Thermostable pectate lyase from Caldicellulosiruptor kronotskyensis provides an efficient addition for plant biomass deconstruction

Journal of Molecular Catalysis B: Enzymatic 121 (2015) 104–112

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Thermostable pectate lyase from Caldicellulosiruptor kronotskyensis provides an efficient addition for plant biomass deconstruction Hong Su a,b , Weihua Qiu a , Qing Kong c , Shuofu Mi a , Yejun Han a,∗ a b c

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Sciences, Beijing, China College of Food Science and Engineering, Ocean University of China, Qingdao, China

a r t i c l e

i n f o

Article history: Received 4 May 2015 Received in revised form 13 August 2015 Accepted 17 August 2015 Available online 21 August 2015 Keywords: Pectate lyase Caldicellulosiruptor Thermostable Degumming Biomass deconstruction

a b s t r a c t To understand the enzymological basis for extremely thermophilic, biomass-degrading genus Caldicellulosiruptor metabolize pectin, a thermostable pectate lyase Pel-863 encoded by a gene cluster for hexose-containing polysaccharide metabolism in genome of C. kronotskyensis was studied. The pectate lyase of Caldicellulosiruptor was highly conserved and the representative Pel-863 was biochemically characterized, and the application for pectin containing biomass degradation was also studied. Pel-863 exhibited an optimal activity at 70 ◦ C and pH 9.0 with Ca2+ as cofactor. It degraded polygalacturonic acid (PGA), methylated pectin and pectic biomass through endo-cleaving action. The respective Vmax and Km for Pel-863 were 172.8 U/mg and 0.60 g/L on PGA. FTIR and SEM analysis indicated that Pel-863 could remove most of pectin in hemp fiber with less damage compared to alkaline degumming. In addition, pre-digestion with Pel-863 improved glucose and xylose yield by 14.2% and 311.6% respectively for corn stalk, 6.5% and 55% for rice stalk compared with sole action of Novozymes Cellic CTec2. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Pectin is a polysaccharide that mainly exists in the primary cell wall and middle lamella of plants and confers integrity and rigidity to plant tissues [1]. Pectin plays an important role in recalcitrance of biomass due to its complex constituents and structure that embeds the cellulose-hemicelluloses network [2]. There are three primary structures in pectin, including HG (homogalacturonan), RG-I (rhamnogalacturonan-I) and RG-II. HG is the liner region consisting of 100–200 d-galacturonic acid linked by ␣-1,4 glycosidic bond with 70–80% of residuals methyl-esterified [3]. RG-I and RG-II both have side chains involving 11 disparate kinds of monosaccharides, but the main chain of RG-I is repetitive disaccharide units composed of galacturonic acid and rhamnose residues while what is HG backbone for RG-II [3]. The enzymatic degradation of pectin is crucial in several industrial and biotechnological processes. Enzymes involved in pectin degradation can be categorized into three main types according to their distinct action modes, i.e. pectin methyl esterase (PME, [EC 3.1.1.11]), polygalacturonase (PG, [EC 3.2.1.15]), and pectin/pectate lyase (PNL, [EC 4.2.2.10] and

∗ Corresponding author at: National Key Laboratory of Biochemical Engineering, Chinese Academy of Sciences, Beijing, China. E-mail address: [email protected] (Y. Han). http://dx.doi.org/10.1016/j.molcatb.2015.08.013 1381-1177/© 2015 Elsevier B.V. All rights reserved.

PEL, [EC 4.2.2.2]). PME eliminates methoxyl groups from pectin; PG cleaves glycosidic bonds in backbone by hydrolysis, and PNL/PEL cut off the main chain generating 4, 5 unsaturated products through trans-elimination mechanism [4–6]. Pectate lyases (Pels) have attracted increasing attention in recent years because of their extensive use in textile processing, paper manufacture, tea or cocoa fermentation, pectic waste water decontamination and plant oil extraction [7,8]. Pectic substances become more soluble at high temperature in alkaline conditions, so Pels derived from alkaliphiles and thermophiles are more competitive due to their high activity and stability at higher pH or temperature [9–11]. Pels so far characterized are mainly originated from genus Bacillus, Pseudomonas and Aspergillus [12,13]. Caldicellulosiruptor is a thermophilic bacterium with optimum temperature of 70–78 ◦ C, and grows on pectin containing natural biomas [14]. The extremely thermophilic, plant biomassdegrading genus Caldicellulosiruptor provides an increasing number of enzymes with desirable potentials for industrial utilization. The Pels encoded by genome of Caldicellulosiruptor have generated an increasing attention. Through genome editing technology, the importance of genes encoding pectin degrading enzymes has been testified, and the ability of C. bescii to grow on both dicot and grass biomass heavily reduced when deleting these genes [16]. Although there is limited biochemical information for the enzymatic activity of Pels in Caldicellulosiruptor, several studies provide indirect evi-

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Fig. 1. Gene constitution and Sequence alignment of Pel-863. A. Gene cluster for hexose-containing polysaccharide metabolism in genome of C. kronotskyensis. It contains catalytic enzymes involving cellulose, mannan, and pectin degradation and other related proteins. Calkro 0863 (in the black box) is annotated as an extracellular pectate lyase belonging to PL3 family. B. Multiple amino acid sequence alignment of Pel-863 with Cbes 1854 (ACM60942.1, PDB entry 3t9 g), KSM-P15 (1ee6 A, PDB entry 1ee6) and Dda3937 00058 (ADM99410.1, PDB entry 3b4n). The predicted catalytic residues and acidic cluster residues for calcium binding were marked with black pentagrams and triangles, respectively.

dence of desirable properties of these Pels. C. bescii PL3 was found to have strong synergy with cellobiohydrolase A (CelA) of C. bescii, since its addition lowered the loadings of CelA by 33% to achieve the same level of unpretreated switchgrass conversion as CelA acted alone [15]. Hence, these Pels have great potentials for both research and application value. As typical strain of genus Caldicellulosiruptor, Caldicellulosiruptor kronotskyensis grew on various pectin containing biomass [17], and was found to have the highest diversity of GH domains (84

GH domains that represent 38 different GH families) among anaerobic thermophiles [18]. Although the structure of C. bescii PL3 was resolved, and the sequence of PL3 was highly conserved of the genus, the biochemical property and the application of PL3 was limitedly reported. In present study, the pectate lyase Pel-863 from C. kronotskyensis was heterologously expressed and biochemically characterized. The applications of Pel-863 in the hemp fiber degumming and straw deconstruction were also investigated.

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in 5 mL LB broth and cultured at 37 ◦ C, 220 rpm overnight. The initial culture was diluted 100-fold in fresh LB (50 ␮g/mL kanamycin and 34 ␮g/mL chloramphenicol) and cultured at 37 ◦ C with shaking (220 rpm) until the absorbance at 600 nm reached 0.6, Isopropyl␤-d-thiogalactopyranoside (IPTG, its final concentration of 0.1 mM) was added to induce Pel-863 expression. The cells, harvested by centrifuge at 3600 rpm for 10 min, were resuspended with binding buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl) and broken by ultrasonication. After centrifugation, the supernatant was loaded onto the His-Tag Ni-affinity resin (National Engineering Research Center for Biotechnology, China) to purify the target protein. Ni2+ -NTA column was pre-equilibrated by double-distilled water and binding buffer. Before using elution buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 500 mM Imidazole) to get target protein, binding buffer was used to wash out nonspecific binding protein. The buffer for eluted protein was replaced by 50 mM pH 7.0 Tris–HCl buffer containing 300 mM NaCl through dialysis and the protein was then subjected to gel filtration chromatography with Superdex 200 column (1.1 × 26.0 cm) for further purification and quaternary structure determination [19]. 2.4. Gel electrophoresis and protein quantification Fig. 2. Purification and quaternary structure analysis of Pel-863. A. Quaternary structure analysis of Pel-863 by gel filtration chromatography (GFC). B. SDS-PAGE of Pel-863 fractions collected from GFC. The bands marked with Arabic numerals 1–7 corresponded to eluents from A.

2. Materials and methods 2.1. Materials C. kronotskyensis (DSM 18902) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). pET-28b (+) (Novagen, USA) was used as vector Escherichia coli Top10 and Rosetta (DE3) were used as host strains for gene cloning and expression, respectively. The PGA, sodium salt, citrus peel pectin, and galacturonic acid used in current study were purchased from Sigma Chemical Co. Hemp fiber, apple pomace, corn stalk and rice stalk were agricultural by-products obtained from suburban Beijing. TIANprep Mini Plasmid Kit was purchased from Tiangen Biotech, Beijing, China. 2.2. Genomic DNA extraction and recombinant plasmid construction For genomic DNA extraction, C. kronotskyensis was cultured in modified DSMZ medium 640 (xylose replaced cellobiose) for 12 h under anaerobic condition with shaking (75 rpm, 75 ◦ C). The collected cells were applied for genomic DNA extraction by using TIANamp Bacteria DNA Kit (Tiangen Biotech, Beijing, China). The genes of Pel-863 was amplified by using the primers 5 -GCCGCGCGGCAGCATGGCGACACTTTTAACA-3 (forward) and 5 GCGGCCGCAAGCGTTTAGTATTGATGTATCTGTG-3 (reverse). The purified PCR product was treated by T4 DNA polymerase (Takara), then ligated to pET-28b (+) based on complementary sticky ends and the recombinant pET-28b-Pel-863 was transformed into competent cells E.coli Top 10. Positive clones were verified by colony PCR and sequencing. 2.3. Expression and purification of pectate lyase The purified recombinant plasmid, was transformed into E. coli Rosetta (DE3) competent cells, and then cultured overnight on Luria–Bertani (LB) agar plates at 37 ◦ C. Single colony was inoculated

The protein was analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [20]. After being stained by Coomassie brilliant blue R-250 (Ourchem, China), protein bands were visualized under the white light by Digital Gel Image Analysis System (Tanon-1600). The protein concentration was measured as described by Bradford [21] by using bovine serum albumin as standard. 2.5. Catalytic properties and kinetic parameters determination The catalytic activity of Pel-863 was measured spectrophotometrically at 235 nm based on the release of 4, 5-unsaturated oligogalacturonides from PGA [22,23]. The reaction mixture, containing 80 ␮L of PGA-NaOH solution (pH 7.0, 2% (W/V), PGA, Alfa Aesar), 320 ␮L of 50 mM Glycine-NaOH buffer (pH 9.0, with 150 mM NaCl and 1.5 mM CaCl2 ), and 10 ␮L of appropriately diluted enzyme, was incubated at 70 ◦ C for 10 min. The reaction was terminated by adding 400 ␮L of 50 mM HCl. The reaction mixture without adding Pel-863 was applied as control. The samples were centrifuged at 13,000 × g for 10 min, and the supernatant was subjected to 4, 5unsaturated oligogalacturonides assay. One unit of Pel-863 activity was defined as the amount of enzyme that produced one micromole of unsaturated oligogalacturonate per minute under assay conditions and the molar extinction coefficient value (235 ) was assumed to be 4075 M−1 cm−1 [24]. The optimum pH of Pel-863 was determined by using 50 mM Citrate buffer (pH 6.0–6.5, containing 150 mM NaCl and 1.5 mM CaCl2 ), 50 mM Tris–HCl buffer (pH 7.0–8.5, containing 150 mM NaCl and 1.5 mM CaCl2 ) and 50 mM Glycine-NaOH buffer (pH 9.0–10.5, containing 150 mM NaCl and 1.5 mM CaCl2 ). The pH stability of Pel-863 was tested by incubating the enzyme with buffers ranged from pH 6.0 to 10.5 at room temperature for 36 h, and the residual activity was measured thereafter. The influence of temperature on Pel-863 activity was assayed within the range of 50–95 ◦ C. Thermostability was tested by incubating the enzyme in GlycineNaOH buffer (pH 9.0) at different temperatures (60, 65, 70, 75 ◦ C) for 12 h, and the residual activity was determined at different intervals. The effect of various metal ions (Cu2+ , Zn2+ , Fe3+ , Fe2+ , Mg2+ , Ca2+ , Co2+ , Ni2+ , Mn2+ , K+ ) and different reagents (EDTA, SDS, ␤-mercaptoethanol) on Pel-863 activity were investigated at the concentration of 1 mM for metal ions and 0.1% (W/V) for reagents. For the kinetic parameters determination, Km and Vmax were determined by collecting activity data at different concentra-

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Fig. 3. Biochemical properties of Pel-863. A. The pH profile of Pel-863. B. The stability of Pel-863 at different pH. C. The temperature profile of Pel-863. D. The thermostability of purified Pel-863. E. Michaelis–Menten kinetics and Lineweaver–Burk plot of fresh purified Pel-863. F. Allosteric Sigmoidal kinetics of Pel-863 stored for one month. Values were shown as the means of three replicates and error bars represent the SD values. G. Effect of different metal ions (with a concentration of 1 mM) and reagents (with a concentration of 0.1%, W/V) on Pel-863 activity. H. Effect of CaCl2 concentration on Pel-863 activity. Values were shown as the means of three replicates and error bars represent the SD values. *: P < 0.05, **: P < 0.03 in statistical analysis with unpaired Student’s t test.

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aration, the plate was left in developing solution (1-butanol/acetic acid/H2 O with a ratio of 5:2:3) for 2 h. The products were visualized by spraying the dried plate with staining solution (0.05% 5-methylresorcinol and 5% sulfuric acid), and incubated at 75 ◦ C for 10 min for color development. Galacturonic acid (GA) was used as standard on TLC plate. 2.7. Enzymatic degumming of hemp fiber using Pel-863 Enzymatic degumming of hemp fiber was conducted as described in Section 2.6. Alkaline scouring was carried out by adding 750 ␮L of 1% (W/V) sodium hydroxide solution to 1.5 mL centrifuge tube containing 15 mg of mechanical pretreated fiber, and incubated at 65 ◦ C for 12 h. The mechanical pretreated fiber without further treatment was set as control-1 and the group with only 750 ␮L of Glycine-NaOH buffer was set as control-2. The control-2 group and Pel-863 and sodium hydroxide treated fibers were washed with distilled water for three times, and then dried at 85 ◦ C for 24 h in drying oven. The resulting fibers were then analyzed by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). Samples for FTIR were prepared by mixing about 1 mg of pulverized fiber with 200 mg of KBr and pressed into a small disc about 1 mm thick. FTIR spectrum was acquired under transmittance mode in the 4000–600 cm−1 range with a 4 cm−1 resolution using spectrometer of Tensor 27 (Bruker, Germany). Electron micrographs of differently treated fibers were collected by a Field Emission Gun–Scanning Electron Microscopy (FEG–SEM) (JSM 6700F, JEOL, Japan) with an acceleration voltage of 5.0 kV. 2.8. Enzymatic deconstruction of pectin-containing biomass by predigestion with Pel-863 Fig. 4. Catalytic properties of Pel-863 on different substrates. A. Unsaturated oligogalacturonate produced by Pel-863 on different substrates. B. End products analyzed by TLC catalyzed by Pel-863. Pel-863 was respectively incubated with PGA, pectin, hemp fiber, apple pomace, corn stalk, and rice stalk overnight at 65 ◦ C. The products of the reaction were analyzed by unsaturated oligogalacturonate and TLC. GA, galacturonic acid; PGA: polygalacturonic acid; “+”: with Pel-863; “–”: without Pel-863. **: P < 0.03 in statistical analysis with unpaired Student’s t test.

tions (0.1–2 g/L) of PGA under optimum conditions and fitting into nonlinear regression (Michaelis–Menten or Allosteric Sigmoidal) using Graph Pad Prism 6 software. 2.6. Catalytic activity of Pel-863 on natural substrates The hemp fiber, apple pomace, corn stalk and rice stalk were pre-processed by mechanical pulverization, washed with distilled water for three times and then dried for 24 h. Enzymatic hydrolysis was conducted in 1.5 mL centrifuge tube containing 15 mg of different substrates (PGA, pectin, hemp fiber, apple pomace, corn stalk, and rice stalk), Pel-863 (12 ␮g/mg substrates) and 750 ␮L of 50 mM Glycine-NaOH buffer (pH 9.0, with 150 mM NaCl and 5 mM CaCl2 ). In preliminary experiment, the activity of Pel-863 with 5 mM Ca2+ was higher than 4 mM Ca2+ on natural substrates, even the difference was not statistically significant. While above the concentration of 5 mM Ca2+ , the activity of Pel-863 on natural substrates keep constant, 5 mM Ca2+ was therefore used for the treatment. The mixtures were incubated at 65 ◦ C for 12 h and corresponding tubes without mixing Pel-863 were set as controls. The samples were centrifuged at 13,000 × g for 10 min, and the supernatant was analyzed by using thin-layer chromatography (TLC) and spectrophotometer at 235 nm as described in Section 2.5. In TLC analysis, 2 ␮L of samples were loaded onto silica gel-coated glass plate (50 × 100 mm) and dried at room temperature. For sep-

Mechanical pretreated corn stalk and rice stalk were used to investigate the role of Pel-863 played in pectin-containing biomass deconstruction. For pre-digestion, the corn stalk and rice stalk were pretreated by Pel-863 as previously stated in Section 2.6 and the group adding the same amount of BSA was set as a control. After pre-digestion at 65 ◦ C for 12 h, the unsaturated oligogalacturonate was analyzed at 235 nm. The reaction mixture was dried in drying oven (∼85 ◦ C), and the residues were mixed with 750 ␮L of acetic acid-sodium acetate buffer (50 mM, pH 5.0) and 7.5 ␮g/mg straw of Novozymes Cellic CTec2, and incubated at 50 ◦ C overnight. The reaction was terminated by boiling for 10 min, and the products in supernatant were determined by High Performance Liquid Chromatography (HPLC) equipped with Hi-Plex Ca column (7.7 × 300 mm, Agilent Technology, USA) and LC-20AT pump (Shimadzu, Japan). 3. Results and discussion 3.1. Gene constitution and structure model prediction of Pel-863 The gene Calkro 0863 of C. kronotskyensis encodes an extracellular pectate lyase (EC 4.2.2.2) of PL3 family with a calculated molecular weight (Mw) of 47.4 kDa, and a computed pI value of 7.61. In genome of C. kronotskyensis, Calkro 0863 was located in a cluster for hexose-containing polysaccharide degradation and metabolism, such as cellulose, mannan, and pectin (Fig. 1A). The modular architecture of Pel-863 consists of a catalytic module PL3 and a carbohydrate binding module CBM 66. The amino acid sequence of Pel-863 was highly conserved in Caldicellulosiruptor and shares 87–99% identities with other annotated Pels in the genus, while showed lower identities with Pels in genus Bacillus (52–58%) and Paenibacillus (50–54%).

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Fig. 5. FTIR spectrum and SEM graphs of hemp fibers treated with NaOH and Pel-863. A. FTIR spectrum of hemp fibers treated with NaOH and Pel-863. Control-1: the mechanical pretreated hemp fiber; Control-2: mechanical pretreated hemp fiber incubated in Glycine-NaOH buffer. B. SEM graphs of hemp fibers (Control-1). C. SEM graphs of hemp fibers (Control-2). D. SEM graphs of hemp fibers treated with Pel-863. E. SEM graphs of hemp fibers treated with sodium hydroxide.

According to CAZy (http://www.cazy.org/) statistical data, there are three structures of pectate lyases belonging to PL3 family have been resolved, and they are Pel-15 (1ee6 A, PDB entry 1ee6) from Bacillus sp. KSM-P15, Cbes 1854 (ACM60942.1, PDB entry 3t9 g) from Caldicellulosiruptor bescii DSM 6725 and Dda3937 00058 (ADM99410.1, PDB entry 3b4n) from Dickeya dadantii 3937. Pel863 is respectively 55%, 99%, and 36% identical to the three proteins. The amino acid sequence of Pel-863 was aligned with the three proteins by using the ClustalX2 software and depicted by ESPrit 3.0 (http://espript.ibcp.fr/). The catalytic residues (marked with black pentagrams) as well as residues formed acidic cluster for Ca2+ binding (marked with black triangles) were all conserved (Fig. 1B). The

␤-elimination reaction for Pels is assumed to be initiated by an essential basic amino acid that abstracts the proton from the substrate carbon C5 [26]. It has been reported that Arg132 for Pel-15 [27], Lys108 for Cbes 1854 [15,25], Lys224 for Dda3937 00058 [26] play the significant role. According to multiple sequence alignment and structure comparison, Lys362 was presumed to be key catalytic residue responsible for capturing proton in Pel-863. Based on the structure of C. bescii PL3, the structure of Pel-863 was predicted to be a parallel ␤-helix, which was shared by almost all of crystallized Pels [8]. The catalytic residues (K362, K384 and R387) and Ca2+ binding site (D318, E338 and D339) were located in a spatial cleft.

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Fig. 6. Enzymatic hydrolysis of pectin containing substrate pre-digested by Pel-863. A. Degradation of corn stalk with Novozymes Cellic CTec2 predigested by Pel-863. B. Degradation of rice stalk with Novozymes Cellic CTec2 predigested by Pel-863. The amount of glucose and xylose was plotted as left vertical axis, and unsaturated oligogalacturonate was plotted as right. C. HPLC analysis of products from degradation of corn stalk and rice stalk. D. Detailed HPLC chromatograph for section of black box in C. Peak G1 for glucose; peak X1 for xylose. Values were shown as the means of three replicates and error bars represent the SD values. **: P < 0.03 in statistical analysis with unpaired Student’s t test.

3.2. Purification and quaternary structure analysis of Pel-863 The quaternary structure of purified recombinant Pel-863 was analyzed through gel filtration chromatography. The molecular mass (Mw) of Pel-863 at two peaks was estimated by the calibration curve of log (Mw) vs. elution volume. The calculated Mw of protein at peak1 (148 kDa) was almost three times of peak 2 (50 kDa) (Fig. 2A). In addition, fractions collected from two peaks showed the same band in SDS-PAGE (Fig. 2B) and it suggested that Pel863 exists as monomer and homotrimer in buffer. The collections of peak1 and peak2 were also subjected to native-PAGE analysis, and two corresponded bands of 150 kDa and 50 kDa were observed respectively, which was consistent with that of gel filtration chromatography (Figure not shown).

3.3. Biochemical properties of Pel-863 The purified Pel-863 was active in pH range of 7.0–10.5, and exhibited an optimum pH of 9.0, around 30% and 40% relative activity was observed at pH 8.0 and 10.0, respectively with CaCl2 loading of 1.5 mM (Fig. 3A). In stability assay in different pH solution, over 60% activity was retained after being incubated in a pH range of 6.0–9.5 for 36 h, even higher stability was observed in pH 7.0–9.0 (Fig. 3B). Under CaCl2 loading of 1.5 mM in the reaction mixture, the optimum pH and stability was also conformed. The pH profile of Pel863 suggested that the enzyme was active in alkaline conditions. In temperature characteristic assay, Pel-863 was active in the range of 55–95 ◦ C, and displayed maximum activity at 70 ◦ C. More than 50% activity was observed in temperature range of 40–85 ◦ C, which

suggested Pel-863 was a thermostable enzyme (Fig. 3C). In thermostability assay, Pel-863 maintained 71% activity at 60 ◦ C after being incubated for 8 h, while only 10% activity was remained when incubated at 75 ◦ C for 1 h (Fig. 3D). In present study, fresh prepared and stored Pel-863 was respectively applied for kinetic parameter determination. The Vmax and Km values of newly purified Pel-863 were calculated to be 172.8 ± 5.6 U/mg and 0.60 ± 0.05 g/L when fitted with Michaelis–Menten equation (R2 = 0.9961) (Fig. 3E). However, after being stored at 4 ◦ C for about one month, Pel-863 tended to have kinetic properties of allosteric enzymes, and the respective Vmax and Khalf values were 80.9 ± 4.9 U/mg and 0.36 ± 0.03 g/L when fitted with Allosteric Sigmoidal equation (R2 = 0.9531) (Fig. 3F). Considering that Pel-863 was observed as monomer and homotrimer in solution, it might be the rising proportion of homotrimer that led to the change of kinetic properties of Pel863. Unlike allosteric modulation of monomeric proteins, such as sperm whale myoglobin, human serum albumin and human ␣thrombin, which was carried out by heterotropic allosteric effect [28], Pel-863 was a multimeric allosteric protein. It’s worth to note that the allosteric effect of pectate lyase was rarely reported previously. The activation effect of metal ions on Pel-863 activity was arrayed as Ca2+ > Fe3+ > Fe2+ > Co2+ , while no activation was observed with the presence of Cu2+ , Zn2+ , Mg2+ , Ni2+ , Mn2+, and slight activation was observed with K+ (Fig. 3G). Among the tested ions, Ca2+ displayed highest activation effect for the activity, and was essential for the catalysis. With the presence of EDTA, the activation of Ca2+ for Pel-863 disappeared, suggesting that the chelated

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Ca2+ cannot activate the catalysis, which was in consistence with the results reported earlier [23,29]. However, different studies reported that Fe2+ , Mn2+ , Co2+ , Ni2+ could be a better cofactor than Ca2+ for Pels activities [30–32]. Activation of enzyme activity was brought by 0.1% ␤-mercaptoethanol, indicating that sulfhydryl groups have positive effect for activity. Furthermore, about 50% activity of Pel-863 was inhibited by 0.1% SDS in presence of Ca2+ and it suggested that Pel-863 might be denaturized by SDS. In statistics analysis, the activity difference between with Ca2+ solely and with Ca2+ and ␤-mercaptoethanol, EDTA, and SDS reached significant level. To determine the optimum Ca2+ concentration for Pel-863 activity, the relation of activity and concentration of Ca2+ was tested under optimum conditions. As shown in Fig. 3H, the activation of Pel-863 increased with the increasing loading of Ca2+ , and a maximum activity was observed at the concentration of 4.0 mM, indicating the ion binding sites were saturated. In statistics analysis, significant level of activity difference was observed with the increase of Ca2+ loading (0.5 mM each) except for 4.0 mM. In the assay, the flowability of PGA solution decreased significantly with the increase of Ca2+ concentration, and it was too sticky to detect the activity at the concentration of 5 mM. 3.4. Catalytic properties of Pel-863 on pectin-containing natural substrates To analyze the catalytic activity of Pel-863 on methylated pectin and natural pectin of biomass, different substrates (PGA, pectin, hemp fiber, apple pomace, corn stalk, and rice stalk) were analyzed respectively. As shown in Fig. 4A, unsaturated oligogalacturonate was detected from all of the applied substrates catalyzed by Pel863. The enzyme displayed maximum activity on PGA, while minimum activity on methylated citrus peel pectin, indicating methylation blocked the catalysis of Pel-863. In statistics analysis, the activity difference between PGA and the other five substrates reached significant level. While interestingly, the catalytic activity of Pel-863 on apple pomace was higher than that of citrus peel pectin and other three pectin-containing natural substrates, and the difference reached significant level in statistics analysis. In addition to unsaturated oligogalacturonate determination, the catalytic mode of Pel-863 was analyzed by analyzing final products from the substrates in TLC. As shown in Fig. 4B, unsaturated GA and digalacturonate was observed from the degradation of PGA, suggesting that Pel-863 was an endo-cleaving lyase. Taking citrus peel pectin as substrate, mono and digalacturonate were observed in the control, and both of which increase slightly with the catalysis of Pel-863. Taking hemp fiber as substrate, the major product was GA, while no digalacturonate was produced. In apple pomace and corn stalk degradation, GA was the main product, with a small quantity of digalacturonate, while digalacturonate was still observed in the control. The chromatographic characteristic of rice stalk was different from the other substrates, in addition to mono and digalacturonate produced, a compound moved even faster than GA was observed. The catalytic properties of Pel-863 suggested that the enzyme was an endo-type lyase, and cleave the internal ␣-1,4linkages of polygalacturonate randomly in both methylated and unmethylated pectin, and generating a mixture of 4,5-unsaturated oligogalacturonates, including unsaturated mono and oligomers. 3.5. Application of Pel-863 in the enzymatic degumming of hemp fiber Pel-863 was active on natural hemp fiber, and soluble products of 4, 5-unsaturated oligogalacturonates were produced. To determine the microcosmic structure changes of hemp fiber by Pel-863 digestion, the substrate before and after treatment was applied for

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FTIR spectrum and SEM assay. The degumming of hemp fiber was normally conducted by using NaOH, which is applied as a positive control in the determination. FTIR spectrum of hemp fiber treated with Pel-863 and NaOH was showed in Fig. 5A. The characteristic absorption peak at 897.2 cm−1 was for ␤-glycosidic linkages in cellulose [33] and it changed slightly, which meant that the structure of cellulose in hemp fiber was undestroyed after treated by both Pel-863 and NaOH. The vibration peak at 1737.7 cm−1 , attributed to the C O stretching of methyl ester and carboxylic acid in pectin or the acetyl group in hemicelluloses [33], which was disappeared after been treated with NaOH, and decreased dramatically after Pel-863 treatment. The peak transformation at 1737.7 cm−1 suggested that the characteristics of pectin changed after treating with Pel-863 and 1% NaOH. In addition, the absorbance peak at 1637.3 cm−1 for stretching vibration of C O in lignin [33] drastically weakened in 1% NaOH treatment, while decreased slightly with Pel-863 treatment. The results suggested that lignin was partly removed by Pel-863 or 1% NaOH treatment. The SEM results also verified that Pel-863 could be an alternate to alkaline solution for hemp fiber degumming. As showed in Fig. 5, Pel-863 (Fig. 5D) and 1% NaOH (Fig. 5E) treated samples were smoother and more even in color intensity. However, there were a lot of dark protuberances, supposed to be gum-like materials, in untreated (Fig. 5B) and Pel-863 buffer treated (Fig. 5C) fibers. Moreover, enzymatic degumming caused less damage to the fiber compared with alkaline treatment under the conditions examined (Fig. 5D and E). 3.6. Facilitating enzymatic conversion of pectin containing biomass by Pel-863 pre-digestion The thermostable and alkaline Pel-863 was active on the pectin-containing natural biomass, and produced unsaturated oligogalacturonate from corn stalk and rice stalk. In enzymatic degradation of biomass, pretreatment is an important step to increase the accessibility of substrates. In most pretreatment, chemicals are applied to remove the non-cellulose components of biomass, such as hemicelluloses, lignin, and pectin. To evaluate the potential of Pel-863 for biomass degradation, the enzyme was applied as predigestion for enzymatic hydrolysis of biomass with commercial Novozymes Cellic CTec2. In the two steps degradation, corn stalk or rice stalk was predigested with Pel-863 in alkaline condition, and then hydrolyzed by Cellic CTec2. The unsaturated oligogalacturonate produced from corn stalk and rice stalk was 1.26 mM and 1.08 mM, respectively. Compared with the corn stalk hydrolyzed with Cellic CTec2 alone, the production of glucose and xylose was increased by 14.2% and 311.6%, respectively, when predigested with Pel-863 (Fig. 6A and D). For enzymatic hydrolysis of rice stalk, the glucose and xylose concentration was improved by respectively 6.5% and 55% with Pel-863 predigestion (Fig. 6B and D). Taking both corn stalk and rice stalk as substrates, the difference of xylose and unsaturated oligogalacturonate production between with and without Pel-863 predigestion reached significant level (P < 0.03). In general, Pel-863 predigestion improved the reducing sugar yield markedly. The improvement of xylose yield was even more significantly, and it could be attributed to the easily release of d-xylose from hemicelluloses. Pectin is a complex structural component and cross-linking with cellulose and hemicelluloses to form recalcitrant plant cell walls [34], and its removal by Pel-863 predigestion consequently make hemicelluloses more sensitive to enzymatic hydrolysis. In addition to biomass degradation, alkaline pectinases have been extensively applied in the industries of textile, retting and degumming of plant fibers, and pulping by complete or partial

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removal of the non-cellulosic components. Compared to traditional plant pretreatment and degumming, which is conducted through a series of chemical and extreme treatments, the enzymatic treatment with thermostable alkaline Pel-863 is a more environmentally friendly method. Therefore, Pel-863 would be a desirable candidate for novel enzyme cocktails aimed at degumming and degradation of lignocellulosic biomass. 4. Conclusions To summarize, an alkaline and thermostable pectate lyase Pel863 from pectic biomass degrading strain C. kronotskyensis was heterologously expressed and biochemically characterized. Pel863 was an endo-cleaving enzyme, and active on PGA, methylated pectin and pectic natural biomass, with unsaturated oligogalacturonates as products. Pel-863 requires divalent cation as cofactor, which was located in a spatial cleft in predicted structure. In quaternary structure assay, Pel-863 exists as a mixture of monomer and homotrimer. The biochemical properties of Pel-863 make it a preferable environmental substitution to alkaline treatment in hemp fiber degumming and notably facilitated the enzymatic hydrolysis of pectic biomass with cellulase. Acknowledgements This work was financially supported by the National “863” High-Tech Research and Development Program of China (2014AA021905) and 100 Talents Program of Institute of Process Engineering, Chinese Academy of Sciences. References [1] W.G. Willats, L. McCartney, W. Mackie, J.P. Knox, Plant Mol. Biol. 47 (2001) 9–27. [2] V. Lionetti, F. Francocci, S. Ferrari, C. Volpi, D. Bellincampi, R. Galletti, R. D’Ovidio, G. De Lorenzo, F. Cervone, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 616–621. [3] M. McNeil, A.G. Darvill, S.C. Fry, P. Albersheim, Annu. Rev. Biochem. 53 (1984) 626–663. [4] J.A. Gerlt, P.G. Gassman, J. Am. Chem. Soc. 114 (1992) 5928–5934. [5] J.A. Gerlt, P.G. Gassman, J. Am. Chem. Soc. 115 (1993) 11552–11568. [6] J.A. Gerlt, J.W. Kozarich, G.L. Kenyon, P.G. Gassman, J. Am. Chem. Soc. 113 (1991) 9667–9669. [7] D.R. Kashyap, P.K. Vohra, S. Chopra, R. Tewari, Bioresour. Technol. 77 (2001) 215–227.

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