Preliminary Characterization of a New Processive Endoglucanase from Clostridium alkalicellulosi DSM17461 ^†

Abstract

The Clostridium alkalicellulosi DSM17461^T genome contains several glucoside hydrolase encoding genes essential for cellulose degradation. Herein, the family 9 glycoside hydrolase enzyme (CalGH9_2089) was cloned and expressed. The enzyme contains one GH9 catalytic module, a family 3 carbohydrate-binding module, and one Type I dockerin at its C-termini. The optimal pH and temperature for CalGH9_2089 to hydrolyze CMC were 55 °C and pH 6.0, with the remaining activity of more than 60% at pH 10.0. CalGH9_2089 produced a series of cello-oligomers (G2-G6) from CMC, suggesting that the enzyme has an endo-acting capability. When regenerated amorphous cellulose was hydrolyzed with CalGH9_2089, the ratio of reducing ends in the soluble fraction to that in the insoluble pellets was 4.8, suggesting that this enzyme acts processively on RAC. This work extends our knowledge of the behavior of the GH9 endoglucanase from the microorganism living in an alkaline environment.

1. Introduction

Cellulose is an unbranched glucose polymer that is linked by the β-1,4-glycosidic bond. Individual cellulose chains are closely associated through intra- and inter-hydrogen bonds, forming a tightly packed structure with a few amorphous regions (semi-crystalline). The crystalline structure makes cellulose insoluble, thus posing difficulty in hydrolysis [1,2]. To break down cellulose, three main enzymes, i.e., endo-1,4-β-glucanases, exo-glucanases/cellobiohydrolases, and β-glucosidases, are required to work in concert to degrade crystalline cellulose to simple sugar successfully. The endo-1,4-β-glucanases are important enzymes. They attack the β-1,4-glycosidic bonds at random sites along the cellulose chain, freeing sections of the chain from the crystal surface and creating free ends upon which exo-glucanases can act, thereby generating cellobioses, which are a substrate for β-glucosidases for producing glucose molecules [1].

According to the carbohydrate-active enzyme (CAZy) database, glycosidases are classified on the basis of similarities in the amino acid sequence and three-dimensional structure. Cellulases belong to glycoside hydrolase families (GH) 5-10, 12, 26, 44, 45, 48, 51, 61, 74, etc. [3]. Most endo-1,4-β-glucanases belong to GH9. GH9 endoglucases catalyze the hydrolysis of β-1,4 linkages with the inversion of anomeric carbon configuration. Several GH9 endoglucanases are tightly associated with a carbohydrate-binding module (CBM) classified in family 3 [4].

In bacterial cellulase systems, GH9 endoglucanases are distinctive from other cellulase families. GH9 endoglucanases have shown either non-processive or processive activity. The processivity is the ability to remain in adsorbing the substrate in between subsequent hydrolytic reactions. This ability is important for the degradation of crystalline cellulose [5,6]. Therefore, the processive glucanases, behaving similarly to exoglucanases, are thought to be a major cellulose-degrading factor in addition to the exoglucanase component [5,6]. A recent study showed that a single GH9 endoglucanase is essential for cellulose degradation by Clostridium phytofermentans, despite its production of several other cellulases [7].

Carbohydrate-binding modules (CBMs) are non-catalytic modules found in some carbohydrate-active enzymes. They play an important role in degrading insoluble substrates [8,9]. Based on the structure of the binding site and functional similarity, CBMs are grouped into three types: Type A CBMs that bind the planar surfaces of crystalline polysaccharides; Type B CBMs bind internally on the glycan chains (endo-type); Type C CBMs bind the termini of glycan (exo-type). CBMs have been classified into 81 families based on their amino acid similarities (http://www.cazy.org, accessed on 12 August 2021) [10], and a family 3 CBM has been shown to bind cellulose effectively. There are numerous examples of fusing CBMs to catalytic modules, subsequently resulting in 2- to 10-fold increase in enzyme activities on insoluble substrates but not on soluble polysaccharides [11,12].

Clostridium spp. have received considerable attention as a source of cellulolytic and enzymes [13]. A number of endo-1,4-β-glucanases from several Clostridial species have been studied in recent years, some of which show promising applications in biofuel and biochemical production due to their high activity and high thermostability [13]. The Clostridium alkalicellulosi DSM17461^T genome contains several glycoside hydrolase encoding genes important for cellulose degradation. Herein, the family 9 glycoside hydrolase enzyme (CalGH9_2089) was cloned and expressed. The recombinant enzyme’s biochemical properties (i.e., pH and temperature optima, mode of action, and processivity) were determined.

2. Materials and Methods

2.1. Cloning and Protein Expression

Genomic DNA of C. alkalicellulosi DSM17461^T was purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Germany).

PCR amplification of the putative endoglucanase gene (CalGH9_2089) was performed using the upper primer (5′-AATTAGCTAGCTCAACTTTTAACTACGGAGA-3′), and the lower primer (5′-AATTACTCGAGTTACTGGTTTACTGGGAAAA-3′) using the genomic DNA as the template and the Phusion Taq High-Fidelity PCR-System (ThermoFisher Scientific, Waltham, MA, USA). The PCR-amplified material was cloned in pET28a(+) (Novagen, Madison, WI, USA) by digestion with restriction enzymes NheI and XhoI.

The purified PCR products and pET28a(+) vectors were doubly digested with NheI and XhoI. The digested PCR products and pET28a(+) vectors were ligated with T4 DNA ligase (New England Biolabs, Hitchin, UK). The ligated plasmids were transformed into NEB 5-alpha E. coli cells.

The positive clones with the target insert were transformed into E. coli BL21 (DE3) for protein expression following the manufacturer protocols (New England BioLabs). The recombinant CalGH9_2089 protein was purified from a 400 mL transformed E. coli BL21 (DE3) culture.

2.2. Enzyme Activity Assay

The enzyme assay was performed in a total reaction volume of 200 µL. The reaction mixture consisted of 1% (w/v) substrate (i.e., CMC and RAC) and 0.25 µM of CalGH9_2089 in 50 mM citrate buffer pH 6.0. The reaction mixture was incubated at 55 °C for 15 min (and 3 h for RAC). The reaction was terminated by immersion in ice water and centrifuged at 13,000× g at 4 °C for 10 min to separate the substrate from the soluble fraction. One hundred microliters of the soluble fraction was taken to a new 1.5 mL microcentrifuge tube, and 150 µL of dinitrosalicylic acid (DNS) [14] was added. The reaction mixture was boiled for 10 min and centrifuged at high speed. A sample was taken and measured at an absorbance of 540 nm. The amount of reducing sugar released in the mixture was estimated using a glucose standard curve. One unit of the enzyme was defined as the amount of enzyme that produces 1 µMol of glucose equivalent in 1 min under the assayed conditions.

2.3. Effect of pH and Temperature

The influence of pH on enzyme activity was determined. The activity at different pH values from pH 3.0 to 11.0 was measured at 55 °C for 15 min. Citrate–sodium citrate buffer for pH 3.0–7.0, Tris-HCl buffer for pH 7.0–9.0, and glycine-NaOH for pH 9.0–11.0 with a concentration of 50 mM were used as a reaction buffer. The maximum activity at the corresponding pH was taken as 100%. A pH stability assay was performed by incubating 5.5 µL of the 11.5 µM enzyme in 10 mM of the buffers mentioned above at 30 °C for 60 min. After that, the enzyme solution was diluted, and the residual activity was measured under optimal assay conditions.

The effect of temperature on enzyme activity was tested by incubating 0.25 µM enzyme in a 200 µL reaction mixture containing 1% (w/v) substrate at a temperature range of 40–70 °C, pH 6.0 (citrate buffer), for 10 min. The maximum activity at the corresponding temperature was taken as 100%. The thermal stability of the enzyme was determined by incubating 5.5 µL of the 11.5 µM enzyme at temperatures of 37, 55, 60, and 70 °C for 0–24 h. The enzyme solution was diluted, and the residual activity was determined under optimal assay conditions.

2.4. Analysis of the Hydrolysis

The reaction mixture (total volume of 200 µL) containing 0.25 µM enzyme and 1% (w/v) substrate (i.e., CMC) was assayed under optimal conditions with varying times from 15 min to 16 h. The reaction was stopped at specific time intervals by boiling for 5 min and centrifuging at 13,000× g at 4 °C for 10 min to remove the undigested substrate. The soluble fraction was transferred to a new tube, and 4 µL of the soluble fraction was mixed with 4 µL of absolute ethanol. The mixed sample (8 µL) was spotted on TLC plates, and the plates were immersed in a TLC chamber containing n-butanol:acetic acid:water at a ratio of 2:1:1 as the mobile phase and heated at 90 °C in a hot air oven for visualization [15].

2.5. Processivity Assay

The content of reducing ends in the soluble and insoluble fractions was determined using RAC as a substrate [16]. Different concentrations of enzyme were added to a 1.5 mL microcentrifuge tube containing 1% (w/v) RAC in citrate buffer (pH 6.0) (final volume of 200 mL). The reaction mixture was incubated at 55 °C for different incubation times. After incubation, the reaction tube was centrifuged at 13,000× g at 4 °C for 10 min. The supernatant was removed, of which 100 µL was taken to measure reducing ends (soluble fraction) by the DNS method. The pellet was washed three times with 500 µL citrate buffer (pH 6.0) by centrifugation at 13,000× g at 4 °C for 5 min. After washing, the pellet was resuspended in 100 µL citrate buffer (pH 6.0) and taken to measure reducing ends (insoluble fraction) by the DNS method.

3. Results and Discussion

3.1. Bioinformatic Analysis

The draft genome sequence of C. alkalicellulosi DSM17461 was sequenced, revealing several putative endoglucanase encoding genes essential for cellulose hydrolysis in its genome. One ORF possessing 2899 bp was designated as CalGH9_2089. This gene was predicted to encode a putative glycoside hydrolase family 9 endoglucanase connected with a family 3 CBM and a type I dockerin according to BlastP analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 6 July 2021). This gene was predicted to have 748 amino acid residues with a theoretical molecular weight of 82.86 kDa and pI of 5.1 (https://web.expasy.org/cgi-bin/protparam/protparam/, accessed on 6 July 2021) (Figure 1). CalGH9_2089 was successfully expressed in E. coli BL21 (DE3), and it was determined for its molecular weight and purity, showing a single band with a molecular weight of about 83 kDa.

3.2. Effects of pH and Temperature

The effect of pH on CalGH9_2089 activity was tested using CMC as a substrate. CalGH9_2089 exhibited an activity from pH 4.0 to 11.0, with maximal activity at pH 6.0–7.0. The enzyme showed more than 50% activity from pH 5.0 to 10.0 (Figure 2A). For pH stability, the enzyme was pre-incubated in different buffers at 55 °C for 60 min and was assayed at the optimal conditions. It was found that the enzyme showed good stability from pH 5.0 to 10.0 with residual activity of more than 60% (Figure 2B). The pH profile suggested that CalGH9_2089 is active in a broad pH range and tolerates alkaline pHs.

The effect of temperature on the activity of CalGH9_2089 was examined with the CMC. CalGH9_2089 exhibited the highest activity of 1804.98 μmol product/min/μmol protein on CMC. The optimal temperature of CalGH9_2089 toward CMC was 55-60 °C (Figure 3A). At 80 °C, the enzyme was almost completely inactivated. The thermal stability analysis of CalGH9_2089 was investigated at pH 6.0 with different temperatures (37, 55, 60, and 70 °C) (Figure 3B). The result showed that CalGH9_2089 was stable at 37 °C, retaining more than 80% activity for 24 h. At 55 °C, the residual activity remained about 60% when the enzyme was incubated for 30 min. The enzyme activity remained 40% of its maximal activity from 6 to 24 h incubation. At 60 °C and 70 °C, the activity of CalGH9_2089 lost rapidly, retaining 20% and 10% of its maximal activity, respectively, when it was incubated for 30 min. This result likely indicates that CalGH9_2089 is a mesophilic enzyme and tolerates a moderate temperature of around 50 °C.

3.3. Mode of Action

To investigate the mode of action of CalGH9_2089, the hydrolysis of CMC was tested at pH 6.0 and 55 °C, and the hydrolysis products at different incubation times were determined by TLC (Figure 4). It was found that CalGH9_2089 was able to hydrolyze CMC. At 1 min, small amounts of oligosaccharides were present around cellotriose (G3) and cellotetraose (G4) compared to the standard. From 3 to 10 min, more oligosaccharides, particularly cellobiose (G2), G3, and G4, were accumulated, and they were clearly observed on the TLC plate. After 15 min to 16 h, the amounts of G2, G3, and G4 greatly increased, together with the accumulation of glucose (G1). This hydrolysis pattern likely indicates the endo-acting mode of CalGH9_2089.

3.4. Processivity

Some GH9 endoglucanases have been shown to have processivity similar to exo-glucanases [17]. The processivity of endoglucanase is usually determined by the ratio of the generated soluble reducing ends to insoluble reducing ends [16]. Here, the distribution of reducing sugars generated by CalGH9_2089 on RAC was studied. It was found that the ratio of reducing ends in the soluble fraction to that in the insoluble fraction increased from 1.6 to 4.8 as incubation time was prolonged from 30 to 180 min (Figure 5). This result indicates that CalGH9_2089 is a processive endoglucanase, differing from the common endoglucanases, for which their actions randomly cut and produce more reducing ends in the insoluble fraction than in the soluble fraction.

Figure 4. TLC analysis of hydrolysis product from CMC by CalGH9_2089.

Figure 4. TLC analysis of hydrolysis product from CMC by CalGH9_2089.

4. Conclusions

Herein, we successfully expressed a putative GH9 endoglucanase possessing a modular structure containing a GH9 catalytic module, a family 3 CBM, and one Type I dockerin (CalGH9_2089) from C. alkalicellulosi genome. The enzyme was functionally active from pH 5.0 to 10.0 with an optimal pH around 6.0–7.0. The optimal temperature for this enzyme is 55 °C; however, its half-life was around 1 h. The production of a series of oligosaccharides from CMC indicates an endo-acting mode of CalGH9_2089. Moreover, the ratio of reducing ends in the soluble fraction to that in the insoluble fraction using RAC as a substrate was 4.8 (3 h incubation), suggesting this CalGH9_2089 endoglucanase possesses processive activity.