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Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity
Journal Pre-proof Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity
Li-Nian Cai, Sheng-Nan Xu, Tao Lu, Dong-Qiang Lin, Shan-Jing Yao PII:
S1004-9541(19)30931-0
DOI:
https://doi.org/10.1016/j.cjche.2019.11.012
Reference:
CJCHE 1593
To appear in:
Chinese Journal of Chemical Engineering
Received date:
10 October 2019
Revised date:
21 November 2019
Accepted date:
29 November 2019
Please cite this article as: L.-N. Cai, S.-N. Xu, T. Lu, et al., Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity, Chinese Journal of Chemical Engineering(2019), https://doi.org/10.1016/j.cjche.2019.11.012
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© 2019 Published by Elsevier.
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Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity
Li-Nian Caia, Sheng-Nan Xua, Tao Lub, Dong-Qiang Lina, Shan-Jing Yaoa*
a
Key Laboratory of Biomass Chemical Engineering of Ministry of Education,
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
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College of Environment, Zhejiang University of Technology, Hangzhou 310014, China
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b
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*Corresponding author:
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Prof. Shan-Jing Yao College of Chemical and Biological Engineering
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Zhejiang University Hangzhou 310027, China
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Tel/Fax: +86-571-87951982
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E-mail: [email protected]
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E-mail address of each author:
Li-Nian Cai: [email protected] Sheng-Nan Xu: [email protected] Tao Lu: [email protected]
Dong-Qiang Lin: [email protected] Shan-Jing Yao: [email protected]
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Abstract The cellulase cocktail produced by marine Aspergillus niger exhibits a property of salt-tolerance, which is of great potential in cellulose degradation in high salt environment. In order to explain the mechanism on the salt-tolerance of the cellulase cocktail produced by marine A. niger, six cellulase components (AnCel6, AnCel7A, AnCel7B, AnEGL, AnBGL1 and AnBGL2) were obtained by directed expression.
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Studies on their enzymatic properties revealed that one β-glucosidase (AnBGL2) and
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one endoglucanase (AnEGL) exhibited an outstanding salt-tolerant property, and one
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cellobiohydrolase (AnCel7B) exhibited a certain salt-tolerant property. Subsequent
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study revealed that the salt-tolerant AnEGL and AnCel7B endowed the cellulase cocktail with stronger salt-tolerant property, while the salt-tolerant AnBGL2 had no
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positive effect. Moreover, after overexpression of AnCel6, AnCel7A, AnCel7B and
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AnEGL, the activity of cellulase cocktail increased by 80%, 70%, 63% and 68%,
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respectively. However, the activity of cellulase cocktail was not improved after overexpression of AnBGL1 and AnBGL2. After mixed-strain fermentation with cellobiohydrolase recombinants (cel6a, cel7a and cel7b recombinants) and endoglucanase recombinant (egl recombinant), the the activity of cellulase cocktail increased by 114%, 102% and 91%, respectively. Keywords: marine Aspergillus niger; cellulase component; directed expression; salttolerance; cellulase activity
Abbreviations: endoglucanase (EG); cellobiohydrolase (CBH); β-glucosidase (BG); lytic polysaccharide mono-oxygenase (LPMO); Agrobacterium tumefaciens-mediated transformation (AMT); sodium carboxymethylcellulose (CMC-Na); p-nitrophenol glucopyranoside (pNPG); pnitrophenol (pNP); 3,5-dinitrosalicylic acid (DNS)
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1. Introduction Cellulase is an enzyme cocktail that can catalyze the hydrolysis of cellulose to glucose [1]. According to the function and mode of action, it can be divided into four categories, including endoglucanase (EG), cellobiohydrolase (CBH), β-glucosidase (BG) and lytic polysaccharide mono-oxygenase (LPMO) [1]. Efficient degradation of
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cellulose is dependent on the synergistic effect of the four cellulase components.
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Briefly, EGs act on the amorphous region of cellulose and hydrolyze the long
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cellulose chain into short ones; LPMOs act on the crystallization region of cellulose
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and oxidize and cut the long cellulose chain into short ones; CBHs act on the reducing
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and non-reducing ends of the cellulose chain and continuously hydrolyze the ends into cello-oligosaccharides (mainly for cellobioses); BGs hydrolyze the soluble cello-
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oligosaccharides into glucose [1-3]. Cellulase is extensively applied in the production
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and life of human beings. The most potential application field of cellulase lies in the
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utilization of lignocellulosic biomass resources [4]. The cheap and readily available lignocellulosic materials are converted by cellulase into soluble small molecules that can be directly used, which are then converted by other processes into high valueadded chemicals [5]. In addition, cellulase is also of great application value in food, textile, animal breeding, paper industry and other fields [6-9]. The efficient catalysis of cellulase is an important guarantee to realize its industrial application. However, the cellulase produced by wild-type strains is always lack of sufficient activity, and the application often results in the embarrassing situation that the cost is greater than the income. Therefore, measures must be taken to
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improve the activity of cellulase produced by wild-type strains. As mentioned above, the efficient degradation of cellulose by cellulase depends on the synergistic effect of each cellulase component. Therefore, in order to improve the activity of cellulase cocktail, in addition to improving the production of cellulase, the ratio of cellulase components should also be optimized. There are six approaches to improve the
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cellulase activity: mutagenesis [10], medium optimization [11-12], complex of
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cellulase [13-14], mixed-strain fermentation [15-16], overexpression of weak
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cellulase components [17-20] and regulation of the cellulase expression pathway [21-
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23].
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In practical application, except for cellulase activity, the effect of specific reaction conditions on cellulase activity during hydrolysis should also be considered,
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i.e., the activity may be reduced at the practical conditions. Thus, the introduction of
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cellulases possessing outstanding properties can further improve the utilization of
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lignocellulosic biomass, in which the salt-tolerant cellulase is a significant member [24]. Salt ions have little influence on or can improve the activity of salt-tolerant cellulase. Therefore, salt-tolerant cellulase is particularly suitable for catalytic hydrolysis of cellulose in high salt concentration. Compared with the ordinary cellulase, salt-tolerant cellulase has more extensive application potential. Salt-tolerant cellulase has a great application prospect in the improvement of saline-alkali soil. Lignocellulosic biomass in saline-alkali soil is difficult to be degraded by microorganisms due to its high salinity, which leads to the poor circulation of organic material and further aggravates soil salinization [25]. If the salt-tolerant strain
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producing salt-tolerant cellulase is introduced, the salt-tolerant cellulase can degrade the lignocellulosic biomass, and the acid generated in the process of using lignocellulose can neutralize the alkali in saline-alkali soil, then improving the carbon cycle and reducing the soil alkalinity. In addition, salt-tolerant cellulase is more suitable for degradation of lignocellulosic biomass after pretreatment through acid
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hydrolysis, alkali hydrolysis or ionic liquid. The lignocellulosic biomass requires pH
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adjustment procedure after pretreatment through by acid or alkali, in which a large
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amount of salt will be inevitably introduced [26]. The ionic liquid is a kind of salt and
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there will be some residue after pretreatment [27]. If the pre-treated lignocellulosic
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material is not desalted, the catalytic efficiency of non-salt-tolerant cellulase will be greatly reduced. At this point, salt-tolerant cellulase can eliminate or reduce the
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adverse impacts of salt on enzyme activity.
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Our laboratory has screened a strain of A. niger from the sludge in the East China
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Sea [28] and some potential application has been developed [29-30]. Compared with the terrestrial A. niger, it exhibits a stronger property of salt-tolerant growth. Studies on its cellulase activity revealed that the cellulase cocktail was more salt-tolerant than those from terrestrial T. reesei and A. niger. However, as most A. niger, the activity of EG and CBH is insufficient, which results the insufficient total cellulase activity for further application. In this study, six cellulase components of marine A. niger cellulase cocktail would be obtained by directed expression for the study on the stronger salt-tolerance. Besides, the effect of overexpression of cellulase components on the activity of cellulase cocktail would be evaluated.
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2. Materials and methods 2.1 Strains, plasmids and media Marine A. niger ZJUBE-1 was stored at the China Center for Type Culture Collection (Conservation No. CCTCC M2010132) and used for directed expression of cellulase components. Agrobacterium tumefaciens AGL-1 was used for the
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transformation of marine A. niger. The plasmids (Mini-Ti vectors pCAMBIA-hph-
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cel6/cel7a/cel7b/egl/bgl1/bgl2) and the corresponding cellulase genes used in this
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study were listed in Table 1 and the diagrams of plasmids were shown in Fig. 1. MN
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medium (40 g/L wheat bran, 2 g/L NH4Cl) was used for propagation of hyphae and
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inducible expression of cellulase cocktail. OGPY medium (40 g/L glucose, 40 g/L peptone, 5 g/L yeast extract, 30 g/L NaCl, 4 g/L KH2PO4) was used for directed
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expression of cellulase components. The induced medium and minimal medium used
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with the protocol [31].
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for A. tumefaciens-mediated transformation (AMT) of marine A. niger were identical
Table 1 Plasmids and cellulase genes in this study
Plasmids
Cellulase genes
GenBank accession numbers
pCAMBIA-hph-bgl1
bgl1
MH744149
pCAMBIA-hph-bgl2
bgl2
MH744150
pCAMBIA-hph-egl
egl
MK587440
pCAMBIA-hph-cel6
cel6
MK898285
pCAMBIA-hph-cel7a
cel7a
MK898286
pCAMBIA-hph-cel7b
cel7b
MK898287
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Fig. 1 Plasmids used in this study
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2.2 Acquisition of recombinants
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A. tumefaciens AGL-1 was transformed with the Mini-Ti vectors pCAMBIAhph-cel6/cel7a/cel7b/egl/bgl1/bgl2 using freeze-thaw method [32]. AMT was
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employed for the transformation of marine A. niger. Optimized procedures of AMT
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were improved from the protocol described by Michielse et al [31]. Briefly, the fresh
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A. tumefaciens recombinant was inoculated in IM broth, cultivated until OD600 reached 0.6-0.8. About 100 μl induced A. tumefaciens recombinant and 106 spores of A. niger were mixed and spread on cellophane of IM agar, cultivated for 2 d at 23 °C. Then the cellophane as well as the strains was transferred onto MM agar with 200 μg/ml hygromycin B and 200 μg/ml cefotaxime sodium. Additional MM agar with equal concentration of antibiotics was poured onto the cellophane to enhance the screening effect. This interlayer medium was cultivated at 30 °C until recombinants grew out. Positive recombinants were verified by PCR identification using primers, hph-F and hph-R. Single conidiospore isolation was performed for the acquisition of
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2.3 Directed expression of cellulase components
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About 107 spores of recombinants were inoculated into 100 ml MN medium in
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250 ml shake flasks and cultivated at 36 °C, 200 rpm for 2 d. Subsequently, about 5
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ml hyphae suspension was inoculated into 100 ml OGPY medium in 250 ml shake
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flasks and cultivated at 36 °C, 250 rpm for 7 d. After fermentation, the hyphae were
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removed by filtration with two layers of gauze and the supernatant was stored at 4 °C.
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2.4 Purification of cellulase components
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The supernatant was rich in salt ions and pigments, which had adverse impacts on subsequent ion-exchange chromatography. Hence, the salt ions and pigments were removed by ultrafiltration with MinimateTM TFF Capsule (Pall corporation). Then the pH of ultrafiltrate was adjusted to 7.0 with 0.1 M NaOH and then loaded on DEAESepharose column pre-equilibrated with 20 mM pH 7.0 sodium phosphate. The fractions were eluted with the gradient of 0-0.5 M NaCl (20 mM sodium phosphate, pH 7.0). The purity and content of target cellulase components in each eluent was determined by SDS-PAGE. Finally, the purified cellulase components were concentrated and the buffer was replaced by 20 mM pH 4.0 sodium acetate by
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ultrafiltration. The resulting enzyme solution was stored at 4 °C. The protein concentration of resulting enzyme solution was quantified using Bradford method [33].
2.5 Definition of enzyme activity unit Cellulase activity mainly comprised of CBH, EG, BG and total cellulase activity.
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Avicel, sodium carboxymethylcellulose (CMC-Na), p-nitrophenol glucopyranoside (pNPG) and Walman No. 1 filter paper were chosen for their substrates, respectively.
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One unit of CBH, EG and total cellulase activity was defined as the amount of
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enzyme required for release 1 µmol glucose equivalent per minute. One unit of BG
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nitrophenol (pNP) per minute.
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activity was defined as the amount of enzyme required for release 1 µmol p-
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2.6 Determination on salt-tolerance of cellulase components
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In terms of CBHs, the enzymatic reaction mixtures (500 μl) including 50 μl properly diluted enzyme solution, 450 μl avicel suspension (50 mg/ml avicel, 0-5 M NaCl, 0.1 M sodium citrate/citric acid buffer, pH 4.5) were incubated in Thermo Shaker (Thermo Scientific) at 50 °C, 1000 rpm for 30 min. The amount of reducing sugar released was determined by 3,5-dinitrosalicylic acid (DNS) assay [34]. Briefly, 500 μl DNS reagent was added into the mixtures. After blending and centrifugation, the supernatant was incubated in boiling water bath for 10 min. The reducing sugar was quantified according to glucose standard curve by determining the absorbance at 540 nm. The activity in the absence of NaCl was defined as 100% relative activity.
Journal Pre-proof In terms of EG, the enzymatic reaction mixtures (500 μl) including 50 μl properly diluted enzyme solution, 450 μl CMC-Na solution (10 mg/ml CMC-Na, 0-5 M NaCl, 0.1 M sodium citrate/citric acid, pH 4.0) were incubated in water bath at 50 °C for 5 min. The determination of activity was the same as that of CBH activity. The activity in the absence of NaCl was defined as 100% relative activity. In terms of BGs, the enzymatic reaction mixtures (500 μl) including 50 μl
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properly diluted enzyme solution, 450 μl pNPG solution (10 mM pNPG, 0-5 M NaCl,
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0.1 M sodium citrate/citric acid, pH 4.0) were incubated in water bath at 50 °C for 5
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min. Subsequently, 500 μl 2 M Na2CO3 was added into the mixtures and the generated
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pNP was quantified according to pNP standard curve by determining the absorbance at 405 nm. The activity in the absence of NaCl was defined as 100% relative activity.
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2.7 Salt-tolerance analysis on mixed cellulases
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For the study of salt-tolerance, the total cellulase activity of mixed cellulases was
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measured at different salt concentrations (0-4 M NaCl). The enzymatic reaction mixtures (500 μl) comprised of 100 μl properly diluted cellulase cocktail of original strains (0-4 M NaCl), 6 μM purified cellulase component (0-4 M NaCl), 300 μl buffer (0-4 M NaCl, 0.1 M sodium citrate/citric acid, pH 4.5), two pieces of 1 × 1 cm2 filter paper, appropriate volume of NaCl solution (0-4 M). The reaction conditions were the same as the determination of CBH activity described in 2.6. The activity in the absence of NaCl was defined as 100% relative activity.
2.8 Inducible expression of cellulase cocktail
Journal Pre-proof About 107 spores of recombinants were inoculated into 100 ml MN medium in 250 ml shake flasks and cultivated at 30 °C, 200 rpm for 7 d. After fermentation, the hyphae and residual wheat bran was removed by filtration with two layers of gauze and centrifugation at 10000 × g for 5 min. The cellulase cocktail was stored at 4 °C.
2.9 Determination of cellulase activity to cellulase cocktail
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In terms of CBH recombinants, the CBH and total cellulase activity were determined. In terms of EG recombinants, the EG and total cellulase activity were
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determined. In terms of BG recombinants, the BG and total cellulase activity were
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determined.
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The enzymatic reaction mixtures (500 μl) for the determination of CBH activity comprised of 50 μl properly diluted enzyme solution, 450 μl avicel suspension (50
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mg/ml avicel, 0.1 M sodium citrate/citric acid, pH 4.5). The enzymatic reaction
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mixtures (500 μl) for the determination of EG activity comprised of 50 μl properly
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diluted enzyme solution, 450 μl CMC-Na solution (10 mg/ml CMC-Na, 0.1 M sodium citrate/citric acid, pH 4.5). The enzymatic reaction mixtures (500 μl) for the determination of BG activity comprised of 50 μl properly diluted enzyme solution, 450 μl pNPG solution (10 mM pNPG, 0.1 M sodium citrate/citric acid, pH 4.0). The enzymatic reaction mixtures (500 μl) for the determination of total cellulase activity comprised of 50 μl properly diluted enzyme solution, two pieces of 1 × 1 cm2 filter paper, 450 μl reaction buffer (0.1 M sodium citrate/citric acid, pH 4.5). For the determination of CBH, EG and BG activity, the reaction conditions were the same as the methods described in 2.6. For the determination of total cellulase activity, the
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reaction condition was the same as the determination of CBH activity described in 2.6.
2.10 Mixed-strain fermentation The CBH recombinants with highest CBH activity and the EG recombinant with highest EG activity were mix-cultured in MN medium for further improvement of total cellulase activity. In detail, 107 spores of CBH recombinants and EG
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recombinant with different ratios (8:1, 4:1, 2:1, 1:1, 1:2, 1:4 and 1:8) were inoculated in 100 ml WN medium in 250 ml shake flasks, cultivated at 30 °C, 180 rpm for 7 d.
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determined by the methods described in 2.9.
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After fermentation, the CBH, EG and total cellulase activity of cellulase cocktail were
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3. Results and discussion
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3.1 Directed expression of cellulase components
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Six recombinants were cultivated in OGPY medium for homologous constitutive
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expression of cellulase components, which included three CBHs (AnCel6, AnCel7A and AnCel7B), one EG (AnEGL) and two BGs (AnBGL1 and AnBGL2). The expression results were analyzed by SDS-PAGE (Fig. 2). The apparent molecular weights of AnCel6, AnCel7A, AnCel7B, AnEGL, AnBGL1 and AnBGL2 were about 65, 75, 58, 26, 110 and 130 kDa. Among these six cellulase components, AnCel7A, AnCel7B, AnEGL, AnBGL1 and AnBGL2 have been studied in our previous researches [35-37]. The concept of directed expression has been put forward in our previous study on AnBGL1 and AnBGL2. Briefly, “directed expression” contains two implications. One is homologous expression for correct glycosylation. Overexpression
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is a universal method to obtain target protein, such as the widely-used overexpression in Escherichia coli and Pichia pastoris. However, difference in glycosylation of target protein is the fatal flaw in heterologous expression [38], which may not reflect the original properties of nature protein. In order to obtain natural protein, homologous expression is recommended. And the other is constitutive expression for obtaining a
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single enzyme from a complex inducible enzyme system. Cellulase is an enzyme
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cocktail and can be produced in the presence of inducer. As a result, there are always
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various components in fermentation broth. Constitutive expression is adopted to
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prevent the expression of induced cellulases. In other words, only the introduced
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constitutive expressed cellulase can be produced without inducer. In this way, we can study any of the inducible enzymes conveniently. As was shown in Fig. 2, the target
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cellulase components were the predominant protein ingredient, which greatly reduced
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the burden of separation and purification for a single cellulase component from the
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complex cellulase cocktail.
Fig. 2 Directed expression of marine A. niger cellulase components (a) Expression of CBHs; (b) Expression of EG; (c) Expression of BGs. M: protein marker；lane 1, 5 and 8: Culture supernatant of original strain; lane 2-4: Culture
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supernatant of cel, cel7a and cel7b recombinants；lane 6: Culture supernatant of egl recombinant; lane 7 and 9: Culture supernatant of bgl1 and bgl2 recombinants.
3.2 Purification of cellulase components After concentrated and desalinated by ultrafiltration, six purified cellulase components were obtained by anion exchange chromatography. Linear and step
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gradient elution with 0-0.5 M NaCl were used in anion exchange chromatography and the eluents were analyzed by SDS-PAGE (Fig. 3, Fig. 4 and Fig. 5). AnCel6,
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AnCel7A, AnCel7B, AnBGL1 and AnBGL2 were eluted with 0-0.1, 0.15-0.25, 0.15-
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0.3, 0.1-0.175, 0.1-0.2 M NaCl, respectively. AnEGL was eluted with 20 mM sodium
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phosphate.
Fig. 3 Gradient elution to three CBHs
(a) Gradient elution to AnCel6; (b) Gradient elution to AnCel7A; (c) Gradient elution to AnCel7B. M: protein marker; lane 1-8, 9-16, 17-24; Linear gradient elution with 00.4 M NaCl, the concentration difference between adjacent lanes was 0.05 M.
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Fig. 4 Separation and purification of EG
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M: protein marker; lane 1: concentrated supernatant; lane 2: elution with 20 mM pH
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7.0 sodium phosphate; lane 3-6: elution with 0.1, 0.2, 0.3 and 0.5 M NaCl.
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Fig. 5 Separation and purification of two BGs
(a) Separation and purification of AnBGL1; (b) Separation and purification of AnBGL2. M: protein marker; lane 1-3: elution with 0.1, 0.175 and 0.5 M NaCl; lane 4-6: elution with 0.1, 0.2 and 0.5 M NaCl. 3.3 Salt-tolerance of cellulase components Marine A. niger cellulase has a stronger salt-tolerant property than terrestrial A. niger cellulase. Therefore, some cellulase components with salt-tolerant property probably exist in marine A. niger cellulase cocktail. In this study, we obtained a total of six cellulase components. The results of salt-tolerance analysis were shown in Fig.
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6. Among them, AnEGL and AnBGL2 have strong salt-tolerant property, and their activity was improved with the increase of salt concentration. In the presence of 4.5 M NaCl, AnEGL activity increased by 29% compared with that in the absence of NaCl. In the presence of 3.6 M NaCl, AnBGL2 activity increased by 36% compared with that in the absence of NaCl. Compared to AnCel6 and AnCel7A, AnCel7B has
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stronger salt-tolerance. At 4.5 M NaCl, AnCel7B retained approximately 80% of the
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maximum enzyme activity, while AnCel6 and AnCel7A retained only about 60%. Of
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the six cellulase components, three have salt-tolerant property. This result revealed
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that the ratio of salt-tolerant cellulases was considerable in marine A. niger cellulase
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cocktail. It was found that A. niger has hundreds of secreted proteins in the study on secretory proteome [39]. It is likely that a high proportion of salt-tolerant proteins are
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present in these secreted proteins from marine A. niger. Therefore, marine A. niger is
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probably a treasure trove of salt-tolerant enzymes.
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Fig. 6 Salt-tolerance of marine A. niger cellulase components (a) Salt-tolerance of three CBHs; (b) Salt-tolerance of one EG; (c) Salt-tolerance of two BGs.
3.4 Salt-tolerance of cellulase cocktail Among the six cellulase components, AnCel7B has certain salt-tolerant property, AnEGL and AnBGL2 have outstanding salt-tolerant property. Which cellulase
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component endows marine A. niger cellulase with the stronger salt-tolerant property? The cellulase cocktail of original strain and the purified single cellulase components were mixed in a specific ratio, and the salt-tolerance of the mixed cellulases was examined. The results were shown in Fig. 7. After the cellulase cocktail of original strain was mixed with AnCel6, AnCel7A, AnBGL1 and AnBGL2, the salt-tolerance of
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the mixed cellulases largely remained the same or slightly decreased. When the
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cellulase cocktail of original strain was mixed with AnCel7B and AnEGL, the salt-
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tolerance was improved, especially for AnEGL. In fact, AnBGL2 has better salt-
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tolerance than AnEGL, but the addition of AnBGL2 did not enhance the salt-tolerance
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of mixed cellulase, which was probably due to the higher content of BG in cellulase
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cocktail of original strain.
Fig. 7 Salt-tolerance of mixed cellulases
3.5 Inducible expression of cellulases The expression of the introduced cellulase genes is driven by gpdA promoter. The gpdA promoter is a strong constitutive promoter, which keep active either under non-inducible condition (OGPY medium) or inducible condition (MN medium). As
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was shown in Fig. 8, the introduced cellulase constitutive expression cassettes were expressed in inducible medium containing bran as main carbon and nitrogen source. It was found that the apparent molecular weight of the target cellulase components were identical to those expressed under non-inducible condition. Different from the noninducing conditions, when using bran as the cellulase inducer, the inherent
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lignocellulolytic enzymes of marine A. niger were also expressed, which resulted of
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the complex protein components in the fermentation supernatant.
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Fig. 8 Inducible expression of marine A. niger cellulases
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(a) Expression of CBHs; (b) Expression of EG; (c) Expression of BGs. M: protein marker；lane 1, 5 and 7: Culture supernatant of original strain; lane 2-4: Culture supernatant of cel, cel7a and cel7b recombinants；lane 6: Culture supernatant of egl recombinant; lane 8 and 9: Culture supernatant of bgl1 and bgl2 recombinants
3.6 Changes on cellulase activity of recombinants The activity of cellulase cocktail fermented with cel6, cel7a and cel7b recombinants was shown in Fig. 9a. After overexpression of three CBHs, the CBH activity of all recombinants was greatly improved. Among them, the CBH activity of cellulase cocktail fermented with cel6, cel7a and cel7b recombinants increased by 54-
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130%, 54-123% and 50-105%, respectively. Moreover, with the improvement of CBH activity, the total cellulase activity was also improved. Among them, the total cellulase activity of cellulase cocktail fermented with cel6, cel7a and cel7b recombinants were increased by 24-80%, 38-70% and 20-63%, respectively. In addition, by comparing the changes in CBH and total cellulase activity, it was found
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that the cellulase cocktail with higher CBH activity exhibited higher total cellulase
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activity, which reflected that the overexpression of extra cellulase components had
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little effect on the expression of inherent cellulases. These results indicated that
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among the marine A. niger cellulase cocktail, CBH was the weak enzyme component
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and was the key factor limiting its total cellulase activity. The activity of cellulase cocktail fermented with egl recombinants was shown in
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Fig. 9b. Similar to the overexpression of CBHs, the EG activity and total enzyme
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activity of cellulase cocktail fermented with egl recombinants were improved
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compared with those of original strain. Among them, the EG activity increased by 6492%, and the total cellulase activity increased by 37-68%. These results indicated that EG was also relatively weak in marine A. niger cellulase cocktail and was the other key factor limiting its total cellulase activity. The activity of cellulase cocktail fermented with bgl1 and bgl2 recombinants was shown in Fig. 9c. After overexpression of BGs, the BG activity of cellulase cocktail fermented with bgl1 and bgl2 recombinants was slightly increased. The BG activity of cellulase cocktail fermented with bgl1 and bgl2 recombinants increased by 4-29% and 1-24%, respectively, compared with that of the original strain. Unlike the
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overexpression of CBHs and EG, the total cellulase activity of cellulase cocktail fermented with bgl1 and bgl2 recombinants decreased somewhat. These results indicated that BG was not the limiting factor on the total cellulase activity in marine A. niger cellulase cocktail. The decrease in total cellulase activity after overexpression of BGs may result from that the increase in BG activity reduces the cellobiose (cellulase
f
inducer) concentration in the medium, resulting in a decrease in cellulase induction
oo
during fermentation, and consequently reducing the production of cellulase cocktail.
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Overexpression of the relatively weak cellulase components is one of the
e-
methods to improve the total cellulase activity. Overexpression method is to integrate
Pr
the cellulase expression cassettes into genome to fundamentally optimize the composition of cellulase, and then improve the total cellulase activity. Examples of
al
overexpression method to improve cellulase activity are shown in Table 2, which
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rn
were mainly to improve the activity of T. reesei cellulase cocktail.
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Pr
e-
pr
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f
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Fig. 9 Changes of cellulase activity in different recombinants (a) Changes of cellulase activity in cel6, cel7a and cel7b recombinants; (b) Changes of cellulase activity in egl recombinants; (c) Changes of cellulase activity in bgl1 and bgl2 recombinants
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Table 2 Enhanced total cellulase activity by overexpression Cellulase name
Source
Host
Relative activitya (%)
Reference
cellobiase
A. niger
T. reesei
144
[17]
β-glucosidase
Periconia sp.
T. reesei
158
[40]
CBHII
T. reesei
T. reesei
430
[19]
β-glucosidase
Penicillium
T. reesei
130
[18]
T. reesei
167
[41]
Aspergillus
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β-glucosidase 1
aculeatus Piromyces
A．niger 750
rhizinflata A. niger
A. niger
AnCel7A
A. niger
A. niger
AnCel7B
A. niger
AnEGL
A. niger
[20]
180
This study
170
This study
Pr
e-
AnCel6
pr
Cellulase
f
decumbens
163
This study
A. niger
168
This study
al
A. niger
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a. The activity of cellulase cocktail from original strain was defined as 100%.
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3.7 Mixed-strain fermentation
After the overexpression of CBHs and EG, the total cellulase activity of marine A. niger cellulase cocktail was improved. The method of overexpressing the weak cellulase component can effectively optimize the proportion of the cellulase components in marine A. niger cellulase cocktail, thereby increasing the total cellulase activity. In order to further optimize the proportion of the cellulase components, mix-strain fermentation was performed. Unlike the reported cases that used different species of strains, we employed the two marine A. niger recombinants, which could eliminate the adverse impacts caused by the competition between
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different species of strains [42]. The changes in cellulase activity of the cellulase cocktail fermented with mixed recombinants were shown in Fig. 10. With the increase of the ratio of CBH recombinants to EG recombinant, the CBH activity was continuously increased, while the EG activity was gradually decreased. The total cellulase activity showed a trend of first increasing and then decreasing. In the three
f
co-culture systems, the highest total cellulase activity was achieved when the
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inoculation ratio of CBH recombinants to EG recombinant was 2:1. Compared with
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the original strain, the total cellulase activity of the cellulase cocktail in the three
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optimal co-culture systems (cel6 + egl, cel7a + egl and cel7b + egl) was increased by
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114%, 102% and 91%, respectively. According to the optimal inoculation ratio in mixed-strain fermentation, the CBH exhibited a greater restriction effect on the total
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cellulase activity than EG.
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Fig. 10 Changes of cellulase activity by mixed-strain fermentation MA: marine A. niger; C6: cel6 recombinant; C7A: cel7a recombinant; C7B: cel7b recombinant.
Conclusions
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In this study, six cellulase components were obtained by directed expression. Studies on their enzymatic properties revealed that AnBGL2 and AnEGL exhibited an outstanding salt-tolerant property, and AnCel7B exhibited a certain salt-tolerant property. It was proved that the salt-tolerant AnEGL and AnCel7B endowed the cellulase cocktail with stronger salt-tolerant property, while the salt-tolerant AnBGL2
f
had no positive effect. Moreover, the activity of cellulase cocktail increased after
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overexpression of CBH and EG components and it was further improved by mixed-
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strain fermentation with CBH recombinants and EG recombinant.
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Acknowledgements
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This work was supported by National Natural Science Foundation of China (21576233, 21878263) and "the Fundamental Research Funds for the Central
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Universities".
References
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Declarations of interest: none.
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Figure captions
Fig. 1 Plasmids used in this study
Fig. 2 Directed expression of marine A. niger cellulase components (a) Expression of CBHs; (b) Expression of EG; (c) Expression of BGs. M: protein marker；lane 1, 5 and 8: Culture supernatant of original strain; lane 2-4: Culture
oo
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supernatant of cel, cel7a and cel7b recombinants；lane 6: Culture supernatant of egl
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recombinant; lane 7 and 9: Culture supernatant of bgl1 and bgl2 recombinants.
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Fig. 3 Gradient elution to three CBHs
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(a) Gradient elution to AnCel6; (b) Gradient elution to AnCel7A; (c) Gradient elution to AnCel7B. M: protein marker; lane 1-8, 9-16, 17-24; Linear gradient elution with 0-
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0.4 M NaCl, the concentration difference between adjacent lanes was 0.05 M.
Fig. 4 Separation and purification of EG
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M: protein marker; lane 1: concentrated supernatant; lane 2: elution with 20 mM pH 7.0 sodium phosphate; lane 3-6: elution with 0.1, 0.2, 0.3 and 0.5 M NaCl.
Fig. 5 Separation and purification of two BGs (a) Separation and purification of AnBGL1; (b) Separation and purification of AnBGL2. M: protein marker; lane 1-3: elution with 0.1, 0.175 and 0.5 M NaCl; lane 4-6: elution with 0.1, 0.2 and 0.5 M NaCl.
Fig. 6 Salt-tolerance of marine A. niger cellulase components (a) Salt-tolerance of three CBHs; (b) Salt-tolerance of one EG; (c) Salt-tolerance of
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two BGs.
Fig. 7 Salt-tolerance of mixed cellulases
Fig. 8 Inducible expression of marine A. niger cellulases (a) Expression of CBHs; (b) Expression of EG; (c) Expression of BGs. M: protein marker；lane 1, 5 and 7: Culture supernatant of original strain; lane 2-4: Culture
oo
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supernatant of cel, cel7a and cel7b recombinants；lane 6: Culture supernatant of egl
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recombinant; lane 8 and 9: Culture supernatant of bgl1 and bgl2 recombinants.
e-
Fig. 9 Changes of cellulase activity in different recombinants
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(a) Changes of cellulase activity in cel6, cel7a and cel7b recombinants; (b) Changes of cellulase activity in egl recombinants; (c) Changes of cellulase activity in bgl1 and
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bgl2 recombinants.
Fig. 10 Changes of cellulase activity by mixed-strain fermentation
recombinant.
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MA: marine A. niger; C6: cel6 recombinant; C7A: cel7a recombinant; C7B: cel7b
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Table 1 Plasmids and cellulase genes in this study Cellulase genes
GenBank accession numbers
pCAMBIA-hph-bgl1
bgl1
MH744149
pCAMBIA-hph-bgl2
bgl2
MH744150
pCAMBIA-hph-egl
egl
MK587440
pCAMBIA-hph-cel6
cel6
MK898285
pCAMBIA-hph-cel7a
cel7a
pCAMBIA-hph-cel7b
cel7b
f
Plasmids
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MK898286
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Pr
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pr
MK898287
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Table 2 Enhanced total cellulase activity by overexpression Cellulase name
Source
Host
Relative activitya (%)
Reference
cellobiase
A. niger
T. reesei
144
[17]
β-glucosidase
Periconia sp.
T. reesei
158
[40]
CBHII
T. reesei
T. reesei
430
[19]
β-glucosidase
Penicillium
T. reesei
130
[18]
T. reesei
167
[41]
Aspergillus
oo
β-glucosidase 1
aculeatus Piromyces
A．niger 750
rhizinflata A. niger
A. niger
AnCel7A
A. niger
A. niger
AnCel7B
A. niger
AnEGL
A. niger
[20]
180
This study
170
This study
Pr
e-
AnCel6
pr
Cellulase
f
decumbens
163
This study
A. niger
168
This study
al
A. niger
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a. The activity of cellulase cocktail from original strain was defined as 100%.
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1. Six cellulase components were obtained from marine Aspergillus niger. 2. Three cellulase components were salt-tolerant. 3. The salt-tolerant components endowed with the salt-tolerance of cellulase cocktail.
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Pr
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4. Cellulase activity was improved by overexpression and mixed-strain fermentation.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Li-Nian Cai, Sheng-Nan Xu, Tao Lu, Dong-Qiang Lin, Shan-Jing Yao PII:
S1004-9541(19)30931-0
DOI:
https://doi.org/10.1016/j.cjche.2019.11.012
Reference:
CJCHE 1593
To appear in:
Chinese Journal of Chemical Engineering
Received date:
10 October 2019
Revised date:
21 November 2019
Accepted date:
29 November 2019
Please cite this article as: L.-N. Cai, S.-N. Xu, T. Lu, et al., Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity, Chinese Journal of Chemical Engineering(2019), https://doi.org/10.1016/j.cjche.2019.11.012
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Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity
Li-Nian Caia, Sheng-Nan Xua, Tao Lub, Dong-Qiang Lina, Shan-Jing Yaoa*
a
Key Laboratory of Biomass Chemical Engineering of Ministry of Education,
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
f
College of Environment, Zhejiang University of Technology, Hangzhou 310014, China
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b
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*Corresponding author:
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Prof. Shan-Jing Yao College of Chemical and Biological Engineering
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Zhejiang University Hangzhou 310027, China
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Tel/Fax: +86-571-87951982
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E-mail: [email protected]
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E-mail address of each author:
Li-Nian Cai: [email protected] Sheng-Nan Xu: [email protected] Tao Lu: [email protected]
Dong-Qiang Lin: [email protected] Shan-Jing Yao: [email protected]
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Abstract The cellulase cocktail produced by marine Aspergillus niger exhibits a property of salt-tolerance, which is of great potential in cellulose degradation in high salt environment. In order to explain the mechanism on the salt-tolerance of the cellulase cocktail produced by marine A. niger, six cellulase components (AnCel6, AnCel7A, AnCel7B, AnEGL, AnBGL1 and AnBGL2) were obtained by directed expression.
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Studies on their enzymatic properties revealed that one β-glucosidase (AnBGL2) and
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one endoglucanase (AnEGL) exhibited an outstanding salt-tolerant property, and one
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cellobiohydrolase (AnCel7B) exhibited a certain salt-tolerant property. Subsequent
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study revealed that the salt-tolerant AnEGL and AnCel7B endowed the cellulase cocktail with stronger salt-tolerant property, while the salt-tolerant AnBGL2 had no
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positive effect. Moreover, after overexpression of AnCel6, AnCel7A, AnCel7B and
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AnEGL, the activity of cellulase cocktail increased by 80%, 70%, 63% and 68%,
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respectively. However, the activity of cellulase cocktail was not improved after overexpression of AnBGL1 and AnBGL2. After mixed-strain fermentation with cellobiohydrolase recombinants (cel6a, cel7a and cel7b recombinants) and endoglucanase recombinant (egl recombinant), the the activity of cellulase cocktail increased by 114%, 102% and 91%, respectively. Keywords: marine Aspergillus niger; cellulase component; directed expression; salttolerance; cellulase activity
Abbreviations: endoglucanase (EG); cellobiohydrolase (CBH); β-glucosidase (BG); lytic polysaccharide mono-oxygenase (LPMO); Agrobacterium tumefaciens-mediated transformation (AMT); sodium carboxymethylcellulose (CMC-Na); p-nitrophenol glucopyranoside (pNPG); pnitrophenol (pNP); 3,5-dinitrosalicylic acid (DNS)
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1. Introduction Cellulase is an enzyme cocktail that can catalyze the hydrolysis of cellulose to glucose [1]. According to the function and mode of action, it can be divided into four categories, including endoglucanase (EG), cellobiohydrolase (CBH), β-glucosidase (BG) and lytic polysaccharide mono-oxygenase (LPMO) [1]. Efficient degradation of
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cellulose is dependent on the synergistic effect of the four cellulase components.
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Briefly, EGs act on the amorphous region of cellulose and hydrolyze the long
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cellulose chain into short ones; LPMOs act on the crystallization region of cellulose
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and oxidize and cut the long cellulose chain into short ones; CBHs act on the reducing
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and non-reducing ends of the cellulose chain and continuously hydrolyze the ends into cello-oligosaccharides (mainly for cellobioses); BGs hydrolyze the soluble cello-
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oligosaccharides into glucose [1-3]. Cellulase is extensively applied in the production
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and life of human beings. The most potential application field of cellulase lies in the
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utilization of lignocellulosic biomass resources [4]. The cheap and readily available lignocellulosic materials are converted by cellulase into soluble small molecules that can be directly used, which are then converted by other processes into high valueadded chemicals [5]. In addition, cellulase is also of great application value in food, textile, animal breeding, paper industry and other fields [6-9]. The efficient catalysis of cellulase is an important guarantee to realize its industrial application. However, the cellulase produced by wild-type strains is always lack of sufficient activity, and the application often results in the embarrassing situation that the cost is greater than the income. Therefore, measures must be taken to
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improve the activity of cellulase produced by wild-type strains. As mentioned above, the efficient degradation of cellulose by cellulase depends on the synergistic effect of each cellulase component. Therefore, in order to improve the activity of cellulase cocktail, in addition to improving the production of cellulase, the ratio of cellulase components should also be optimized. There are six approaches to improve the
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cellulase activity: mutagenesis [10], medium optimization [11-12], complex of
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cellulase [13-14], mixed-strain fermentation [15-16], overexpression of weak
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cellulase components [17-20] and regulation of the cellulase expression pathway [21-
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23].
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In practical application, except for cellulase activity, the effect of specific reaction conditions on cellulase activity during hydrolysis should also be considered,
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i.e., the activity may be reduced at the practical conditions. Thus, the introduction of
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cellulases possessing outstanding properties can further improve the utilization of
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lignocellulosic biomass, in which the salt-tolerant cellulase is a significant member [24]. Salt ions have little influence on or can improve the activity of salt-tolerant cellulase. Therefore, salt-tolerant cellulase is particularly suitable for catalytic hydrolysis of cellulose in high salt concentration. Compared with the ordinary cellulase, salt-tolerant cellulase has more extensive application potential. Salt-tolerant cellulase has a great application prospect in the improvement of saline-alkali soil. Lignocellulosic biomass in saline-alkali soil is difficult to be degraded by microorganisms due to its high salinity, which leads to the poor circulation of organic material and further aggravates soil salinization [25]. If the salt-tolerant strain
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producing salt-tolerant cellulase is introduced, the salt-tolerant cellulase can degrade the lignocellulosic biomass, and the acid generated in the process of using lignocellulose can neutralize the alkali in saline-alkali soil, then improving the carbon cycle and reducing the soil alkalinity. In addition, salt-tolerant cellulase is more suitable for degradation of lignocellulosic biomass after pretreatment through acid
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hydrolysis, alkali hydrolysis or ionic liquid. The lignocellulosic biomass requires pH
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adjustment procedure after pretreatment through by acid or alkali, in which a large
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amount of salt will be inevitably introduced [26]. The ionic liquid is a kind of salt and
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there will be some residue after pretreatment [27]. If the pre-treated lignocellulosic
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material is not desalted, the catalytic efficiency of non-salt-tolerant cellulase will be greatly reduced. At this point, salt-tolerant cellulase can eliminate or reduce the
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adverse impacts of salt on enzyme activity.
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Our laboratory has screened a strain of A. niger from the sludge in the East China
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Sea [28] and some potential application has been developed [29-30]. Compared with the terrestrial A. niger, it exhibits a stronger property of salt-tolerant growth. Studies on its cellulase activity revealed that the cellulase cocktail was more salt-tolerant than those from terrestrial T. reesei and A. niger. However, as most A. niger, the activity of EG and CBH is insufficient, which results the insufficient total cellulase activity for further application. In this study, six cellulase components of marine A. niger cellulase cocktail would be obtained by directed expression for the study on the stronger salt-tolerance. Besides, the effect of overexpression of cellulase components on the activity of cellulase cocktail would be evaluated.
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2. Materials and methods 2.1 Strains, plasmids and media Marine A. niger ZJUBE-1 was stored at the China Center for Type Culture Collection (Conservation No. CCTCC M2010132) and used for directed expression of cellulase components. Agrobacterium tumefaciens AGL-1 was used for the
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transformation of marine A. niger. The plasmids (Mini-Ti vectors pCAMBIA-hph-
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cel6/cel7a/cel7b/egl/bgl1/bgl2) and the corresponding cellulase genes used in this
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study were listed in Table 1 and the diagrams of plasmids were shown in Fig. 1. MN
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medium (40 g/L wheat bran, 2 g/L NH4Cl) was used for propagation of hyphae and
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inducible expression of cellulase cocktail. OGPY medium (40 g/L glucose, 40 g/L peptone, 5 g/L yeast extract, 30 g/L NaCl, 4 g/L KH2PO4) was used for directed
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expression of cellulase components. The induced medium and minimal medium used
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with the protocol [31].
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for A. tumefaciens-mediated transformation (AMT) of marine A. niger were identical
Table 1 Plasmids and cellulase genes in this study
Plasmids
Cellulase genes
GenBank accession numbers
pCAMBIA-hph-bgl1
bgl1
MH744149
pCAMBIA-hph-bgl2
bgl2
MH744150
pCAMBIA-hph-egl
egl
MK587440
pCAMBIA-hph-cel6
cel6
MK898285
pCAMBIA-hph-cel7a
cel7a
MK898286
pCAMBIA-hph-cel7b
cel7b
MK898287
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Fig. 1 Plasmids used in this study
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2.2 Acquisition of recombinants
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A. tumefaciens AGL-1 was transformed with the Mini-Ti vectors pCAMBIAhph-cel6/cel7a/cel7b/egl/bgl1/bgl2 using freeze-thaw method [32]. AMT was
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employed for the transformation of marine A. niger. Optimized procedures of AMT
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were improved from the protocol described by Michielse et al [31]. Briefly, the fresh
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A. tumefaciens recombinant was inoculated in IM broth, cultivated until OD600 reached 0.6-0.8. About 100 μl induced A. tumefaciens recombinant and 106 spores of A. niger were mixed and spread on cellophane of IM agar, cultivated for 2 d at 23 °C. Then the cellophane as well as the strains was transferred onto MM agar with 200 μg/ml hygromycin B and 200 μg/ml cefotaxime sodium. Additional MM agar with equal concentration of antibiotics was poured onto the cellophane to enhance the screening effect. This interlayer medium was cultivated at 30 °C until recombinants grew out. Positive recombinants were verified by PCR identification using primers, hph-F and hph-R. Single conidiospore isolation was performed for the acquisition of
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2.3 Directed expression of cellulase components
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About 107 spores of recombinants were inoculated into 100 ml MN medium in
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250 ml shake flasks and cultivated at 36 °C, 200 rpm for 2 d. Subsequently, about 5
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ml hyphae suspension was inoculated into 100 ml OGPY medium in 250 ml shake
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flasks and cultivated at 36 °C, 250 rpm for 7 d. After fermentation, the hyphae were
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removed by filtration with two layers of gauze and the supernatant was stored at 4 °C.
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2.4 Purification of cellulase components
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The supernatant was rich in salt ions and pigments, which had adverse impacts on subsequent ion-exchange chromatography. Hence, the salt ions and pigments were removed by ultrafiltration with MinimateTM TFF Capsule (Pall corporation). Then the pH of ultrafiltrate was adjusted to 7.0 with 0.1 M NaOH and then loaded on DEAESepharose column pre-equilibrated with 20 mM pH 7.0 sodium phosphate. The fractions were eluted with the gradient of 0-0.5 M NaCl (20 mM sodium phosphate, pH 7.0). The purity and content of target cellulase components in each eluent was determined by SDS-PAGE. Finally, the purified cellulase components were concentrated and the buffer was replaced by 20 mM pH 4.0 sodium acetate by
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ultrafiltration. The resulting enzyme solution was stored at 4 °C. The protein concentration of resulting enzyme solution was quantified using Bradford method [33].
2.5 Definition of enzyme activity unit Cellulase activity mainly comprised of CBH, EG, BG and total cellulase activity.
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Avicel, sodium carboxymethylcellulose (CMC-Na), p-nitrophenol glucopyranoside (pNPG) and Walman No. 1 filter paper were chosen for their substrates, respectively.
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One unit of CBH, EG and total cellulase activity was defined as the amount of
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enzyme required for release 1 µmol glucose equivalent per minute. One unit of BG
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nitrophenol (pNP) per minute.
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activity was defined as the amount of enzyme required for release 1 µmol p-
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2.6 Determination on salt-tolerance of cellulase components
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In terms of CBHs, the enzymatic reaction mixtures (500 μl) including 50 μl properly diluted enzyme solution, 450 μl avicel suspension (50 mg/ml avicel, 0-5 M NaCl, 0.1 M sodium citrate/citric acid buffer, pH 4.5) were incubated in Thermo Shaker (Thermo Scientific) at 50 °C, 1000 rpm for 30 min. The amount of reducing sugar released was determined by 3,5-dinitrosalicylic acid (DNS) assay [34]. Briefly, 500 μl DNS reagent was added into the mixtures. After blending and centrifugation, the supernatant was incubated in boiling water bath for 10 min. The reducing sugar was quantified according to glucose standard curve by determining the absorbance at 540 nm. The activity in the absence of NaCl was defined as 100% relative activity.
Journal Pre-proof In terms of EG, the enzymatic reaction mixtures (500 μl) including 50 μl properly diluted enzyme solution, 450 μl CMC-Na solution (10 mg/ml CMC-Na, 0-5 M NaCl, 0.1 M sodium citrate/citric acid, pH 4.0) were incubated in water bath at 50 °C for 5 min. The determination of activity was the same as that of CBH activity. The activity in the absence of NaCl was defined as 100% relative activity. In terms of BGs, the enzymatic reaction mixtures (500 μl) including 50 μl
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properly diluted enzyme solution, 450 μl pNPG solution (10 mM pNPG, 0-5 M NaCl,
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0.1 M sodium citrate/citric acid, pH 4.0) were incubated in water bath at 50 °C for 5
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min. Subsequently, 500 μl 2 M Na2CO3 was added into the mixtures and the generated
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pNP was quantified according to pNP standard curve by determining the absorbance at 405 nm. The activity in the absence of NaCl was defined as 100% relative activity.
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2.7 Salt-tolerance analysis on mixed cellulases
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For the study of salt-tolerance, the total cellulase activity of mixed cellulases was
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measured at different salt concentrations (0-4 M NaCl). The enzymatic reaction mixtures (500 μl) comprised of 100 μl properly diluted cellulase cocktail of original strains (0-4 M NaCl), 6 μM purified cellulase component (0-4 M NaCl), 300 μl buffer (0-4 M NaCl, 0.1 M sodium citrate/citric acid, pH 4.5), two pieces of 1 × 1 cm2 filter paper, appropriate volume of NaCl solution (0-4 M). The reaction conditions were the same as the determination of CBH activity described in 2.6. The activity in the absence of NaCl was defined as 100% relative activity.
2.8 Inducible expression of cellulase cocktail
Journal Pre-proof About 107 spores of recombinants were inoculated into 100 ml MN medium in 250 ml shake flasks and cultivated at 30 °C, 200 rpm for 7 d. After fermentation, the hyphae and residual wheat bran was removed by filtration with two layers of gauze and centrifugation at 10000 × g for 5 min. The cellulase cocktail was stored at 4 °C.
2.9 Determination of cellulase activity to cellulase cocktail
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In terms of CBH recombinants, the CBH and total cellulase activity were determined. In terms of EG recombinants, the EG and total cellulase activity were
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determined. In terms of BG recombinants, the BG and total cellulase activity were
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determined.
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The enzymatic reaction mixtures (500 μl) for the determination of CBH activity comprised of 50 μl properly diluted enzyme solution, 450 μl avicel suspension (50
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mg/ml avicel, 0.1 M sodium citrate/citric acid, pH 4.5). The enzymatic reaction
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mixtures (500 μl) for the determination of EG activity comprised of 50 μl properly
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diluted enzyme solution, 450 μl CMC-Na solution (10 mg/ml CMC-Na, 0.1 M sodium citrate/citric acid, pH 4.5). The enzymatic reaction mixtures (500 μl) for the determination of BG activity comprised of 50 μl properly diluted enzyme solution, 450 μl pNPG solution (10 mM pNPG, 0.1 M sodium citrate/citric acid, pH 4.0). The enzymatic reaction mixtures (500 μl) for the determination of total cellulase activity comprised of 50 μl properly diluted enzyme solution, two pieces of 1 × 1 cm2 filter paper, 450 μl reaction buffer (0.1 M sodium citrate/citric acid, pH 4.5). For the determination of CBH, EG and BG activity, the reaction conditions were the same as the methods described in 2.6. For the determination of total cellulase activity, the
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reaction condition was the same as the determination of CBH activity described in 2.6.
2.10 Mixed-strain fermentation The CBH recombinants with highest CBH activity and the EG recombinant with highest EG activity were mix-cultured in MN medium for further improvement of total cellulase activity. In detail, 107 spores of CBH recombinants and EG
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recombinant with different ratios (8:1, 4:1, 2:1, 1:1, 1:2, 1:4 and 1:8) were inoculated in 100 ml WN medium in 250 ml shake flasks, cultivated at 30 °C, 180 rpm for 7 d.
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determined by the methods described in 2.9.
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After fermentation, the CBH, EG and total cellulase activity of cellulase cocktail were
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3. Results and discussion
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3.1 Directed expression of cellulase components
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Six recombinants were cultivated in OGPY medium for homologous constitutive
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expression of cellulase components, which included three CBHs (AnCel6, AnCel7A and AnCel7B), one EG (AnEGL) and two BGs (AnBGL1 and AnBGL2). The expression results were analyzed by SDS-PAGE (Fig. 2). The apparent molecular weights of AnCel6, AnCel7A, AnCel7B, AnEGL, AnBGL1 and AnBGL2 were about 65, 75, 58, 26, 110 and 130 kDa. Among these six cellulase components, AnCel7A, AnCel7B, AnEGL, AnBGL1 and AnBGL2 have been studied in our previous researches [35-37]. The concept of directed expression has been put forward in our previous study on AnBGL1 and AnBGL2. Briefly, “directed expression” contains two implications. One is homologous expression for correct glycosylation. Overexpression
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is a universal method to obtain target protein, such as the widely-used overexpression in Escherichia coli and Pichia pastoris. However, difference in glycosylation of target protein is the fatal flaw in heterologous expression [38], which may not reflect the original properties of nature protein. In order to obtain natural protein, homologous expression is recommended. And the other is constitutive expression for obtaining a
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single enzyme from a complex inducible enzyme system. Cellulase is an enzyme
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cocktail and can be produced in the presence of inducer. As a result, there are always
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various components in fermentation broth. Constitutive expression is adopted to
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prevent the expression of induced cellulases. In other words, only the introduced
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constitutive expressed cellulase can be produced without inducer. In this way, we can study any of the inducible enzymes conveniently. As was shown in Fig. 2, the target
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cellulase components were the predominant protein ingredient, which greatly reduced
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the burden of separation and purification for a single cellulase component from the
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complex cellulase cocktail.
Fig. 2 Directed expression of marine A. niger cellulase components (a) Expression of CBHs; (b) Expression of EG; (c) Expression of BGs. M: protein marker；lane 1, 5 and 8: Culture supernatant of original strain; lane 2-4: Culture
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supernatant of cel, cel7a and cel7b recombinants；lane 6: Culture supernatant of egl recombinant; lane 7 and 9: Culture supernatant of bgl1 and bgl2 recombinants.
3.2 Purification of cellulase components After concentrated and desalinated by ultrafiltration, six purified cellulase components were obtained by anion exchange chromatography. Linear and step
oo
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gradient elution with 0-0.5 M NaCl were used in anion exchange chromatography and the eluents were analyzed by SDS-PAGE (Fig. 3, Fig. 4 and Fig. 5). AnCel6,
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AnCel7A, AnCel7B, AnBGL1 and AnBGL2 were eluted with 0-0.1, 0.15-0.25, 0.15-
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0.3, 0.1-0.175, 0.1-0.2 M NaCl, respectively. AnEGL was eluted with 20 mM sodium
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phosphate.
Fig. 3 Gradient elution to three CBHs
(a) Gradient elution to AnCel6; (b) Gradient elution to AnCel7A; (c) Gradient elution to AnCel7B. M: protein marker; lane 1-8, 9-16, 17-24; Linear gradient elution with 00.4 M NaCl, the concentration difference between adjacent lanes was 0.05 M.
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Fig. 4 Separation and purification of EG
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M: protein marker; lane 1: concentrated supernatant; lane 2: elution with 20 mM pH
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Pr
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7.0 sodium phosphate; lane 3-6: elution with 0.1, 0.2, 0.3 and 0.5 M NaCl.
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Fig. 5 Separation and purification of two BGs
(a) Separation and purification of AnBGL1; (b) Separation and purification of AnBGL2. M: protein marker; lane 1-3: elution with 0.1, 0.175 and 0.5 M NaCl; lane 4-6: elution with 0.1, 0.2 and 0.5 M NaCl. 3.3 Salt-tolerance of cellulase components Marine A. niger cellulase has a stronger salt-tolerant property than terrestrial A. niger cellulase. Therefore, some cellulase components with salt-tolerant property probably exist in marine A. niger cellulase cocktail. In this study, we obtained a total of six cellulase components. The results of salt-tolerance analysis were shown in Fig.
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6. Among them, AnEGL and AnBGL2 have strong salt-tolerant property, and their activity was improved with the increase of salt concentration. In the presence of 4.5 M NaCl, AnEGL activity increased by 29% compared with that in the absence of NaCl. In the presence of 3.6 M NaCl, AnBGL2 activity increased by 36% compared with that in the absence of NaCl. Compared to AnCel6 and AnCel7A, AnCel7B has
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stronger salt-tolerance. At 4.5 M NaCl, AnCel7B retained approximately 80% of the
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maximum enzyme activity, while AnCel6 and AnCel7A retained only about 60%. Of
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the six cellulase components, three have salt-tolerant property. This result revealed
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that the ratio of salt-tolerant cellulases was considerable in marine A. niger cellulase
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cocktail. It was found that A. niger has hundreds of secreted proteins in the study on secretory proteome [39]. It is likely that a high proportion of salt-tolerant proteins are
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present in these secreted proteins from marine A. niger. Therefore, marine A. niger is
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probably a treasure trove of salt-tolerant enzymes.
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Pr
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Fig. 6 Salt-tolerance of marine A. niger cellulase components (a) Salt-tolerance of three CBHs; (b) Salt-tolerance of one EG; (c) Salt-tolerance of two BGs.
3.4 Salt-tolerance of cellulase cocktail Among the six cellulase components, AnCel7B has certain salt-tolerant property, AnEGL and AnBGL2 have outstanding salt-tolerant property. Which cellulase
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component endows marine A. niger cellulase with the stronger salt-tolerant property? The cellulase cocktail of original strain and the purified single cellulase components were mixed in a specific ratio, and the salt-tolerance of the mixed cellulases was examined. The results were shown in Fig. 7. After the cellulase cocktail of original strain was mixed with AnCel6, AnCel7A, AnBGL1 and AnBGL2, the salt-tolerance of
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the mixed cellulases largely remained the same or slightly decreased. When the
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cellulase cocktail of original strain was mixed with AnCel7B and AnEGL, the salt-
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tolerance was improved, especially for AnEGL. In fact, AnBGL2 has better salt-
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tolerance than AnEGL, but the addition of AnBGL2 did not enhance the salt-tolerance
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of mixed cellulase, which was probably due to the higher content of BG in cellulase
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cocktail of original strain.
Fig. 7 Salt-tolerance of mixed cellulases
3.5 Inducible expression of cellulases The expression of the introduced cellulase genes is driven by gpdA promoter. The gpdA promoter is a strong constitutive promoter, which keep active either under non-inducible condition (OGPY medium) or inducible condition (MN medium). As
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was shown in Fig. 8, the introduced cellulase constitutive expression cassettes were expressed in inducible medium containing bran as main carbon and nitrogen source. It was found that the apparent molecular weight of the target cellulase components were identical to those expressed under non-inducible condition. Different from the noninducing conditions, when using bran as the cellulase inducer, the inherent
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lignocellulolytic enzymes of marine A. niger were also expressed, which resulted of
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Pr
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the complex protein components in the fermentation supernatant.
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Fig. 8 Inducible expression of marine A. niger cellulases
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(a) Expression of CBHs; (b) Expression of EG; (c) Expression of BGs. M: protein marker；lane 1, 5 and 7: Culture supernatant of original strain; lane 2-4: Culture supernatant of cel, cel7a and cel7b recombinants；lane 6: Culture supernatant of egl recombinant; lane 8 and 9: Culture supernatant of bgl1 and bgl2 recombinants
3.6 Changes on cellulase activity of recombinants The activity of cellulase cocktail fermented with cel6, cel7a and cel7b recombinants was shown in Fig. 9a. After overexpression of three CBHs, the CBH activity of all recombinants was greatly improved. Among them, the CBH activity of cellulase cocktail fermented with cel6, cel7a and cel7b recombinants increased by 54-
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130%, 54-123% and 50-105%, respectively. Moreover, with the improvement of CBH activity, the total cellulase activity was also improved. Among them, the total cellulase activity of cellulase cocktail fermented with cel6, cel7a and cel7b recombinants were increased by 24-80%, 38-70% and 20-63%, respectively. In addition, by comparing the changes in CBH and total cellulase activity, it was found
f
that the cellulase cocktail with higher CBH activity exhibited higher total cellulase
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activity, which reflected that the overexpression of extra cellulase components had
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little effect on the expression of inherent cellulases. These results indicated that
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among the marine A. niger cellulase cocktail, CBH was the weak enzyme component
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and was the key factor limiting its total cellulase activity. The activity of cellulase cocktail fermented with egl recombinants was shown in
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Fig. 9b. Similar to the overexpression of CBHs, the EG activity and total enzyme
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activity of cellulase cocktail fermented with egl recombinants were improved
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compared with those of original strain. Among them, the EG activity increased by 6492%, and the total cellulase activity increased by 37-68%. These results indicated that EG was also relatively weak in marine A. niger cellulase cocktail and was the other key factor limiting its total cellulase activity. The activity of cellulase cocktail fermented with bgl1 and bgl2 recombinants was shown in Fig. 9c. After overexpression of BGs, the BG activity of cellulase cocktail fermented with bgl1 and bgl2 recombinants was slightly increased. The BG activity of cellulase cocktail fermented with bgl1 and bgl2 recombinants increased by 4-29% and 1-24%, respectively, compared with that of the original strain. Unlike the
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overexpression of CBHs and EG, the total cellulase activity of cellulase cocktail fermented with bgl1 and bgl2 recombinants decreased somewhat. These results indicated that BG was not the limiting factor on the total cellulase activity in marine A. niger cellulase cocktail. The decrease in total cellulase activity after overexpression of BGs may result from that the increase in BG activity reduces the cellobiose (cellulase
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inducer) concentration in the medium, resulting in a decrease in cellulase induction
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during fermentation, and consequently reducing the production of cellulase cocktail.
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Overexpression of the relatively weak cellulase components is one of the
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methods to improve the total cellulase activity. Overexpression method is to integrate
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the cellulase expression cassettes into genome to fundamentally optimize the composition of cellulase, and then improve the total cellulase activity. Examples of
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overexpression method to improve cellulase activity are shown in Table 2, which
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were mainly to improve the activity of T. reesei cellulase cocktail.
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Pr
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Fig. 9 Changes of cellulase activity in different recombinants (a) Changes of cellulase activity in cel6, cel7a and cel7b recombinants; (b) Changes of cellulase activity in egl recombinants; (c) Changes of cellulase activity in bgl1 and bgl2 recombinants
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Table 2 Enhanced total cellulase activity by overexpression Cellulase name
Source
Host
Relative activitya (%)
Reference
cellobiase
A. niger
T. reesei
144
[17]
β-glucosidase
Periconia sp.
T. reesei
158
[40]
CBHII
T. reesei
T. reesei
430
[19]
β-glucosidase
Penicillium
T. reesei
130
[18]
T. reesei
167
[41]
Aspergillus
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β-glucosidase 1
aculeatus Piromyces
A．niger 750
rhizinflata A. niger
A. niger
AnCel7A
A. niger
A. niger
AnCel7B
A. niger
AnEGL
A. niger
[20]
180
This study
170
This study
Pr
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AnCel6
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Cellulase
f
decumbens
163
This study
A. niger
168
This study
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A. niger
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a. The activity of cellulase cocktail from original strain was defined as 100%.
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3.7 Mixed-strain fermentation
After the overexpression of CBHs and EG, the total cellulase activity of marine A. niger cellulase cocktail was improved. The method of overexpressing the weak cellulase component can effectively optimize the proportion of the cellulase components in marine A. niger cellulase cocktail, thereby increasing the total cellulase activity. In order to further optimize the proportion of the cellulase components, mix-strain fermentation was performed. Unlike the reported cases that used different species of strains, we employed the two marine A. niger recombinants, which could eliminate the adverse impacts caused by the competition between
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different species of strains [42]. The changes in cellulase activity of the cellulase cocktail fermented with mixed recombinants were shown in Fig. 10. With the increase of the ratio of CBH recombinants to EG recombinant, the CBH activity was continuously increased, while the EG activity was gradually decreased. The total cellulase activity showed a trend of first increasing and then decreasing. In the three
f
co-culture systems, the highest total cellulase activity was achieved when the
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inoculation ratio of CBH recombinants to EG recombinant was 2:1. Compared with
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the original strain, the total cellulase activity of the cellulase cocktail in the three
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optimal co-culture systems (cel6 + egl, cel7a + egl and cel7b + egl) was increased by
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114%, 102% and 91%, respectively. According to the optimal inoculation ratio in mixed-strain fermentation, the CBH exhibited a greater restriction effect on the total
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cellulase activity than EG.
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Fig. 10 Changes of cellulase activity by mixed-strain fermentation MA: marine A. niger; C6: cel6 recombinant; C7A: cel7a recombinant; C7B: cel7b recombinant.
Conclusions
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In this study, six cellulase components were obtained by directed expression. Studies on their enzymatic properties revealed that AnBGL2 and AnEGL exhibited an outstanding salt-tolerant property, and AnCel7B exhibited a certain salt-tolerant property. It was proved that the salt-tolerant AnEGL and AnCel7B endowed the cellulase cocktail with stronger salt-tolerant property, while the salt-tolerant AnBGL2
f
had no positive effect. Moreover, the activity of cellulase cocktail increased after
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overexpression of CBH and EG components and it was further improved by mixed-
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strain fermentation with CBH recombinants and EG recombinant.
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Acknowledgements
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This work was supported by National Natural Science Foundation of China (21576233, 21878263) and "the Fundamental Research Funds for the Central
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Universities".
References
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Declarations of interest: none.
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Figure captions
Fig. 1 Plasmids used in this study
Fig. 2 Directed expression of marine A. niger cellulase components (a) Expression of CBHs; (b) Expression of EG; (c) Expression of BGs. M: protein marker；lane 1, 5 and 8: Culture supernatant of original strain; lane 2-4: Culture
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supernatant of cel, cel7a and cel7b recombinants；lane 6: Culture supernatant of egl
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recombinant; lane 7 and 9: Culture supernatant of bgl1 and bgl2 recombinants.
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Fig. 3 Gradient elution to three CBHs
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(a) Gradient elution to AnCel6; (b) Gradient elution to AnCel7A; (c) Gradient elution to AnCel7B. M: protein marker; lane 1-8, 9-16, 17-24; Linear gradient elution with 0-
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0.4 M NaCl, the concentration difference between adjacent lanes was 0.05 M.
Fig. 4 Separation and purification of EG
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M: protein marker; lane 1: concentrated supernatant; lane 2: elution with 20 mM pH 7.0 sodium phosphate; lane 3-6: elution with 0.1, 0.2, 0.3 and 0.5 M NaCl.
Fig. 5 Separation and purification of two BGs (a) Separation and purification of AnBGL1; (b) Separation and purification of AnBGL2. M: protein marker; lane 1-3: elution with 0.1, 0.175 and 0.5 M NaCl; lane 4-6: elution with 0.1, 0.2 and 0.5 M NaCl.
Fig. 6 Salt-tolerance of marine A. niger cellulase components (a) Salt-tolerance of three CBHs; (b) Salt-tolerance of one EG; (c) Salt-tolerance of
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two BGs.
Fig. 7 Salt-tolerance of mixed cellulases
Fig. 8 Inducible expression of marine A. niger cellulases (a) Expression of CBHs; (b) Expression of EG; (c) Expression of BGs. M: protein marker；lane 1, 5 and 7: Culture supernatant of original strain; lane 2-4: Culture
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supernatant of cel, cel7a and cel7b recombinants；lane 6: Culture supernatant of egl
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recombinant; lane 8 and 9: Culture supernatant of bgl1 and bgl2 recombinants.
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Fig. 9 Changes of cellulase activity in different recombinants
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(a) Changes of cellulase activity in cel6, cel7a and cel7b recombinants; (b) Changes of cellulase activity in egl recombinants; (c) Changes of cellulase activity in bgl1 and
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bgl2 recombinants.
Fig. 10 Changes of cellulase activity by mixed-strain fermentation
recombinant.
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MA: marine A. niger; C6: cel6 recombinant; C7A: cel7a recombinant; C7B: cel7b
Journal Pre-proof Declaration of interests
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Table 1 Plasmids and cellulase genes in this study Cellulase genes
GenBank accession numbers
pCAMBIA-hph-bgl1
bgl1
MH744149
pCAMBIA-hph-bgl2
bgl2
MH744150
pCAMBIA-hph-egl
egl
MK587440
pCAMBIA-hph-cel6
cel6
MK898285
pCAMBIA-hph-cel7a
cel7a
pCAMBIA-hph-cel7b
cel7b
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Plasmids
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MK898286
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MK898287
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Table 2 Enhanced total cellulase activity by overexpression Cellulase name
Source
Host
Relative activitya (%)
Reference
cellobiase
A. niger
T. reesei
144
[17]
β-glucosidase
Periconia sp.
T. reesei
158
[40]
CBHII
T. reesei
T. reesei
430
[19]
β-glucosidase
Penicillium
T. reesei
130
[18]
T. reesei
167
[41]
Aspergillus
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β-glucosidase 1
aculeatus Piromyces
A．niger 750
rhizinflata A. niger
A. niger
AnCel7A
A. niger
A. niger
AnCel7B
A. niger
AnEGL
A. niger
[20]
180
This study
170
This study
Pr
e-
AnCel6
pr
Cellulase
f
decumbens
163
This study
A. niger
168
This study
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A. niger
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a. The activity of cellulase cocktail from original strain was defined as 100%.
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1. Six cellulase components were obtained from marine Aspergillus niger. 2. Three cellulase components were salt-tolerant. 3. The salt-tolerant components endowed with the salt-tolerance of cellulase cocktail.
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4. Cellulase activity was improved by overexpression and mixed-strain fermentation.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10