Inulinase Gene Research Articles

A novel transcriptional activation mechanism of inulinase gene in Kluyveromyces marxianus involving a glycolysis regulator KmGcr1p with unique and functional Q-rich repeats.

Kluyveromyces marxianus is the most suitable fungus for inulinase industrial production. However, the underlying transcriptional activation mechanism of the inulinase gene (INU1) is hitherto unclear. Here, we undertook genetic and biochemical analyses to elucidate that a glycolysis regulator KmGcr1p with unique Q-rich repeats is the key transcriptional activator of INU1. We determined that INU1 and glycolytic genes share similar transcriptional activation patterns and that inulinase activity is induced by fermentable carbon sources including the hydrolysis products of inulin (fructose and glucose), which suggests a novel model of product feedback activation. Furthermore, all four CT-boxes in the INU1 promoter are important for KmGcr1p DNA-binding in vitro, but the most downstream CT-box 1 primarily confers upstream activating sequence activity in vivo. More intriguingly, the use of artificial and natural GCR1 mutants suggests that the Q-rich repeats act as a functional module to maintain KmGcr1p transcriptional activity by contributing to its solubility and DNA-binding affinity. Altogether, this study uncovers a novel transcriptional activation mechanism for the inulinase gene, that is different from the previous understanding for filamentous fungi, but might have universal significance among inulinase-producing yeasts, thereby leading to a better understanding of the regulation mechanism of yeast inulinase genes.

One-step utilization of inulin for docosahexaenoic acid (DHA) production by recombinant Aurantiochytrium sp. carrying Kluyveromyces marxianus inulinase.

This study aimed to express an inulinase gene from the yeast Kluyveromyces marxianus (KmINU) in Aurantiochytrium sp. and realized one-step utilization of inulin resource for DHA production without any chemical pretreatment. An expression cassette with a length of 6052bp for expressing the inulinase gene was constructed by a fast two-step PCR method and then was transferred into the Aurantiochytrium sp. cells. The Aurantiochytrium sp. recombinant T39 was selected with an inulinase activity up to 50.1 U/mL in 72h. In a 5-l fed-batch fermentation, as high as 148.9g/L of inulin was directly used within 120h, and only 1.2g/L of total sugar was left in the medium at the end of fermentation. The biomass of 51.4g/L with a lipid content of 69.2% DCW and a DHA yield of 14.9g/L was obtained.

Identification of endophytic actinobacteria from Jerusalem artichoke and examination of inulinase gene and enzyme properties

Identification of endophytic actinobacteria from Jerusalem artichoke and examination of inulinase gene and enzyme properties

Inulinase hyperproduction by Kluyveromyces marxianus through codon optimization, selection of the promoter, and high-cell-density fermentation for efficient inulin hydrolysis

This study aimed to overexpress an inulinase gene in Kluyveromyces marxianus to achieve the inulinase overproduction and preparation of ultra-high-fructose syrup. First, the inulinase gene (INU1 gene) was overexpressed through codon optimization and selection of a suitable promoter. Then, the inulinase was overproduced by high-cell-density fermentation. Finally, ultra-high-fructose syrup was prepared. It was found that optimization of the codons of the native INU1 gene encoding inulinase from Kluyveromyces marxianus KM-0 made a recombinant strain KM-N70 carrying the optimized INU1N gene produce 251.4 U/mL of the inulinase activity. Furthermore, inulinase activity produced by a recombinant KM-KN16 strain carrying the optimized INU1N gene directed by the native TPS1 promoter from K. marxianus KM-0 reached 338.5 U/mL and expression level of the optimized INU1N gene in the recombinant KM-KN16 strain was also greatly enhanced. During a 10-L fermentation, the recombinant KM-KN16 strain could produce 374.3 U/mL of inulinase activity within 24 h, while during a high-cell-density fed-batch fermentation, the recombinant KM-KN16 strain could produce 896.1 U/mL of inulinase activity and OD600nm value of its culture reached 108. The crude inulinase preparation obtained in this study had an inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation. It was found that 90.3% of 332.4 g/L of inulin was hydrolyzed to produce 41.0 g/L of glucose and 256.0 g/L of fructose and 91.1% of 328.2 g/L of inulin in the extract of the tubers of Jerusalem artichoke was hydrolyzed to produce 48.3 g/L of glucose and 250.5 g/L of fructose by the crude inulinase preparation (75 U/g of the substrate) within 8 h. The hydrolysates contained major monosaccharides and a trace amount of trisaccharides and the monosaccharides were composed of around 85% fructose and 15% glucose. So far, any other yeasts available have produced only up to 120 U/mL of inulinase activity. Together, this made the recombinant KM-KN16 strain be the best inulinase producer at this moment. The inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation and the ultra-high-fructose syrup with 41.0 g/L of glucose and 256.0 g/L of fructose were obtained. The inulinase activity obtained in this study was the highest among all the inulinase activities produced by yeast, fungal, and bacterial strains obtained so far.

Production of high titer of citric acid from inulin

BackgroundCitric acid is considered as the most economically feasible product of microbiological production, therefore studies on cheap and renewable raw materials for its production are highly desirable. In this study citric acid was synthesized by genetically engineered strains of Yarrowia lipolytica from widely available, renewable polysaccharide – inulin. Hydrolysis of inulin by the Y. lipolytica strains was established by expressing the inulinase gene (INU1 gene; GenBank: X57202.1) with its native secretion signal sequence was amplified from genomic DNA from Kluyveromyces marxianus CBS6432. To ensure the maximum citric acid titer, the optimal cultivation strategy–repeated-batch culture was applied.ResultsThe strain Y. lipolytica AWG7 INU 8 secreted more than 200 g dm− 3 of citric acid during repeated-batch culture on inulin, with a productivity of 0.51 g dm− 3 h− 1 and a yield of 0.85 g g− 1.ConclusionsThe citric acid titer obtained in the proposed process is the highest value reported in the literature for Yarrowia yeast. The obtained results suggest that citric acid production from inulin by engineered Y. lipolytica may be a very promising technology for industrial citric acid production.

Cloning of Phytase Genes from Pantoea Sp. 3.5.1 and Bacillus ginsengihumi M2.11 in Pichia pastoris

Phytases (myo-inositol hexakisphosphate hydrolase) catalyze the hydrolysis of phytate to inorganic phosphate and less phosphorylated myo-inositol derivatives and are widely used as feed additives in animal nutrition. Nevertheless, nowadays, there is a constant search for new phytases and new expression systems for better production of these important enzymes. In this study, we report cloning of two novel bacterial phytases belonging to the different enzyme classes and having different properties in the methylotrophic yeast Pichia pastoris. Sequences of agpP and phyC genes, encoding histidine acid phytase from Pantoea sp. 3.5.1 and β-propeller phytase from Bacillus ginsengihumi M2.11, respectively, were optimized and chemically synthesized according to the P. pastoris codon usage bias. The optimized genes were cloned into the yeast vectors pPINK-HC and pPINK-LC under the control of the inducible promoter AOX1 and two different signal peptides—signal sequence of α-amylase gene from Aspergillus niger and presequence of inulinase gene from Kluyveromyces maxianus. PCR analysis, restriction analysis, and DNA sequencing confirmed correct integration of agpP and phyC genes into P. pastoris genome. As a result, recombinant strains of P. pastoris with codon-optimized bacterial phytase genes (agpP and phyC), encoding histidine acid and β-propeller phytases, integrated into the genome under the alcohol oxidase promoter AOX1 and two different signal peptides, were obtained. Recombinant phytase AgpP was stably expressed and secreted into the culture medium of yeasts, whereas the expression of phyC gene was only confirmed at transcription level.

Gene regulation in Kluyveromyces marxianus in the context of chromosomes.

Eukaryotes, including the unicellular eukaryotes such as yeasts, employ multiple levels of gene regulation. Regulation of chromatin structure through chromatin compaction cascades, and influenced by transcriptional insulators, might play a role in the coordinated regulation of genes situated at adjacent loci and expressed as a co-regulated cluster. Subtelomeric gene silencing, which has previously been described in the yeast Saccharomyces cerevisiae, is an example of this phenomenon. Transcription from a common regulatory element located around a shared intergenic region is another factor that could coordinate the transcription of genes at adjacent loci. Additionally, the presence of DNA binding sites for the same transcription factor may coordinate expression of multiple genes. Yeasts such as the industrially important Kluyveromyces marxianus may also display these modes of regulation, but this has not been explored to date. An exploration was done using a complete genome and RNA-seq data from a previous study of the transcriptional response to glucose or xylose as the carbon source in a defined culture medium, and investigating whether the species displays clusters of co-localised differentially expressed genes. Regions of possible subtelomeric silencing were evident, but were non-responsive to the carbon sources tested here. Additionally, glucose or xylose responsive clusters were discovered far from telomeres which contained some of the most significantly differentially expressed genes, encoding enzymes involved in the utilisation of alternative carbon sources such as the industrially important inulinase gene INU1. These clusters contained putative binding sites for the carbon source responsive transcription factors Mig1 and Adr1. Additionally, we investigated the potential contribution of common intergenic regions in co-regulation. Some observations were also made in terms of the evolutionary conservation of these clusters among yeast species and the presence of potential transcriptional insulators at the periphery of these clusters.

Extracellular β-Glucosidase Production from bglp15.2 Gene Carrying Inulinase Signal Peptide in Saccharomyces cerevisiae BY4741

One of the important enzymes in cellulase complex is β-glucosidase. In this research, adding signal peptide of inulinase gene from Kluyveromyces marxianus, cloning, and expressing of bglp15.2 gene in S. cerevisiae BY4741 had been done. Gene of bglp15.2 encoding β-glucosidase has 90% identity to nucleotide sequence of Shewanella frigidimarina NCIMB 400 bacteria. Adding nucleotide sequence of signal peptide was aimed to secrete β-glucosidase and had been done with PCR (Polymerase Chain Reaction) method. The addition of nucleotide sequence of signal peptide in bglp15.2 gene had been done succesfully that indicated from nucleotide sequencing result and the increment of amplicon band size in electroferogram of the last addition PCR step. The bglp15.2 and bglp15.2INU gene (the bglp15.2 gene that has signal peptide nucleotide sequence) were cloned in Escherichia coli DH5α using pGEM-T-Easy vector and pBEVY-GL shuttle vector. The pBEVY-GL shuttle vector was used for transforming S. cerevisiae BY4741 with bglp15.2 and bglp15.2INU. The recombinant S. cerevisiae BY4741 carrying bglp15.2INU gene and growing in 48 hours had extracellularly β-glucosidase enzyme activity of 0,0178 U/ml and the intracellularly activity was 0,0181 U/ml. The β-glucosidase enzyme without signal peptide was not secreted. With K. marxianus inulinase signal peptide, about 50% Bglp15.2INU protein could be secreted. The protein molecular weight of secreted Bglp15.2INU was 44 kDa in SDS-PAGE result.

Disposition and bioavailability of inulin and free sugar in untreated and dilute acid pretreated Agave tequilana leaves

Industrially, the Agave tequilana plant is most commonly associated with beverage production, which requires it to be grown for 8–10 year before harvesting. However, its robust growth characteristics and low water usage requirements suggest that it could also be utilized as a substrate for alternative fermentation processes that do not require such long crop turnover times. Therefore, a study was undertaken to characterize the feasibility of utilizing the leaves from two year-old plants as a microbial fermentation biomass substrate. Quantitative saccharification revealed the carbohydrate concentration of two year-old A. tequilana leaves to be 633 mg g−1 dry weight biomass. HPLC analysis identified the primary carbohydrates as glucans, with minor contributions from xylans, galactans, arabinanas, and mannans. Fructose was observed predominantly as inulin. The direct use of unpretreated A. tequilana leaf biomass as a sole carbon source could support yeast growth, however, dilute acid pretreatment removed sufficient levels of carbohydrates to prevent the growth of wild type or inulinase-expressing yeast strains. Up to 133.5 g kg−1 of soluble carbohydrates and 69.6 g kg−1 of soluble inulin were extracted in the pretreatment soakate and 4.0 g kg−1 of soluble carbohydrates and 1.3 g kg−1 of soluble inulin remained in the biomass post processing. Inulinase gene expression did not significantly improve yeast growth rates, but permitted up to 6.3 g kg−1 of the available soluble inulin from the dilute acid pretreatment soakate to be metabolized towards cellular growth.

Heavy oils (mainly alkanes) over-production from inulin by Aureobasidium melanogenum 9-1 and its transformant 88 carrying an inulinase gene

After screening the yeast-like strains isolated from a mangrove ecosystem at Dongzaikang, Haikou, Hainan Province of China, it was found that Aureobasidium melanogenum 9-1 could produce 28.5 g l−1 of crude heavy oils when it was grown in the medium containing 120.0 g l−1 glucose. It is interesting to note that when grown in a medium containing 160.0 g l−1 inulin, it could produce 30.2 g l−1 of crude heavy oils. After an inulinase gene from Kluyveromyces marximus was actively expressed in A. melanogenum 9-1, a transformant 88 could produce 70.8 U ml−1 of inulinase activity within 72 h whereas A. melanogenum 9-1 only produced 17.4 U ml−1 of inulinase activity. During the 10-L fermentation, A. melanogenum 9-1 could yield 38.24 g l−1 of the crude heavy oils and cell dry weight was 25.2 g l−1 within 120 h while the transformant 88 could yield 43.0 g l−1 of the crude heavy oils and cell dry weight was 24.7 g l−1 under the same conditions. However, 9.97 g l−1 of total sugar and 2.55 g l−1 of reducing sugar were left in the culture of A. melanogenum 9-1 while only 3.2 g l−1 of total sugar and 0.48 g l−1 of reducing sugar were maintained in the culture of the transformant 88. The extracellular heavy oils produced by A. melanogenum 9-1 contained 87.12% alkanes and 12.88% fatty acids whereas the extracellular heavy oils produced by the transformant 88 contained 89.8% hydrocarbons and 10.2% fatty acids. In contrast, the intracellular oils produced A. melanogenum 9-1 contained 99.3% fatty acids and 0.7% alkanes while the intracellular oils produced by the transformant 88 contained 95.44% fatty acids and 4.56% alkanes. All these results showed that most of the secreted heavy oils were alkanes and most of the synthesized alkanes were secreted into the medium while most of the synthesized fatty acids remained in the cells.

Cloning and characterization of an inulinase gene from the marine yeast Candida membranifaciens subsp. flavinogenie W14-3 and its expression in Saccharomyces sp. W0 for ethanol production.

The INU1 gene encoding an exo-inulinase from the marine-derived yeast Candida membranifaciens subsp. flavinogenie W14-3 was cloned and characterized. It had an open reading frame of 1,536 bp long encoding an inulinase. The coding region of it was not interrupted by any intron. The cloned gene encoded 512 amino acid residues of a protein with a putative signal peptide of 23 amino acids and a calculated molecular mass of 57.8 kDa. The protein sequence deduced from the inulinase gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP FS and Q. The protein also had six conserved putative N-glycosylation sites. The deduced inulinase from the yeast strain W14-3 was found to be closely related to that from Candida kutaonensis sp. nov. KRF1, Kluyveromyces marxianus, and Cryptococcus aureus G7a. The inulinase gene with its signal peptide encoding sequence was subcloned into the pMIRSC11 expression vector and expressed in Saccharomyces sp. W0. The recombinant yeast strain W14-3-INU-112 obtained could produce 16.8 U/ml of inulinase activity and 12.5 % (v/v) ethanol from 250 g/l of inulin within 168 h. The monosaccharides were detected after the hydrolysis of inulin with the crude inulinase (the yeast culture). All the results indicated that the cloned gene and the recombinant yeast strain W14-3-INU-112 had potential applications in biotechnology.

Enhanced expression of the codon-optimized exo-inulinase gene from the yeast Meyerozyma guilliermondii in Saccharomyces sp. W0 and bioethanol production from inulin.

In the present study, after the exo-inulinase gene INU1 from Meyerozyma guilliermondii was optimized according to the codon usage bias of Saccharomyces cerevisiae, both the optimized gene INU1Y and the native gene INU1 were ligated into the homologous integration expression vector pMIRSC11 and expressed in Saccharomyces sp. W0. It was determined that the inulinase activity of the recombinant yeast Y13 with the optimized gene INU1Y was 43.84 U/mL, which was obviously higher than that (31.39 U/mL) produced by the recombinant yeast EX3 with the native gene INU1. Moreover, it was indicated that the recombinant yeast Y13 could produce 126.30 mg/mL ethanol from 300.0 g/L inulin while the recombinant yeast EX3 and Saccharomyces sp. W0 produced 122.75 mg/mL and 114.15 mg/mL ethanol, respectively, under the same conditions. In addition, the ethanol productivity of the recombinant yeast Y13 was 2.25 mg/mL/h within 48 h of the fermentation, which was obviously higher than that of the recombinant yeast EX3 (1.97 mg/mL/h) and Saccharomyces sp. W0 (1.77 mg/mL/h) within the same period. The results demonstrated that the recombinant yeast Y13 had higher ethanol production and productivity than the recombinant yeast EX3 and Saccharomyces sp. W0. Therefore, it was concluded that the codon optimization of the exo-inulinase gene from M. guilliermondii effectively enhanced inulinase activity and improved ethanol production from inulin by Saccharomyces sp. W0 carrying the optimized inulinase gene.

Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase.

Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity.

Inulinase production by the yeast Kluyveromyces marxianus with the disrupted MIG1 gene and the over-expressed inulinase gene

Kluyveromyces marxianus has been widely used in food industries and inulinase produced by the yeast has many applications. In order to greatly enhance inulinase production by the yeast, it is very important to both derepress glucose repression and overexpress the native inulinase gene. The MIG1 gene in the yeast K. marxianus KM-0 was disrupted. It was found that a glucose-derepressed mutant (KM-69) of K. marxianus KM-0 could produce 94.6 units of inulinase activity per ml in the inulin medium within 36h. In order to further increase inulinase production, the native inulinase gene was over-expressed in the glucose-derepressed mutant. During the 10-l fermentation, the recombinant strain KM-526 yielded 133.5 units of inulinase activity per ml within very short time (24h). The results also showed that inulinase produced by the recombinant strain KM-526 could more effectively convert inulin into monosaccharides than that produced by K. marxianus KM-0. The results indicate that it is very important to delete the MIG1 gene in K. marxianus KM-0 and over-express the native INU1 gene in order to further enhance inulinase yield within the short time by this yeast and for efficient hydrolysis of inulin. The recombinant strain KM-526 and its inulinase have highly potential applications in food industries.

Gene cloning, expression, and characterization of an exo-inulinase from Paenibacillus polymyxa ZJ-9.

An inulinase-producing strain, Paenibacillus polymyxa ZJ-9, was isolated from natural sources to produce R,R-2,3-butanediol via one-step fermentation of raw inulin extracted from Jerusalem artichoke tubers. The inulinase gene from P. polymyxa ZJ-9 was cloned and overexpressed in Escherichia coli BL21 (DE3), and the purified recombinant inulinase was estimated to be approximately 56kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. This result suggests that the active form of the inulinase is probably a monomer. Terminal hydrolysis fructose units from the inulin indicate that enzymes are exo-inulinase. The purified recombinant enzyme showed maximum activity at 25°C and pH6.0, which indicate its extreme suitability for industrial applications. Zn(2+), Fe(2+), and Mg(2+) stimulated the activity of the purified enzyme, whereas Co(2+), Cu(2+), and Ni(2+) inhibited enzyme activity. The K m and V max values for inulin hydrolysis were 1.72mM and 21.69μmolmin(-1)mg(-1) protein, respectively. The same parameters toward sucrose were 41.09mM and 78.7μmolmin(-1)mg(-1) protein, respectively. Considering its substrate specificity and other enzymatic characteristics, we believe that this inulinase gene from P. polymyxa ZJ-9 could be transformed into other special bacterial strains to allow inulin conversion to other biochemicals and bioenergy through one-step fermentation.

Engineering an industrial Saccharomyces cerevisiae strain with the inulinase gene for more efficient ethanol production from Jerusalem artichoke tubers

Engineering industrial microbial strains with inulinase production for developing a consolidated bioprocessing (CBP) strategy is a desirable solution to biofuel production from Jerusalem artichoke tubers. In this study, the integrative vector pFA6a‐rDNA‐PGK‐INU was generated by fusing the 3.3‐kb ribosomal DNA fragment with the phosphoglycerate kinase promoter to regulate the expression of the inulinase gene isolated from Kluyveromyces marxianus, which was then integrated into the chromosome of an industrial Saccharomyces cerevisiae strain for ethanol production from Jerusalem artichoke tubers. Compared to the host strain, no significant difference was observed in the growth of the recombinant yeast, but its inulinase production was enhanced: 22.9 versus 10.6 U/mL for aerobic seed cultures and 14.5 versus 10.0 U/mL for ethanol fermentation, which consequently facilitated the CBP process for ethanol production: 72.5 g/L ethanol produced in a fermentation time of 48 h, while only 67.0 g/L ethanol was produced by the host strain in a fermentation time of 60 h. Thus, the ethanol productivity was increased from 1.12 to 1.51 g/L/h, presenting an increase of 34.8%.

Improved ethanol production in Jerusalem artichoke tubers by overexpression of inulinase gene in Kluyveromyces marxianus

Ethanol production from Jerusalem artichoke tubers through a consolidated bioprocessing (CBP) strategy using the inulinase-producing yeast Kluyveromyces marxianus is an economical and competitive than that from a grainbased feedstock. However, poor inulinase production under ethanol fermentation conditions significantly prolongs the fermentation time and compromises ethanol productivity. Improvement of inulinase activity appears to be promising for increasing ethanol production from Jerusalem artichoke tubers by CBP. In the present study, expression of the inulinase gene INU with its own promoter in K. marxianus (K/INU2) was explored using the integrative cassette. Overexpression of INU was explored using chromosome integration via the HO locus of the yeast. Inulinase activity and ethanol were determined from inulin and Jerusalem artichoke tubers under fed-batch operation. Inulinase activity was 114.9 U/mL under aerobic conditions for K/INU2, compared with 52.3 U/mL produced by the wild type strain. Importantly, inulinase production was enhanced in K/INU2 under ethanol fermentation conditions. When using 230 g/L inulin and 220 g/L Jerusalem artichoke tubers as substrates, inulinase activities of 3.7 and 6.8 U/mL, respectively, were measured using K/INU2, comparing favorably with 2.4 and 3.1 U/mL, respectively, using the wide type strain. Ethanol concentration and productivity for inulin were improved by the recombinant yeast to 96.2 g/L and 1.34 g/L/h, respectively, vs 93.7 g/L and 1.12 g/L/h, respectively, by the wild type strain. Ethanol concentration and productivity improvements for Jerusalem artichoke tubers were 69 g/L and 1.44 g/L/h, respectively, from the recombinant strain vs 62 g/L and 1.29 g/L/h, respectively, from the wild type strain.

Cloning, characterization and heterelogous expression of the INU1 gene from Cryptococcus aureus HYA

The INU1 gene (Accession number: JX073660) encoding exo-inulinase from Cryptococcus aureus HYA was cloned and characterized. The gene had an open reading frame (ORF) of 1653bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 551 amino acid residues of a protein with a putative signal peptide of 23 amino acids and the calculated molecular mass of 59.5kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP, FS and Q. It also had two conserved putative N-glycosylation sites. The inulinase from C. aureus HYA was found to be closely related to that from Kluyveromyces marxianus and Pichia guilliermondii. The inulinase gene without the signal sequence was subcloned into pPICZaA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum inulinase activity of 16.3±0.24U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. The optimal temperature and pH for action of the enzyme were 50°C and 5.0, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified recombinant inulinase.

Properties of the inulinase gene levH1 of Lactobacillus casei IAM 1045; cloning, mutational and biochemical characterization

Though some genetic features of lactobacillar fructan hydrolases were elucidated, information about their enzymology or mutational analyses were scarce. Lactobacillus casei IAM1045 exhibits extracellular activity degrading inulin. After partial purification of the inulin-degrading protein from the spent culture medium, several fragments were obtained by protease digestion. Based on their partial amino-acid sequences, oligonucleotide primers were designed, and its structural gene (levH1) was determined using the gene library constructed in the E. coli system. The levH1 gene encoded a protein (designated as LevH1), of which calculated molecular mass and pI were 138.8-kDa and 4.66, respectively. LevH1 (1296 amino-acids long) was predicted to have a four-domain structure, containing (i) an N-terminal secretion signal of 40 amino-acids, (ii) variable domain of about 140 residues whose function is unclear, (iii) a catalytic domain of about 630 residues with glycoside-hydrolase activity consisting of two modules, a five-blade β-propeller module linked to a β-sandwich module, (iv) a C-terminal domain of about 490 residues comprising five nearly perfect repeat sequences of 80 residues homologous to equivalents of other hypothetical cell surface proteins, followed by 37-residues rich in Ser/Thr/Pro/Gly, a pentad LPQAG (the LPXTG homologue). When overproduced in E. coli, the putative variable-catalytic domain region of about 770 residues exhibited exo-inulinase activity. Deletion analyses demonstrated that the variable-catalytic domain region containing two modules is important for enzymatic activity. Presence of eight conserved motifs (I–VIII) was suggested in the catalytic domain by comparative analysis, among which motif VIII was newly identified in the β-sandwich module in this study. Site-directed mutagenesis of conserved amino-acids in these motifs revealed that D198, R388, D389 and E440, were crucial for inulinase activity. Moreover, mutations of D502A and D683A in motif VI and VIII respectively caused significant decrease in the activity. These results suggested that the variable domain and β-sandwich module, besides the β-propeller module, are important for inulin-degrading activity of LevH1.

Production and Properties of Microbial Inulinases: Recent Advances

Microbial inulinases find application in enzymatic hydrolysis of inulin for production of fructose (sweetener) and inulooligosaccharides (prebiotics). Inulin is one of the most abundant nonstructural polysaccharides found widely dispersed in plants and awaits judicious utilization. Demands for an alternative healthy sweetener and multifunctional fructooligosaccharides have prompted investigators to explore microorganisms for inulinase production and to develop bioprocesses for production of high-fructose syrup and oligosaccharides based on enzymatic hydrolysis of raw inulin. Inulinases have been characterized in several molds, yeasts and a few bacteria. Recently, there has been a spurt of interest in finding novel inulinase producers, cloning and expression of inulinase gene in heterologous hosts and use of crude plant inulin and agro-industrial media in both submerged and solid state fermentation for inulinase production. The review discusses current knowledge on production, properties and applications of microbial inulinases and looks into the recent advances in the field.