Neurospora crassa tox-1 Gene Encodes a pH- and Temperature

May 27, 2016 - Cellulases that endure extreme conditions are essential in various industrial sectors. This study reports a mini-cellulase gene tox-1 f...
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Neurospora crassa tox‑1 Gene Encodes a pH- and TemperatureTolerant Mini-Cellulase Yue Xiao,† Qiongsi Zhang,† Yiquan Luo,† Ying Zhang,∥ Xi Luo,†,‡ Yuchuan Wang,§ Weiguo Cao,⊥ Vito De Pinto,#,∇ Qiuyun Liu,*,†,‡ and Gang Li*,† †

School of Life Sciences, ‡MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, and §School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China ∥ Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China ⊥ Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634, United States # Department of Biomedicine and Biotechnology BIOMETEC, Section of Biology and Genetics, University of Catania, 95125 Catania, Italy ∇ National Institute for Biomembranes and Biosystems, Section of Catania, 95125 Catania, Italy S Supporting Information *

ABSTRACT: Cellulases that endure extreme conditions are essential in various industrial sectors. This study reports a minicellulase gene tox-1 from Neurospora crassa. The gene tox-1 was cloned in Escherichia coli after chimerization with the YebF gene and substitutions of certain isoleucine and valine with leucine residues. The yeast transformants could grow on rice straw−agar medium. The 44-amino acid peptide and its two mutant variants displayed potent cellulase activities in Congo Red assay and enzymatic assays. Conservative replacements with leucine have substantially increased the stabilities and half-lives of the peptides at alkaline pH and low and high temperatures and also the tolerance to organic solvents and surfactants, on the basis of activities toward cellose. The small size of the mini-cellulase would allow for commercially viable automatic chemical peptide synthesis. This work suggests that conservative leucine replacements may serve as a general strategy in the engineering of more robust enzymes with special features with little loss of activities. KEYWORDS: mini-cellulase, conservative amino acid replacements, enzyme assays, pH and thermal tolerance

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INTRODUCTION The worldwide shortage of nonrenewable fuel and greenhouse gas emissions have drawn increasing attention to the development of alternative, environmentally friendly biofuels.1 Biotechnological conversion of cellulosic biomass is a potentially sustainable approach to develop novel bioprocesses and products.2 Efficient cellulose hydrolysis requires at least three classes of cellulase: endo-(1,4)-β-D-glucanase (EC 3.2.1.4), exo-(1,4)-β-D-glucanase (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21).3,4 Cellulases are inducible enzymes synthesized by archaea, bacteria, fungi, plants, and animals5 and have been commercially available for more than 30 years.4,6 Cellulases demonstrated their potential applications in various industries, including food processing,7,8 animal feed,9 brewing and wine making,10 agriculture,11,12 biomass refining,13−15 pulp and paper,16,17 textile, and laundry.18,19 However, the improvement of cellulase activities has to target extreme conditions, such as high temperatures, alkaline pH, and high concentrations of metal ions and organic solvents. The tox-1 gene is located in the cosmids 7:12B and 21:10D of a pSV50 library of Neurospora crassa,20 but it is recalcitrant to subcloning. It was cloned as two separate fragments only after splitting the 3.7 kb EcoRI fragment of the cosmids with BglII endonuclease in the middle of the tox-1 open reading frame (orf). A nine-amino acid segment is found to be homologous to the endoglucanase of Cellvibrio japonicas after a BLASTP search [Figure S1, Supporting Information (SI)], which prompted us © XXXX American Chemical Society

to interrogate the function of this small peptide, which may have high bioavailability. The TOX-1 peptide of fungal origin does not have a prokaryotic secretion signal for cellulose hydrolysis. The tox-1 gene was cloned in Escherichia coli after chimerization with the YebF gene and introduction of conservative isoleucine and valine to leucine replacements. YebF with unknown function is secreted into the periplasm normally and then exported into the culture media. A previous report indicated that the protein attached to the carboxyl terminus of YebF was efficiently secreted. YebF could serve as a tool to circumvent toxicity and other issues associated with protein production in E. coli, as carboxymethyl cellulose (CMC) are macromolecules inaccessible to intracellularly distributed enzymes.21 Given the small size of the TOX-1 peptides and the potentially attendant high bioavailability and stability, the prospects for commercial peptide synthesis and animal fodder preparation using transgenic yeast are promising. An investigation was initiated to characterize the properties of these peptides and the coding genes, and conservative amino acid replacements were attempted to improve the attributes of enzymes. Received: January 5, 2016 Revised: May 25, 2016 Accepted: May 27, 2016

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DOI: 10.1021/acs.jafc.6b00043 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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for 30 s, and 63 °C for 1 min 20 s, followed by 30 cycles of 93 °C for 30 s, 30 °C for 30 s, and 63 °C for 1 min 20 s. The final PCR amplicons were directly electroporated to S. cerevisiae wild type strain GIM2.207 after ethanol precipitation for integration into the yeast chromosomes. Transformants were selected on 1% CMC plates. Colony PCRs were conducted to identify putative positive clones. The positive clones were subsequently propagated in YPD media. When the OD600 of cells reached 1.5, equivalent amounts of cells were harvested and washed once using sterile water. The different cells with equal wet weight were inoculated on CMC plates and stained with Congo Red after 2 days of incubation at 30 °C. The yeast transformants of tox-1, tox-v2, and tox-v3 genes were streaked on plates (0.5% rice straw only ground in liquid nitrogen, 2% agar, 50 μg/ mL ampicillin) and incubated for 5 days at 30 °C. Enzymatic Activity Assays of TOX-1 Peptide and Its Two Mutant Variants. As the expressions of TOX-1 and its two mutant variants in both prokaryotic and eukaryotic cells were undetectable, the peptides encoded by tox-1, tox-v2, and tox-v3 were synthesized via the Fmoc strategy and purified with RP-HPLC by Shanghai BOOTECH Bioscience & Technology Co., Ltd. Product characterizations were performed using HPLC and mass spectral analysis. Peptides were at least 98% pure. The sequences of the two mutant peptides were as follows: TOX-v2, MMLLYVAELCLSVWLWDGLGDKKKGYAMLGWARPLILGCGSKNQ; TOX-v3, MMLLYLAELCLSVWLWDGLGDKKKGYAMLGWARPLILGCGSKNQ (the amino acid substitutions are shown in italic). One unit (U) of enzyme activity was defined as the activity required for the formation of 1 μg of glucose per min under the assay conditions. Enzymatic activities were measured using a substrate mixture consisting of 2.92 mM cellose and 50 mM phosphate-buffered saline (PBS, pH 7.4). The reaction was initiated by adding 10 μL of chemically synthesized peptide (100 ng/μL) to the mixture and incubated at 50 °C for 15 min. The reaction was stopped by adding DNS and boiling for 5 min. The amount of glucose liberated was monitored at 540 nm using an Infinite 200 PRO NanoQuant microplate reader. All the assays were performed in triplicate. Substrate Specificity and Determination of Enzyme Kinetic Constants. Substrate specificities of these enzymes were determined for polysaccharides, including CMC, agar, and soluble starches, and oligosaccharides, including cellose, maltose, and sucrose, as substrates in the standard assays. The Km and Vmax values of the peptides (100 ng/μL) were evaluated by linear regression from a double-reciprocal plot with different concentrations (20−200 μM) prepared using the optimum substrate. The cellulolytic activities were determined after addition of the enzymes followed by incubation for 15 min. Effects of Temperature and pH on Enzyme Activities and Stabilities. The optimal temperature yielding maximal activities of these enzymes (100 ng/μL) was determined by assaying the enzyme activities at different incubation temperatures (4−70 °C) using cellose as substrate. The specific optima of the three peptides were obtained by taking 2 °C increments starting from the temperature with highest activities. The stabilities of these peptides were examined by measuring residual enzyme activities at various temperatures (4−70 °C) after incubation for 6 h. The activity before incubation was defined as 100%. Residual enzyme activities were expressed as a percentage of the enzyme activity. In order to study the effects of pH on the cellulolytic activities, the enzyme (100 ng/μL) assays were performed at different pH values (4.0−10.0) using cellose as substrate. The buffer system was PBS over a range of pH from 5.0 to 12.0. The specific optimal pH values of the three peptides were obtained by taking 0.2 pH unit increments. Stabilities of the enzymes at various pH values were also determined by measuring the residual activities at pH 7.4 after incubating the enzymes in the aforementioned different buffer systems (pH 4.0−8.0) for 6 h at room temperature (RT) (25 ± 2 °C), respectively. Effects of Metal Ions, Organic Solvents, and Surfactants on Enzyme Activities and Stabilities. The effects of metal ions on enzymatic activities were obtained by preincubating the enzyme (100 ng/μL) with metal ions at a concentration of 10 mM at RT (25 ± 2 °C) for 2 h. The metal ions used were Na+, Zn2+, Ca2+, and Fe2+. The

MATERIALS AND METHODS

Chemicals, Reagents, and Equipment. PrimerSTAR DNA polymerase and restriction enzymes were manufactured by Takara (Dalian, China). Yeast nitrogen base (YNB) was purchased from Invitrogen (Shanghai, China). The Infinite 200 PRO NanoQuant was a product of Tecan (Melbourne, Australia). A goniometer (BI-200SM, Brookhaven Instruments, Holtzville, NY) was used for dynamic light scattering (DLS) measurements. All other chemicals and reagents were of analytical grade and were purchased from commercial sources, unless otherwise stated. Microbial Strains and Vectors. E. coli DH5α and Saccharomyces cerevisiae INVSc1 strains were provided by Invitrogen (Carlsbad, CA). E. coli MG1655 was the kind gift of E. coli Genetic Stock Center. S. cerevisiae wild type strain GIM2.207 was obtained from Guangdong Microbiology Culture Center (Guangzhou, China). pET-32a (+) was supplied by Novagen (Madison, WI). The N. crassai strain used was FGSC 1858T (WFCC Global Catalogue of Microorganisms). The Cloning of tox-v2 Gene. The DNA sequence of tox-1 wild type gene is ATGATGTTAATTTACGTAGCTGAGATCTGTCTGTCTGTCTGGCTTTGGGATGGGTTAGGAGACAAGAAAAAAGGCTATGCTATGCTAGGCTGGGCTAGGCCACTTATTCTAGGTTGTGGAAGCAAAAATCAATAA (GenBank accession number ALA65344.1). The encoded amino acid sequence is as follows: MMLIYVAEICLSVWLWDGLGDKKKGYAMLGWARPLILGCGSKNQ (amino acids for leucine replacements in tox-v2 or tox-v3 are italicized). Primers used in this study are listed in Table S1 (SI). Genomic DNA of N. crassai was amplified with PrimerSTAR DNA polymerase with primers tF and tR. The tF primer harbored two isoleucine to leucine replacements in the orf. To increase the PCR amplification efficiency, the annealing temperature was set as 28 °C at the first 8 cycles and 55 °C in the following 28 cycles. Then, the PCR amplicons were ethanol-precipitated and subjected to double digestions by BamHI and HindIII. The restricted DNA fragment was ligated with linearized vector pUC-118 and pET-32a overnight, respectively, and electroporated to E. coli DH5α cells. Putative positive clones were propagated and the plasmid DNAs were sequenced by Invitrogen (Shanghai, China). Chimerization of tox Genes with the YebF Gene and the Constructions of E. coli Vectors. To fuse tox genes with the YebF gene, primers YebtF1, YebtF2, and YebtF3 were paired with TQR at 1:10 molar ratio, respectively, and the genomic DNA of N. crassai was amplified as described above. The YebtF2 and YebtF3 primers allowed two isoleucine to leucine replacements or two replacements of isoleucine with a valine and with a leucine in the orf, respectively. TQF was added as upstream primer and paired with PCR amplicons above individually (Figure S2, SI). PCR was conducted using E. coli DH5α cells as templates to amplify the YebF-tox gene chimeras in the presence of 0.25% formamide with the following conditions: 53 cycles of incubation at 93 °C for 30 s, 42 °C for 30 s, and 72 °C for 30 s. The purified PCR amplicons harboring the chimeric YebF-tox genes were double-digested with EcoRI and HindIII, and the restricted DNA fragments and linearized vector pUC-118 were respectively ligated and electroporated to E. coli DH5α cells. Transformants were selected on CMC plates and subjected to colony PCR and plasmid sequencing (Figure S3, SI). The Functions of tox-1 and Its Two Mutant Variants in Yeast Cells. In order to examine the function of tox-1 and its two mutant variants in yeast cells, two runs of PCR amplifications were performed in succession. Primers GPDt1, GPDt2, and GPDt3 were paired with TOXRN, respectively, with a molar ratio of 1:5 for asymmetric PCR, and the three vectors constructed above were used as templates. PCR amplifications were carried out according to the protocol of Premix PrimeSTAR HS. The PCR amplicons from the initial amplifications were used as downstream primers and paired with GPDF forward primer with a molar ratio of 1:5. PCR amplifications were conducted with the S. cerevisiae INVSc1 strain with 0.31 U of PrimerSTAR DNA polymerase and 0.94 U of exTaq polymerase to construct the GPD promoter-tox chimeras, and 0.25% (v/v) deionized formamide was added in the reactions to increase the specificity of PCR amplifications. The PCR conditions were as follows: 8 cycles of 93 °C for 30 s, 16 °C B

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Figure 1. Functions of S. cerevisiae transformants integrated with tox-1, tox-v2, and tox-v3 gene variants harboring GPD promoter. (A) The Congo Red assay of yeast transformants on CMC plate. Punched holes were 9 mm in diameter. (B) The rice straw utilization experiment. Equal yeast transformants were streaked on a rice-straw-only plate and incubated for 5 days at 30 °C. Growth is magnified on the two side-panels. activity of the enzyme solution without any metal was used as a control (100% of relative activity). Tolerance to organic solvents was examined with methanol (10% v/v), DMSO (10% v/v), Tween-80 (1% v/v), and Triton X-100 (1% v/v). These solvents were diluted in PBS solution, and enzymatic activities were measured under the optimal temperature and pH. The activity of each peptide without any additive was defined as 100%. Dynamic Light Scattering. Three milligrams of peptides was used for DLS measurements. TOX-1 was dissolved in 50% ethanol, and TOX-v2 and TOX-v3 were dissolved in 30% ethanol. TOX-v2 solution was heated at 60 °C for 2 h to enhance solubilization. DLS measurements were performed using a BI-200SM goniometer. Circular Dichroism (CD) Spectrum. One milligram of peptides was used for CD experiments. The preparation of peptides was the same as for DLS, and the final concentration of each peptide was 0.1 mg/mL. CD spectra were recorded at temperatures of 10, 30, and 50 °C and wavelength ranging from 260 to 190 nm. The measurements were conducted via Chirascan CD spectrometer (Applied Photophysics Ltd.). The absorption cuvette was a 1 mm NIR rectangular cell with lid (quartz SUPRASIL 300), with a light path of 1 mm. Data were obtained via a Chirascan Control Panel (Version 4.1.7), Pro-Data Viewer (Version 4.1.6), and CDNN software (Version 2.1, Norma J. Greenfield). Statistical Analysis. All experiments were repeated a minimum of three times. Data are reported as the mean ± standard errors. All data were normal-distributed or approximately normal-distributed, as examined by the Shapiro−Wilk normality test. Experiments were

evaluated using the Tukey HSD posthoc test of the univariate general linear model on SPSS 22.0 software (SPSS, Chicago, IL). A P-value of less than 0.05 was considered statistically significant.

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RESULTS AND DISCUSSION The Cloning of tox-v2 Gene in Prokaryotic Cells. A cloning experiment was conducted to explore the function of the tox-v2 gene. Primers tF and tR were used to amplify the genomic DNA of N. crassai, and an intense band was visualized around 150 bp. The tox-v2 gene was then ligated to pUC-118 and pET-32a vector, respectively, and electroporated to E. coli DH5α cells. The sequencing results (Figure S3a,b, SI) indicated that the tox-v2 gene could be cloned in E. coli cells. The sizes of previously reported cellulases vary over a wide range of molecular masses, and the smallest cellulase documented was 6.3 kDa from Cytophaga,22 while the cellulase from Fusarium solani was up to 400 kDa.23 The full-length tox-1 orf is 132 bp, and the estimated molecular weight of TOX-1 was 4.94 kDa, which was the smallest protein among known endoglucanases, the molecular masses of which range from 35.9 to 659 kDa.24,25 The Constructions of Vectors Harboring Chimeric tox Genes. For gene chimerization with YebF, the amplicons of the initial PCR amplifications involving the tox-1 gene were used as the downstream primers of the second PCR. The restricted 500 C

DOI: 10.1021/acs.jafc.6b00043 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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U/mg). A double-reciprocal plot was used to determine Km and Vmax of the peptides using cellose as substrates (Table 1). The lowest value assayed for Km was 0.41 mM, and the highest kcat/ Km value was 3298.17 s−1·mM−1 for TOX-v3 among the three peptides. It indicated that TOX-v3 possesses higher affinity for the substrate and higher velocity of substrate degradation compared with TOX-1 wild type peptide. Temperature−Activity Profile of TOX Peptides. The optimal temperatures of TOX peptides were determined in the temperature range from 4 to 70 °C at pH 7.4 (Figure 3A). The

bp DNA fragments were inserted into the pUC-118 vector, respectively. Subsequently, E. coli DH5α transformants were selected on CMC-supplemented plates after electroporations. Colonies with different sizes appeared on the CMC plates after 3 days. The differences in the numbers of colonies transformed with the three gene variants on CMC plates were statistically significant (Table S2, SI). The mutant variants, especially toxv3, displayed better growth than the transformants of the wild type gene. Sequencing confirmed the presence of the chimeric genes (Figure S2c−e, SI). The Functions of the tox-1 Gene and Its Two Mutant Variants in Yeast Transformants. Linear PCR amplicons fused with GPD promoters were directly electroporated to S. cerevisiae wild type strain GIM2.207 and selected on CMC plates. Transformants growing on CMC plates were subjected to PCR amplifications. The yeast transformant of tox-v3 gene exhibited the largest transparent zone in a Congo Red assay (Figure 1A). Furthermore, the tox-1 and its two mutant variants could grow on the plates containing only rice straw and agar (Figure 1B). The two variants, especially transformants with tox-v3, formed larger single colonies on the plates. These results confirmed the cellulolytic activities of tox genes and indicated that the amino acid replacements of tox genes could increase cellulolytic activities. The results on yeast transformants demonstrate their potential utility in animal fodder preparation. Substrate Specificity and Kinetic Constants of TOX Peptides. The substrate specificities of TOX peptides were investigated by testing the enzymatic activities against nine saccharides at pH 7.4, 50 °C (Figure 2). These peptides

Figure 3. Temperature and pH profiles of the TOX protein variants. (A). The activities at different temperatures were measured by using cellose as substrate. (B). The peptides were incubated at 4, 30, 50, 60, and 70 °C, respectively, for 6 h followed by determination of residual activities. The standard errors were below 5%. (C). The activities of the peptides in buffers at different pH were measured by using cellose as substrate at the optimum temperatures. Results are the mean from three independent experiments. (D). Stabilities at various pH were measured as the residual activities at their optimal temperature after incubation in different buffer systems (pH 4.0−8.0) for 6 h, respectively. Results are mean ± standard error from three independent experiments. ***P ≤ 0.001, **P < 0.01, *P < 0.05 versus TOX-1 group; #P < 0.05 and ##P < 0.01, TOX-v2 versus TOXv3.

Figure 2. Substrate specificities of the three peptides. Activities were determined with polysaccharides (CMC, Agar, Soluble Starches, Rice straw, Avicel, Filter paper) and oligosaccharides (cellose, maltose, sucrose) as substrates, at pH 7.4 and 50 °C. The solution without any additives was used as a negative control. Results are mean ± standard error from three independent experiments. ***P < 0.001 versus TOX1 group; ##P < 0.01, TOX-v2 versus TOX-v3.

TOX-v2 and TOX-v3 exhibited over 70% relative activities across all tested temperatures. The specific optimal temperature of the three peptides was 64, 72, and 68 °C, respectively. The thermal stabilities of TOX peptides were also investigated by preincubating the enzymes at different temperatures for 6 h followed by detection of the residual activities (Figure 3B). Incubation at 4 and 30 °C for 6 h did not result in any

exhibited a clear preference for cellose (TOX-1, 394.37 ± 0.02 U/mg; TOX-v2, 504.49 ± 0.04 U/mg; TOX-v3, 413.97 ± 0.06

Table 1. Kinetic Parameters of Three TOX-1 Peptides for the Hydrolysis of Cellosea name

Km (mM)

Vmax (μmol·min−1·mg−1)

kcat (s−1)

kcat/Km (s−1·mM−1)

TOX-1 TOX-v2 TOX-v3

0.54 ± 0.14 0.49 ± 0.09 0.41 ± 0.12

1587.80 ± 3.64 1336.93 ± 4.12b 2171.05 ± 1.68b,c

998.62 ± 2.54 840.83 ± 1.79b 1365.44 ± 3.26b,c

1856.17 ± 4.23 2031.01 ± 1.08b 3298.17 ± 2.64b,c

a

The Km and Vmax values of the peptides were evaluated by linear regression from a double reciprocal plot with different concentrations using cellose as substrate. Results are mean ± standard error from three independent experiments. bP < 0.001 versus TOX-1 group. cP < 0.001 TOX-v2 versus TOX-v3. D

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Journal of Agricultural and Food Chemistry Table 2. Half-Lives (h) of the Three TOX-1 Peptides at Different Temperaturesa half-lives (h) peptide

4 °C

30 °C

50 °C

60 °C

70 °C

TOX-1 TOX-v2 TOX-v3

47.23 ± 3.58 59.97 ± 1.93b 158.40 ± 4.82d,e

29.65 ± 1.32 17.42 ± 3.84c 47.03 ± 2.83d,e

8.45 ± 2.04 10.84 ± 2.57 12.85 ± 2.04b

4.61 ± 1.95 7.01 ± 2.06 8.49 ± 1.57

1.87 ± 0.84 4.25 ± 0.48 5.53 ± 1.28c

a Results are mean ± standard error from three independent experiments. bP < 0.05 versus TOX-1 group. cP < 0.01 versus TOX-1 group. dP ≤ 0.001 versus TOX-1 group. eP ≤ 0.001 TOX-v2 versus TOX-v3.

Table 3. Effects of Various Metal Ions, Organic Solvents, and Surfactants on the Three TOX Peptidesa additive (concn) NaCl (10 mM) CaCl2 (10 mM) ZnSO4 (10 mM) FeSO4 (10 mM) EDTA (10 mM) methanol (10%) DMSO (10%) Triton X-100 (1%) Tween-80 (1%)

TOX-1 100.18 88.93 101.26 219.04 90.75 87.51 96.78 118.04 107.56

± ± ± ± ± ± ± ± ±

TOX-v2

0.62 1.14 0.87 3.12 2.78 0.34 0.52 0.93 0.22

105.15 81.94 96.11 121.56 98.33 97.56 113.04 119.42 108.15

± ± ± ± ± ± ± ± ±

1.18 0.87 0.93 0.24b 3.15 0.99 2.35c 0.67 0.32

TOX-v3 108.01 81.51 95.04 213.56 109.45 98.81 94.94 131.25 121.39

± ± ± ± ± ± ± ± ±

0.45 0.92 0.35 0.43d 1.62c 0.23 1.17e 0.63 0.51

a

The metal ions, organic solvents, and surfactants were diluted in PBS solution and measured at the optimal temperatures and pH. The activity of each TOX peptide without any additive was defined as 100%. Results are mean ± standard error from three independent experiments. bP < 0.001 versus TOX-1 group. cP < 0.05 versus TOX-1 group. dP < 0.001 TOX-v2 versus TOX-v3. eP < 0.01 TOX-v2 versus TOX-v3.

treatment of typical surfactants (1% Triton X-100 and 1% Tween-80). These results indicated that TOX peptides were effective in the presence of some heavy metal ions or organic solvents. The characteristics of the three peptides and some reported glycosyl hydrolases are summarized in Table S4 (SI).27−31 The three peptides were more thermophilic than most of the other listed glycosyl hydrolases, and the addition of surfactants (Tween-80 and Triton X-100) could enhance their activities. The optimal pH of TOX-v3 shifted to alkaline and the chelator EDTA could gently stimulate the activity of TOX-v3. The cold, thermal, and pH tolerances conferred by the conservative leucine replacements may be explained by the longer aliphatic tail of the residue,32 which reduces the hydrophilicity of the amino acid and perhaps that of the peptides in this study. As previously reported, β-branched aliphatic amino acids had high propensities for β-sheet formation. This may be achieved through enhanced hydrogen bonds.33 The van der Waals interaction of the side group of leucine with the carbonyl group is intermediate in strength.34 Leucine was more inhibitory of acid corrosion of cobalt than isoleucine and valine were.35 Due to the longer hydrophobic side group and weaker wan der Waals interaction with the carbonyl group, the carbonyl oxygen and perhaps amino nitrogen of leucine residues may form weaker secondary chemical bonds with the surrounding milieu than the latter two amino acids, which results in weak interference by ions, water, and other ambient factors. Consequently, non-β-branched leucine replacements resulted in better pH, cold, and thermal tolerances. Gene chimerization may reduce peptide toxicity via neutralization of electric charges and disruption of salt bridges, hydrogen bonding, secondary structures, etc. DLS and CD. Particles of TOX-1, TOX-v2, and TOX-v3 peptides with an average diameter 4.2 ± 0.026, 65.5 ± 1.49, and 46.0 ± 0.65 nm (±standard deviation), respectively, were detected via DLS (Figure S4, SI), suggesting that leucine replacements at isoleucine and valine sites enhanced hydro-

significant loss of the enzyme activities, especially for TOX-v3 (Table 2). TOX-v2 and TOX-v3 displayed higher enzyme activities than TOX-1 at 70 °C. These results suggest that the TOX peptides, especially the peptides containing amino acids substitutions, are stable across a wide temperature range. pH−Activity Profile of TOX Peptides. The optimal pHs of TOX peptides were determined over a pH range of 4.0− 10.0, at optimal temperature (Figure 3C). The optimal activities of the three peptides appeared at pH 6.4, 6.8, and 7.2, respectively. At pH 6.0, the TOX-v3 had lower activity compared to its other two isoforms, but it was more active at pH 7.0 to 10.0. The pH stabilities of TOX peptides were examined by preincubating the enzymes at different pH values for 6 h followed by the detection of the residual activities (Figure 3D). Similar activity profiles were observed at pH 4.0 and 5.0. With treatment of the enzyme at pH 6.0−8.0 for 6 h, the half-lives of the TOX-v2 and TOX-v3 were 2 and 4 times that of TOX-1 at pH 7.0, respectively. These two variants also showed longer half-lives than TOX-1 (Table S3, SI) at pH 8.0. Hence, the mutants of TOX peptides were biologically active under alkaline conditions. As well-recognized, potent cellulase activity under alkaline conditions with the tolerance of surfactants could be valuable in manufacturing detergents, because ideal alkaline cellulases as potential detergent additives were confirmed to be effective in a milieu of more conventional detergent ingredients, which mainly includes anionic or nonionic surfactant, citric acid, or a water-soluble salt.6,26 The optimal pHs of most reported cellulases were acidic, whereas the residual activity of TOX-v3 remained over 165.26 ± 0.03 U/mg at pH 8.0 after 6 h incubation, constituting a promising candidate for detergent additives. Effects of Metal Ions, Organic Solvents, and Surfactants on Enzymatic Activity. The effects of metal ions and organic solvents on the activities of TOX isoforms were determined (Table 3). The activities of the three peptides were increased under the treatment of Fe2+ to varying degrees. The activities of the three peptides were also enhanced under E

DOI: 10.1021/acs.jafc.6b00043 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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phobicity and intermolecular interactions. TOX-v2 and TOXv3 peptides may have formed oligomers in solutions. As judged by the CD experiment, the α-helix was the principle secondary structure in all three peptides (Table S5, SI). Besides, the αhelix content in the three peptides changed little across different temperatures (Figure S5, SI). The reduced α-helix content in TOX-v2 and TOX-v3 may be associated with the increased hydrophobicity of peptides. The CD data indicated that the peptides were compact and folded as α-helix is a key component of protein folding.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00043. Tables S1−S5 and Figure S1−S5 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Q.L.: e-mail, [email protected]; phone, +86 20 84110296. *G.L.: e-mail, [email protected]; phone, +86 20 84110296. Funding

This work was supported by the grants of National Science Foundation of China (31170117, 31270156, 31300669), National Marine Research Funds for Public Welfare Projects of China (201205020), Science and Technology Plan Project in Guangdong Province (2012B010300021), Natural Science Foundation of Guangdong Province (S2012010010464) to G.L. and Guangdong Natural Science Foundation (S2011010004264), Guangdong Science and Technology Program (No. 2008B020100001), Open Fund of MOE Key Laboratory of Aquatic Product Safety, Open Fund of Laboratory (20150119) and 2016 Key Project Budget at Sun Yat-Sen University, Foreign Expert Program at Sun Yat-Sen University, The Public Welfare and Competence Program of Guangdong Province (2016B020204001), and National Natural Science Foundation of China (J1310025) to Q.L. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Yuqing Li, Xingqiang Lai, Cui Yang, Yiqi Li, and Shaoyun Song for technical help. We appreciate Yan Shi’s help with languages. This paper does not contain any studies with human participants or animals performed by any of the authors.

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ABBREVIATIONS USED E. coli, Escherichia coli; N. crassa, Neurospora crassa; CMC, carboxymethyl cellulose; DLS, dynamic light scattering; orf, open reading frame

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REFERENCES

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