Production Optimization of an Active <span class="nowrap"><svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-4.5269pt" id="M1" height="16.7875pt" version="1.1" viewBox="-0.0657574 -12.2606 10.1388 16.7875" width="10.1388pt"><g transform="matrix(.017,0,0,-0.017,0,0)"><path id="g113-224" d="M558 587C558 666 497 712 432 712C379 712 330 691 284 650C212 586 178 508 148 348L71 -65C49 -185 35 -229 23 -235L27 -261C49 -259 101 -251 124 -224C131 -216 138 -178 159 -24C171 66 197 200 227 356C264 550 295 668 393 668C443 668 479 632 479 575C479 516 446 458 383 418C361 404 344 398 318 397L296 350C395 338 460 281 460 192C460 110 411 40 300 40C258 40 215 55 192 69L181 51C191 18 222 -16 266 -16C308 -16 351 1 397 26C471 67 545 142 545 221C545 315 486 365 401 395C469 437 558 498 558 587Z"/></g></svg>-</span>Galactosidase of<i> Bifidobacterium animalis</i> in Heterologous Expression Systems

Xu, Xinxin; Fan, Xiaohu; Fan, Chao; Qin, Xing; Liu, Bo; Nie, Chunming; Sun, Ning; Yao, Qingzhi; Zhang, Yuhong; Zhang, Wei

doi:https://doi.org/10.1155/2019/8010635

BioMed Research International

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Abstract Introduction Materials and Methods Results Discussion Conclusions Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2019 | Article ID 8010635 | https://doi.org/10.1155/2019/8010635

Production Optimization of an Active -Galactosidase of Bifidobacterium animalis in Heterologous Expression Systems

Xinxin Xu,¹Xiaohu Fan,^1,2Chao Fan,¹Xing Qin,¹Bo Liu,¹Chunming Nie,¹Ning Sun,¹Qingzhi Yao,²Yuhong Zhang,¹and Wei Zhang¹

Academic Editor: Sherry L. Mowbray

Received10 Jul 2018

Revised31 Dec 2018

Accepted25 Jan 2019

Published20 Feb 2019

Abstract

β-Galactosidase (E.C.3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose and the synthesis of galacto-oligosaccharides as well. The β-galactosidases from bacteria, especially lactobacilli, and yeast have neutral pH and are much more likely to be developed as food additives. However, the challenges of cumbersome purification, product toxicity, and low yield in protein production have limited the commercialization of many excellent candidates. In this study, we identified a β-galactosidase gene (bg42-106) in Bifidobacterium animalis ACCC05790 and expressed the gene product in Escherichia coli BL21(DE3) and Pichia pastoris GS115, respectively. The recombinant bG42-106 purified from E. coli cells was found to be optimally active at pH 6.0 and 60°C and had excellent stability over a wide pH range (5.0–8.0) and at high temperature (60°C). The specific activity of bG42-106 reached up to 2351 U/mg under optimal conditions. The galacto-oligosaccharide yield was 24.45 g/L after incubation with bG42-106 at 60°C for 2 h. When recombinant bG42-106 was expressed in Pichia pastoris GS115, it was found in the culture medium but only at a concentration of 1.73 U/ml. To increase its production, three strategies were employed, including codon optimization, disulfide formation, and fusion with a Cherry tag, with Cherry-tag fusion being most effective. The culture medium of P. pastoris that expressed Cherry-tagged bG42-106 contained 24.4 U/mL of β-galactosidase activity, which is 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106.

1. Introduction

The β-galactosidase (EC 3.2.1.23) is an enzyme that breaks the β-1,4-D-galactosidic linkages of lactose to produce glucose and galactose. This enzymatic action makes it a food supplement to make lactose-free milk special for the lactose-intolerant people, which comprise more than 50% of the world’s population [1]. Moreover, the enzyme has transglycosylation activity to synthesize galacto-oligosaccharides, the prebiotics to stimulate the growth and/or activity of beneficial bacteria in the colon [2]. Besides the application in food industry, β-galactosidase is attracting much attention owing to its ability to synthesize β-galactosyl derivatives that are involved in many biological processes [3]. Thus β-galactosidase represents a high-value biocatalyst, and its mass production is of importance for the commercial purpose.

Commercial β-galactosidases are mainly from microorganisms, especially from yeast and bacteria. Of them, Kluyveromyces lactis represents the major microbial source due to the high lactose hydrolysis activity of its β-galactosidase in milk. However, K. lactisβ-galactosidase has poor thermal and pH stabilities [4, 5] and is not a secreted protein, which means that its large-scale application is hampered by the high purification cost. Therefore, identification of β-galactosidases that have good stability and excellent catalytic properties is of practical significance. β-Galactosidases from many different species, including Aspergillus niger [6], Thermus sp. [7], Lactobacillus reuteri [8], and Bifidobacteria infantis [9], have been found. The excellent stability under a wide range of conditions and substantial catalytic activity make Bifidobacteriaβ-galactosidase a compelling candidate for application in food industry. The only bottleneck is its intracellular enzyme in nature, and its removal would present a further stage of cells disruption and purification, which are a costly process. Production of Bifidobacteriaβ-galactosidase in a recombinant expression system, e.g., Escherichia coli or Pichia pastoris, may overcome these limitations.

For the study reported herein, bg42-106 from Bifidobacterium animalis ACCC05790, which encodes a β-galactosidase with advantageous properties, was cloned and expressed in E. coli and P. pastoris. To improve gene expression and recombinant protein secretion in P. pastoris, several strategies were employed, including codon optimization and coexpression with a protein disulfide isomerase and fusion with a Cherry tag. Among these, codon optimization is widely and successfully used to increase the expression levels of foreign proteins in P. pastoris [10]. Protein disulfide isomerase is an endoplasmic reticulum-associated molecular chaperone involved in the rearrangement of incorrect disulfide pairings via its isomerase activity and also facilitates folding, assembly, and posttranslational modifications of eukaryotic proteins in general [11]. Several studies have shown that coexpression of pdi in P. pastoris improves heterologous protein expression [12, 13]. The commercial Cherry tag is a portion of the cytochrome heme-binding domain that can increase the solubility of the tagged protein. Our results showed that all these strategies increased the yield of B. animalisβ-galactosidase, with Cherry-tag fusion being most effective at protein secretion level.

2. Materials and Methods

2.1. Strains and Growth Conditions

B. animalis ACCC05790 from the Agricultural Culture Collection of China (Beijing, China) was grown anaerobically at 37°C in deMan, Rogosa and Sharpe (MRS) medium (2% glucose, 1% peptone, 1% meat extract, 0.5% yeast extract, 0.5% sodium acetate, 0.2% K₂HPO₄, 0.2% diammonium citrate, 0.1% (v/v) Tween 80, 0.02% MgSO₄·7H₂O, and 0.005% MnSO₄·H₂O, pH 6.2). E. coli strains TOP10 (TransGen, Beijing, China) and BL21(DE3) (Novagen, Darmstadt, Germany) were cultured in Luria-Bertani (LB) medium (0.5% yeast extract, 1% tryptone, and 1% NaCl) containing 100 μg/mL ampicillin. Saccharomyces cerevisiae and P. pastoris GS115 were cultivated at 30°C in yeast extract/peptone dextrose medium (2% peptone, 2% glucose, and 1% yeast extract). Buffered glycerol complex medium, buffered methanol complex medium, regeneration dextrose medium, minimal dextrose medium, and minimal methanol medium were prepared according to manuals of Pichia expression kits (Invitrogen, Carlbad, CA).

2.2. Plasmids, Enzymes, and Chemicals

pEASY-T3 (Transgen) was used for gene cloning. pET-30a(+) (Novagen), pPICZA (Invitrogen), and pPIC9 (Invitrogen) served as expression vectors. Restriction and other enzymes used for DNA manipulations were obtained from TaKaRa (Dalian, China). Chemicals and reagents for high-performance liquid chromatography (HPLC) were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. o-Nitrophenyl-β-D-galactopyranoside (oNPG) was purchased from AppliChem (Gatersleben, Germany).

2.3. Cloning of the β-Galactosidase Gene bg42-106

Standard procedures for DNA extraction, plasmid isolation, restriction-enzyme digestion, and ligation were performed as described [26]. Bacterial β-galactosidase sequences were retrieved from GenBank by an Entrez search (http://www.ncbi.nlm.nih.gov/Entrez/); consensus regions in an alignment of these sequences were identified by ClustalW [27] and used to design the degenerate primers bG42F and bG42R (Table 1).

The core region of bg42-106 was obtained via touchdown PCR with the B. animalis genomic DNA (50 ng) as the template and primers bG42F and bG42R (final concentrations, 5 μM each primer). The PCR program was 94°C for 3 min; 10 cycles of 94°C for 30 s, 55°C for 30 s with a decrease of 1°C per cycle, and 72°C for 30 s; 30 cycles at 94°C for 30 s, 45°C for 30 s, and 72°C for 30 s. The gene fragment was purified using the reagents of a gel extraction kit (Tiangen, Beijing, China) and ligated into pEASY-T3 vectors, which were individually transformed into E. coli TOP10. Positive transformants were screened on LB agar plates containing 0.8 mg/mL X-gal, 3 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and 100 μg/mL ampicillin for 16 h at 37°C. White colonies containing the bg42-106 fragment were confirmed by DNA sequencing. Based on the known sequence, the up- and down-stream flanking regions of bg42-106 were obtained with genome-walking thermal asymmetric interlaced (TAIL)-PCR [28] with specific primers Dsp1, Dsp2, Dsp3, Usp1, Usp2, and Usp3 (Table 1).

2.4. Sequence Analysis

Verification of the nucleotide and deduced amino acid sequences, an open-reading frame search, multiple sequence alignment, and sequence assembly were performed using Vector NTI 10.3 software. The sequences of the DNA fragments obtained by touchdown PCR were compared with those of known β-galactosidases in GenBank by BLASTx. SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP) was used to determine if bg42-106 contained a signal sequence.

2.5. Expression and Purification of Recombinant bg42-106 in E. coli

Primers bG42-106f and bG42-106r (Table 1) harboring the restriction sites EcoRV and NotI were used to amplify the full-length bg42-106 from B. animalis genomic DNA. The PCR product was purified, enzyme digested, and inserted into pET-30a(+) to construct the recombinant plasmid pET30-bG42-106, which was further transformed into chemically competent E. coli BL21(DE3) cells by heat-shock at 42°C. Positive E. coli cells were cultured at 37°C in LB medium, 50 μg/mL kanamycin until the of the culture reached 0.6. The cells were then induced with 0.4 mM IPTG at 28°C for an additional 4 h. Recombinant bG42-106 carried a C-terminal (His)₆ tag.

The cells were harvested by centrifugation (12,000 × g, 5 min, 4°C), washed with 50 mM sodium phosphate (pH 6.5), and disrupted by ultrasonication. Cell debris was removed by centrifugation (12,000 × g, 10 min, 4°C), and the crude enzyme extract was loaded onto a Ni²⁺-NTA affinity column (Qiagen, Hilden, Germany) that had been equilibrated with start buffer (20 mM sodium phosphate pH 6.5, 500 mM NaCl, and 5 mM imidazole). The protein was eluted at 1 mL/min with elution buffer (20 mM sodium phosphate pH 6.5, 500 mM NaCl, and 200 mM imidazole). Fractions containing β-galactosidase activity were pooled, desalted, and concentrated.

Protein concentration was determined using Bradford protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. The contents of total cellular protein and purified bG42-106 were examined by SDS-PAGE (12%, w/v). To estimate the native molecular mass, purified bG42-106 was detected in an 8% (w/v) nondenaturing PAGE. The activity was then examined by incubating the gel in an X-gal (4 mg/mL) solution.

2.6. β-Galactosidase Activity Assay

The β-galactosidase activity was monitored with oNPG hydrolysis at . Reactions contained 200 μL of enzyme solution and 800 μL of substrate (0.25% (w/v) oNPG in 100 mM Na₂HPO₄-citric acid, pH 6.0). Each reaction was incubated at 60°C for 15 min and terminated by addition of 1 mL of 10% (w/v) trichloroacetic acid and 2 mL of 1 M Na₂CO₃. One unit of β-galactosidase activity was defined as the amount of enzyme that liberated 1 μmol of o-nitrophenol per min. The specific activity of the enzyme was expressed as units/mg protein.

2.7. Characterization of Recombinant bG42-106 from E. coli

To determine the pH optimum of bG42-106, its activity was measured at 60°C between pH 3.0 and 7.0 in 100 mM Na₂HPO₄-citric acid, pH 7.0 and 9.0 in 100 mM Tris-HCl, and pH 9.0 and 10.0 in 100 mM Na₂CO₃-NaHCO₃. To measure its pH stability, the enzyme was first incubated in the aforementioned buffers at 37°C for 60 min and then assayed under standard conditions (pH 6.0, 60°C, 15 min). To determine the optimal temperature for enzyme activity, reactions were measured between 30 and 75°C at optimal pH for 15 min. The effect of temperature on stability was measured by incubating bG42-106 at 60°C or 65°C. Samples were removed at various times and assayed under standard conditions. To determine the half-life of bG42-106, the enzyme was incubated at 60°C, and activity was determined periodically for 72 h under standard conditions.

The effects of various reagents on enzyme activity were examined by assaying the enzyme in the presence of 1 or 10 mM of Na⁺, K⁺, Ca²⁺, Cu²⁺, Mn²⁺, Co²⁺, Cd²⁺, Fe²⁺, Ni²⁺, Mg²⁺, Zn²⁺, Pb²⁺, Ag⁺, Triton X-100, Na₂EDTA, SDS, or CTAB in 100 mM Na₂HPO₄-citric acid, pH 6.0. Enzyme activity measured in the absence of any reagent was defined as 100%. Kinetic parameters (, , and ) were obtained from the Michaelis-Menten equation using GraphPad Prism software. The substrate (oNPG) concentration ranged from 1.38 to 25 mM. All experiments were carried out at 60°C and pH 6.0 in triplicate.

2.8. Lactose Hydrolysis and Galacto-Oligosaccharide Synthesis by Recombinant bG42-106 from E. coli

To assess the extent of lactose hydrolysis by bG42-106, reactions were performed at 50°C or 60°C in 100 mM Na₂HPO₄-citric acid (pH 6.0) containing 200 g/L lactose and purified bG42-106 for various times and terminated by heating for 10 min at 100°C. Formation of galacto-oligosaccharides and other saccharides was quantified by HPLC through a 6.5 × 300 mm Sugar-Pak I column (Waters, Milford, MA). This column can separate oligosaccharide molecules of the same size but different linkage type. Samples were eluted in 50 mg/mL CaNaEDTA at 500 μL/min and 85°C. The yield of galacto-oligosaccharides (g/L) was calculated as [GOS] = [] – [] – [] – [] [29], in which GOS is the yield of galacto-oligosaccharides, is the initial amount of lactose, is the final amount of lactose, is the final yield of glucose, and is the final yield of galactose.

2.9. Expression of bg42-106 in P. pastoris

Restriction enzyme-digested bg42-106 as described above was ligated into pPIC9 to form the recombinant plasmid pPIC9-bG42-106, which was then transformed into E. coli TOP10 to maintain the plasmid. The construct pPIC9-bG42-106 was linearized by BglII and then transformed into P. pastoris GS115 competent cells by electroporation. Transformed cells were selected according to the protocols in the Pichia expression kit manual (Invitrogen). Recombinant P. pastoris cells were incubated in 25 mL of buffered methanol complex medium at 30°C, followed by 48-h induction with 0.5% methanol. The culture supernatant was assayed for β-galactosidase activity, and the total protein in the culture medium was subjected to SDS-PAGE analysis. After disrupting cell integrity by grinding the cells in liquid nitrogen by metallic bead, intracellular β-galactosidase activity was also measured. The P. pastors strain containing the highest β-galactosidase was denoted as wild-type GS115/ bG42-106.

2.10. Codon Optimization and Expression of Codon-Optimized bg42-106m

To improve bg42-106 expression in P. pastoris, low-usage (<15% frequency) codons were replaced with the common ones found in the P. pastoris genome [30]. In addition, the G+C content of bg42-106 was adjusted to be similar to those of highly expressed P. pastoris genes. The optimized gene, denoted as bg42-106m, was synthesized by AugCt (Beijing, China). bg42-106m was ligated into pPIC9 and expressed in P. pastoris as described above. Intra- and extracellular β-galactosidase activities were assayed. The yeast strain containing bg42-106m was denoted as GS115/bG42-106m.

2.11. Coexpression of scpdi in P. pastoris GS115/bG42-106

The gene coding for the protein disulfide isomerase of Saccharomyces cerevisiae (scpdi) was PCR amplified with FastPfu DNA polymerase (Transgen) using S. cerevisiae genomic DNA as the template and primers ScPDIf and ScPDIr (Table 1). The PCR product was purified and ligated into pPICZA to form pPICZA-scpdi, which was transformed into E. coli TOP10 competent cells for sequencing. Recombinant pPICZA-scpdi was electroporated into P. pastoris GS115/bG42-106. The transformants were screened on yeast extract/peptone dextrose agar plates that contained 100 μg/mL Zeocin. The P. pastoris strain that contained both scpdi and bg42-106 was denoted as GS115/ScPDI-bG42-106. After coexpression of both proteins as described above for bGF42-106 expression, bG42-106 activity was measured as described above.

2.12. Fusion of bg42-106 with a Cherry Tag

Primers CherryF and CherryR (Table 1) were used to clone the Cherry-tag coding sequence in the Cherry Express vector pSCherry1 (Delphi Genetics SA, Gosselies, Belgium). A 15-bp extension homologous to the sequence flanking the multiple cloning site in pPIC9-bG42-106 was added to both ends of the Cherry-tag sequence by PCR. The primer pairs P1 and P2 and P3 and P4 (Table 1) were used to individually amplify pPIC9/bG42-106. The 12- to 19-bp overlaps at the ends of the vector were used as the homologous recombinant arms. Using the CloneEZ recombinant cloning kit (Genscript, Nanjing, China), the Cherry tag and the vectors were fused via the Cherry-tag extensions and the homologous sequences in the vector to yield pPIC9-Cherry-bG42-106. In addition, a KEX2 site (Glu-Lys-Arg Glu-Ala-Glu-Ala, where is the cleavage site) coding sequence was inserted between Cherry and bG42-106 genes for subsequent removal of the Cherry tag during secretion from P. pastoris. The plasmid pPIC9-Cherry-bG42-106 was electroporated into P. pastoris GS115 as described above. β-Galactosidase activity in the culture medium and yeast cells was measured, respectively. The yeast strain containing pPIC9-Cherry-bG42-106 is denoted as GS115/Cherry-bG42-106.

2.13. Nucleotide Sequence Accession Number

The nucleotide sequence for the B. animalis ACCC05790 β-galactosidase gene bg42-106 was deposited in the GenBank database under accession number JX188444.

3. Results

3.1. Cloning and Sequence Analysis of bg42-106

The full-length bg42-106 was obtained from the genomic DNA of B. animalis ACCC05790 through touchdown PCR and TAIL-PCR. The open-reading frame contained 2088 bp that encoded a polypeptide of 695 amino acids and a stop codon. No signal peptide sequence was identified. The calculated molecular mass of bG42-106 was 78 kDa. Sequence comparison indicated that bG42-106 belonged to family 42 of glycosyl hydrolases (GH). The deduced amino acid sequence was 98% and 33% identical to that of a putative β-galactosidase from B. animalis subsp. lactis HN019 (ZP_02963414) and a characterized β-galactosidase from Bifidobacterium adolescentis DSM20083 [16].

3.2. Expression and Purification of Recombinant bG42-106 from E. coli

The recombinant plasmid, pET30-bG42-106, was constructed by cloning bG42-106 into vector pET-30(+) and then transferred into E. coli. After induction with IPTG for 4 h, cells were sonicated, and the intracellular β-galactosidase activity was determined to be ~50 U/mL. The crude enzyme extract was purified to apparent homogeneity with Ni²⁺-NTA affinity chromatography (Figure 1(a)). Purified bG42-106 had an apparent molecular mass of ~80 kDa on SDS-PAGE, which was consistent with the theoretical mass of bG42-106 (78 kDa). The native molecular mass of bG42-106 was also analyzed, and the result showed that purified bG42-106 migrated one single band of ~180 kDa on native PAGE (Figure 1(b)), suggesting that bG42-106 existed as a homodimer in solution. It was higher than the theoretical value, which might be ascribed to the imprecision of the gel electrophoresis.

(a)

(b)

Figure 1

Electrophoresis analysis of recombinant bG42-106 expressed in E. coli. (a) SDS-PAGE profiles of recombinant bG42-106. Lanes: M, the protein molecular mass standards; 1, the lysate of induced E. coli BL2(DE3) harboring empty pET-30a(+); 2, the intracellular protein of uninduced E. coli BL21(DE3) harboring pET-bg42-106; 3, the intracellular protein from E. coli BL21(DE3) harboring pET-bg42-106 with IPTG induction (4 mM and 28°C for 4 h); 4, the proteins eluted in 200 mM histidine wash from the Ni²⁺-NTA affinity resin. The arrow indicates the position of recombinant bG42-106. (b) Activity staining of purified bG42-106 on nondenaturing polyacrylamide gel. Lanes: M, the protein molecular mass standards; 1, purified bG42-106 from E. coli.

3.3. Characterization of Recombinant bG42-106 from E. coli

The pH and temperature optima for bG42-106 activity were pH 6.0 and 60°C, respectively (Figures 2(a) and 2(b)). The enzyme retained almost all of the activity after incubation for 1 h between pH 5.0 and 8.0 at 37°C, but lost activity sharply below pH 4.0 or above pH 9.0 (Figure 2(c)). bG42-106 was thermostable at 60°C, retaining approximately 100% of the activity after 1-h incubation at 60°C and pH 6.0 (Figure 2(d)). The half-life at 60°C was determined to be 53.7 h. After incubation at 65°C for 30 min, the enzyme only retained about 50% activity.

(a)

(b)

(c)

(d)

The effects of various metal ions and reagents on bG42-106 activity were assessed (Table 2). Cu²⁺, Cd²⁺, Fe²⁺, and SDS (each at 10 mM) inhibited the enzymatic activity by 44% to 65%, but only SDS at lower concentration (1 mM) affected the activity. Conversely, 1 mM Pb²⁺ and Ag⁺ completely inhibited the activity. Other metal ions (K⁺, Na⁺, Co²⁺, Zn²⁺, and Ni²⁺) and reagents (CTAB and Na₂EDTA) had no substantial effects on the activity.

With the use of oNPG as the substrate for a steady-state kinetic study, the , and values were found to be 720 μmol/min/mg, 5.14 mM and 186.77 s⁻¹ mM⁻¹, respectively. The specific activity of purified bG42-106 was 2351 U/mg as determined under standard assay conditions.

3.4. Lactose Hydrolysis and Galacto-Oligosaccharide Synthesis of Recombinant bG42-106 from E. coli

The hydrolysis ability of bG42-106 to degrade lactose into galactose and glucose was assessed at two temperatures (50°C or 60°C) and different enzyme concentrations (2 to 40 U/mL) for 24 h. When the initial concentration of lactose was >200 g/L, hydrolysis was nearly complete after 24 h in the presence of 40 U/mL bG42-106 (Figure 3(a)). The rate of lactose hydrolysis was also monitored at different enzyme concentrations for up to 24 h (Figure 3(b)). The lactose concentration decreased from the initial 200 g/L to ~150 g/L at 24 h in the presence of 2 or 4 U/mL of bG42-106. At a concentration of 40 U/mL, however, bG42-106 degraded ~50% of the lactose within 2 h; then, the lactose hydrolysis rate decreased, and nearly ~90% of the lactose was hydrolyzed at 16 h.

(a)

(b)

The production of galacto-oligosaccharides in the presence of different amounts of bG42-106 was also assessed at 60°C (Figure 3(b)). In the presence of 2 or 4 U/ml of bG42-106 and 200 g/L lactose, the amount of galacto-oligosaccharides increased up to 15 g/L at 24 h. The maximal amount of galacto-oligosaccharides (24.45 g/L) was achieved with 40 U/mL bG42-106 at 60°C for 2 h. Thereafter, the amount of galacto-oligosaccharides decreased to almost zero at 24 h. When the bG42-106 concentration was 10 U/mL, a hydrolysis and synthesis balance of galacto-oligosaccharides was obtained, in which the amount of galacto-oligosaccharides was ~10 g/L.

3.5. Effect of Codon Optimization

A total of 451 nucleotides of bg42-106 involving 387 amino acids were replaced according to the optimized codon usage (Supplementary Table 1). The nucleotide sequence identity of wild-type and modified bg42-106 was 77.9%. The G+C content was adjusted from 61.1% to 50.2% to mimic that of the P. pastoris genome. bg42-106 and bg42-106m were expressed in P. pastoris GS115 with methanol induction. The highest enzyme activity found in the culture medium was 1.73 U/mL for bG42-106 and 2.64 U/ml for bG42-106m (Figure 4), respectively, reflecting a 1.5-fold improvement in the yield.

3.6. Effect of Coexpression of ScPDI and bG42-106

ScPDI and bG42-106 were successfully coexpressed in P. pastoris. After induction as described above, the β-galactosidase activity in the culture medium was 3.15 U/mL, 1.8 folds of that of bG42-106 alone. The intracellular β-galactosidase activity for ScPDI-bG42-106 was 9.23 U/ml, which was less than that of bG42-106 (11.97 U/mL) (Figure 4). ScPDI, therefore, acted as a molecular chaperone that may assist bG42-106 folding and subsequent secretion.

3.7. Effect of Fusing a Cherry Tag

A 326-bp Cherry-tag sequence was PCR amplified from pSCherry1 and fused with bg42-106 in vector pPIC9. The Cherry tag was removed from bG42-106 upon secretion. The extracellular activity of Cherry-bG42-106 was 24.4 U/mL, which was 14-fold greater than that for bG42-106. Moreover, the intracellular activity of Cherry-bG42-106 was less than that of its extracellular activity (Figure 4). The Cherry tag, therefore, significantly improved the yield of bG42-106 in P. pastoris. When bG42-106, bG42-106m, and bG42-106 were coexpressed with ScPDI, their intracellular levels were 3- to 7-fold higher than the extracellular ones (Figure 4).

4. Discussion

In this study we identified a β-galactosidase in B. animalis ACCC05790 and successfully produced it in both prokaryotic and eukaryotic expression systems. Compared with the β-galactosidases of various microbial sources that have been characterized (Table 3), bG42-106 under study represents relatively high β-galactosidase activity. Moreover, it has excellent enzyme properties, such as stability over a wide pH range (5.0–8.0) and at high temperature (60°C), and high yield of galacto-oligosaccharides (24.45 g/L). These features are particularly useful for biotechnological and industrial applications.

P. pastoris is well established for the rapid and cost-effective expression of recombinant proteins from discovery through commercialization. Proteins are secreted from P. pastoris under a strictly regulated control system. To improve the yield of bG42-106 in P. pastoris, we employed three strategies including codon optimization, coexpression of a protein disulfide isomerase, and fusion of a Cherry tag. Of them, protein disulfide isomerase has unpredictable effect on heterologous protein secretion from P. pastoris [31]. For example, coexpression of a protein disulfide isomerase increased the secretion of a malaria vaccine candidate (Pfs25) by 2- to 5-fold [32] and increased the yield of a human parathyroid hormone (hPTH) from 127 mg/L to 349 mg/L [33]. Because hPTH contains no cysteine, the protein disulfide isomerase may act as a molecular chaperone instead of a disulfide isomerase. In other studies, however, protein disulfide isomerase had little effect on the expression of targeted protein, and in one instance the production level of an immunoglobulin-binding protein was actually reduced [34]. bG42-106 contains 11 cysteines, some of which might form disulfides and thus require protein disulfide isomerase for formation of disulfide bonds, protein folding, and export from the cell. Thus we coexpressed bg42-106 and a protein disulfide isomerase from S. cerevisiae in P. pastoris and increased the yield of bG42-106 to 1.8 folds. Compared with previous studies, i.e., references [19, 20], the effect of protein disulfide isomerase on bG42-106 is less significant; it may be because this is not the most important limiting factor affecting the secretion of bG42-106 and other strategies are also expected to improve the yield of bG42-106.

Gene fusion, with glutathione S-transferase, maltose-binding protein, as examples, has been used to promote secretion of heterologous proteins in recombinant expression systems. Still, each fusion is assessed empirically, and thus the optimum results for any single case are not necessarily generalizable to other heterologously expressed proteins [35]. The Cherry tag, a red light-producing polypeptide composed of the cytochrome heme-binding domain, has been used to attain high levels of soluble protein in E. coli when fused to the N-terminus of a targeted protein. In some cases, Cherry tags have been used as a cost-efficient tool for protein purification by imparting heat stability [36]. In our study, the Cherry-tag gene was fused with bg42-106 in vector pPIC9. Cherry-bG42-106 was secreted at a higher level, 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106. This favorable secretion level may be a consequence of the relatively high solubility of the Cherry tag itself.

In certain cases the conformation of an expressed protein may preclude its passage through cell membrane, suggesting the need to compare the conformation of the targeted protein with that of proteins secreted naturally. In addition, the expression of exogenous protein in P. pastoris usually depends on the natural host of the target gene; the wild-type unsecreted protein is often difficult to achieve secretion expression in recombinant host. bG42-106 from B. animalis ACCC05790 is an intracellular protein, which may explain its relatively low-level secretion in P. pastoris in the absence of the Cherry tag or another expression “trick” for heterologous proteins.

5. Conclusions

A novel β-galactosidase was identified in B. animalis ACCC05790 and expressed in both prokaryotic and eukaryotic systems. The recombinant protein showed high specific activity and good pH and thermal stability and produced high galacto-oligosaccharide yield. Three strategies including codon optimization, coexpression of a protein disulfide isomerase, and fusion of a Cherry-tag expression were carried out to promote the yield of bG42-106 from P. pastoris. Fusion protein approach improved the secretion level of Cherry-tagged bG42-106.

Data Availability

No data were used to support this study.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors’ Contributions

Xinxin Xu and Xiaohu Fan are equal contributors.

Acknowledgments

The authors sincerely thank Dr. Rui Ma at the Key Laboratory of Feed Biotechnology of the Ministry of Agriculture for her help in English polishing. This work was supported by the Fundamental Research Funds for Central Nonprofit Scientific Institution (Y2018LM02; 1610392018006).

Supplementary Materials

Supplementary Table1. Comparison of the codon usage of bg42-106 and the optimized bg42-106m gene. (Supplementary Materials)

References

R. K. Montgomery, S. D. Krasinski, J. N. Hirschhorn, and R. J. Grand, “Lactose and lactase—who is lactose intolerant and why?” Journal of Pediatric Gastroenterology and Nutrition, vol. 45, no. Suppl 2, pp. S131–S137, 2007.
View at: Publisher Site | Google Scholar
I. Rowland and R. Tanaka, “The effects of transgalactosylated oligosaccharides on gut flora metabolism in rats associated with a human faecal microflora,” Journal of Applied Microbiology, vol. 74, no. 6, pp. 667–674, 1993.
View at: Publisher Site | Google Scholar
L. Lu, M. Xiao, X. Xu, Z. Li, and Y. Li, “A novel β-galactosidase capable of glycosyl transfer from Enterobacter agglomerans B1,” Biochemical and Biophysical Research Communications, vol. 356, no. 1, pp. 78–84, 2007.
View at: Publisher Site | Google Scholar
C. S. Kim, E.-S. Ji, and D.-K. Oh, “Expression and characterization of Kluyveromyces lactisβ-galactosidase in Escherichia coli,” Biotechnology Letters, vol. 25, no. 20, pp. 1769–1774, 2003.
View at: Publisher Site | Google Scholar
Q. Z. Zhou and X. D. Chen, “Effects of temperature and pH on the catalytic activity of the immobilized β-galactosidase from Kluyveromyces lactis,” Biochemical Engineering Journal, vol. 9, no. 1, pp. 33–40, 2001.
View at: Publisher Site | Google Scholar
S. O’Connell and G. Walsh, “A novel acid-stable, acid-active β-galactosidase potentially suited to the alleviation of lactose intolerance,” Applied Microbiology and Biotechnology, vol. 86, no. 2, pp. 517–524, 2010.
View at: Publisher Site | Google Scholar
B. C. Pessela, A. Vian, C. Mateo et al., “Overproduction of thermus sp. Strain T2 β-galactosidase in escherichia coli and preparation by using tailor-made metal chelate supports,” Applied and Environmental Microbiology, vol. 69, no. 4, pp. 1967–1972, 2003.
View at: Publisher Site | Google Scholar
T.-H. Nguyen, B. Splechtna, M. Yamabhai, D. Haltrich, and C. Peterbauer, “Cloning and expression of the β-galactosidase genes from Lactobacillus reuteri in Escherichia coli,” Journal of Biotechnology, vol. 129, no. 4, pp. 581–591, 2007.
View at: Publisher Site | Google Scholar
M. N. Hung and B. H. Lee, “Purification and characterization of a recombinant β-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96,” Applied Microbiology and Biotechnology, vol. 58, no. 4, pp. 439–445, 2002.
View at: Publisher Site | Google Scholar
A.-S. Xiong, Q.-H. Yao, R.-H. Peng et al., “High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris,” Applied Microbiology and Biotechnology, vol. 72, no. 5, pp. 1039–1047, 2006.
View at: Publisher Site | Google Scholar
B. Wilkinson and H. F. Gilbert, “Protein disulfide isomerase,” Biochimica et Biophysica Acta—Proteins and Proteomics, vol. 1699, no. 1-2, pp. 35–44, 2004.
View at: Publisher Site | Google Scholar
Z. Li, A. Moy, S. R. Gomez, A. H. Franz, J. Lin-Cereghino, and G. P. Lin-Cereghino, “An improved method for enhanced production and biological activity of human secretory leukocyte protease inhibitor (SLPI) in Pichia pastoris,” Biochemical and Biophysical Research Communications, vol. 402, no. 3, pp. 519–524, 2010.
View at: Publisher Site | Google Scholar
S. Zhang, H. Fang, L. Zhao et al., “Co-overexpression of PpPDI Enhances Secretion of Ancrod in Pichia pastoris,” Applied Biochemistry and Biotechnology, vol. 164, no. 7, pp. 1037–1047, 2011.
View at: Publisher Site | Google Scholar
S. H. Yi, I. Alli, K. H. Park, and B. Lee, “Overexpression and characterization of a novel transgalactosylic and hydrolytic β-galactosidase from a human isolate Bifidobacterium breve B24,” New Biotechnology, vol. 28, no. 6, pp. 806–813, 2011.
View at: Publisher Site | Google Scholar
P. Katrolia, Q. Yan, H. Jia, Y. Li, Z. Jiang, and C. Song, “Molecular cloning and high-level expression of a β-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris,” Journal of Molecular Catalysis B: Enzymatic, vol. 69, no. 3-4, pp. 112–119, 2011.
View at: Publisher Site | Google Scholar
S. W. Hinz, L. A. van den Broek, G. Beldman, J. Vincken, and A. G. Voragen, “β-Galactosidase from Bifidobacterium adolescentis DSM20083 prefers β(1,4)-galactosides over lactose,” Applied Microbiology and Biotechnology, vol. 66, no. 3, pp. 276–284, 2004.
View at: Publisher Site | Google Scholar
M. Rhimi, N. Aghajari, B. Jaouadi et al., “Exploring the acidotolerance of β-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus: an attractive enzyme for lactose bioconversion,” Research in Microbiology, vol. 160, no. 10, pp. 775–784, 2009.
View at: Publisher Site | Google Scholar
M. Ladero, A. Santos, J. García, A. Carrascosa, B. Pessela, and F. García-Ochoa, “Studies on the activity and the stability of β-galactosidases from Thermus sp strain T2 and from Kluyveromyces fragilis,” Enzyme and Microbial Technology, vol. 30, no. 3, pp. 392–405, 2002.
View at: Publisher Site | Google Scholar
T.-T. Nguyen, H.-M. Nguyen, B. Geiger et al., “Heterologous expression of a recombinant lactobacillal β-galactosidase in Lactobacillus plantarum: Effect of different parameters on the sakacin P-based expression system,” Microbial Cell Factories, vol. 14, no. 1, p. 30, 2015.
View at: Google Scholar
S. Iqbal, T. Nguyen, T. T. Nguyen, T. Maischberger, and D. Haltrich, “β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides,” Carbohydrate Research, vol. 345, no. 10, pp. 1408–1416, 2010.
View at: Publisher Site | Google Scholar
W. Chen, H. Chen, Y. Xia, J. Zhao, F. Tian, and H. Zhang, “Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from bacillus stearothermophilus,” Journal of Dairy Science, vol. 91, no. 5, pp. 1751–1758, 2008.
View at: Publisher Site | Google Scholar
M. Moracci, A. La Volpe, J. F. Pulitzer, M. Rossi, and M. Ciaramella, “Expression of the thermostable β-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression,” Journal of Bacteriology, vol. 174, no. 3, pp. 873–882, 1992.
View at: Publisher Site | Google Scholar
J. A. Coker, P. P. Sheridan, J. Loveland-Curtze, K. R. Gutshall, A. J. Auman, and J. E. Brenchley, “Biochemical characterization of a β-galactosidase with a low temperature optimum obtained from an Antarctic Arthrobacter isolate,” Journal of Bacteriology, vol. 185, no. 18, pp. 5473–5482, 2003.
View at: Publisher Site | Google Scholar
R. Karan, M. D. Capes, P. DasSarma, and S. DasSarma, “Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi,” Bmc Biotechnology, vol. 13, no. 1, p. 3, 2013.
View at: Google Scholar
S. Campuzano, B. Serra, D. Llull, J. L. García, and P. Garcí, “Cloning, expression, and characterization of a peculiar choline-binding β-galactosidase from Streptococcus mitis,” Applied and Environmental Microbiology, vol. 75, no. 18, pp. 5972–5980, 2009.
View at: Publisher Site | Google Scholar
J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2nd edition, 1989.
J. D. Thompson, D. G. Higgins, and T. J. Gibson, “CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice,” Nucleic Acids Research, vol. 22, no. 22, pp. 4673–4680, 1994.
View at: Publisher Site | Google Scholar
Y. Liu and R. F. Whittier, “Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking,” Genomics, vol. 25, no. 3, pp. 674–681, 1995.
View at: Publisher Site | Google Scholar
F. Jorgensen, O. C. Hansen, and P. Stougaard, “High-efficiency synthesis of oligosaccharides with a truncated β-galactosidase from Bifidobacterium bifidum,” Applied Microbiology and Biotechnology, vol. 57, no. 5-6, pp. 647–652, 2001.
View at: Publisher Site | Google Scholar
X. Zhao, K. K. Huo, and Y. Y. Li, “Synonymous codon usage in Pichia pastoris,” Chinese Journal of Biotechnology, vol. 16, no. 3, pp. 308–311, 2000.
View at: Google Scholar
D. Mattanovich, B. Gasser, H. Hohenblum, and M. Sauer, “Stress in recombinant protein producing yeasts,” Journal of Biotechnology, vol. 113, no. 1-3, pp. 121–135, 2004.
View at: Publisher Site | Google Scholar
C. W. Tsai, P. F. Duggan, R. L. Shimp, L. H. Miller, and D. L. Narum, “Overproduction of Pichia pastoris or Plasmodium falciparum protein disulfide isomerase affects expression, folding and O-linked glycosylation of a malaria vaccine candidate expressed in P. pastoris,” Journal of Biotechnology, vol. 121, no. 4, pp. 458–470, 2006.
View at: Publisher Site | Google Scholar
R. Vad, E. Nafstad, L. A. Dahl, and O. S. Gabrielsen, “Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone,” Journal of Biotechnology, vol. 116, no. 3, pp. 251–260, 2005.
View at: Publisher Site | Google Scholar
L. M. Damasceno, K. A. Anderson, G. Ritter, J. M. Cregg, L. J. Old, and C. A. Batt, “Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris,” Applied Microbiology and Biotechnology, vol. 74, no. 2, pp. 381–389, 2007.
View at: Publisher Site | Google Scholar
Y. Shih, W. Kung, J. Chen, C. Yeh, A. H. Wang, and T. Wang, “High-throughput screening of soluble recombinant proteins,” Protein Science, vol. 11, no. 7, pp. 1714–1719, 2002.
View at: Publisher Site | Google Scholar
K. M. P. Das, S. Barve, S. Banerjee, S. Bandyopadhyay, and S. Padmanabhan, “A novel thermostability conferring property of cherry tag and its application in purification of fusion proteins,” Journal of Microbial & Biochemical Technology, vol. 1, no. 1, pp. 59–63, 2009.
View at: Publisher Site | Google Scholar

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Copyright © 2019 Xinxin Xu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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