An alkaline pectate lyase D from Dickeya dadantii DCE-01: clone,expression,characterization,and potential application in ramie bio-degumming

Abstract

Pectinase plays a crucial role in ramie bio-degumming. A pectate lyase gene (pel4J4) from the high-efficiency degumming bacteria Dickeya dadantii DCE-01 of bast fibers was cloned and connected to pET28a, and then the recombinant plasmid was successfully transformed into Escherichia coli BL21(DE3). The pectate lyase (Pel4J4) induced was purified by ultrafiltration and Sephadex G-100 gel chromatography. The enzymatic properties of Pel4J4 were studied in detail. pel4J4 (GenBank accession number: KC900167) had a sequence length of 1179 bp, encoding 392 amino acids. The extracellular pectate lyase activity of pET28a-pel-BL was up to 204.4 IU/mL. The optimal temperature and pH of the purified Pel4J4 were 55℃ and 8.5, respectively. The stable temperature and pH of Pel4J4 activity were 45℃ and 8.5–10.0, respectively. The catalytic activity is Ca²⁺ dependent and promoted by 1 mmol/L Zn²⁺, Fe³⁺, Ca²⁺, and NH₄⁺, but seriously inhibited by Cu²⁺ and Pb²⁺. The optimal substrate is citrus pectin with more than 85% esterification. The heat-resistant alkaline Pel4J4 could strongly degrade natural ramie pectin, indicating a promising application prospect in ramie bio-degumming.

Keywords

pectate lyase ramie biological degumming

Pectin is polymerized by galacturonic acids with different esterification degrees and α-1,4 glucosidic bonds, which interweaves with cellulose, hemicelluloses, lignin, and proteins in plant tissues to maintain the intrinsic morphology of cells and tissue structures. The side chain often carries rhamnose, arabinose, galactose, xylose, trehalose, and apiose. Free carboxyls partially or completely combine with Ca²⁺, K⁺, and Na⁺, especially boron compounds.¹ Thorough pectin degradation requires the synergistic effect of a series of enzymes. According to the action substrate and mode, pectin-degrading enzymes can be divided into three types: pectate lyase (Pel), pectinesterase, and pectin hydrolase.

Pel (EC 4.2.2.2) randomly depolymerizes the pectic acid polymers or α-1,4-glucosidic bonds of pectin through the β-transelimination mechanism, generating C₄-C₅ unsaturated oligomerization polygalacturonic acid. The optimal pH generally falls within the alkaline range, and the catalysis is Ca²⁺ dependent.² Pels are widely applied in food processing,³ the papermaking industry,⁴ the textile industry,⁵ and for environmental protection.⁶

There exists about 30% non-cellulosic substances, called “gum,” in raw ramie bast fiber, including 4–8% pectin, 12–18% hemicellulose, and 0.8–1.5% lignin.⁷ Ramie fiber should be degummed before high-quality textile product processed. The traditional chemical degumming method has problems of high labor intensity, severe environment pollution, and unstable product quality, as well as high cost and fiber damage. Biological degumming can overcome the shortages and has the characteristics of high efficiency, low energy consumption, and reduced emissions. Indeed, the development of the ramie processing industry is geared toward biological degumming.⁸ Many microbial resources, including Pseudozyma sp. SPJ, Bacillus pumilus DKS1, and Bacillus sp. NT-39 for biological degumming have been reported.^9–11 The bacteria are able to secrete extracellular pectinase. Pectinase can degrade pectin substances that serve as “bonds” in the early stage of ramie bio-degumming, as well as loosen up cellular structures and promote the effective permeation of hemicellulase and other degumming factors, thereby increasing the efficiency of degradation to the gum complex. Pectinase as a primase is considered as one of the most important factors that can affect ramie biological degumming.¹²

Researches on the gene clone, expression, and functions of Pel for the biological degumming of bast fibers have been reported with the development of genetic engineering and protein technology. Yuan et al.¹³ cloned pel from Streptomyces sp. S27 and proved the significant degumming effect of the encoded Pel in jute. Basu et al.¹⁴ cloned pel from a ramie bio-degumming strain B. pumilus DKS1 and induced the prokaryotic expression of the encoded Pel. The molecular weight, optimal temperature, and pH were 35 kDa, 60℃ and 8.5–9.0, respectively. Both Ca²⁺ and Mn²⁺ could promote Pel activity, whereas Zn²⁺ and ethylenediaminetetraacetic acid (EDTA) significantly inhibited Pel activity.

A high-efficiency degumming strain D. dadantii DCE-01, bred by the authors, can have ramie degummed independently in 6 h.^15,16 In the present work, based on the whole genome DNA sequence and gene function annotation of D. dadantii DCE-01 (data not shown), primers were designed to clone the predicted pel4J4, and a prokaryotic-expression system was constructed, which were then induced to express PelD. The expression products were purified, and the enzymatic properties were studied, which would provide scientific references for elaborating the bio-degumming mechanism of bast fibers and rapidly expanding industrial application of Pel.

Materials and methods

Materials

Strains and plasmids

D. dadantii DCE-01 strain was obtained and deposited in the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences. Plasmid pET28a and E. coli BL21(DE3) were purchased from Novagen (USA).

Chemical reagents and media

Polygalacturonic acid sodium salt (no. P3850), citrus pectin (nos. P9561, P9436, and P9311), and galacturonic acid monohydrate (no. 48280) were purchased from Sigma (USA). The high-fidelity polymerase KOD plus ver. 2.0, BamH I, Hind III, rTaq, and dNTPs were purchased from TOYOBO (Japan). DNA Marker III was purchased from Tiangen (China). Protein molecular weight standard (no. SM0671) was purchased from Fermentas (USA). Sephadex G-100 (ultrafine) was purchased from Pharmacia (USA). Other conventional reagents were analytical pure commercial reagents purchased from China.

Culture media were an improved nutrient broth (glucose 1.0%, NaCl 0.5%, beef extract 0.5%, peptone 0.5%, H₂O 100 mL)¹⁵ and Luria–Bertani (LB) medium.¹⁷

Methods

Bacterial culture and fermentation

D. dadantii DCE-01 preserved by the authors on slopes was inoculated into 5 mL of improved nutrient broth and vortexed well. It was cultured for 5–6 h at 35℃ and 180 r/min in a shaker, diluted, and spread onto plates containing improved solid nutrient medium before culturing for 18–20 h in a 35℃ incubator. A single colony was separated and inoculated into 5 mL of LB medium overnight at 35℃ and 180 r/min in a shaker. It was used to extract genome DNA or other purposes.

The genetically engineered bacteria (50 µL) stored in glycerin at –80℃ were inoculated into 5 mL of LB medium containing 30 mg/L kanamycin (Kan) and cultured overnight at 37℃ and 220 r/min for plasmid DNA extraction or other purposes.

The positive clone of pel4J4 was inoculated into 5 mL of LB medium containing 30 mg/L Kan and cultured overnight at 37℃ and 220 r/min in a shaker. Then, 1 mL of bacteria solution was inoculated into 100 mL of LB medium containing 30 mg/L Kan and cultured at 37℃ and 220 r/min. When OD₆₀₀ reached 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mmol/L. The mixture was induced for 21 h at 28℃ and 120 r/min.

Pel gene clone

The specific primers for amplifying the open reading frame (ORF) of pel4J4 were designed according to the genome DNA sequence of D. dadantii DCE-01. The forward primer was F₁: 5′-CGGGATCCATGAACAACACTCGTGTGTCTTCTG-3′ (BamH I digestion site) and the reverse primer was R₁: 5′-CCCAAGCTTGCTTACAGTTTGCCGTAGCCTGC-3′ (Hind III digestion site). The upstream primer T₇ for the promoter of carrier pET28a was synthesized by Shanghai Sangon Biotechnology Company.

Using the genome DNA of D. dadantii DCE-01 as the template, pel4J4 was amplified with F₁-R₁. The polymerase chain reaction (PCR) system was composed of 5 µL of 10 × buffer, 2 µL of MgSO₄ (25 mmol/L), 1 µL of F₁ (10 mmol/L), 1 µL of R₁ (10 mmol/L), 5 µL of dNTPs (2 mmol/L), 1 µL of genome DNA, and 1 µL of KOD enzyme (1 U/µL). The final volume of 50 µL was adjusted with ddH₂O. The PCR program was pre-denaturation for 4 min at 94℃, followed by 30 s at 94℃ for denaturation, 30 s at 55℃ for annealing, 80 s at 68℃ for extension for 30 cycles, and finally preserving at 68℃ for 10 min. PCR products were tested by 1% agarose-gel electrophoresis.

Construction and induction of prokaryotic expression

The PCR products of pel4J4 were double digested after they were purified. The digestion system was composed of 2 µL of 10 × buffer, 0.5 µL of Hind III, 0.5 µL of BamH I, and 17 µL of PCR products. The system was incubated for 1 h at 37℃, and then the pel4J4 digestion products were purified. They were connected to pET28a subjected to the same double enzyme digestion and then transferred into E. coli BL21(DE3). Positive clones were screened with LB plate containing 50 mg/L Kan.

A PCR was performed to determine whether the target genes in the positive clones were accurate. The PCR reaction system consisted of 10 µL of 2×Power Taq PCR MasterMix, 1.0 µL of T₇ primer (10 mmol/L), 1.0 µL of R₁ primer (10 mmol/L), and 1.0 µL of bacterial-culture liquid. The final volume of 20 µL was adjusted with ddH₂O. The PCR program was pre-denaturation for 4 min at 95℃, followed by denaturation for 30 s at 94℃, annealing for 30 s at 55℃, extension for 1 min at 72℃ for 30 cycles, and final preservation at 72℃ for 10 min. PCR products were tested by 1.0% agarose-gel electrophoresis.

Activity assay of Pel4J4

About 5 g/L polygalacturonic acid sodium solution was prepared using 0.05 mol/L Gly-NaOH buffer (pH 8.5). After preheating 2 mL of substrate to 55℃, 1 mL of Pel with appropriate dilution ratio and 6 µL of 1 mol/LCaCl₂ solution were added. The mixture was mixed well, reacted for 10 min at 55℃, and then boiled to inactivate the enzyme. Inactivated Pel subjected to the same treatment served as the negative control. Pel4J4 activity was measured spectrophotometrically by the release from polygalacturonate of unsaturated oligogalacturonides at 235 nm. One unit of Pel activity (expressed by IU) was the amount of enzyme that liberated 1 mmol of product per minute at 55℃. The extinction coefficient in the given wavelength was 4600/(M·cm).¹⁸

Purification of Pel4J4

The supernatant from the mature fermentation liquor of pET28a-pel-BL was ultrafiltered through Cross Flow Ultra-filtration Cassettes (Sartorius, Germany) with selected membranes of 5 and 50 kDa. Subsequently, the 5–50 kDa macromolecule fractions were collected as crude Pel4J4 for Sephadex G-100 gel chromatography.¹⁹ The solution components collected at the double peak of Pel activity and protein content were subjected to 48 h dialysis at 4℃ and then concentrated with polyethylene glycol 20,000. The purified Pel4J4 was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).²⁰

SDS-PAGE electrophoresis

The SDS-PAGE system was prepared using 5% stacking gel and 12% separation gel. The protein belts were stained with Coomassie brilliant blue (CBB) R250.²⁰ Electrophoreses of the protein pre-dyed marker and samples were performed under the same conditions. The logarithm of protein molecular weight was used as the Y-axis and the relative mobility as the X-axis to calculate the apparent molecular weight of Pel4J4.²¹ The protein molecular weight standards were 170, 130, 95, 72, 55, 43, 34, 26, 17, and 10 kDa.

Enzymatic properties

The optimal pH was assayed at 55℃ in buffer solutions with different pH gradients (pH 7.5–8.0 Na₂HPO₄-citric acid, pH 8.5–10.5 Gly-NaOH, and pH 11.0 Na₂HPO₄-NaOH; interval of pH 0.5) containing 0.5% poly(glycolic acid) (PGA) (w/v) and 1 mmol/L CaCl₂. The optimal temperature was measured at 40–65℃ with an interval temperature of 0.5℃ for 10 min in standard reaction buffer (0.05 mol/L Gly-NaOH; pH 8.5) with 0.5% PGA (w/v) and 1 mmol/L CaCl₂. pH stability was assayed by incubating Pel4J4 in different buffers with pH gradients (pH 7.5–8.0 Na₂HPO₄-citric acid, pH 8.5–10.5 Gly-NaOH, and pH 11.0 Na₂HPO₄-NaOH; interval of pH 0.5) at 4℃ for 60 min. Thermal stability was determined by measuring the residual Pel4J4 activity after incubation at 30–55℃ with an interval of 5℃ for 60 min.

The influence of metal ions on Pel4J4 activity was determined by incubating the enzyme in 1 mmol/L of chloride metal ions (KCl, ZnCl₂, FeCl₃, CaCl₂·2H₂O, PbCl₂·2H₂O, CuCl₂·2H₂O, MgCl₂·6H₂O, NiCl₂·6H₂O, and NH₄Cl) and then measuring residual relative activity under the standard assay conditions. To test the effect of Ca²⁺ on Pel4J4 activity, 0.05 mol/L Gly-NaOH buffers (pH 8.5) containing 0.5% PGA (w/v) and different concentrations of CaCl₂ (0–5 mmol/L) were used for the activity assay.

Substrate specificity was determined by assaying the activity of purified Pel4J4 on different substrates with a concentration of 0.5% (w/v), including citrus pectin with different esterification degrees (20%–34%, 56%–75%, and ≥85%), polygalacturonic acid sodium, and ramie bast pectin powder under standard reaction conditions (pH 8.5 and 55℃).

Effect of Pel4J4 on ramie degumming

The reaction mixtures consisted of 100 mL of 0.05 mol/L Gly-NaOH buffer (pH 8.5), 5 g of ramie fibers, and 150 IU/mL Pel4J4 added with 1 mmol/L Ca²⁺. Degumming was performed at 50℃ for 3 h. The degummed fibers were beaten to remove the residual gum from the surface and washed with water. Drying was performed at 100℃ to achieve constant weight. Ramie fibers without enzyme treatment served as the negative control.^16,22

The chemical oxygen demand (COD) of the degumming solution was measured using the COD–potassium permanganate method (GB/T 15456-2008). All measurements were performed three times. The weight-loss rate, fiber fineness, and breaking strength of fibers were used to evaluate the ramie degumming efficiency of Pel4J4. Scanning electron microscopy was used to observe the surface morphology and microstructure of the bio-degummed ramie fibers using an EVO-18 (ZEISS, German).

Results and discussion

Construction of the prokaryotic-expression system

The genome DNA of D. dadantii DCE-01 was used as the template. The target gene was amplified with F₁-R₁ primers and analyzed by 1% agarose-gel electrophoresis. The fragment size of the target gene was 1179 bp (Figure 1, lanes 1 and 2). The interposition fragment size and direction of the positive clone pET28a-pel-BL were verified using T₇ and R₁. The amplified band size was found to be consistent with the expected size (1.37 kb) (Figure 1, lanes 3 and 4).

Figure 1.

Construction of the prokaryotic-expression system for pel4J4. Note: M, DNA marker III; lane 1, pel4J4-1; lane 2, pel4J4-2; lane 3, polymerase chain reaction (PCR) by positive clone 1; lane 4, PCR by positive clone 2.

The pel4J4 gene sequence (GenBank accession number: KC900167) was obtained. Compared with pel4J4 predicted from the genome sequence of D. dadantii DCE-01, the gene 1179 bp in length was free of mutation, encoding 392 amino acids. The leader protein molecular weight was 41.8 kDa, the mature protein molecular weight was 38.8 kDa, and pI was 7.3. Interproscan analysis revealed that it belonged to the PF00544 family. The enzyme contained the Pec_lyase_C superfamily according to blastp. It had an identity of 100% with Pel from Dickeya sp. MK7, more than 80% with Pels from other species of Dickeya family, and more than 50% with Pels from Xanthomonas spp. and Pseudomonas luteola (Table 1).

Table 1.

Identity of Pel sequences from partial microorganisms

Protein accession number	Source of strain	Identity
WP_038919873.1	Dickeya sp. MK7	100%
WP_038666766.1	Cedecea neteri	99%
WP_029729679.1	Dickeya dianthicola	95%
WP_038901806.1	Dickeya dadantii	94%
WP_023640484.1	Dickeya zeae	88%
WP_040000191.1	Dickeya chrysanthemi	87%
WP_026738391.1	Lonsdalea quercina	66%
WP_046978128.1	Xanthomonas hyacinthi	42%
WP_026004026.1	Pseudomonas luteola	45%
WP_031424386.1	Xanthomonas axonopodis	44%
WP_008576993.1	Xanthomonas perforans	44%

Pel4J4 belonged to PF00544 and contained the PelC structural domain, which conforming to the alkaline Pel structural domain from Streptomyces sp. S27.¹³ Compared with the PelD protein sequence (GenBank accession number: YP003884109.1) of the model strain D. dadantii 3937, 25 different amino acids existed (Figure 2). Among them, 13 loci (e.g. 9, 26, and 28) had amino acids with completely different structures and categories, and 12 loci (e.g. 22, 27, and 29) had amino acids with similar structures.

Figure 2.

Alignment of Pel Ds from D. dadantii 3937 and Dickeya sp. DCE-01. Note: “·” represents a different amino acid pair; “:” represents a structurally similar amino acid pair.

Induction of Pel4J4

Mature fermentation liquor was used to test the activity of extracellular Pel (Table 2). Table 2 shows that under alkaline conditions, Pel activity in pET28a-pel-BL was 204.4 IU/mL, and the specific enzyme activity was 1059.4 IU/mg, which was 11.1 times that of the original strain D. dadantii DCE-01. The activities were higher than those of Pel obtained from Bacillis subtillus (142 IU/mL), Bacillis pumilus BK2 (76.8 IU/mL), and Paenibacillus barcinonensis (104 IU/mL).^23–25

Table 2.

Pel activities from the genetically engineered and the original strains

Strain	Pel activity (IU/mL)	Specific Pel activity (IU/mg)
D. dadantii DCE-01	18.4 ± 0.1	94.8 ± 1.1
pET28a-pel-BL	204.4 ± 8.1	1059.4 ± 9.7

Pel4J4 purification

The extracellular Pel was separated and purified from the fermentation liquor of the positive clone pET28a-pel-BL. Table 3 demonstrates that the activity-recovery rate of the purified Pel after ultrafiltration and Sephadex G-100 gel chromatography was 38.9%, whereas the specific enzyme activity was 15,066.8 U/mg, notably purified 14.2 times.

Table 3.

Separation and purification of Pel4J4

Purification step	Total activity (IU)	Total protein (mg)	Specific activity (IU/mg)	Purification fold	Yield (%)
Supernatant of fermentation liquor	12,425.5	11.7	1059.4	1	100
Ultrafiltration (5–50 kDa)	10,784.6	4.6	2334.3	2.3	86.8
Gel chromatography (Sephadex G-100)	4821.4	0.3	15,066.8	14.2	38.9

The expression and purification effects of Pel4J4 were tested by SDS-PAGE. Figure 3 shows that the whole protein of the positive clone (lane 2) had a distinctive protein band at 34–43 kDa compared with the negative control (lane 1). The purified sample (lane 3) had only one protein band. The apparent molecular weight was 39.2 kDa, which was consistent with the expected mature protein molecular weight (38.8 kDa).

Figure 3.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of crude and purified Pel4J4. Note: M: protein molecular weight ladders; lane 1: crude culture supernatant of pET28a-BL, negative control; lane 2: crude culture supernatant of pET28a-pel-BL; lane 3: purified Pel4J4.

Enzyme properties of Pel4J4

Pel activity at different temperatures was tested. The relative Pel activity peak was observed at 55℃ (Figure 4(a)), indicating that the optimal reaction temperature of the enzyme was 55℃. Residual enzyme activity after incubation was examined at different temperatures for 60 min (Figure 4(b)). At 30–45℃, residual enzyme activity was more than 80%, indicating that the enzyme had high thermostability. When the storage temperature exceeded 50℃, the enzyme was inactivated quickly. Pel activity at different pH values was tested, and the optimal reaction pH was found to be 8.5 (Figure 4(c)). The stable pH was 8.5–10 (Figure 4(d)). Analysis of the enzyme properties revealed that the prepared Pel4J4 was alkaline. Nowadays, many alkaline Pels have been used in bioscouring processes, for example, degumming of ramie, jute, flax, and hemp bast fibers.^9,10,16 Compared with the chemical refining process under high temperature and high alkaline condition, the enzymatic degumming process shows advantages, such as flexible and mild processing conditions, environmentally friendly operation, low cost, and low damage to fibers. Therefore, Pel4J4 is a promising application prospect in ramie bio-degumming.²⁶

Figure 4.

The effect of temperature and pH on Pel activity: (a) the optimum temperature curve; (b) the thermal stability curve; (c) the optimum pH curve; and (d) the pH stability curve.

The effects of 1 mmol/L metal ions on the enzyme activity are shown in Table 4. The enzyme activity was promoted by Zn²⁺, Fe³⁺, Ca²⁺, and

{NH}_{4}^{+}

but significantly inhibited by Cu²⁺ and Pb²⁺, while Ni²⁺, Mn²⁺, and K⁺ had no significant effects on the enzyme activity.

Table 4.

Effect of metal ions on Pel4J4 activity

Metal ions	Relative activity (%)	Metal ions	Relative activity (%)	Metal ions	Relative activity (%)
Negative control	100	Ni²⁺	104.3 ± 2.7	Mn²⁺	96.1 ± 1.5
Zn²⁺	117.0 ± 2.1	${NH}_{4}^{+}$	132.1 ± 3.7	Mg²⁺	91.8 ± 1.2
Cu²⁺	44.6 ± 0.8	K⁺	104.1 ± 2.0	Pb²⁺	68.4 ± 0.9
Fe³⁺	121 ± 1.7	Ca²⁺	230.7 ± 3.3

The Pel activities under different Ca²⁺ concentrations shown in Figure 5 revealed that the optimal Ca²⁺ concentration for the Pel was 1 mmol/L.

Figure 5.

Influence of Ca²⁺ concentration on Pel4J4 activity.

The relative enzyme activities with different substrates shown in Table 5 demonstrated that the optimal substrate for Pel was citrus pectin with more than 85% degree of esterification, and its degradation rate to natural ramie pectin powder reached 98.8%, so it would be inferred that Pel4J4 had a strong degumming ability to ramie bast fiber.²⁷

Table 5.

Relative activity for Pel4J4 with different substrates

Substrates	Relativity activity (%)
Polygalacturonic acid sodium (0% esterified)	40.5 ± 0.82
Citrus pectin (20–34% esterified)	79.5 ± 1.7
Citrus pectin (55–70% esterified)	92.8 ± 2.5
Citrus pectin (≥85% esterified)	100
Ramie bast pectin	98.8 ± 2.1

Compared with the PelD of D. dadantii 3937, the proposed Pel had different optimal reaction temperatures (55℃ versus 50℃), optimal pH (8.5 versus 8.8), and optimal Ca²⁺ concentration (1 mmol/L versus 0.1 mmol/L).²⁸ After first-level structural analysis of gene sequences and corresponding expression products, the two types of Pel were found to have different types of amino acids in eight loci: 37, 227, 355, 363, 365, 155, 165, and 220. Further studies are needed to determine which loci play decisive roles, and to try to the increase heat and alkaline resistance through point mutation.²⁹

Effect of Pel4J4 on ramie degumming

The study examined the effects of crude Pel4J4 on ramie degumming. The COD value of the ramie bio-degumming solution was 2.9 g/L, much higher than that of the negative control (1.1 g/L). As shown in Table 6, the weight-loss rate of ramie fiber increased from 9.8% to 20.4%, and the breaking strength and fiber fineness were doubled compared with those of the negative control.

Table 6.

Mechanical characteristics of degummed ramie fiber

	Weight-loss rate (%)	Breaking strength (cN/dtex)	Fiber fineness (dtex)
Ramie fiber degummed by Pel4J4	20.4 ± 0.4	795.7 ± 10.2	5.15 ± 0.1
Negative control	9.8 ± 0.1	400.6 ± 3.4	2.5 ± 0.05

The surface morphology of the ramie fibers was then observed with a scanning electron microscope. Figures 6(a) and (b) show that the ramie fiber treated with Pel4J4 was more dispersed than the negative control group, which revealed the degumming effect for Pel4J4. However, a small amount of non-cellulosic components remained on the surfaces of the treated ramie fibers. This finding may be due to the fact that Pel degraded pectin and some residual hemicellulose and other gummy material components still covered the surfaces of the degummed ramie fibers. Thus, Pel4J4 could be used to degum ramie, and the degumming effect could be improved by hemicellulase (xylanase and/or mannanase), which causing further enzymolysis and gum loss in the late degumming period.^12,30

Figure 6.

Fiber morphology scanned with a scanning electron microscope: (a) ramie fiber without enzyme treatment (negative control); and (b) ramie fiber with Pel4J4 treatment.

Conclusion

An important pel4J4 (GenBank accession number: KC900167) was cloned from the high-efficiency degumming strain D. dadantii DCE-01 for bast fibers. The extracellular Pel activity of the prokaryotic expression was 204.4 IU/mL. The pure Pel obtained was a kind of heat-resistant alkaline Pel with an optimal reaction temperature and pH of 55℃ and 8.5, respectively. The enzyme activity measured using natural ramie pectin as the substrate was 98.8% of that using the optimal substrate, indicating the promising application prospects of the alkaline Pel in the biological degumming of ramie.

Footnotes

Declaration of conflicting interests

The authors hereby declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Natural Science Foundation of China (Grant Number 31700438), the Chinese Agricultural Science and Technology Innovation Project (Grant Number ASTIP-IBFC08), the Natural Science Foundation of Hunan Province (Grant Number 2016jj3126), the China Agriculture Research System for Bast and Leaf Fiber Crops (Grant Number CARS-16-E22), and the Fundamental Research and Incremental Budget of Chinese Academy of Agricultural Sciences (Grant Number Y2016PT36).

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