Heterologous expression of a novel β-1, 4-glucosidase originated from Aspergillus fresenii and its enzymatic characters

Background: β-1, 4-glucosidases play important roles in the degradation of lignocellulosic biomass. It helps generate glucose from cellobiose and oligosaccharides, which could enhance the productivity in biorefinery and bioconversion process for energy and chemicals. Discovering novel β-1, 4-glucosidases can provide broader possibilities and understanding. The purpose of this study was to find a new β-1, 4-glucosidase in Aspergillus fresenii by an efficient method based on the high throughput sequencing technique. Results: With the high throughput sequencing technique, a novel β-1, 4-glucosidase, named bgl T2, was cloned from Aspergillus fresenii , which was 2586 bp encoding 862 amino acid residues based on the sequencing analysis. Its amino acid sequence 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 β-glucosidase bgl T2 gene was optimized according to the codon bias of Komagataella phaffii (≡ Pichia pastoris (nom. illeg.)) and synthetized. The optimized bgl T2 gene was inserted into plasmid pPICZαA, and transformed into K.phaffii X33 for its heterologous expression and enzymatic characters determination. The heterologous expressed β-glucosidase bgl T2 presented the highest activity at 55 °C and pH 5.5. When bgl T2 treated in citric acid- disodium hydrogen phosphate buffer (from pH 2.5 to pH 8.0) for one hour, the enzymatic activity was stable for pH 3.0 to pH 8.0 treatment, while the enzymatic activity dropped down to 22% with the

Background β-1, 4-glucosidase (bgl; EC 3.2.1.21) catalyzes 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 [1,2]. Cellulase enzymes, as the most important and costly part of the generating glucose from lignocellulose biomass [3,4], are always interested to be understood, especially a novel one.
β-1, 4-glucosidase exists in plenty of organisms, which gives it a variety properties from each other.
The study of β-1, 4-glucosidase started as early as in 1837, found by Wöhler and Liebig from almond emulsion [5]. Through years of studying, plentiful β-1, 4-glucosidase were discovered [6]. Acquiring a new β-1, 4-glucosidase might help with the treatments of renewable agricultural, industrial and municipal cellulosic wastes for biofuels, chemicals, or animal feed. The conflict between humans and animals for consuming food is becoming critical with the growing population. Alternative feed material is required to subdue this problem. Glucose obtained from lignocellulosic biomass might be an energy source for animals if the lignocellulose biomass wastes digest properly [7,8]. Since monogastric animals lack the capacity to convert cellobiose into glucose, β-1, 4-glucosidase must play important role in the lignocellulose biomass digestion process.
This study focused on a novel β-1, 4-glucosidase (bgl T2) that predicted by the result of the high throughput sequencing for mRNA. To understand the potential of bgl T2 for further utilization, it was heterologous expressed by K.phaffii. The bgl T2 was identified as a member of glycoside hydrolase family 3 (GH3) and its properties were determined. This study broadened the knowledge of β-1, 4glucosidase from A.fresenii and provided a candidate for applications related to cellulose degradation.

Results
Identification of the β-1, 4-glucosidase bgl T2 According to the result of mRNA high throughput sequencing, assembled bgl T2 mRNA (GenBank accession number: MK986475) contain 2586 nucleotides including initiation codon and termination codon. Thus, the heterologous expressed bgl T2 contains 861 amino acids with a predicted molecular mass of 93.55 kDa, and had a theoretical pI of pH 5.11 disregarding the 6 × His-tag [9] Aspergillus aculeatus (P48825.1), and Aspergillus fumigatus A1163 (B0XPE1.1), respectively. The alignment of amino acid sequences among these β-glucosidases was presented in Fig. 1.

Construction of the expression plasmid and strain
The optimized bgl T2 coding sequence for K.phaffii (MK965547) and original bgl T2 sequence were presented in Supplementary file 2. 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. .09% in order to avoid rare codons in K.phaffii.
The optimized bgl T2 coding sequence was successfully inserted into pPICZαA plasmid and 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). They were induced in flask by methanol under 28 °C with 250 rpm. The recombinant K.phaffii labeled bgl T2-7 gave the highest β-1, 4-glucosidase activity.
Thus, the optimized bgl T2 coding sequence were successfully recombined with the K.phaffii X-33 genome.
Although the predicted theoretical molecular mass of bgl T2 was 93.55 kDa, the heterologous expressed protein appeared a band of approximately 130 kDa (Fig. 2).
Character study of bgl T2 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 pH5.5. There was a mere 2% difference between pH 5.0 and pH 5.5 for the bgl T2 relative activity. 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 °C to 55 °C, the bgl T2 activity increased gradually, while it dropped down dramatically started from 55 °C to a higher temperature. The dynamic graph between pH and bgl T2 activity was presented in Fig. 3, while the relationship between temperature and its activity was presented in Fig. 4.
When the bgl T2 treated in a range of pH 3.0 to pH 8.0 at 4 °C for one hour, bgl T2 kept stable. Its activity became the same or even more than the controls (Fig. 5). The treatment of pH 5.0 gave 60% more relative activity than the control. Treating bgl T2 in pH 2.5 for one hour resulted in residue activity drop down to 22%.
The half-life 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 1). The K m and V max of bgl T2 against pNPG were 0.0007 mol/L and 9 × 10 − 8 mol/L/s, respectively. Table 1 Effects of eleven chemicals on the activity of bgl T2.

Chemicals
Relative

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 its characters 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 show more of guarantee.
Traditionally, having a new enzyme and its gene sequence requires the enzyme purification, isolation, and identification to initialize the enzyme discovery [10,11], 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 [12,13], which is a time consuming, costly, and risky process. Identification of a new enzyme through LC-MS/MS may present 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, the case of this study got the mRNA sequence directly by the high throughput sequencing, which made the gene cloning much more convenient. Normally, a partial mRNA sequence of the new enzyme would be found by the high throughput sequencing instead of the full length mRNA. Using the information of partial mRNA sequence to cloning the new enzyme gene is easier than having it by a partial peptide sequence. To uncover new enzyme under the help of high throughput sequencing is not like the transcriptome resequencing study that require at least 3 replicates, but a sufficient clean reads quantity. For the case of this study, 6 GB clean reads were enough.
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 βglucosidases of Aspergillus steynii IBT 23096 (XP_024702113.1), it still should be considered as a novel β-glucosidases, because the β-glucosidases of Aspergillus steynii is merely a putative one that is not confirmed yet. 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 the optimal catalytic pH range from 4.0 to 6.0 [14,15]. Most members of β-1, 4-glucosidases in GH3 originated from fungi have the optimal temperature between 50 °C to 65 °C, which fit the case of bgl T2 who has the optimal temperature at 55 °C. For the kinetic properties, the K m of bgl T2 is lower, which means it has higher affinity to substrates. Table 2 present the comparison of some main properties of bgl T2 with other β-1, 4glucosidases. It is hard to get a convincing explanation.
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 to obtain 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.

Strains, vectors, media and chemicals
The

Enzyme assay
The enzyme activity of β-1, 4-glucosidase was assayed according to the description of Parry et al. [25] with some modifications. Using pNPG as the hydrolytic reaction substrate, the release of p-nitrophenol

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 broken down into small fragments. Their cDNA were synthesized by reverse transcriptase reactions using random hexamers and double-stranded DNA were synthesized by polymerase chain reaction (PCR). After purification, selection, and amplification of their doublestranded DNA obtained from mRNA, the library for high throughput sequencing was constructed. The library was sequenced by Illumina Hiseq 4000, PE150. Clean reads were obtained at least 6GB for each treatment and assembled by Trinity [26].
The genomic DNA of A. fresenii was extracted as the template to amplify the bgl T2 gene. The primers Once the encoding sequence of bgl T2 was confirmed, its amino acids sequence was also compared within NCBI protein BlastX for the similarity study [28].

Construction of the expression plasmid and strain
The bgl T2 coding sequence (CDS) was optimized according to the code bias of (K.phaffii) and synthesized with a 6×His-tag and a restriction sites of EcoRI at 5' end while a restriction sites of Xba I at 3' end. Plasmid of pPICZαA and optimized bgl T2 coding sequence were double digested with EcoRI and Xba I. The digested products were 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 [29]. A zeocin-resistant colony was confirmed by sequencing harboring the recombinant plasmid and 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 zeocin-resistance (1000 μg/ml The supernatant of bgl T2-7 fermentation was collected by centrifugation. It 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 expression culture was centrifuged at 12,000 rpm (13,105 g, rcf

Declarations
Ethics approval and consent to participate  the dynamic graph between pH and bgl T2 activity Results are the mean of three replicates. Figure 4 the dynamic graph between temperature and bgl T2 activity Results are the mean of three replicates.