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. 2020 May 8;10(6):239. doi: 10.1007/s13205-020-02229-x

The enzymatic characters of heterologous expressed novel β-1, 4-glucosidase originated from Aspergillus fresenii

Yongzhi Yang 1, Jian Wang 1, Henan Guo 1, Yunhe Cao 1,✉
PMCID: PMC7210331  PMID: 32405443

Abstract

β-1, 4-glucosidases generate glucose from cellobiose and oligosaccharides, enhancing the productivity in biorefinery and the bioconversion process as well as the nutritional value in food and feed. With the high-throughput sequencing technique, a novel β-1, 4-glucosidase, named bgl T2, containing 861 amino acid residues, was found from Aspergillus fresenii. bgl T2 belongs to the glycosyl hydrolase (GH) family 3. The bgl T2 that expressed by Komagataella phaffii X33 presented the highest activity at 55 °C and pH 5.5. The half-lives of bgl T2 under 50 °C, 55 °C, 60 °C, and 65 °C were 9 min 36 s, 4 min 22 s, 117 s, and 68 s, respectively. The bgl T2 was stable between pH 3.0 to pH 8.0. The Michaelis constant (Km) and the theoretical maximum rate (Vmax) of bgl T2 were 0.0007 mol/L and 9 × 10−8 mol/L/s, respectively. In a 5 L fermentation vessel, the recombinant K.phaffii X33 could yield a β-1, 4-glucosidase activity of 4.45 U/mL after 96 h methanol inducement. As an important member of cellulases, the novel bgl T2 might contribute to bioenergy, food processing, feed enrichment, and nutritional study, etc. This study also developed a path to obtain new enzymes depending on high-throughput sequencing technique.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02229-x) contains supplementary material, which is available to authorized users.

Keywords: β-1, 4-glucosidase; Aspergillus fresenii; Heterologous expression; Komagataella phaffii

Introduction

β-1, 4-glucosidases (bgl; EC 3.2.1.21) catalyse the cellobiose and oligosaccharides hydrolysis into glucose. It could remit the inhibition action of cellobiose against endoglucanases and cellobiohydrolases during the enzymatic catalysing cellulose hydrolysis (Mateusz et al. 2018; Saha et al. 1994). Cellulase enzymes, as the most important and costly part of generating glucose from lignocellulose biomass (Gupta et al. 2011; Capolupo and Faraco 2016), are always interesting to understand, especially a novel one. Acquiring a new β-1, 4-glucosidase might help with the treatments of renewable agricultural, industrial and municipal cellulosic wastes, as biofuels, chemicals, or animal feed resources.

β-1, 4-glucosidases exist in plenty of organisms, having a variety properties. The study of β-1, 4-glucosidase got its start as early as in 1837, discovered by Wöhler and Liebig from almond emulsion (Wöhler and Liebig 1838). Through years of study, plentiful β-1, 4-glucosidases were discovered (Payne et al. 2015). The majority of β-glucosidases have been classified into the GH1 and the GH3 families (Salgado et al. 2018). β-glucosidases in the GH3 family are comprised of GH3 N-terminal domain (pfam00933) and GH3 C-terminal domain (pfam01915) as catalytic domains. Although β-glucosidases in the GH3 family catalyse a broader range of substrates than those in the GH1 family, they are less tolerant toward glucose and glucolactone (Rudakiya et al. 2019). This shortcoming might be neglected in feed additives applications since glucose and glucolactone would be absorbed fast and nearly complete in digestion tract. On the other hand, β-glucosidases were inhibited by hematite, kaolinite, and montmorillonite due to the inactivation or hindrance of enzyme active sites (Yang et al. 2019). Montmorillonite is used as an adsorbent to deal with the fungal contamination in the feed industry. Furthermore, the conflict between humans and animals for consuming food is becoming critical with the growing population (Erb et al. 2016). Alternative feed material is required to solve this problem. Glucose obtained from lignocellulosic biomass could be a potential energy source for animals, if the lignocellulose biomass were digested properly (Zhao et al. 2013; Choct 2015). Since monogastric animals lack the capacity to convert cellobiose into glucose, β-1, 4-glucosidases must be essential for them to digest the lignocellulose biomass to get glucose from it.

Higher activity and stability of fungal β-1, 4-glucosidases make them more convenient for industrial applications (Rudakiya and Gupte 2017; Narra et al. 2012). However, extracting enzymes from A.fresenii culture media is not favorable because A.fresenii produces ochratoxin (Visagie et al. 2014). Besides, in the pre-study of this work, a non-detectable amount of β-1, 4-glucosidases of A.fresenii were found on the inducing culture media. Heterologous expression of bgl T2 by K.phaffii can not only avoid the mycotoxin-related health hazard in the practical production phase, but also can facilitate the enzymatic study of bgl T2 with less interfering factors. Thus, this study focused on heterologous expressing the novel β-1, 4-glucosidase (bgl T2) that predicted by the result of the mRNA high-throughput sequencing, not only to understand the properties and potentials of bgl T2 for further utilization, but also to determine if the high-throughput sequencing of mRNA technique could provide sufficient information to discover new enzymes from a non-reported genome of a fungus. As results, the bgl T2 that was identified as a member of GH3 family, was successfully expressed by K.phaffii and its properties were determined. This study broadened the knowledge of β-1, 4-glucosidase from A.fresenii by providing a candidate for applications related to cellulose degradation, and confirmed that high-throughput sequencing of mRNA facilitated enzyme mining work.

Results

Identification of the β-1, 4-glucosidase bgl T2

On Avicel induction plates, A. fresenii grew poorly with fewer and shorter hyphae, but spore formation happened comparatively sooner. It caused difficulties for enzyme isolation and protein sequencing to discover new enzyme. However, there was no problem with the mRNA high-throughput sequencing.

Four cellulases’ mRNA sequences of A. fresenii, one endoglucanase, two cellobiohydrolases, and bgl T2 were able to be assembled into full-length (sequences in Supplementary file 4). All of them were up-regulated on the Avicel induction plate (Table 1). Assembled bgl T2 mRNA (GenBank accession number: MK986475) contains 2586 nucleotides including initiation codon and termination codon. Thus, the bgl T2 contains 861 amino acids with a predicted molecular mass of 93.55 kDa, and a theoretical pI of pH 5.11 (Kozlowski 2016).

Table 1.

A. fresenii expression differences of cellulases assembled into full-length mRNA

Cellulase name Annotation Expression differences (log2 fold change)a
bgl T2 β-1,4-glucosidase 26.33
CBH T4 Cellobiohydrolase 51.22
CBH T5 Cellobiohydrolase 5.05
EG T1 Endoglucanase 8.90
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aThe expression differences of cellulases in A. fresenii were compared between A. fresenii grown on Avicel plate against it grown on glucose plate

In comparing the assembled bgl T2 mRNA with the sequence of PCR amplification product from A. fresenii genomic DNA, 5 introns were found in the bgl T2 open reading frame containing 2901 bp (MK986476). The mRNA sequence of bgl T2, the bgl T2 open reading frame, and the amino acids sequences of bgl T2 are in Supplementary file 1.

Conserved domains of GH3 N-terminal domain, BglX, GH3 C-terminal domain, and fibronectin type III-like domain were found in the bgl T2. Due to these conserved domains in bgl T2, it should be considered as a member of GH3 family. The amino acid sequence of bgl T2 shared 91%, 80%, 80%, and 78% identity with the β-glucosidases of Aspergillus steynii IBT 23096 (XP_024702113.1), Aspergillus oryzae (5FJJ_A), Aspergillus aculeatus (P48825.1), and Aspergillus fumigatus A1163 (B0XPE1.1), respectively. The alignment of amino acid sequences among these β-glucosidases was provided in supplementary file 2.

Construction of the expression plasmid and strain

The optimized bgl T2 coding sequence (MK965547) for K.phaffii and original bgl T2 sequence were presented in Supplementary file 3. The codon adaptation index (CAI) value was increased from 0.63 to 0.93 by the optimization for K.phaffii expression, while the CG content decreased from 57.12% to 42.09% to avoid rare codons in K.phaffii.

The optimized bgl T2 coding sequence was successfully inserted into pPICZαA plasmid. It was preserved in E. coli TOP 10, confirmed by sequencing and double digestion. Eight zeocin-resistant clones of recombinant K.phaffii X-33 were picked up and preserved on YPD plates (1% yeast extract, 2% peptone, 2% dextrose and 2% agar). The recombinant K.phaffii labeled bgl T2-7 gave the highest β-1, 4-glucosidase activity after methanol inducement in flask. Thus, the optimized bgl T2 coding sequence was successfully recombined with the K.phaffii X-33 genome.

Expression bgl T2 by recombinant K.phaffii X-33

After 96 h inducement in 500 mL flask, the supernatant of bgl T2-7 transformant was collected and determined for its β-1, 4-glucosidase activity. The bgl T2-7 transformant gave 0.23 U/mL toward p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrate. While the bgl T2-7 transformant was induced in 5 L fermentation vessel for 96 h, it yielded 4.45 U/mL. The specific activity of bgl T2 was calculated as 3.6 U/mg.

Although the predicted theoretical molecular mass of bgl T2 was 93.55 kDa, the heterologous expressed protein appeared in a band at approximately 130 kDa (Fig. 1).

Fig. 1.

Fig. 1

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SDS-PAGE of the recombinant bgl T2. Lane M: molecular weight marker. Lane 1: heterologous expressed bgl T2

Character study of bgl T2

Although very weak activities were observed against CMC-Na, pNPC, and Avicel, bgl T2 only showed reliable activity against pNPG among the substrates tested.

bgl T2 showed highest activity at pH 5.5 and 55 °C. At pH 5.0, the bgl T2 activity was very close to its activity at pH 5.5, merely 2% difference. At the range of pH 4.5 to pH 6.5, bgl T2 stood more than 50% of its activity, while its activity was almost inhibited completely below pH 3.5 or above pH 8.0. From 25 to 55 °C, the bgl T2 activity increased gradually, while it dropped down dramatically from 55 °C to a higher temperature. The dynamic graph between pH and bgl T2 activity was presented in Fig. 2, while the relationship between temperature and its activity was presented in Fig. 3.

Fig. 2.

Fig. 2

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The dynamic graph between pH and bgl T2 activity. The activities of bgl T2 in different pH McIlvaine buffer indicated that the optimal pH for bgl T2 was at pH 5.5. Results are the mean of three replicates

Fig. 3.

Fig. 3

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The dynamic graph between temperature and bgl T2 activity. The activities of bgl T2 under different temperature indicated that the optimal temperature was at 55 °C. Results are the mean of three replicates

When the bgl T2 was treated in a range of pH 3.0 to pH 8.0 at 4 °C for 1 h, bgl T2 kept stable. Its activity became the same or even more than the controls (Fig. 4). The treatment of pH 5.0 gave 60% higher relative activity than the control. Treating bgl T2 in pH 2.5 buffer for one hour resulted in residue activity dropping down to 22%.

Fig. 4.

Fig. 4

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The tolerance of bgl T2 against pH value. bgl T2 showed tolerance to a pH range from pH 3.0 to pH 8.0. Treating bgl T2 at pH 5.0 enhanced more than 60% relative activity of it, whereas only 22% activity was recovered from pH 2.5 treatment. Results are the mean of three replicates

The half-lives of bgl T2 were 9 min 36 s, 4 min 22 s, 117 s, and 68 s under 50 °C, 55 °C, 60 °C, 65 °C, respectively.

The bgl T2 was slightly affected by the chemicals of sodium sulphate, copper sulphate, calcium chloride, ammonium sulphate, potassium chloride, sodium chloride, magnesium chloride, sodium nitrate, manganese sulphate, while it lost 7% activity with zinc sulphate and cobalt sulphate (Table 2). The Km and Vmax of bgl T2 against pNPG were 0.0007 mol/L and 9 × 10−8 mol/L/s, respectively.

Table 2.

Effects of eleven chemicals on the activity of bgl T2

Chemicals Relative activity (%)a
Control 100 ± 0.31
Sodium sulphate 99.36 ± 0.36
Copper sulphate 102.25 ± 0.10
Ammonium sulphate 100.16 ± 0.38
Manganese sulphate 94.53 ± 0.58
Zinc sulphate 93.57 ± 0.25
Cobalt sulphate 92.93 ± 0.75
Calcium chloride 94.21 ± 0.29
Potassium chloride 96.95 ± 0.48
Sodium chloride 94.37 ± 0.20
Magnesium chloride 94.69 ± 0.22
Sodium nitrate 98.71 ± 0.37
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aControl was set as 100% relative activity. These results are the mean of three replicates with standard deviation

Discussion

This study discovered a novel β-1, 4-glucosidase, bgl T2, and its gene from Aspergillus fresenii depending on the results of high-throughput sequencing of mRNA. When the full length of bgl T2 mRNA was assembled, its ORF was amplified from the genomic DNA of A.fresenii to confirm the accuracy of the assembled bgl T2 mRNA. It turned out that the bgl T2 mRNA sequence obtained by high-throughput sequencing was solid. Furthermore, the bgl T2 was heterologous expressed by K. phaffii X33, and the enzymatic characters of bgl T2 were determined. Mastering this method to acquire a new functional enzyme was pretty reliable based on the success in this study. Comparing the traditional method to obtain a new enzyme and its gene, the route that this study took appears to have more guarantee. Traditionally, to uncover a new enzyme and its gene sequence requires the enzyme purification, isolation, and identification to initialize the enzyme discovery (Liu et al. 2012; Jeya et al. 2010), which seems difficult to achieve. Without knowing the nature of the new enzyme, purification and isolation of the new enzyme normally requires several attempts through very complicated steps using combinations of various chromatographic columns (Yan et al. 2008; Kim et al. 1992), which is a time consuming, costly, and risky process. Identification of a new enzyme through LC–MS/MS may provide partial peptide sequences of the enzyme, which would be the basic knowledge for the enzyme gene cloning. As an amino acid could share multiple codon, the primers design to amplify the full-length gene of the new enzyme would sometimes be problematic. On the other hand, this study obtained the mRNA sequence directly by the high-throughput sequencing, which made the gene cloning much more convenient. Even though, a partial mRNA sequence of the new enzyme would normally be found by the high-throughput sequencing instead of the full length mRNA, using the information of partial mRNA sequence to clone the new enzyme gene is easier than doing it based on partial peptide sequence. To uncover new enzymes under the help of high-throughput sequencing is not like the transcriptome resequencing study that requires at least 3 replicates, but a sufficient clean reads quantity. For the case of this study, 6 GB clean reads were enough. The approach this study applied seems to be a more general solution for enzyme discovering work.

Since the genome of A. fresenii is not yet reported, this study shall be the first one to uncover the characters and the encoding sequence of bgl T2. Although bgl T2 having 91% identity to the β-glucosidases of Aspergillus steynii IBT 23096 (XP_024702113.1), it still should be considered as a novel β-glucosidases, because the β-glucosidases of A. steynii is merely a putative one that is not confirmed yet. Fibronectin type III-like domain is often found in association with GH3 N-terminal domain and GH3 C-terminal domain. This is also found in bgl T2, although its function is as yet unknown.

Larger molecular mass is usually found with K. phaffii expression due to the protein post-translational modification, for instance, glycosylation and phosphorylation. One N-linked glycosylation site in protein would contribute 1–3 kDa extra weight by K. phaffii. The difference of the bgl T2 theoretical molecular mass and heterologous expressed bgl T2 was about 35 kDa, while there are 12 N-linked glycosylation sites in bgl T2, which may give about 12–36 kDa larger molecular mass by K. phaffii. Thus, the larger molecular mass of heterologous expressed bgl T2 seems to be mostly caused by the glycosylation of the yeast.

bgl T2 stands in the same line of many other β-1, 4-glucosidases for its optimal catalytic conditions. β-1, 4-glucosidases are commonly seen that the optimal catalytic pH range is from 4.0 to 6.0 (Leite et al. 2007; Joo et al. 2010). Most of β-1, 4-glucosidases in GH3 originated from fungi have the optimal temperature between 50 and 65 °C, which fits the case of bgl T2 that has the optimal temperature at 55 °C. For the kinetic properties, the Km of bgl T2 is lower, which means it has higher affinity to substrates. The main properties comparison of bgl T2 with otherβ-1, 4-glucosidases is presented in Table 3.

Table 3.

Properties of β-glucosidase from various fungi

Mr (kDa) Km (mM) Opt. pH Opt. Temp. (°C) Vmax (U mg−1) References
Aspergillus niger 105 21.70 5 55 124.4 Yan and Lin (1997)
Aspergillus oryzae 110 Not reported 5.5 50 Not reported Tang et al. (2014)
Aspergillus oryzae 77 0.74 5.0 60 19.4 Zhang et al. (2007)
Aspergillus oryzaea 90 2.91 4.5 55 0.138b Jing et al. (2013)
Protoplast fusant of Aspergillus oryzae and Aspergillus niger 125 0.04 5.4 65 215.2 Zhu et al. (2010)
Ceriporiopsis subvermispora 110 3.29 3.5 60 0.113b Magalhães et al. (2006)
Phanerochaete chrysosporium 114 0.10 4.0–5.2 Not reported Not reported Lymar (1995)
Daldinia eschscholzii 64 1.52 5.0 50 3.2 Karnchanatat et al. (2010)
Penicillium purpurogenum 110 5.10 5.0 65 934 Jeya et al. (2010)
Aspergillus fresenii 130 0.70 5.5 55 5.4b This study
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aThe properties were determined against cellobiose as the substrate

bThe unit of Vmax was reported as μmol/min

bgl T2 is tolerant to a wide range of the pH value, from pH 3.0 to pH 8.0, which means that bgl T2 might adapt to some applications that have varied pH, such as feed additives. Interestingly, treating bgl T2 under pH 5.0 for 1 h gives it 60% higher activity. It seems that the McIlvaine buffer of pH 5.0 somehow enhances the enzyme protein. This might be because of the favourably crystallization of bgl T2 under pH 5.0 by its dimer(s), being the preferred biological arrangement in the asymmetric unit (Agirre et al. 2016). It is hard to get a convincing explanation as yet.

Metal ions and chemicals may affect the activity of an enzyme. This study tested the effect of eleven common chemicals on the bgl T2. bgl T2 showed a stable property to these chemicals, which only zinc sulphate and cobalt sulphate inhibited about 7% of the bgl T2 activity.

Conclusion

This paper successfully uncovered a novel β-1, 4-glucosidase bgl T2 and its ORF from Aspergillus fresenii under the help of high-throughput sequencing of mRNA technique. This method is more convenient than the traditional method of obtaining a new enzyme and its genetic information, as explained in the discussion section.

The optimized bgl T2 gene was heterologous expressed by K.phaffii X33. The properties of bgl T2 were tested including optimal catalysis pH and temperature, pH tolerance, thermostability, effects of common chemicals, and kinetic properties against pNPG. The low Km of bgl T2 means that bgl T2 has higher affinity to its substrates. Combining with the stability properties of bgl T2, it could be a useful candidate for further applications.

Materials and methods

Strains, vectors, media and chemicals

The Aspergillus fresenii (JCM 01963) was purchased from RIKEN BioResource Center, Japan Collection of Microorganisms. Escherichia coli TOP 10 and K. phaffii X-33 (Invitrogen, USA) were used as host strains. The bgl T2-opt gene was synthesized according to the codon bias of K. phaffii and constructed into the pPICZαA vector (Invitrogen, USA) with the EcoRI and Xba I restriction enzyme sites. The p-nitrophenyl-β-d-glucopyranoside (pNPG) was purchased from Sigma (USA). Other chemicals that are not specifically mentioned are of analytical grade, and are available on commercial supplier.

Enzyme assay

The enzyme activity of β-1, 4-glucosidase was assayed according to the description of Parry et al. (Parry et al. 2001) with some modifications. Using pNPG as the hydrolytic reaction substrate, the quantity of the released p-nitrophenol per minute stands for the activity of β-1, 4-glucosidase. The reaction mixture contained 100 μL of pNPG (10 mM) in McIlvaine buffer, and 100 μL of enzyme solution. After incubating the reaction mixture for 10 min, the reaction was stopped by adding 800 μL of 1.0 M sodium carbonate. The absorbance of the final reaction solution was read at 405 nm, and the reading was calculated to know the amount of p-nitrophenol generated according to the standard graph which prepared under the same conditions.

One β-1, 4-glucosidase enzyme unit (U) was defined as the amount of β-1, 4-glucosidase that released 1 μmol of p-nitrophenol out of pNPG per minute.

Identification of the β-1, 4-glucosidase bgl T2

The A. fresenii (JCM 01963) was cultured on the induction plate (replace the sucrose in Czapek–Dox medium by Avicel PH-101 (Sigma)) and control plate (replace the sucrose in Czapek–Dox medium by glucose) for 7 days. The mycelium of the A. fresenii on both plates were collected. Their RNA were extracted and used to construct the cDNA libraries by standard methods. The libraries were sequenced by Illumina Hiseq 4000, PE150. Clean reads were obtained at least 6 GB for each treatment and assembled by Trinity (Mao et al. 2005). The gene function was predicted by seven databases, GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups), NR (Non-Redundant Protein Database databases), TCDB (Transporter Classification Database), Swiss-Prot, and, TrEMBL. Carbohydrate-Active enzymes were predicted by the Carbohydrate-Active enzymes Database. Through the assembled mRNA result, the cellulases’ full-length mRNA was collected. Their gene expression differences between treatments were analysed by DESeq (Anders and Huber 2010).

The genomic DNA of A. fresenii was extracted as the template to amplify the bgl T2 gene. The primers, designed based upon the assembled mRNA result, were bgl T2-F (5′-ATGAAGTTTGGTTGGTTCGAGGCGGCG-3′) and bgl T2-R (5′-TTAAACCACCACGGGCAACGAGCCCTG-3′). The PCR mixture contained 5 μl of 5 × HF buffer, 4 μl of 10 mM dNTPs mix, 0.5 μg of genomic DNA of A. fresenii, 0.5 μL of 10 pmol/μL each primers, and 0.5 U of Phusion DNA polymerase (Thermo Scientific) in a total volume of 50 μL. The conditions and procedures of the bgl T2 gene amplifications were set as follows: one initialization step at 98 °C for 5 min, 30 times of the amplification cycles (denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 2 min), and one final elongation step at 72 °C for 10 min. The DNA sequencing of amplification products were detected by chain-termination methods (Sanger and Coulson 1975). The results were compared within National Center for Biotechnology Information (NCBI) databases as well as the assembled mRNA result for similarity analysis and intron detection.

Once the encoding sequence of bgl T2 was confirmed, its amino acids sequence was also compared within NCBI protein BlastX for the similarity study (Altschul et al. 1990).

Construction of the expression plasmid and strain

The bgl T2 coding sequence (CDS) was optimized according to the code bias of K.phaffii, and it was synthesized with a 6 × His-tag and a restriction site of EcoRI at 5′ end while a restriction site of Xba I at 3′ end. Plasmid of pPICZαA and the synthesized optimized bgl T2 sequence were double digested with EcoRI and Xba I. The digested product was purified and ligated as bgl T2 opt-pPICZαA recombinant plasmid. The recombinant plasmid of bgl T2-opt-pPICZαA were transformed into TOP 10 E. coli competent cells by chemical methods (Green and Sambrook 2012). A zeocin-resistant colony harboring the recombinant plasmid was confirmed by sequencing, and it was used to reproduce the recombinant plasmid. 5 μg of the recombinant plasmid was linearized with Sac I, purified, and transformed in K.phaffii X-33 strain by electroporation. The positive expression strains were selected by their zeocin-resistance (1000 μg/ml).

Expression bgl T2 by recombinant K.phaffii X-33 and purification

The recombinants K.phaffii X-33 were induced by methanol to express bgl T2 in flask according to the EasySelect™ Pichia Expression Kit manual (invitrogen™). The recombinant that presented highest β-1, 4-glucosidase activity, called bgl T2-7, was used for follow up study.

bgl T2 was also expressed in a 5 L fermentation vessel to exam its productivity. After a conventional enrichment phase, the fermentation conditions were set at 30 °C and 350 rpm, while the dissolved oxygen in the fermentation vessel was maintained at 20–60% by methanol supplement during 96 h inducement phase.

The supernatant of bgl T2-7 fermentation was purified by Ni–NTA magnetic beads. The purified bgl T2 enzyme activity was tested, and its protein concentration was determined by the BAC protein assays kit (ThermoFisher scientific, USA).

Character study of bgl T2

The activities of bgl T2 were tested toward pNPG, CMC-Na, pNPC, and Avicel. The pNPG was used as the substrate for its character study.

The optimal pH of bgl T2 was performed on every 0.5 pH in a range of pH 2.5 to pH 8.0 with McIlvaine buffer. The optimal temperature of bgl T2 was performed at every 5 °C from 25 to 80 °C. For examining the bgl T2 resistance against different pH, ultra-filtrated bgl T2 was treated in McIlvaine buffer from pH 2.5 to pH 8.0 for one hour, and then diluted 100 times by the pH 5.5 Mcllvaine buffer, while the control bgl T2 was mixed with relative McIlvaine buffer and diluted right before the enzymatic activity residue assay. The thermostability of bgl T2 was tested at five time points to calculate the its half-life under 50 °C, 55 °C, 60 °C, and 65 °C. bgl T2 mixed with final concentration of 10 mM of tested chemicals to understand the effects toward bgl T2 activity. Determining the activity of bgl T2 against 2.5 mM, 2 mM, 1.5 mM, and 1.2 mM pNPG under optimal reaction conditions, the Km and Vmax were calculated according to Eadie-Hofstee plots (Hofstee 1952).

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 17 kb) (17.6KB, docx)
Supplementary material 2 (DOCX 594 kb) (594.9KB, docx)
Supplementary material 3 (DOCX 16 kb) (16.4KB, docx)
Supplementary material 4 (DOCX 16 kb) (16.2KB, docx)

Acknowledgements

Appreciate Austin James FAUST and Paul DELMAIN for correcting this paper’s writing.

Author contributions

YY did most of the experiments, data analysis, and the manuscript writing. JW quantified the bgl T2 protein. HG helped the SDS-PAGE. YC supervised this study. All authors read and approved the manuscript.

Funding

This research was financially supported by funds of National Natural Science Foundation of People’s Republic of China (no. 31572437).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Contributor Information

Yongzhi Yang, Email: B20153040151@cau.edu.cn.

Jian Wang, Email: S20173040498@cau.edu.cn.

Henan Guo, Email: ghn_657@cau.edu.cn.

Yunhe Cao, Email: caoyh@cau.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1 (DOCX 17 kb) (17.6KB, docx)
Supplementary material 2 (DOCX 594 kb) (594.9KB, docx)
Supplementary material 3 (DOCX 16 kb) (16.4KB, docx)
Supplementary material 4 (DOCX 16 kb) (16.2KB, docx)

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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