Endo-inulinase Gene Research Articles

Bioprocess Optimisation for High Cell Density Endoinulinase Production from Recombinant Aspergillus niger.

Endoinulinase gene was expressed in recombinant Aspergillus niger for selective and high-level expression using an exponential fed-batch fermentation. The effects of the growth rate (μ), glucose feed concentration, nitrogen concentration and fungal morphology on enzyme production were evaluated. A recombinant endoinulinase with a molecular weight of 66 kDa was secreted. Endoinulinase production was growth associated at μ> 0.04 h-1, which is characteristic of the constitutive gpd promoter used for the enzyme production. The highest volumetric activity (670 U/ml) was achieved at a growth rate of 93% of μmax (0.07 h-1), while enzyme activity (506 U/ml) and biomass substrate yield (0.043 gbiomassDW/gglucose) significantly decreased at low μ (0.04 h-1). Increasing the feed concentration resulted in high biomass concentrations and viscosity, which necessitated high agitation to enhance the mixing efficiency and oxygen. However, the high agitation and low DO levels (ca. 8% of saturation) led to pellet disruption and growth in dispersed morphology. Enzyme production profiles, product (Yp/s) and biomass (Yx/s) yield coefficients were not affected by feed concentration and morphological change. The gradual increase in the concentration of nitrogen sources showed that, a nitrogen limited culture was not suitable for endoinulinase production in recombinant A. niger. Moreover, the increase in enzyme volumetric activity was still directly related to an increase in biomass concentration. An increase in nitrogen concentration, from 3.8 to 12 g/L, resulted in volumetric activity increase from 393 to 670 U/ml, but the Yp/s (10053 U/gglucose) and Yx/s (0.049 gbiomasDWs/gglucose) did not significantly change. The data demonstrated the potential of recombinant A. niger and high cell density fermentation for the development of large-scale endoinulinase production system.

Identification, soluble expression, and characterization of a novel endo-inulinase from Lipomyces starkeyi NRRL Y-11557

Studies on endo-inulinases from yeast are scarce, compared to those from other microbial sources. In this study, a novel endo-inulinase from Lipomyces starkeyi NRRL Y-11557 was identified, expressed in its soluble form, and characterized its physicochemically properties, together with its enzymatic activity and production of fructooligosaccharides (FOSs). A putative endo-inulinase gene inu3 was identified through rational genome mining. Through enzymatic activity and SDS-PAGE analysis, the endo-inulinase putative function of the protein encoded by inu3B gene (INU3B) was confirmed, and its soluble expression was achieved with pET22b (+) in Escherichia coli. INU3B showed effective catalytic activity and high thermostability. To our knowledge, the specific activity of INU3B against inulin reported in this study, 2262.8 ± 82.3 U·mg−1, at 70 °C and pH 5.0–6.0, is the highest reported to date. When the enzyme catalyzed FOSs production, the main products were DP3, DP4 and DP5. Overall, this report describes a novel yeast-derived endo-inulinase with optimal enzymatic properties, and thus, the reported enzyme has great potential for industrial production of FOSs.

Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.

Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33U/mL within 72h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33U/mL within 72h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63g/L within 78h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.

High-efficient production of fructo-oligosaccharides from inulin by a two-stage bioprocess using an engineered Yarrowia lipolytica strain

A convenient and efficient two-stage bioprocess was established for fructo-oligosaccharides (FOSs) production, during which the endo-inulinase was first produced and subsequently the inulin supplemented was directly hydrolyzed by the produced endo-inulinase, in the meantime the generated non-prebiotic saccharides was assimilated by the yeast cells. This process was implemented by an engineered Yarrowia lipolytica strain Enop56, in which an optimized endo-inulinase gene from Aspergillus niger was overexpressed. When the strain Enop56 was fermented with an inulin concentration of 600g/L in a 10-L bioreactor, the inulinase activity, FOSs titer, yield and productivity reached to 551.6U/mL, 546.6g/L, 0.91gFOS/gInulin, and 15.18g/L/h, respectively. Besides, the hydrolysis products were mainly FOSs with polymerization degrees of 3–5 and the total amount of non-prebiotic mono- and disaccharides was only 4.97% in the final fermentation broth. This study demonstrated that the two-stage bioprocess using the strain Enop56 was a promising strategy to produce FOSs on an industrial scale.

Genetic modification and optimization of endo-inulinase for the enzymatic production of oligofructose from inulin

The enzymatic hydrolyzation of inulin by endo-inulinase to produce oligofructoses, a new type of food additive and health product, is a promising, “green”, and environmentally friendly technique. To identify novel genetic sources of endo-inulinase genes and facilitate their industrial application for oligofructose production, we cloned an endo-inulinase gene from a Fusarium oxysporum strain and achieved high-level expression in the genetically modified Pichia pastoris strain in a pilot-scale bioreactor by using strategies such as C-terminal truncation and mutagenesis of protease-sensitive sites. We then optimized the parameters of the inulinase reaction and the amount of enzyme used to inulin hydrolysis and oligofructose production. The results of this study should facilitate the bulk production of inulinase and provide a reference for the industrial production of oligofructose from inulin.

A one-step bioprocess for production of high-content fructo-oligosaccharides from inulin by yeast

Commercial fructo-oligosaccharides (FOS) are predominantly produced from sucrose by transfructosylation process that presents a maximum theoretical yield below 0.60gFOSgSucrose−1. To obtain high-content FOS, costly purification is generally employed. Additionally, high-content FOS can be produced from inulin by using endo-inulinases. However, commercial endo-inulinases have not been extensively used in scale-up production of FOS. In the present study, a one-step bioprocess that integrated endo-inulinase production, FOS fermentation, and non-FOS sugars removal into one reactor was proposed to produce high-content FOS from inulin. The bioprocess was implemented by a recombinant yeast strain JZHΔS-TSC, in which a heterologous endo-inulinase gene was expressed and the inherent invertase gene SUC2 was disrupted. FOS fermentation at 40°C from 200g/L chicory inulin presented the maximun titer, yield, and productivity of 180.2±0.8g/L, 0.9gFOSgInulin−1, and 7.51±0.03g/L/h, respectively. This study demonstrated that the one-step bioprocess was simple and highly efficient.

Synergistic effect between the recombinant exo-inulinase and endo-inulinase on inulin hydrolysis

An exo-inulinase gene from Kluyveromyces marxianus and an endo-inulinase gene from Aspergillus niger were cloned and expressed in Yarrowia lipolytica, respectively. Then, the purified recombinant exo-inulinase (rEXINU) and endo-inulinase (rENINU), either individual or combination with another, were used for hydrolysis of inulin and inulin in tuber meal of Jerusalem artichoke. After the analysis of the dynamic hydrolysis process, it was found that the rEXINU and the rENINU acted synergistically with each other, and the maximum degree of synergistic effect on the level of fructose formation (DSEFru) could be obtained with a molar ratio of the rEXINU to the rENINU at 1:1. Furthermore, the investigation of the hydrolysis product in the synergistic hydrolysis process showed that a wide range of oligosaccharides were first generated and then converted to fructose. Based on the enzyme kinetics, substrate specificity and product inhibition of the rEXINU and the rENINU, a synergism mechanism of the them for the inulin hydrolysis was proposed. During the process of the synergistic action, the rENINU preferentially degraded the inulin with high DP, but was inhibited by the produced oligosaccharides. Then the released oligosaccharides which were the optimum substrates of the rEXIUN were rapidly hydrolyzed by the enzyme to produce fructose and glucose. Meanwhile, this process relieved the inhibition of the produced oligosaccharides to the rENINU. Besides, in the synergistic hydrolysis of the inulin in tuber meal of Jerusalem artichoke, the depolymerization of the rENINU could result in the increase of the subtrate solubility to improve the hydrolysis efficiency. Therefore, this kind of the synergistic effect between the exo-inulinase and the endo-inulinase may provide a potential driving force in the industrial application of inulin and inulin-containing materials.

Cloning, overexpression, and characterization of a highly active endoinulinase gene from Aspergillus fumigatus Cl1 for production of inulo-oligosaccharides.

In this study, an endoinulinase gene from Aspergillus fumigatus was cloned and overexpressed in Pichia pastoris. A maximum activity, 3860 U/ml, of the recombinant endoinulinase was obtained by using a high cell-density fermentation approach. The recombinant endoinulinase with a molecular weight of 58 kDa was purified for further enzymatic investigation. The pH and temperature optima were pH 6.0 and 55 °C, and the K m and V max values toward inulin were 2.18 mM and 1590 μmol min(-1) mg(-1), respectively. The degree of polymerization (DP) for inulo-oligosaccharides product 3, 4, 5, and >5 was determined by thin-layer chromatography (TLC) and high-performance liquid chromatograph (HPLC). Immobilized endoinulinases were prepared and characterized, which showed higher stability than the free endoinulinase under various temperature levels. A residual activity of 81.2 % could be still obtained after ten reaction cycles. Thus, the present recombinant endoinulinase exhibited great potential for scale-up production of inulo-oligosaccharides.

Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production

In the present study, the endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620 was optimized according to the codon usage of Pichia pastoris and both the native and the optimized gene were expressed in P. pastoris. Use of the optimized gene resulted in the secretion of recombinant endoinulinase activity that reached 1,349Uml(-1), 4.18 times that observed using the native gene. This is the highest endoinulinase activity reported to date. The recombinant enzyme was optimally active at pH 6.0 and 60°C. Moreover, inulooligosaccharides production from inulin was studied using the recombinant enzyme produced from the optimized gene. After 8h under optimal conditions, which included 400gl(-1) inulin, an enzyme concentration of 40Ug(-1) substrate, 50°C and pH 6.0, the inulooligosaccharide yield was 91%. The high substrate concentration and short reaction time described here should reduce production costs distinctly, compared with the conditions used in previous studies. Thus, this study may provide the basis for the industrial use of this recombinant endoinulinase for the production of inulooligosaccharides.

Ethanol production from inulin and unsterilized meal of Jerusalem artichoke tubers by Saccharomyces sp. W0 expressing the endo-inulinase gene from Arthrobacter sp.

After the endo-inulinase gene from Arthrobacter sp. was ligated the expression vectors pMIDSC31 and pMIRSC31, the endo-inulinase gene was inserted into the chromosomal DNA of Saccharomyces sp. W0. It was found that the inulinase activity of the recombinant yeast D5 in which the endo-inulinase gene was inserted into the delta sequence was higher than that of the recombinant yeast R1 in which the endo-inulinase gene was inserted into 18S rDNA sequence. More ethanol from inulin was produced by the recombinant yeast D5 than by the recombinant yeast R1. But Saccharomyces sp. W0 produced the lowest inulinase activity and concentration of ethanol. During the 3-l fermentation, the recombinant yeast D5 could produce 13.6 ml of ethanol per 100ml of the fermented medium from 30% inulin. The recombinant yeast D5 could actively convert the unsterilized meal of Jerusalem artichoke tubers, yielding 10.1 ml of ethanol per 100ml of the fermented medium.

Improved ethanol fermentation by heterologous endoinulinase and inherent invertase from inulin by Saccharomyces cerevisiae

It is hypothesized that introduction of an endoinulinase gene into Saccharomyces cerevisiae will improve its inulin utilization and ethanol fermentation through collaboration between the heterologous endoinulinase and the inherent invertase SUC2. The aim of this work was to test the hypothesis by introducing the endoinulinase gene inuA from Aspergillus niger into S. cerevisiae. The results showed that heterologous inuA expressed in S. cerevisiae selectively digested long chains of inulin into short fructooligosaccharides and parts of these fructooligosaccharides could be efficiently utilized by the yeast. This study demonstrated that collaboration between heterologous endoinulinase and inherent invertase improved inulin degradation and ethanol fermentation in S. cerevisiae.

Overexpression of the endo-inulinase gene from Arthrobacter sp. S37 in Yarrowia lipolytica and characterization of the recombinant endo-inulinase

Abstract The endo-inulinase gene (EnIA) from Arthrobacter sp. S37 was ligated into the expression vector pINA1317 and over-expressed in Yarrowia lipolytica Po1h. It was found that the endo-inulinase activity and specific endo-inulinase activity produced by the transformant 1317-EnIA were 16.7 U/mL and 93.4 U/mg, respectively. The recombinant EnIA was purified and characterized. The molecular weight of the purified rEnIA was 78.9 kDa. The optimal pH and temperature of the purified rEnIA were 4 and 50 °C, respectively. The purified rEnIA was stable in the temperature range of 4–40 °C and in the pH range of 2–8. The activity of rEnIA was greatly stimulated in the presence of Li+. The purified rEnIA could actively convert inulin into disaccharides.

Quantitative expression analysis of inulinase gene cluster of Penicillium sp. strain TN-88

The filamentous fungus Penicillium sp. strain TN-88 carries the endoinulinase gene inuC and the exoinulinase gene inuD that are linked head-to-head on the genome and divergently transcribed from an 859-bp intergenic region [Moriyama et al., Biosci. Biotechnol. Biochem., 66, 1887-1896 (2002)]. Quantitative real-time PCR amplification revealed that the transcription levels of the inuC and inuD genes increased 42- and 3260-fold in inulin-grown mycelia, respectively. Sucrose as well as fructose did not induce the expression of the inuC or inuD gene at all. The levels of inuC and inuD transcripts in mycelia grown on the glucose/inulin mixture were both below their basal levels in glucose-grown mycelia. Thus, glucose exerts a strong carbon catabolite repression on the expression of the two genes.

Purification and properties of an extracellular exoinulinase from Penicillium sp. strain TN-88 and sequence analysis of the encoding gene.

An exoinulinase, P-I, was purified from the culture filtrate of Penicillium sp. strain TN-88 grown on inulin. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis with an apparent Mr of 81 kDa. The purified enzyme had extremely high specific activity, 743 U/mg, toward inulin. Inulinase activity was optimal at pH 4.0 and 55 degrees C. A genomic DNA and cDNAs encoding this protein were cloned and sequenced. The exoinulinase gene (inuD) was present as a single copy in the genome. An open reading frame of 2,106 bp was interrupted by a single intron of 56 bp, and encoded a 25-amino acid signal peptide and a 677-amino acid mature protein. The mature protein contained two Cys residues and eight potential N-linked glycosylation sites. The 5'-noncoding region had a putative CAAT box at position -239. Four distinct transcription start points were observed at positions -98 (A), -91 (A), -80 (A), and -76 (A) from the start codon. The exoinulinase gene inuD was located 860-bp upstream of the previously reported endoinulinase gene inuC in the opposite direction of transcription.

Molecular Cloning and Sequence Analysis of an Endoinulinase Gene from Penicillium sp. Strain TN-88

A genomic DNA segment and cDNAs encoding an extracellular endoinulinase of Penicillium sp. strain TN-88 were cloned and sequenced. Southern blot analysis indicated that the endoinulinase gene (inuC) was present as a single copy in the genome. An open reading frame, consisting of 1,545 bp, was not interrupted by introns, and it encoded a 25 amino acid signal peptide and a 490 amino acid mature protein. The mature protein contained three Cys residues and ten potential N-linked glycosylation sites. Three distinct transcriptional start points were observed at positions -242 (A), -215 (A), and -75 (C) from the start codon. The 5'-noncoding region had a putative TATA box at position -120 (TATATATA) and two contiguous CAAT sequences at -159 to -151. The deduced amino acid sequence showed 72 and 85% identities with those of Aspergillus niger and Penicillium purpurogenum endoinulinase genes, respectively. A neighbor-joining tree showed that fungal endoinulinases form a distinct cluster from other members of the beta-fructofuranosidase superfamily and that they are more closely related to bacterial levanases than to a fungal fructosyltransferase, yeast invertases, or a yeast exoinulinase.

Production of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity

To utilize intracellular endoinulinase for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was successfully cloned into the plasmid pBR322 by using EcoRI restriction endoinulinase and E. coli HB101 as a host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, showing similar reaction modes as compared to those of the original strain. However, some critical differences existed in optimal reaction conditions and oligosaccharide compositions between the two products catalyzed by the native enzyme of original strain and those by intact cells from recombinant cells. The IOS compositions produced by recombinant E. coli were quite different due to the diffusional restriction of the substrate and products within the cell wall. Optimal reaction conditions for batchwise production of IOS were as follow : optimum temperature, 55 °C; pH, 7.5; substrate concentration, 100 g/l inulin; enzyme dosage, 20 units/g substrate. Continuous production of IOS from inulin was also carried out at 50 °C using a bioreactor packed with the recombinant cells immobilized on calcium alginate gel. The optimal feed concentration and the feed flow rate were 100 g/l inulin and 0.6 h−1 as a superficial space velocity, respectively. Under the optimum operation conditions, continuous production of IOS was successfully performed with productivity of 166.7 g/l·h for 15 days at 50 °C without significant loss of initial activity.

Microbial production of inulo-oligosaccharides by an endoinulinase from Pseudomonas sp. Expressed in Escherichia coli

In an attempt to utilize the whole cell as a biocatalyst for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene ( inu1) of Pseudomonas sp. was cloned into the plasmid pBR322 using EcoRI restriction endonuclease and Escherichia coli HB101 as the host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, producing a high yield of IOS (78%). In a batchwise reaction, the initial enzyme concentration determined the total oligosaccharide yield, and excess enzyme decreased the total oligosaccharide yield due to the formation of high amounts of free sugars such as glucose and fructose. The recombinant E. coli expressing endoinulinase activity were immobilized on a polystyrene carrier material, resulting in a dramatically enhanced thermal stability of the enzyme. Continuous production of IOS from inulin was also carried out at 50°C using a bioreactor packed with the immobilized cells. Under the optimal operation conditions, continuous production of IOS was achieved with a productivity of 150 g/ l·h for 17 d at 50°C without significant loss of initial activity.

Transcriptional analysis of two endoinulinase genes inuA and inuB in Aspergillus niger and nucleotide sequences of their promoter regions

Aspergillus niger 12 contained two copies of endoinulinase genes ( inuA and inuB) in the genome (K. Ohta et al., Biosci. Biotechnol. Biochem., 62, 1731–1738, 1998). The inuA- and inuB-specific DNA probes were constructed according to the respective 3′-noncoding sequences that diverged from each other. Poly(A) + RNA was prepared from mycelia grown on inulin, fructose, or glucose in submerged culture. Three endoinulinase cDNA sequences that corresponded to the coding regions and their 5′- and 3′-flanking regions were obtained by reverse transcription and subsequent polymerase chain reaction. Southern blot analysis revealed that the amplified cDNA 3′-noncoding sequences hybridized to the inuB probe but not to the inuA probe, regardless of the carbon source. The data suggest that only the inuB gene was transcribed constitutively. Four distinct 5′ ends of the transcripts were observed at positions −80(A), −72(G), −69(A), and −65(A) from the start codon. The inuB mRNAs were polyadenylated at various sites between 94 and 297 bp downstream of the stop codon. We have determined the nucleotide sequences of the 1201- and 1017-bp 5′-noncoding regions of the inuA and inuB genes, respectively. The inuB promoter region included a putative TATA box at −116 (TATATA).

Molecular cloning and sequence analysis of an endo-inulinase gene from Arthrobacter sp.

A 3.0-kb DNA fragment containing an endo-inulinase gene was cloned from Arthrobacter sp. S37. It contained a single open reading frame of 2439 bp, encoding a polypeptide composed of signal peptide of 53 amino acids and mature protein of 759 amino acids. From the comparison with amino acids sequences of fructan hydrolases and invertase, five highly conserved regions including the β-fructosidase motif were found. The sequence of the endo-inulinase had the identity in the range of 13.3% to 16.0%.

Molecular cloning and sequence analysis of two endoinulinase genes from Aspergillus niger.

Two genomic DNAs encoding endoinulinase from Aspergillus niger 12 were cloned and sequenced. Open reading frames (ORFs) of the endoinulinase genes, inuA and inuB, both consisted of 1,548 nucleotides encoding 516 amino acids. It was suggested that the coding regions were not interrupted by introns. The ORFs differed from each other by 23 nucleotide substitutions or by eight amino acid replacements, indicating that the inuA and inuB genes arose by gene duplication. Each mature enzyme of 493 amino acids was preceded by a hydrophobic signal peptide of 23 amino acids. The enzymes contained two Cys residues and five potential sites for N-linked glycosylation. Partial amino acid sequences of the secreted enzyme suggested that it originated from the inuB gene product. The deduced amino acid sequences of the mature A. niger enzymes showed 73% identity with that of the Penicillium purpurogenum endoinulinase.