Mining and Expression of a Metagenome-Derived Keratinase

Subscriber access provided by UNIV OF DURHAM

Article

Mining and expression of a metagenome-derived keratinase responsible for biosynthesis of silver nanoparticles Li-Yan Tao, Jin-Song Gong, Chang Su, Min Jiang, Heng Li, Hui Li, Zhen-Ming Lu, Zheng-Hong Xu, and Jin-Song Shi ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00687 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on March 3, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Mining and expression of a metagenome-derived keratinase responsible for biosynthesis of silver nanoparticles Li-Yan Tao,† Jin-Song Gong,† Chang Su,† Min Jiang,† Heng Li,† Zhen-Ming Lu,†,‡ Zheng-Hong Xu,†,‡ and Jin-Song Shi*,†

†

‡

School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, PR China; National Engineering Laboratory for Cereal Fermentation Technology, School of

Biotechnology, Jiangnan University, Wuxi 214122, PR China

*Corresponding author. Tel./Fax: +86-510-85328177 E-mail: [email protected] (J.S. Shi)

1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

ABSTRACT: A keratinase gene kerBv was mined from soil metagenomes. The open reading frame consisted of 1149 bp and potentially encoded a protein of 382 amino acid residues. It shared the same active site with several reported typical keratinases via analysis of the amino acid sequence. The keratinase was successfully expressed in B. subtilis WB600 with pMA5 expression vector. The maximum activity of 164.8 U/mL in the fermentation supernatant was observed after incubating for 30 h in Terrifc Broth (TB) medium. The keratinase exhibited outstanding resistance to metal ions and was surfactant-stable. Besides, the enzyme displayed broad substrate specificity especially towards insoluble substrate feather meal due to its disulfide bond-reducing activity. Furthermore, the reducing power of the recombinant keratinase was investigated. It showed that the protein exhibited a relatively high reducing power, which was subsequently used in the biosynthesis of silver nanoparticles (AgNPs). The biosynthesized AgNPs were characterized by ultraviolet-visible (UV-vis) spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), as well as Fourier transform infrared spectroscopy (FTIR) and displayed obvious antibacterial activities towards Escherichia coli. KEYWORDS: keratinase; Bacillus subtilis; biochemical characteristics; silver nanoparticles; biosynthesis

2 ACS Paragon Plus Environment

Page 3 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION In recent years, keratinases have attracted considerable attention due to their unique capability superior to conventional proteolytic enzymes to degrade insoluble hard keratin substrates. Keratinases have been widely investigated from a variety of microorganisms but the majority of works still focus on screening of keratinase-producing microorganisms, purification and characterization of keratinases. To date, only a few keratinase genes from Bacillus species1-2, Stenotrophomonas maltophilia3, Pseudomonas aeruginosa4-5, Streptomyces fradiae6, Actinomadura viridilutea7 have been cloned and expressed in heterologous hosts, which is not sufficient for the commercial use as the market awareness of keratinases has gradually increased8. It is estimated that less than 1% of the microorganisms in nature can be cultivated by traditional culture techniques. The soil metagenome is emerging as a large reservoir for exploitation of genetic diversity and mining of genes with commercial interests9. Metagenomic approaches accessing to functional genes are generally composed of sequence-based screening and activity-based screening10. The present study aims to mine novel keratinases for heterologous expression from soil metagenomes with sequence-based screening method. B. subtilis is generally recognized as safe (GRAS) and has efficient extracellular secretory system, which can largely simplify the downstream processing of protein purification. It is reported that intracellular overexpression of keratinase has a toxic effect on host cells11. Additionally, microbial keratinases are predominantly 3

ACS Paragon Plus Environment


Page 4 of 33

extracellular12, B. subtilis is thus considered as a suitable expression host for keratinase. On the other hand, the application potential of keratinases has far from been fully explored. In previous reports, keratinases find applications in keratin wastes treatment13, feed additives, leather dehairing14, detergent15, and textiles16. In recent years, some new applications such as cosmetic17, and CIP process18 are tapped by scientists. In this work, high reducing power was detected from our recombinant keratinase and it can be applied into reduction reaction like the biosynthesis of silver nanoparticles from silver ions. Silver nanoparticles have attracted considerable interests of researchers because of their unique antimicrobial properties. They exhibit wide applications in medical and pharmaceutical industries. Nevertheless, the non-toxic, green and eco-friendly methods for the synthesis and assembly of AgNPs still need to be developed. Previous report revealed that several bacteria and plants could effectively uptake metal ions from soils to form insoluble complexes in the form of nanoparticles during the detoxification process19. Moreover, biological systems have been reported employed in the biosynthesis of AgNPs via these microorganisms and plants20-21. The microbial enzymes and phytochemicals with reducing potential play a critical role in the reduction process. The recent study by Lateef et al. confirmed the feasibility of AgNPs biosynthesis by keratinase22. This study would provide a potential and alternative approach for green synthesis of AgNPs.

4


Page 5 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2. MATERIALS AND METHODS 2.1. Strains, plasmids and materials. All strains and plasmids used in this study were listed in Table 1. Soil DNA Kit -D5625 was purchased from OMEGA (America). All primers and kits for plasmid extraction, PCR products purification and gel extraction were supplied by Generay Biotech (Shanghai, China). Restriction endonucleases, PrimeSTAR™ HS DNA Polymerase, T4 DNA ligase were obtained from TaKaRa Biotech (Dalian, China). Sequencing was performed by Sangon Biotech (Shanghai, China). The 5% soluble keratin was purchased from J&K® (China). All other chemicals were of analytical grade and commercially available. Table 1. Strains and plasmids used in this study. Strains/plasmids

Genotype or characteristics

Source

e14- (mcrA) endA1 recA1 hsdR17 (rk-, mk+) Novagen

E. coli JM109 (lac-proAB) lacIqZM15 relA1 B. subtilis WB600

168 derivative, ∆nprE ∆aprE ∆epr ∆bpr ∆mpr ∆nprB

Lab stock

T-vector pMD™19

TA cloning with 3’ cohesive end “T”

TaKaRa

PHpaII ColE1 repB, replicates in E. coli (ApR) or B. pMA5

subtilis (KmR)

pMA5-kerBv

pMA5 derivative, containing the kerBv gene

Lab stock This work

2.2. Sample source and DNA isolation. Several soil and water samples were collected from a leather factory in Jiangsu Province. The metagenomic DNA was directly isolated from the samples by Soil DNA Kit according to the manufacturer’s instructions with minor modification. The concentration of metagenomic DNA was detected by NanoDrop 2000 and quality can be observed by agarose gel 5



Page 6 of 33

electrophoresis.

2.3. Primer design and amplification of targeted sequences. All keratinase protein sequences in National Center for Biotechnology Information (NCBI) database were analyzed

by

Constraint-based

Multiple

Alignment

Tool

(https://www.ncbi.nlm.nih.gov/tools/cobalt/). The metagenomic DNA was used as template and the PCR procedure was carried out with the degenerate primers based on the conserved domain and similar sequences of keratinases. The amplified PCR products were analyzed by 1% agarose gel electrophoresis and the purified DNA fragments were ligated with pMD19-T simple vector at 16°C overnight. Positive transformants were screened for sequencing. Then the DNA sequence was compared to the GenBank database in NCBI BLAST program, new primers were designed to obtain complete DNA sequence. Amino acid sequences alignment was performed by DNAMAN 8.0 software. Signal peptide sequence was predicted by SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/). The MEGA 5.0 software was used for dendrogram analysis of keratinases.

2.4. Expression in B. subtilis WB600. The six extracellular proteases-deficient strain B. subtilis WB60023 and the shuttle vector pMA5 harbouring the constitutively active promoter PHpaII were adopted as the host strain and expression vector, respectively. The complete open reading frame of the gene kerBv including its native signal peptide was amplified by PCR using the oligonucleotide specific primers: 6


Page 7 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5’-CGGGATCCATGAGAGGCAAAAAGGTATGG-3’ (with the restriction site BamH I underlined) and 5’-CGACGCGTTTACTGAGCTGCCGCCTGTA-3’ (with the restriction site Mlu I underlined). The PCR product was gel purified, digested with restriction enzymes and ligated into the corresponding sites of the plasmid pMA5. Competent cells of B. subtilis WB600 were transformed with the recombinant plasmid pMA5-kerBv. Transformants able to produce a transparent zone that carried the desired gene were directly screened on 50 µg/mL kanamycin solid plate with 1% skim milk powder. The recombinant strain was incubated in TB medium for 30 h and then the fermentation broth was centrifuged at 8,000 rpm for 10 min at 4°C. The fermentation supernatant namely crude keratinase was used for activity assay and further analysis.

2.5. Keratinase activity assay. The keratinase activity assay was carried out in accordance with the method modified from Su et al.24 and used the 1% soluble keratin as substrate. In the experimental group, the 100 µL 1% (w/v) soluble keratin (diluted in 50 mM Tris-HCl buffer, pH 9.0) was added to 100 µL dilute crude keratinase solution. While in the control group, the 200 µL 5% (w/v) trichloroacetic acid (TCA) replaced the substrate in the experimental group. All these reaction solutions were incubated at 50°C for 15 min. The incubation was stopped by addition of 200 µL 5% (w/v) TCA in the experimental group and the 100 µL 1% (w/v) soluble keratin was added to the control group. All mixed tubes were centrifuged at 12,000 rpm for 5 min. Then 200 µL supernatant was pipetted into another tube with 1 mL 0.4 M Na2CO3 and 7



Page 8 of 33

200 µL Folin-Phenol reagent. After incubating at 40°C for 20 min, the absorbance at 680 nm was measured. One unit (U) of keratinase activity was defined as an increase of 0.01 absorbance unit at 680 nm under the conditions above mentioned. Protein concentration was determined using the Bradford reagent with bovine serum albumin as a standard.

Sodium

dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) was performed with 12% polyacrylamide gel. After electrophoresis, gels were stained with Coomassie blue R-250. The native polyacrylamide gel electrophoresis (Native-PAGE) of zymography analysis with 0.1% (w/v) casein (dissolved in 50 mM Tris–HCl buffer, pH 9.0) was carried out under non-denaturing conditions in the absence of SDS.

2.6. Biochemical characterization of recombinant keratinase. The influence of temperature on enzyme activity was evaluated at temperatures ranging from 40°C to 70°C. The temperature stability assay was performed by incubating the enzyme at a temperature range of 40–70°C for 30 min and the residual activity was then measured under standard assay condition. The effect of pH on the enzymatic activity was determined at the pH range of 6.0–12.0. The following buffer solutions were used to investigate the effect of pH: 50 mM sodium phosphate buffer (pH 6.0–7.0), 50 mM Tris-HCl buffer (pH 8.0–9.0), 50 mM Glycine-NaOH buffer (pH 10.0-11.0) and 50 mM KCl-NaOH buffer (pH 12.0). The pH stability assay was carried out by incubating the enzyme at a pH range of 6.0–12.0 for 1 h and the residual activity was then measured under standard assay condition. The maximum activity of keratinase in 8


Page 9 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the temperature and pH assays was taken as 100%. To investigate the effect of various metal ions on the kerBv activity, the keratinase activity assay was carried out with keratinase exposure to metal ions at the final concentrations of 1 mM and 5 mM for 30 minutes. To determine the effect of surfactants (Tween, Triton, and SDS), dimethyl sulfoxide (DMSO), H2O2, phenyl methane sulfonyl fluoride (PMSF), ethylene diamine tetraacetic acid (EDTA), dithiothreitol (DTT), and β-mercaptoethanol (β-ME), the procedure used was the same as that for metal ions. The activity without addition of any chemicals was taken as 100% and the samples with only chemicals were also determined as control. Substrate specificity of kerBv was determined by measuring the enzyme activity towards various soluble substrates (w/v, 1%) such as casein, soluble keratin, bovine serum albumin (BSA), azo-casein and insoluble substrates (w/v, 1%) such as wool, feather meal, human hair, keratin azure, collagen and gelatin. All insoluble substrates were crushed by grinding. A reaction mixture containing 5 mg each substrate in 500 µL Tris-HCl buffer (50 mM, pH 9.0) and 500 µL appropriately diluted enzyme solution was incubated at 50°C for 1 h. The enzyme reaction was stopped by adding 500 µL 10% (w/v) TCA and then the mixture was centrifuged at 12,000 rpm for 5 min. In the control group, the 500 µL 10% (w/v) TCA was firstly added before incubation followed by the enzyme. The enzymatic activity was measured by coloring with Folin-Phenol reagent as described above and the maximum activity was taken as 100%.

9



Page 10 of 33

2.7. Reducing power assay. Reducing power assay of crude recombinant keratinase was determined according to the method of Jiang25 with minor modifications. The 300 µL appropriately diluted keratinase solution was mixed with 300 µL 0.2 M phosphate buffer (pH 6.6) and 300 µL 1% (w/v) potassium ferricyanide. The mixture was incubated in 50°C for 20 min and then 300 µL 10% (w/v) TCA was added to the mixture. By centrifugation at 4000 rpm for 10 min, the 200 µL supernatant solution was mixed with 800 µL 0.01% (w/v) ferric trichloride. After 10 min of incubation at 30°C, the absorbance of the solution was measured at 700 nm. A higher absorbance value indicated higher reducing power. The fermentation supernatant of B. subtilis WB600 carrying pMA5 plasmid with the same concentration of protein was processed with a similar manner and taken as the control.

2.8. Synthesis of silver nanoparticles. The recombinant keratinase used for synthesizing AgNPs was filter sterilized. The biosynthesis process of AgNPs was optimized step by step under three factors, namely, concentration of silver nitrate (1, 1.5, 2, 2.5, 5 mM), enzyme activity (100, 200, 300, 400, 500 U) and reaction time (12, 24, 36, 48 h). The synthesis reaction was carried out in 250 mL flasks with 50 mL reaction mixture incubated on rotary shaker (220 rpm) at 37°C in dark for 48 h. The progress of the reduction reaction between silver ions and keratinase was monitored by Ultraviolet-visible (UV–Vis) spectroscopy over a wavelength range of 300-800 nm (Molecular Devices SpectraMax M2e). The generation of AgNPs could be detected by identifying the characteristic excitation of surface plasmon vibrations. In addition, 10


Page 11 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


visual observation of the colour change also indicated the formation of AgNPs. After the reduction reaction, AgNPs were collected by centrifugation at 13,000 rpm for 10 min and washed with distilled water for three times. The AgNPs were then dried for further characterization. The AgNPs synthesized by chemical reducing reagent in this experiment were prepared as the control according to the method of Tejamaya26 with certain modification. The procedure was described below. 90 mg PVP k-30 was added into 50 mL 1 mM silver nitrate solution and the mixture was stirred for 10 min. Then 2 mL 80 mM sodium borohydride solution was slowly dripped into the reaction system with vigorously stirring until the reaction solution remained steady yellow.

2.9. Characterization of silver nanoparticles. Dynamic light scattering (DLS) method was employed for the hydrodynamic diameter, polydispersity index (PDI) and zeta potential analysis of the colloidal AgNPs by PSSNICOMP 380ZLS. The morphological properties of AgNPs were observed by transmission electron microscope (TEM) under the accelerating voltage of 120 kV (Hitachi H-7650). Size distribution and mean diameter were analyzed by Image-Pro Plus 6.0 software. Finally, Fourier transform infrared spectroscopy (FTIR) was used by Boman FALA 2000104 to elucidate the specific functional groups responsible for reduction of silver nitrate to generate silver nanoparticles.

2.10. Antibacterial activity assay. The antibacterial activity of the AgNPs was investigated by standard diffusion method27 against Escherichia coli. Briefly, the 2 11



Page 12 of 33

mL bacterial solution was added to 100 mL nutrient agar solid medium and the medium was solidified at room temperature for 3 h. Then, sterilized paper discs (6 mm diameter) impregnated with filter sterilized AgNPs were placed on inoculated plates. In addition, the sterile water and AgNPs sample via chemical method were processed in the same manner. The plates were incubated at 37°C for 12-16 h until the inhibition zone around paper could be observed. The diameter of inhibition zone was represented as the antibacterial ability of tested samples. Another method by detecting the biomass of tested strains was also used to evaluate antibacterial activity. The liquid medium was inoculated by 1% (v/v) overnight cultured strains in addition with 1 mL filter sterilized ddH2O, biosynthesized AgNPs and control AgNPs solution (both with the concentration of 100 µg/mL), respectively. Then, the bacterial suspensions were grown at 37°C and OD600 was detected after incubation for 2 h, 4 h, 6 h, 8 h, 11 h, 14 h, and 24 h, respectively.

2.11. Statistical analysis. All experiments were repeated for three times. Microsoft Excel was used for the statistical analysis and data were presented as mean ± standard deviation (SD). The One-Samplet-Test of originPro 8.0 software was used and a p value of 0.05 was considered statistically significant.

3. RUSULTS AND DISCUSSION 3.1. Identification of keratinase coding sequence from metagenomic DNA. Extraction of high-quality metagenomic DNA with more complete fragments was a 12


Page 13 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


crucial step. The concentration of metagenomic DNA was determined to be 218 ng/µL and the OD260/OD280 was 1.88. A DNA fragment was successfully amplified from metagenome via several rounds of PCR with degenerate primers. The complete DNA sequence finally obtained consisted of 1149 bp and encoded 382 amino acids residues. Homology comparisons and phylogenetic analysis (Figure 1) showed that the sequence exhibited more than 95% of identity with keratinases from B. velezensis (AGC81872.1) and B. amyloliquefaciens (AKR05134.1). Furthermore, the sequence was found sharing the same active site of 139D, 171H, and 328S with other keratinases (Figure S1).

62 99

96 kerBv 100 keratinase Bacillus velezensis (AGC81872.1) keratinase Bacillus amyloliquefaciens (AKR05134.1) 100 91 keratinolytic serine protease Bacillus subtilis (API68871.1) 67 subtilisin E Bacillus subtilis (WP_009966941.1) 100 keratinase Bacillus pumilus (ADK11996.1) keratinase Bacillus licheniformis (AKI30031.1) keratinolytic protease Aspergillus niger (AJW29052.1) alkaline serine protease Bacillus thuringiensis (WP_088068318.1) keratinase Stenotrophomonas maltophilia (BAQ36632.1) keratinase Geobacillus stearothermophilus (AJD77429.1)

0.2

Figure 1. Phylogenetic analysis of amino acid sequence of various keratinases.

3.2. Heterologous expression of kerBv. B. subtilis possesses efficient secretory systems that can allow the intracellular protein secreted into the culture medium. In this study, the recombinant keratinase can be secreted into extracellular medium by its own signal peptide. The expression plasmid pMA5-kerBv of recombinant keratinase was successfully constructed, the map of which was displayed in Figure 2. The recombinant strain could produce transparent zone on the skim milk LB medium plate 13



Page 14 of 33

after incubating at 37°C for 16-24 h (Figure 3a). When fermented in TB medium, the maximum keratinase activity of 164.8 U/mL in the fermentation supernatant was achieved after incubating for 30 h (Figure 3b). The keratinase production was growth-associated according to the activity-growth curve. Apart from the cell autolysis during microbial growth cycle, the accumulation of active keratinase in the fermentation broth was also inferred as the reason for the decline of the cell density and keratinase activity after 30 h fermentation in TB medium. The protein analysis of recombinant keratinase in the polyacrylamide gel electrophoresis could be obtained in Figure S2.

Figure 2. Map of keratinase expression plasmid pMA5-kerBv

a

b

B. subtilis WB600/ pMA5-kerBv 14


Page 15 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Transparent zone on the skim milk plate of the recombinant strain (a) and diagram of keratinase activity-growth curve (b).

3.3. Biochemical characterization of recombinant keratinase. This keratinase showed the highest activity at 60°C, which was in accordance with several other Bacillus keratinases reported previously28-29. However, kerBv was relatively thermolabile as it lost about 50% of activity after incubation at 55°C for 30 min (Figure 4a). In literature, Suntornsuk28 et al. reported that the keratinase from a thermo tolerant feather-degrading bacterium was found lost about 50% of activity after incubation at 50°C for 60 min. The keratinase was highly active in the alkaline conditions and showed over 80% of relative activity under the pH range of 8.0–11.0. It displayed the maximum activity at pH 10.0. It was stable within a broad pH range of 6.0-12.0 with more than 60% of residual activity retained (Figure 4b).

a

b

15



Page 16 of 33

c

Figure 4. Biochemical characterization of recombinant keratinase. a: Effects of temperature on keratinase activity and temperature stability of recombinant enzyme; b: Effects of pH on keratinase activity and pH stability of recombinant enzyme; c: Substrate specificity of recombinant keratinase. The effect of metal ions on the keratinase activity was displayed in Table 2. Compared with control group, the keratinase activity was slightly enhanced with the existence of ions like Ca2+, Ni2+, Cd2+, and Fe3+ at low concentration of 1 mM, whereas other ions did not significantly affect the enzyme activity. On the other hand, except for Mn2+, Co2+, Fe2+, Cd2+, and Pb2+ with the concentration of 5 mM led to significant inhibitory effect on activity, other metal ions only showed little influence on the keratinase activity. It’ s worth noting that the keratinase could maintain over 80% relative activity even with 5 mM Ag+, which lay the foundation for the biosynthesis of AgNPs. Overall, the keratinase exhibited outstanding stability and resistance to most metal ions probably because it was mined from soil samples that could withstand environmental pressure. Table 2. Effects of metal ions (1 mM and 5 mM) on keratinase activity of recombinant enzyme. 16


Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Relative activity (%) Metal ions

Relative activity (%) Metal ions

1 mM

5 mM

1 mM

5 mM

Control

100.0±1.6

100.0±6.3

Cd2+

142.5±4.2

78.7±1.9

Mg2+

107.9±8.1

113.6±3.0

Ba2+

95.1±9.2

99.1±7.9

Ca2+

114.4±2.2

91.1±7.7

Li+

99.9±2.5

109.2±8.5

Zn2+

103.2±2.1

85.1±2.6

Fe3+

113.8±1.9

97.9±8.0

Mn2+

60.2±4.3

47.6±2.4

Al3+

87.0±0.8

93.4±7.9

Co2+

92.0±7.2

68.4±3.6

Pb2+

86.3±4.2

13.9±0.2

Ni2+

111.8±1.3

92.3±4.1

Ag+

80.5±4.7

84.6±3.6

Fe2+

101.0±1.9

68.3±2.1

In this study, several surfactants like Tween and Triton could slightly improve the keratinase activity. It indicated that the enzyme was surfactant-stable. The enzyme retained only 20% of activity with addition of 1 mM PMSF, a serine protease inhibitor. Besides, the 5 mM PMSF could completely inhibited keratinase activity. It suggested that the enzyme belongs to serine protease. EDTA exhibited no obvious impact on keratinase activity even when at the concentration of 5 mM. Both β-ME and DTT showed prominent inhibitory activity to the enzyme (Table 3). However, previous studies demonstrated that the reductants could facilitate the keratin degradation by their crucial role in assisting the reduction of disulfide bonds of keratin30-32. In this study, another experiment was carried out using feather meal as substrate. The results showed that the keratinase activity could be improved to 1.2-fold and 1.7-fold with addition of 1 mM β-ME and DTT, respectively. Furthermore, 5 mM β-ME and DTT 17



Page 18 of 33

could improve the activity to 1.3-fold and 3.0-fold, respectively (Table S1). The current study extends the previous investigations and confirms that the reducing agents like β-ME and DTT have positive effect on reduction of disulfide bonds. Table 3. Effects of chemical reagents on keratinase activity of recombinant enzyme. Relative activity

Relative activity Chemicals Concentration

Chemicals Concentration (%)

(%)

Control

-

100.0±1.9

PMSF

1 mM

20.7±9.4

Tween 20

1%(v/v)

131.5±6.9

EDTA

1 mM

107.4±6.2

Tween 80

1%(v/v)

113.4±10.0

DTT

1 mM

34.3±5.8

1%(v/v)

115.5±6.3

β-ME

1 mM

53.4±0.6

1%(v/v)

103.5±4.4

PMSF

5 mM

NDa

DMSO

1%(v/v)

100.2±8.9

EDTA

5 mM

78.8±1.3

SDS

1%(w/v)

26.3±2.8

DTT

5 mM

NDa

H2O2

1%(v/v)

80.4±2.8

β-ME

5 mM

29.4±1.6

Triton X-100 Triton X-114

a

ND: Not detected. The controls of only chemicals were also determined and none of

them exhibited keratinase activity. The recombinant keratinase exhibited activity to different substrates not only soluble protein but some recalcitrant keratin substrates, which demonstrated that it had broad range of substrate specificity (Figure 4c). The enzyme showed the highest activity to casein substrate, followed by keratin. This phenomenon was also observed in several other reported keratinases33-34. In insoluble substrates, relatively high hydrolysis ability towards feather meal was observed. It indicates that the keratinase

18


Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


can efficiently degrade β-keratin, which is the major component of feather and contains a large number of disulfide bonds12. The complete degradation of feather includes sulfitolysis (breakdown of disulfide bonds) and protein hydrolysis11. Apart from keratinolytic hydrolysis activity, the disulfide reductase activity was also found from keratinase by some scientists31 and the reducing power of protein might be responsible for the reduction of disulfide bond.

3.4. Synthesis of AgNPs. The silver nanoparticles can be synthesized by physical, chemical and biological methods. The physical methods require advanced equipment and are often expensive. The traditional chemical methods need reducing agent to generate silver atoms and stabilizer to cover the AgNPs, which pose an environmental burden. In recent years, the biological methods for synthesis of AgNPs have attracted increasing attention in the field of nanotechnology. Several previous studies reported biosynthesis of AgNPs by bacteria19, 35, fungi27, 36 and plant37 as reducing and capping agents. These methods inspire the experimental process for the synthesis of AgNPs as it is simple, eco-friendly, cost-effective and has pharmaceutical applications. In this study, a significant reducing power was observed from recombinant keratinase kerBv compared with the control sample of empty plasmid protein (Figure 5a). By virtue of this exclusive characteristic of remarkable reducing power, the enzyme might be applied in reduction reaction for biosynthesis of AgNPs. In our experiment of AgNPs biosynthesis via keratinase, three factors including AgNO3 concentration, enzyme activity and reaction time were investigated for 19



Page 20 of 33

synthesis optimization step by step. As shown in Figure 5b, the surface plasmon resonance (SPR) absorption band which confirmed the generation of AgNPs was only achieved under the AgNPs concentration of 1 mM after 48 h reaction. The effect of enzyme activity on AgNPs synthesis was investigated from 100 U to 500 U with the AgNPs concentration of 1 mM. All groups showed an obvious strong absorbance peak except the sample with 100 U. The absorbance peaks of SPR under different enzyme activity in Figure 5c indicated the biosynthesis was positively correlated with the enzyme activity. Then, the 1 mM AgNO3 and 200 U of enzyme activity were chosen for the reaction time assay of biosynthesis. The AgNPs suspensions had an obvious yellow colouration gradually from 24 h. The maximum absorbance was achieved around 420 nm from 24 h and without shifts with the time going on (Figure 5d). The biosynthesized AgNPs were finally harvested under the reaction of 48 h. The process of AgNPs biosynthesis by keratinase was simple and cost-effective. In previous reports of biosynthesis of AgNPs, 20 g Penicillium aculeatum Su1 biomass in 100 mL27, 50 mg/mL leaf extracts37 or 100 mL culture supernatant of Variovorax guangxiensis19 were used for the reduction reaction. In this study, the 200 U keratinase was used for the reduction in 50 mL reaction mixture, which was probably due to its outstanding reducing power.

20


Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b

a

d

c

Figure 5. Reducing power assay of keratinase (a) and effects of different conditions on the biosynthesis of AgNPs (b, c, d). a: Reducing power analysis of recombinant keratinase. Sample 1: Fermentation supernatant of B. subtilis WB600 only with pMA5 plasmid; Sample 2: Protein of recombinant keratinase kerBv; b: Effects of AgNO3 concentration (1, 1.5, 2.0, 2.5, 5 mM) under the same enzyme activity of 100 U; c: Effects of enzyme activity (100, 200, 300, 400, 500 U) under the same AgNPs concentration of 1 mM; d: Effects of reaction time (12, 24, 36, 48 h) under the 1 mM AgNO3 and enzyme activity of 200 U.

3.5. Characterization of AgNPs. The hydrodynamic diameter of colloidal AgNPs 21



Page 22 of 33

measured by DLS was about 60 nm. The DLS overestimates particle size in several cases, the phenomenon is also reported in many other studies26,

36

. The PDI of

colloidal AgNPs was 0.23, indicating that particles had pretty narrow dispersity since PDI ≤ 0.2 was considered narrow dispersed generally for colloidal particles38. The stability of the AgNPs colloidal was analyzed by zeta potential measurements. The AgNPs had a high negative zeta potential of about -22 mV, which indicated the stability of the AgNPs colloidal was due to electrostatic repulsion among the negative charges. The negative charges were also responsible for preventing AgNPs from aggregation and maintaining the suitable size of the particle37. The TEM micrograph investigated the morphology and distribution of biosynthesized AgNPs. It was observed that the majority of AgNPs were spherical in shape (Figure 6a). The particle size histogram displayed the diameter of AgNPs ranging from 3 to 15 nm and had a calculated mean diameter of 6.54±2.62 nm as represented in Figure 6b. In other reports, the size of nanoparticles biosynthesized by Kannan et al. varied from 3 to 44 nm with average of ~30 nm39. The mechanism for biosynthesis of AgNPs is supposed that the biological substance with reducing power accounts for the reduction of silver ions into silver atoms of a nanosize dimension27. From the FTIR spectrum in Figure 6c, the absorption bands between 3100-3500 cm-1 was presumed as the N-H asymmetric stretch mode of amides. The 1650 cm-1 band was generally identified as amide I35. The results inferred that biomolecules of protein were responsible for the biosynthesis of AgNPs and sterically dominated nanoparticles by colloidal stability. In other words, the keratinase played the role as both reducing agent and stabilizer. 22


Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

b

Figure 6. Characterization of AgNPs. a: TEM micrograph of biosynthesized AgNPs; b: Size distribution histogram of biosynthesized AgNPs; c: FTIR spectrum of biosynthesized AgNPs.

3.6. Evaluation of antibacterial activity. Antibacterial activity assay was estimated by analyzing both inhibition zone and growth inhibition of test strain. As seen in Figure 7, an obvious inhibition zone was observed with the biosynthesized AgNPs while a relatively insignificant inhibition zone could be observed by chemically synthesized AgNPs. A similar result was obtained from the growth inhibition assay, revealing that the biosynthesized AgNPs displayed a more obvious 23



Page 24 of 33

effect of bacterial inhibition compared with the chemically produced AgNPs when at the concentration of 100 µg/mL. It can be explained as the polymeric stabilizers PVP worked for capping AgNPs and preventing them from aggregation may have negative influence on the surface activities of AgNPs, especially antibacterial activities40. Moreover, the zeta potential of chemically produced AgNPs and biosynthesized AgNPs were around -10 mV and -22 mV, respectively. The relatively higher negative zeta potential of biosynthesized AgNPs indicates that they exhibited better stability to serve antibacterial function.

Figure 7. The antibacterial activity of biosynthesized AgNPs. Sample 1: Control of ddH2O; Sample 2: Chemically produced AgNPs; Sample 3: Biosynthesized AgNPs by recombinant keratinase.

4. CONCLUSION In conclusion, a keratinase gene kerBv was mined from soil metagenomes and realized heterologous expression in B. subtilis WB600. The recombinant keratinase exhibited outstanding resistance to metal ions and was surfactant-stable. Furthermore, it showed hydrolysis activity towards wide range of substrate especially the insoluble 24


Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


feather meal probably due to its disulfide bond-reducing activity. Another significant property of keratinase explored in this study was relatively high reducing power. According to the unique properties mentioned above, we attempted to biologically synthesize AgNPs with the recombinant keratinase. The keratinase played the role as both the reducing reagent and the stabilizer in the biosynthesis reaction. The biosynthesized AgNPs were fully characterized and exhibited favorable antibacterial activity towards E. coli. The biosynthesis process of AgNPs via keratinase is simple, mild and eco-friendly, which has the potential to be applied into the medical industry.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Alignment of amino acid sequence of various keratinases; SDS-PAGE and Native-PAGE analysis of recombinant keratinase; Effects of DTT and β-Me on keratinase activity with feather meal as substrate (PDF)

AUTHOR INFORMATION Corresponding Authors *Email: [email protected] (J.S. Shi). Tel: +86-510-85328177 Notes The authors declare no competing financial interest. 25



Page 26 of 33

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21676121) and the National High Technology Research and Development Program of China (No. 2012AA022204C).

REFERENCES (1) Radha, S.; Gunasekaran, P. Sustained expression of keratinase gene under PxylA and PamyL promoters in the recombinant Bacillus megaterium MS941. Bioresour. Technol. 2008, 99, 5528-5537, DOI: 10.1016/j.biortech.2007.10.052. (2) Rajput, R.; Gupta, R. Expression of Bacillus pumilus keratinase rK27 in Bacillus subtilis: enzyme application for developing renewable flocculants from bone meal. Ann. Microbiol. 2013, 64, 1257-1266, DOI: 10.1007/s13213-013-0770-2. (3) Fang, Z.; Zhang, J.; Liu, B.; Jiang, L.; Du, G.; Chen, J. Cloning, heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1. Process Biochem. 2014, 49, 647-654, DOI: 10.1016/j.procbio.2014.01.009. (4) Lin, H. H.; Yin, L. J.; Jiang, S. T. Functional expression and characterization of keratinase from Pseudomonas aeruginosa in Pichia pastoris. J. Agric. Food Chem. 2009, 57, 5321-5325, DOI: 10.1021/jf900417t. (5) Sharma, R.; Gupta, R. Extracellular expression of keratinase Ker P from Pseudomonas aeruginosa

in

E.

coli.

Biotechnol.

Lett.

2010,

32,

1863-1868,

DOI:

10.1007/s10529-010-0361-2. 26


Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6) Li, J.; Shi, P. J.; Han, X. Y.; Meng, K.; Yang, P. L.; Wang, Y. R.; Luo, H. Y.; Wu, N. F.; Yao, B.; Fan, Y. L. Functional expression of the keratinolytic serine protease gene sfp2 from Streptomyces fradiae var. k11 in Pichia pastoris. Protein Expression Purif. 2007, 54, 79-86, DOI: 10.1016/j.pep.2007.02.012. (7) Ben, M.; Zarai, N.; Rekik, H.; Omrane, M.; Mechri, S.; Moujehed, E.; Kourdali, S.; Hattab, M.; Badis, A.; Bejar, S.; Jaouadi, B. Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea DZ50. Int. J. Biol. Macromol. 2016, 92, 299-315, DOI: 10.1016/j.ijbiomac.2016.07.009. (8) Gupta, R.; Rajput, R.; Sharma, R.; Gupta, N. Biotechnological applications and prospective market of microbial keratinases. Appl. Microbiol. Biotechnol. 2013, 97, 9931-9940, DOI: 10.1007/s00253-013-5292-0. (9) Handelsmanl, J.; Rondon, M. R.; Brady, S. F.; Clardy, J.; Goodman, R. M. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol. 1998, 5, 245-249. (10) Ufarte, L.; Laville, E.; Duquesne, S.; Potocki, G. Metagenomics for the discovery of pollutant

degrading

enzymes.

Biotechnol.

Adv.

2015,

33,

1845-1854,

DOI:

10.1016/j.biotechadv.2015.10.009. (11) Gupta, R.; Sharma, R.; Beg, Q. K. Revisiting microbial keratinases: next generation proteases for sustainable biotechnology. Crit. Rev. Biotechnol. 2013, 33, 216-228, DOI: 10.3109/07388551.2012.685051. (12) Gupta, R.; Ramnani, P. Microbial keratinases and their prospective applications: an overview. Appl. Microbiol. Biotechnol. 2006, 70, 21-33, DOI: 10.1007/s00253-005-0239-8. 27



Page 28 of 33

(13) Yusuf, I.; Ahmad, S. A.; Phang, L. Y.; Syed, M. A.; Shamaan, N. A.; Abdul, K.; Dahalan, F. A.; Shukor, M. Y. Keratinase production and biodegradation of polluted secondary chicken feather wastes by a newly isolated multi heavy metal tolerant bacterium-Alcaligenes sp. AQ05-001. J. Environ. Manage. 2016, 183, 182-195, DOI: 10.1016/j.jenvman.2016.08.059. (14) Bouacem, K.; Bouanane, A.; Zarai, N.; Joseph, M.; Hacene, H.; Ollivier, B.; Fardeau, M. L.; Bejar, S.; Jaouadi, B. Novel serine keratinase from Caldicoprobacter algeriensis exhibiting outstanding hide dehairing abilities. Int. J. Biol. Macromol. 2016, 86, 321-328, DOI: 10.1016/j.ijbiomac.2016.01.074. (15) Rai, S. K.; Mukherjee, A. K. Optimization of production of an oxidant and detergent-stable alkaline β-keratinase from Brevibacillus sp. strain AS-S10-II: Application of enzyme in laundry detergent formulations and in leather industry. Biochem. Eng. J. 2011, 54, 47-56, DOI: 10.1016/j.bej.2011.01.007. (16) Liu, B.; Zhang, J.; Li, B.; Liao, X.; Du, G.; Chen, J. Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing. World J. Microbiol. Biotechnol. 2013, 29, 825-832, DOI: 10.1007/s11274-012-1237-5. (17) Sanghvi, G.; Patel, H.; Vaishnav, D.; Oza, T.; Dave, G.; Kunjadia, P.; Sheth, N. A novel alkaline keratinase from Bacillus subtilis DP1 with potential utility in cosmetic formulation. Int. J. Biol. Macromol. 2016, 87, 256-262, DOI: 10.1016/j.ijbiomac.2016.02.067. (18) Paul, T.; Jana, A.; Das, A.; Mandal, A.; Halder, S. K.; Das Mohapatra, P. K.; pati, B. R.; Chandra Mondal, K. Smart cleaning-in-place process through crude keratinase: an eco-friendly cleaning techniques towards dairy industries. J. Cleaner Prod. 2014, 76, 140-153, 28


Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DOI: 10.1016/j.jclepro.2014.04.028. (19) Du, J.; Yi, T. H. Biosynthesis of silver nanoparticles by Variovorax guangxiensis THG-SQL3 and their antimicrobial potential. Mater. Lett. 2016, 178, 75-78, DOI: 10.1016/j.matlet.2016.04.069. (20) Kulkarni, R. R.; Shaiwale, N. S.; Deobagkar, D. N.; Deobagkar, D. D. Synthesis and extracellular accumulation of silver nanoparticles by employing radiation-resistant Deinococcus radiodurans, their characterization, and determination of bioactivity. Int J Nanomedicine 2015, 10, 963-74, DOI: 10.2147/IJN.S72888. (21) Ahmed, M. J.; Murtaza, G.; Mehmood, A.; Bhatti, T. M. Green synthesis of silver nanoparticles using leaves extract of Skimmia laureola: Characterization and antibacterial activity. Mater. Lett. 2015, 153, 10-13, DOI: 10.1016/j.matlet.2015.03.143. (22) Lateef, A.; Adelere, I. A.; Gueguim-Kana, E. B.; Asafa, T. B.; Beukes, L. S. Green synthesis of silver nanoparticles using keratinase obtained from a strain of Bacillus safensis LAU 13. Int. Nano Lett. 2014, 5, 29-35, DOI: 10.1007/s40089-014-0133-4. (23) Wu, X. C.; Wilson L.; Louise T.; Wong, S. Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J. Bacteriol. 1991, 173, 4952-4958. (24) Su, C.; Gong, J. S.; Zhang, R. X.; Tao, L. Y.; Dou, W. F.; Zhang, D. D.; Li, H.; Lu, Z. M.; Xu, Z. H.; Shi, J. S. A novel alkaline surfactant-stable keratinase with superior feather-degrading potential based on library screening strategy. Int. J. Biol. Macromol. 2017, 95, 404-411, DOI: 10.1016/j.ijbiomac.2016.11.045. (25) Jiang, H.; Tong, T.; Sun, J.; Xu, Y.; Zhao, Z.; Liao, D. Purification and characterization 29



Page 30 of 33

of antioxidative peptides from round scad (Decapterus maruadsi) muscle protein hydrolysate. Food Chem. 2014, 154, 158-163, DOI: 10.1016/j.foodchem.2013.12.074. (26) Tejamaya, M.; Romer, I.; Merrifield, R. C.; Lead, J. R. Stability of citrate, PVP, and PEG coated silver nanoparticles in ecotoxicology media. Environ. Sci. Technol. 2012, 46, 7011-7017, DOI: 10.1021/es2038596. (27) Ma, L.; Su, W.; Liu, J. X.; Zeng, X. X.; Huang, Z.; Li, W.; Liu, Z. C.; Tang, J. X. Optimization for extracellular biosynthesis of silver nanoparticles by Penicillium aculeatum Su1 and their antimicrobial activity and cytotoxic effect compared with silver ions. Mater Sci Eng: C 2017, 77, 963-971, DOI: 10.1016/j.msec.2017.03.294. (28) Suntornsuk, W.; Tongjun, J.; Onnim, P.; Oyama, H.; Ratanakanokchai, K.; Kusamran, T.; Oda,

K.

Purification

and

characterisation

of

keratinase

from

a

thermotolerant

feather-degrading bacterium. World J. Microbiol. Biotechnol. 2005, 21, 1111-1117, DOI: 10.1007/s11274-005-0078-x. (29) Zhang, R. X.; Gong, J. S.; Su, C.; Zhang, D. D.; Tian, H.; Dou, W. F.; Li, H.; Shi, J. S.; Xu, Z. H. Biochemical characterization of a novel surfactant-stable serine keratinase with no collagenase activity from Brevibacillus parabrevis CGMCC 10798. Int. J. Biol. Macromol. 2016, 93, 843-851, DOI: 10.1016/j.ijbiomac.2016.09.063. (30) Gradisar, H.; Friedrich, J.; Krizaj, I.; Jerala, R. Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Appl. Environ. Microbiol. 2005, 71, 3420-3426, DOI: 10.1128/AEM.71.7.3420-3426.2005. (31) Prakash, P.; Jayalakshmi, S. K.; Sreeramulu, K. Purification and characterization of 30


Page 31 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


extreme alkaline, thermostable keratinase, and keratin disulfide reductase produced by Bacillus halodurans PPKS-2. Appl. Microbiol. Biotechnol. 2010, 87, 625-633, DOI: 10.1007/s00253-010-2499-1. (32) Rajput, R.; Sharma, R.; Gupta, R. Biochemical characterization of a thiol-activated, oxidation stable keratinase from Bacillus pumilus KS12. Enzyme Res. 2010, 2010, 132-148, DOI: 10.4061/2010/132148. (33) Fakhfakh, N.; Hmidet, N.; Haddar, A.; Kanoun, S.; Nasri, M. A novel serine metallokeratinase from a newly isolated Bacillus pumilus A1 grown on chicken feather meal: biochemical and molecular characterization. Appl. Biochem. Biotechnol. 2010, 162, 329-344, DOI: 10.1007/s12010-009-8774-x. (34) Daroit, D. J.; Corrêa, A. P. F.; Segalin, J.; Brandelli, A. Characterization of a keratinolytic protease produced by the feather-degrading Amazonian bacterium Bacillus sp. P45. Biocatal. Biotransform. 2010, 28, 370-379, DOI: 10.3109/10242422.2010.532549. (35) Ganesh, M. M.; Gunasekaran, P. Production and structural characterization of crystalline silver nanoparticles from Bacillus cereus isolate. Colloids Surf., B 2009, 74, 191-195, DOI: 10.1016/j.colsurfb.2009.07.016. (36) Chan, Y. S.; Matdon, M. Biosynthesis and structural characterization of Ag nanoparticles from white rot fungi. Mater Sci Eng: C 2013, 33, 282-288, DOI: 10.1016/j.msec.2012.08.041. (37) Paosen, S.; Saising, J.; Wira Septama, A.; Piyawan Voravuthikunchai, S. Green synthesis of silver nanoparticles using plants from Myrtaceae family and characterization of their antibacterial activity. Mater. Lett. 2017, 209, 201-206, DOI: 10.1016/j.matlet.2017.07.102. (38) Zhang, Y.; Mintzer, E.; Uhrich, K. E. Synthesis and characterization of PEGylated 31



Page 32 of 33

bolaamphiphiles with enhanced retention in liposomes. J. Colloid Interface Sci. 2016, 482, 19-26, DOI: 10.1016/j.jcis.2016.07.013. (39) Kannan, R. R. R.; Arumugam, R.; Ramya, D.; Manivannan, K.; Anantharaman, P. Green synthesis of silver nanoparticles using marine macroalga Chaetomorpha linum. Appl. Nanosci. 2012, 3, 229-233, DOI: 10.1007/s13204-012-0125-5. (40) Lin, J. J.; Lin, W. C.; Li, S. D.; Lin, C. Y.; Hsu, S. H. Evaluation of the antibacterial activity and biocompatibility for silver nanoparticles immobilized on nano silicate platelets. ACS Appl. Mater. Interfaces 2013, 5, 433-443, DOI: 10.1021/am302534k.

32


Page 33 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Mining and expression of a metagenome-derived keratinase responsible for biosynthesis of silver nanoparticles Li-Yan Tao,† Jin-Song Gong,† Chang Su,† Min Jiang,† Heng Li,† Zhen-Ming Lu,†,‡ Zheng-Hong Xu,†,‡ and Jin-Song Shi*,†

Keratinase

SPR

AgNO3

AgNPs

Antibacterial activity

For Table of Contents Use Only

33


Mining and Expression of a Metagenome-Derived Keratinase

Recommend Documents