbank accession No. NR 027552) compared with Genbank. Comparison of this sequence with those available from the NCBI public databases showed that B. subtilis CH-1 was most closely related to the Bacillus members. This strain was deposited in CGMCC (CCTCC NO. M2011111). The enzyme activities of keratin hydrolysis and disulfide bond reduction The enzyme activities of keratin hydrolysis (KT) and disulfide bond reduction (DRT) of B. subtilis CH-1 in basal feather medium were measured at 7 h, 14 h, 21 h, 28 h, 35 h fermentation respectively. Typical time courses for the DRT and KT of B. subtilis CH-1 in basal feather medium were shown in Fig. 2. The value of DRT is changing from high to low with the lowest peak (9.6 IU ml
1) at around 21 h, then increasing again and reach a new high point (17.4 IU ml
1) at 35 h of the fermentation. Intriguingly, the value of KT is

To study the effect of culture conditions on feather-degrading, growth was carried out in basal medium with varied initial pH, temperature and carbon/nitrogen sources. Firstly, the effects of initial pH and temperature were investigated. B. subtilis CH-1 grew at ranges of 25e50  C and pH 6.0e9.0 (Fig. A1) with an optimum effect at 37  C (data not shown) and pH 8.5 in a basal feather medium. As shown in Table 1, reductase activity and soluble protein concentration were the highest at pH 8.5, whereas cell growth was the fastest at pH 7.5. The effects of carbon compounds were investigated in the basal whole-feather medium at pH 8.5 and 37  C, which are shown in Table 2. DRT was the highest (12.00 ± 0.15 IU ml
1) with mannitol. The cell growth and soluble protein concentration showed the same trend as DRT. A majority of the applied carbon sources showed positive effects, while citric acid showed negative effects (Fig. A2). As shown in Table 3, the additional nitrogen sources of tryptone, yeast extract and beef extract considerably improved the levels of degradation and cell growth compared with the basal medium. DRT reached 16.52 ± 0.24 IU ml
1 with tryptone in the medium, which is about double higher than the yield in the basal feather medium without any additive (8.13 ± 0.05 IU ml
1). In contrast, addition of carbamide or NH4NO3 restrained the levels of degradation (Fig. A3). However, the metal ion test showed indistinguishable effects (Table 4, Fig. A4). DRT was improved slightly by Mg2þ or Ca2þ, was only changed a little by Naþ or Kþ, but was repressed by Cu2þ or Zn2þ. In this work, an improved medium for feather-degrading was established, which contained 10.0 g l
1 mannitol, 10.0 g l
1 tryptone, 0.1 g l
1 MgCl2, 0.4 g l
1 KH2PO4, 0.3 g l
1 K2HPO4, 0.5 g l
1 NaCl, and 2.0 g l
1 intact feather, with pH 8.5 and 37  C. This optimized medium greatly improved DRT and soluble protein concentration. The degradation time of intact black feather was reduced from 36 h to less than 24 h. Purification and identification of enzymes The supernatant after ammonium sulphate precipitation was dialyzed and checked for both keratinolytic and reductase activity, which resulted in an enzyme preparation with a KT of 4.86 IU ml
1 with 1.28 purification fold. This fraction was loaded on to a CM32cellulose cation-exchange chromatography column. The proteins were eluted and fractions were analyzed for the enzyme activities

Table 1 Effect of initial pH on production of keratinase by Bacillus subtilis CH-1 at 18 h in mineral feathers medium. Initial pH

Cell growth (A600)

6.0 6.5 7.0 7.5 8.0 8.5 9.0

0.34 0.33 0.49 0.51 0.32 0.50 0.30

a

Fig. 2. The production of KT and DRT during the course of 35 h of B. subtilis CH-1 fermentation in the basal feather medium.

± ± ± ± ± ± ±

b 0.03 0.04 0.05 0.03 0.03 0.02 0.01

Degradation time (hour)

Soluble protein (mg ml
1)

UDa 36.00 30.00 36.00 30.00 30.00 UDa

4.77 9.70 14.11 12.03 11.51 16.44 5.03

± ± ± ± ±

3.00 3.00 2.00 3.00 4.00

± ± ± ± ± ± ±

0.26 0.33 0.57 0.55 0.52 0.56 0.29

DRT (IU ml
1) 5.04 6.58 8.00 5.26 7.88 8.13 5.06

± ± ± ± ± ± ±

0.12 0.23 0.10 0.05 0.15 0.16 0.01

UD: Undegraded within 36 h. ±: Values are expressed as mean ± standard deviation of four independent experiments. b

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Table 2 Effect of extra carbon source on production of keratinase by Bacillus subtilis CH-1 at 18 h in mineral feathers medium. Extra carbon source (10 g l
1)

Cell growth (A600)

Nonea Glucose Maltose Sucrose Citric acid Mannitol Inositol

0.50 0.23 0.26 1.20 0.06 1.28 1.06

± ± ± ± ± ± ±

0.02 0.02 0.05 0.03 0.01 0.05 0.03

Degradation time (hour) 30.00 36.00 36.00 30.00 UD 24.00 30.00

± ± ± ±

4.00 2.00 4.00 3.00

± 2.00 ± 3.00

Soluble protein (mg ml
1) 16.44 27.60 29.15 41.86 14.89 46.45 42.90

± ± ± ± ± ± ±

0.56 1.53 1.55 1.98 0.70 1.01 2.00

DRT (IU ml
1) 8.13 9.70 10.40 11.32 3.04 12.00 11.26

± ± ± ± ± ± ±

0.16 0.23 0.20 0.17 0.13 0.15 0.16

Table 4 Effect of extra metal ion on production of keratinase by Bacillus subtilis CH-1 at 18 h in mineral feathers medium. Extra metal ion (0.1 g l
1)

Cell growth (A600)

None MgCl₂ NaCl CaCl₂ CuSO₄ ZnSO₄ KCl

0.50 0.44 0.38 0.37 0.15 0.27 0.20

± ± ± ± ± ± ±

0.02 0.02 0.01 0.05 0.01 0.05 0.04

Degradation time (hour) 30.00 30.00 36.00 36.00 UD UD 36.00

± ± ± ±

4.00 2.00 2.00 4.00

± 3.00

Soluble protein (mg ml
1) 16.44 14.37 18.52 19.55 4.77 6.59 11.51

± ± ± ± ± ± ±

0.56 0.90 1.00 1.10 1.00 1.20 1.02

DRT (IU ml
1) 8.13 8.58 8.00 8.64 2.46 2.94 7.90

± ± ± ± ± ± ±

0.16 0.14 0.13 0.15 0.11 0.16 0.22

a

None: CH-1 was cultivated in mineral feather medium without any additive at pH8.5.

of keratin hydrolysis and disulfide bond reduction. The fraction with high keratinolytic and reductase activity (14.4 IU ml
1, 20.3 IU ml
1 respectively) was performed by SDS-PAGE. Coomassie blue stained SDS polyacrylamide gel of the purified enzyme preparations revealed four clear bands with estimated molecular masses of 68 KDa, 64 KDa, 45 KDa, 32 KDa respectively (Fig. 3). Proteomic analysis of protein bands from SDS-PAGE The four main bands were excised, digested and analyzed with LC-MS/MS. A peptide scores are equal to or greater than the significance threshold (P < 0.05) could be considered as positive protein hits. There are totally 13 proteins were identified after LCMS/MS analysis. These identified proteins were further classified according to their GO annotations (Table A2). Noticeably, four proteins (extracellular protease Vpr, glyoxal/methylglyoxal reductase, g-glutamyl transpeptidase, peptidase T) (Table 5) were predicted involved in the progress of feather-degradation according to GO annotations (Fig. 4) (Strauch and Miller, 1983; Kho et al., 2005; Ghosh et al., 2008; Tiwary and Gupta, 2010; Sharma and Gupta, 2012). Discussion

proteolytic enzymes can effect on the substrates. The activity of keratinases from Bacillus pumilus KS12 was enhanced by two and eight folds in presence of 10 mM DTT and b-mercaptoethanol (Rajput et al., 2010). It has been suggested that sulfitolysis, as a prerequisite for keratin degradation, be performed by disulfide reductases, chemical or live cell redox to play an important role in reduction of disulfide bonds of keratin, then the proteolytic attack be accomplished by keratinases (Ramnani et al., 2005; Ramnani and Gupta, 2007; Ghosh et al., 2008; Brandelli et al., 2010). In present study, the two kinds of enzyme types of B. subtilis CH1, keratinases (extracellular protease Vpr and peptidase T) and reductases (g-glutamyl transpeptidase and glyoxal/methylglyoxal reductase) were purified and identified. Extracellular protease Vpr of B. subtilis can function like a keratinase (Kho et al., 2005; Ghosh et al., 2008; Choi et al., 2010) and peptidase T can enhance hydrolysis of protein (Strauch and Miller, 1983). With respect to reductases, g-glutamyl transpeptidase (GGT) is a periplasmic enzyme and plays a key role in the gammaeglutamyl cycle by transferring the glutamyl moiety to a variety of acceptor molecules including certain L-amino acids and peptides to hydrolyze glutathione and then produce cysteinyl-glycine to further reduce disulfide bonds (Mazmanian et al., 2005; Tiwary and Gupta, 2010). It has been identified that the role of GGT-GSH serves as a redox principle during degradation of feather keratin (Sharma and Gupta, 2012). Glyoxal/methylglyoxal reductase belongs to the aldo/keto reductase family and can catalyze methylglyoxal and glutathione to S-

A bacterium isolated from the gut of tarantula C. guangxiensis was identified as B. subtilis CH-1 on the basis of 16S rRNA gene sequence analysis. This isolate possesses high keratin degrading ability. The whole black feather was hydrolyzed completely within 24 h in the optimal medium. We observed DRT was high but KT was low at the first 7 h during the whole feather degradation, the two kinds of enzymes (KT and DRT) vary in a relatively inconsistent manner during the fermentation. Here, we proposed a hypothesis about the relationship between KT and DRT of B. subtilis CH-1: the process of b-keratindegradation begins with reduction especial for disulfide-broken, which loose the compact structure of keratins, so that the

Table 3 Effect of extra nitrogen source on production of keratinase by Bacillus subtilis CH-1 at 18 h in mineral feathers medium. Extra nitrogen source (10 g l
1)

Cell growth (A600)

None beef extract Carbamide Tryptone Yeast Extract Yeast Nitrogen Base NH₄NO₃

0.50 1.73 0.05 1.83 1.91 0.30

± ± ± ± ± ±

0.02 0.07 0.01 0.02 0.07 0.03

0.23 ± 0.04

Degradation time (hour) 30.00 30.00 UD 24.00 30.00 36.00 UD

± 4.00 ± 3.00 ± 3.00 ± 2.00 ± 3.00

Soluble protein (mg ml
1) 16.44 9.18 11.26 9.44 7.85 6.33

± ± ± ± ± ±

0.56 1.08 1.10 0.50 0.90 1.00

5.55 ± 0.90

DRT (IU ml
1) 8.13 12.26 3.82 16.52 13.00 7.68

± ± ± ± ± ±

0.16 0.17 0.14 0.24 0.21 0.23

2.84 ± 0.11

Fig. 3. Coomassie blue-stained SDS-polyacrylamide gel after electrophoresis of the purification of crude enzyme. Lanes: 1, Marker proteins; 2, Crude enzyme; 3, The 0.1 M NaCl elution solution. The enzyme system with extracellular protease Vpr (Epv), gglutamyl transpeptidase (g-Glu), peptidase T (Pep T) and glyoxalmethylglyoxal reductase(MetR) was performed in the lane 3.

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Table 5 The four key enzymes of LC-MS/MS analyses with GO annotation. Sequence name

Seq description

Length

Number of unique peptides

gij16080943jrefjNP_391771.1j

Peptidase t

410

5

gij2635853jembjCAB15345.1j

Glyoxal methylglyoxal reductase

276

2

gij320017761jgbjADV92747.1j

Gammaglutamyltranspeptidase

587

14

gij62511911jgbjAAX84518.1j

Extracellular protease vpr

646

42

lactoyl-glutathione by BIOCYC software (Fig. A5). Herein, we suggest that glyoxal/methylglyoxal reductase and g-glutamyl transpeptidase firstly catalyze a couple of reduction in keratin including reduction of disulfide bond (Gupta et al., 2013). Then, the combination of extracellular protease Vpr and peptidase T play a crucial role in the degradation of subsequent proteins and peptides. In addition, we tentatively put forward the possible explanation of the phenomena of the cell growth is inhibited when reducing sugars (glucose and maltose) in medium in this article: the reducing sugar containing polyhydroxyaldehyde (aldehyde group) can be converted to corresponding alcohol catalyzed by aldose reductase using NADPH as the electron donor (der Jagt et al., 1992; Del Corso et al., 2000; Van). That may inhibit methylglyoxal reductase to degrade methylglyoxal by competing NADPH (Inoue et al., 1988). As a result, the accumulation of cytotoxic methylglyoxal inhibit cell growth (Nguyen et al., 2009). Some recent studies focus on bacterial gut symbionts of the termites (Russell et al., 2009), and quite a few of the symbionts were isolated, which showed that bacterial gut symbionts play a crucial role in helping termites to digest by secreting various enzymes (Shinzato et al., 2007). Correlatively in this study, the strain

GO descriptions

Enzyme descriptions

C:cytoplasm; F:tripeptide aminopeptidase activity; P:peptide metabolic process; P:proteolysis; F:zinc ion binding; F:metallopeptidase activity F:methylglyoxal reductase (NADPH-dependent) activity; F:2,5-didehydrogluconate reductase activity; P:oxidation-reduction process

(3.4) Acting on peptide bonds (peptide hydrolases)

97

(1.1) Acting on the CHeOH group of donors; (1.1) Acting on the CHeOH group of donors (2.3) Acyltransferases

176

F:gammaeglutamyltransferase activity; C:extracellular region; F:glutathione hydrolase activity; P:glutathione biosynthetic process C:extracellular region; P:negative regulation of catalytic activity; P:proteolysis; F:identical protein binding; F:serine-type endopeptidase activity

(3.4) Acting on peptide bonds (peptide hydrolases)

Mascot score

460

4301

B. subtilis CH-1 isolated from the gut of tarantula C. guangxiensis is high-efficient and safe. Our work also provides a new way for understanding spider's digestive process. In our opinion, the bacterial gut symbionts play an important role in digestion of prey by secreting digestive enzyme. Moreover, symbiotic microorganisms help greatly to the balance between the internal ecosystem, health, and even immune system (Mazmanian et al., 2005). Therefore, the isolate B. subtilis CH-1, a B. subtilis, which known as probiotics, is more desirable for the application of keratinase in feather meal with live cells. Conclusions In summary, B. subtilis CH-1 is an effective and environmentally benign feather-degrading microorganism in moderate reaction condition. The enzymes involved feather degradation are complicated and at least including oxidoreductase and proteolytic activities in B. subtilis CH-1. We identified four main roles including extracellular protease Vpr, peptidase T as proteolytic activities and g-glutamyl transpeptidase, glyoxalmethylglyoxal reductase as oxidoreductase activities respectively. This study develops an

Fig. 4. Bar Chart of the combined GO annotation with respect to molecular function for four key predicted enzymes (A: peptidase t B: extracellular protease vpr C: glyoxal/ methylglyoxal reductase D: gamma-glutamyltranspeptidase).

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understanding for the degradation mechanism of keratin. This feather-degrading B. subtilis CH-1 would be potentially developed for wide applications. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30900242), the Hunan Provincial Natural Science Foundation (14JJ3093), the Science Research Fund of the Hunan Provincial Education Department (10B049), the Science and Technology Plan of Changsha City (K1307106-31), the National Natural Science Foundation of China (31071943, 31272339) and Hunan Provincial Science and Technology Department (2012NK4048, 2013NK4003, 2014NK3048). We thank Professor Zhilong Wang for his critical reading of the manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibiod.2014.08.008. References Alvarado-Cuevas, Z.D., Acevedo, L.G., Salas, J.T., De Leon-Rodriguez, A., 2013. Nitrogen sources impact hydrogen production by Escherichia coli using cheese whey as substrate. New. Biotechnol. 30, 585e590. Brandelli, A., Daroit, D.J., Riffel, A., 2010. Biochemical features of microbial keratinases and their production and applications. Appl. Microbiol. Biotechnol. 85, 1735e1750. CAaC, M., 2008. The use of fermented feather meal for replacement fish meal in the diet of oreochromis niloticus. Environ. Nat. Environ. Nat. Resour. J. 6, 13e24. Cai, S.B., Huang, Z.H., Zhang, X.Q., Cao, Z.J., Zhou, M.H., Hong, F., 2011. Identification of a keratinase-producing bacterial strain and enzymatic study for its improvement on shrink resistance and tensile strength of wool- and polyesterblended fabric. Appl. Biochem. Biotechnol. 163, 112e126. Cedrola, S.M.L., Melo, A.C.N., Mazotto, A.M., Lins, U., Zingali, R.B., Rosado, A.S., Peixoto, R.S., Vermelho, A.B., 2011. Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste. World J. Microbiol. Biotechnol. 28, 1259e1269. Chitte, R.R., Dey S, N.V., 1999. Keratinolytic activity from the broth of a featherdegrading thermophilic Streptomyces thermoviolaceus strain SD8. Lett. Appl. Microbiol. 28, 131e136. Choi, N.-S., Chung, D.-M., Park, C.-S., Ahn, K.-H., Kim, J.S., Song, J.J., Kim, S.-H., Yoon, B.-D., Kim, M.-S., 2010. Expression and identification of a minor extracellular fibrinolytic enzyme (Vpr) from Bacillus subtilis KCTC 3014. Biotechnol. Bioprocess Eng. 15, 446e452. Conville, P.S., Witebsky, F.G., 2001. Lack of usefulness of carbon utilization tests for identification of Mycobacterium mucogenicum. J. Clin. Microbiol. 39, 2725e2728. Del Corso, A., Costantino, L., Rastelli, G., Buono, F., Mura, U., 2000. Aldose reductase does catalyse the reduction of glyceraldehyde through a stoichiometric oxidation of NADPH. Exp. Eye Res. 71, 515e521. el-Naghy, M.A., el-Ktatny, M.S., Fadl-Allah, E.M., Nazeer, W.W., 1998. Degradation of chicken feathers by Chrysosporium georgiae. Mycopathologia 143, 77e84. Gerholdt, J.E., 1995. Tarantula Spiders. ABDO, USA. Ghosh, A., Chakrabarti, K., Chattopadhyay, D., 2008. Degradation of raw feather by a novel high molecular weight extracellular protease from newly isolated Bacillus cereus DCUW. J. Ind. Microbiol. Biotechnol. 35, 825e834. Gousterova, A., Braikova, D., Goshev, I., Christov, P., Tishinov, K., VasilevaTonkova, E., Haertle, T., Nedkov, P., 2005. Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis. Lett. Appl. Microbiol. 40, 335e340. Gradisar, H., Friedrich, J., Krizaj, I., Jerala, R., 2005. Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Appl. Environ. Microbiol. 71, 3420e3426. Gupta, R., Ramnani, P., 2006. Microbial keratinases and their prospective applications: an overview. Appl. Microbiol. Biotechnol. 70, 21e33. Gupta, R., Sharma, R., Beg, Q.K., 2013. Revisiting microbial keratinases: next generation proteases for sustainable biotechnology. Crit. Rev. Biotechnol. 33, 216e228. Inoue, Y., Rhee, H., Watanabe, K., Murata, K., Kimura, A., 1988. Metabolism of 2oxoaldehyde in mold. Purification and characterization of two methylglyoxal reductases from Aspergillus niger. Eur. J. Biochem./FEBS 171, 213e218.

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