Properties of the extracellular alkaline inulinase produced by haloalkaliphilic phototrophic bacteria Ectothiorodospirea mobilis.

The studies have revealed alkaline exoinulinase produced by haloalkaliphilic phototrophic bacteria Ectothiorhodospirea mobilis Al-2 for the first time. A new method for the isolation of a homogeneous exoinulinase from the culture broth was developed and the properties of this enzyme have been investigated. It was shown that specified exoinulinase in contrast to the studied exoinulinases produced by microorganisms exhibits catalytic activity at the wide range of pH (7.0-10) and a temperature (20-60 °C) with a maximum of the inulolitic activity at pH 9.0 and 50 °C. The studied exoinulinase possessing also invertase activity (I/S1.4) is a monomeric protein with molecular mass 57Kda, as well as Km and Vmax for inulin 3.8 mM/ml and 10 µmol/ml/min-1, respectively. The studies of the influence of different metal ions on enzyme activity have shown that Mn+2, Cu+2, Co+2, Mg+2, NaCl 5-7% promote relatively higher catalytic activity while Zn+2, Cu+2 and Fe+2 partially suppress the enzyme activity and Hg2+completely inactivates the enzyme.The formation of only fructose and glucose at the enzymatic hydrolysis of inulin confirms that the studied exoinulinase belongs to the exo-type of enzymes. The obtained results supplement our fundamental knowledge in biochemistry-enzymology, as well as the biodiversity of microorganisms expressing exoinulinase. The studied exoinulinase exhibits activity at salinity of the medium and can potentially be used in the biotechnology of inulin bioconversion into bioproducts under alkaline conditions.

Characterization of inulolytic enzymes from the Jerusalem artichoke-derived Glutamicibacter mishrai NJAU-1.

The rhizosphere context of inulin-accumulating plants, such as Jerusalem artichoke (Helianthus tuberosus), is an ideal starting basis for the discovery of inulolytic enzymes with potential for bio fructose production. We isolated a Glutamicibacter mishrai NJAU-1 strain from this context, showing exo-inulinase activity, releasing fructose from fructans. The growth conditions (pH 9.0; 15 °C) were adjusted, and the production of inulinase by Glutamicibacter mishrai NJAU-1 increased by 90% (0.32 U/mL). Intriguingly, both levan and inulin, but not fructose and sucrose, induced the production of exo-inulinase activity. Two exo-inulinase genes (inu1 and inu2) were cloned and heterologously expressed in Pichia pastoris. While INU2 preferentially hydrolyzed longer inulins, the smallest fructan 1-kestose appeared as the preferred substrate for INU1, also efficiently degrading nystose and sucrose. Active site docking studies with GFn- and Fn-type small inulins (G is glucose, F is fructose, and n is the number of ß (2-1) bound fructose moieties) revealed subtle substrate differences between INU1 and INU2. A possible explanation about substrate specificity and INU's protein structure is then suggested. KEY POINTS: • A Glutamicibacter mishrai strain harbored exo-inulinase activity. • Fructans induced the inulolytic activity in G. mishrai while the inulolytic activity was optimized at pH 9.0 and 15 °C. • Two exo-inulinases with differential substrate specificity were characterized.

Fusion and secretory expression of an exo-inulinase and a d-allulose 3-epimerase to produce d-allulose syrup from inulin.

BACKGROUND: This study developed a feasible catalytic method for d-allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers. RESULTS: d-Allulose 3-epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo-inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing 12 residues (GSAGSAAGSGEF). At 55 °C and pH 8.0, and as with the addition of 1 mmol L-1 Mn2+ , the CSCA-linkerE-DAE fusion enzyme obtained through high cell-density cultivation displayed a maximal exo-inulinase activity of 21.8 U mg-1 and resulted in a yield of 6.3 g L-1 d-allulose and 39.2 g L-1 d-fructose using 60 g L-1 inulin as the raw material. Catechol-modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA-immobilized CSCA-linkerE-DAE (8 U g-1 ) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L-1 d-allulose and 32.7 g L-1 D-fructose from 60 g L-1 inulin. CONCLUSION: This study shed light on a feasible and cost-effective approach for the production of syrup containing d-allulose and D-fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. © 2020 Society of Chemical Industry.

Inulin Fermentation by Lactobacilli and Bifidobacteria from Dairy Calves.

Prebiotics are increasingly examined for their ability to modulate the neonate gut microbiota of livestock, and products such as inulin are commonly added to milk replacer used in calving. However, the ability of specific members of the bovine neonate microbiota to respond to inulin remains to be determined, particularly among indigenous lactobacilli and bifidobacteria, beneficial genera commonly enriched by inulin. Screening of Bifidobacterium and Lactobacillus isolates obtained from fresh feces of dairy calves revealed that lactobacilli had a higher prevalence of inulin fermentation capacity (58%) than bifidobacteria (17%). Several Ligilactobacillus agilis (synonym Lactobacillus agilis) isolates exhibited vigorous growth on, and complete degradation of, inulin; however, the phenotype was strain specific. The most vigorous inulin-fermenting strain, L. agilis YZ050, readily degraded long-chain inulin not consumed by bifidobacterial isolates. Comparative genomic analysis of both L. agilis fermenter and nonfermenter strains indicated that strain YZ050 encodes an inulinase homolog, previously linked to extracellular degradation of long-chain inulin in Lacticaseibacillus paracasei, that was strongly induced during growth on inulin. Inulin catabolism by YZ050 also generates extracellular fructose, which can cross-feed other non-inulin-fermenting lactic acid bacteria isolated from the same bovine feces. The presence of specific inulin-responsive bacterial strains within calf gut microbiome provides a mechanistic rationale for enrichment of specific lactobacilli and creates a foundation for future synbiotic applications in dairy calves aimed at improving health in early life.IMPORTANCE The gut microbiome plays an important role in animal health and is increasingly recognized as a target for diet-based manipulation. Inulin is a common prebiotic routinely added to animal feeds; however, the mechanism of inulin consumption by specific beneficial taxa in livestock is ill defined. In this study, we examined Lactobacillus and Bifidobacterium isolates from calves fed inulin-containing milk replacer and characterized specific strains that robustly consume long-chain inulin. In particular, novel Ligilactobacillus agilis strain YZ050 consumed inulin via an extracellular fructosidase, resulting in complete consumption of all long-chain inulin. Inulin catabolism resulted in temporal release of extracellular fructose, which can promote growth of other non-inulin-consuming strains of lactic acid bacteria. This work provides the mechanistic insight needed to purposely modulate the calf gut microbiome via the establishment of networks of beneficial microbes linked to specific prebiotics.

Genomics- and Metabolomics-Based Investigation of the Deep-Sea Sediment-Derived Yeast, Rhodotorula mucilaginosa 50-3-19/20B.

Red yeasts of the genus Rhodotorula are of great interest to the biotechnological industry due to their ability to produce valuable natural products, such as lipids and carotenoids with potential applications as surfactants, food additives, and pharmaceuticals. Herein, we explored the biosynthetic potential of R. mucilaginosa 50-3-19/20B collected from the Mid-Atlantic Ridge using modern genomics and untargeted metabolomics tools. R. mucilaginosa 50-3-19/20B exhibited anticancer activity when grown on PDA medium, while antimicrobial activity was observed when cultured on WSP-30 medium. Applying the bioactive molecular networking approach, the anticancer activity was linked to glycolipids, namely polyol esters of fatty acid (PEFA) derivatives. We purified four PEFAs (1-4) and the known methyl-2-hydroxy-3-(1H-indol-2-yl)propanoate (5). Their structures were deduced from NMR and HR-MS/MS spectra, but 1-5 showed no anticancer activity in their pure form. Illumina-based genome sequencing, de novo assembly and standard biosynthetic gene cluster (BGC) analyses were used to illustrate key components of the PEFA biosynthetic pathway. The fatty acid producing BGC3 was identified to be capable of producing precursors of PEFAs. Some Rhodotorula strains are able to convert inulin into high-yielding PEFA and cell lipid using a native exo-inulinase enzyme. The genomic locus for an exo-inulinase enzyme (g1629.t1), which plays an instrumental role in the PEFA production via the mannitol biosynthesis pathway was identified. This is the first untargeted metabolomics study on R. mucilaginosa providing new genomic insights into PEFA biosynthesis.

Cross-Feeding among Probiotic Bacterial Strains on Prebiotic Inulin Involves the Extracellular exo-Inulinase of Lactobacillus paracasei Strain W20.

Probiotic gut bacteria employ specific metabolic pathways to degrade dietary carbohydrates beyond the capabilities of their human host. Here, we report how individual commercial probiotic strains degrade prebiotic (inulin type) fructans. First, a structural analysis of commercial fructose oligosaccharide-inulin samples was performed. These ß-(2-1)-fructans differ in termination by either glucose (GF) or fructose (FF) residues, with a broad variation in the degrees of polymerization (DPs). The growth of individual probiotic bacteria on short-chain inulin (sc-inulin) (Frutafit CLR), a ß-(2-1)-fructan (DP 2 to DP 40), was studied. Lactobacillus salivarius W57 and other bacteria grew relatively poorly on sc-inulin, with only fractions of DP 3 and DP 5 utilized, reflecting uptake via specific transport systems followed by intracellular metabolism. Lactobacillus paracasei subsp. paracasei W20 completely used all sc-inulin components, employing an extracellular exo-inulinase enzyme (glycoside hydrolase family GH32 [LpGH32], also found in other strains of this species); the purified enzyme converted high-DP compounds into fructose, sucrose, 1-kestose, and F2 (inulobiose). The cocultivation of L. salivarius W57 and L. paracasei W20 on sc-inulin resulted in cross-feeding of the former by the latter, supported by this extracellular exo-inulinase. The extent of cross-feeding depended on the type of fructan, i.e., the GF type (clearly stimulating) versus the FF type (relatively low stimulus), and on fructan chain length, since relatively low-DP ß-(2-1)-fructans contain a relatively high content of GF-type molecules, thus resulting in higher concentrations of GF-type DP 2 to DP 3 degradation products. The results provide an example of how in vivo cross-feeding on prebiotic ß-(2-1)-fructans may occur among probiotic lactobacilli.IMPORTANCE The human gut microbial community is associated strongly with host physiology and human diseases. This observation has prompted research on pre- and probiotics, two concepts enabling specific changes in the composition of the human gut microbiome that result in beneficial effects for the host. Here, we show how fructooligosaccharide-inulin prebiotics are fermented by commercial probiotic bacterial strains involving specific sets of enzymes and transporters. Cross-feeding strains such as Lactobacillus paracasei W20 may thus act as keystone strains in the degradation of prebiotic inulin in the human gut, and this strain-exo-inulinase combination may be used in commercial Lactobacillus-inulin synbiotics.

High level production of a recombinant acid stable exoinulinase from Aspergillus kawachii.

Exoinulinases-enzymes extensively studied in recent decades because of their industrial applications-need to be produced in suitable quantities in order to meet production demands. We describe here the production of an acid-stable recombinant inulinase from Aspergillus kawachii in the Pichia pastoris system and the recombinant enzyme's biochemical characteristics and potential application to industrial processes. After an appropriate cloning strategy, this genetically engineered inulinase was successfully overproduced in fed-batch fermentations, reaching up to 840 U/ml after a 72-h cultivation. The protein, purified to homogeneity by chromatographic techniques, was obtained at a 42% yield. The following biochemical characteristics were determined: the enzyme had an optimal pH of 3, was stable for at least 3 h at 55 °C, and was inhibited in catalytic activity almost completely by Hg+2. The respective Km and Vmax for the recombinant inulinase with inulin as substrate were 1.35 mM and 2673 µmol/min/mg. The recombinant enzyme is an exoinulinase but also possesses synthetic activity (i. e., fructosyl transferase). The high level of production of this recombinant plus its relevant biochemical properties would argue that the process presented here is a possible recourse for industrial applications in carbohydrate processing.

Biotechnological potential of microbial inulinases: Recent perspective.

Among microbial enzymes, inulinases or fructo-furanosylhydrolases have received considerable attention in the past decade, and as a result, a variety of applications based on enzymatic hydrolysis of inulin have been documented. Inulinases are employed for generation of fructose and inulo-oligosaccharides (IOS) in a single-step reaction with specificity. The high fructose syrup can be biotransformed into value-added products such as ethanol, single cell protein, while IOS are indicated in nutraceutical industry as prebiotic. Myriad microorganisms produce inulinases, and a number of exo- and endo-inulinases have been characterized and expressed in heterologous hosts. Initially, predominated by Aspergilli, Penicillia, and some yeasts (Kluyveromyces spp.), the list of prominent inulinase producers has gradually expanded and now includes extremophilic prokaryotes and marine-derived microorganisms producing robust inulinases. The present paper summarizes important developments about microbial inulinases and their applications made in the last decade.

Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

Single substitution in α-helix of active center enhanced thermostability of Aspergillus awamori exo-inulinase.

Exo-inulinases are applied in inulin hydrolysis and production of feed additives and need to be stable at temperatures of 60-95 °C. Aspergillus awamori exo-inulinase Inu1 is considerably thermostable, with a Tm of 73.2 °C. However, the thermostability of the enzyme should be improved. A single substitution G338A in α-helix in the active center of the enzyme provided a 3.5 °C improvement in Tm. The time of half-life at 70 °C and 80 °C was increased in 5.7- and 2.7-times, respectively, compared to wild-type. Molecular dynamics simulations demonstrated that the substitution G338A caused a decrease in RMSF not only for the α-helix 337-YAANI-341, but also for the catalytically active residues D41 and E241 and the amino acid residues forming the cleft of the active center. Calculations with Constraint Network Analysis for the variant G338A showed the increase in the stability of intramolecular clusters.

Aspergillus welwitschiae inulinase enzyme cocktails obtained on agro-material inducers for the purpose of fructooligosaccharides production.

Production of fructooligosaccharides (FOS) is a trending topic due to their prebiotic effect becoming increasingly important for the modern human diet. The most suitable process for FOS production is the one using fungal inulinases. Introduction of new fungal inulinase producers and their implementation in production of inulinase enzymes is therefore gaining interest. This study provides a new approach to FOS synthesis by fungal enzyme complex without prior separation of any specific enzyme. Inulinase enzyme complexes could be used for the synthesis of FOS in two possible ways - hydrolysis of inulin (FOSh) and transfructosylation process of sucrose (FOSs), as demonstrated here. Depending on the fungal growth inducing substrate, a variety of inulinase enzyme complexes was obtained - one of which was most successful in production of FOSh and another one of FOSs. Substrates derived from crops: triticale, wheat bran, Jerusalem artichoke and Aspergillus welwitschiae isolate, previously proven as safe for use in food, were utilized for production of inulinase enzyme cocktails. The highest FOSs production was obtained by enzyme complex rich in ß-fructofuranosidase, while the highest FOSh production was obtained by enzyme complex rich in endoinulinase. Both FOSh and FOSs showed antioxidant potential according to ABTS and ORAC, which classifies them as a suitable additive in functional food. Simultaneous zymographic detection of inulinase enzymes, which could contribute to expansion of the knowledge on fungal enzymes, was developed and applied here. It demonstrated the presence of different inulinase isoforms depending on fungal growth substrate. These findings, which rely on the innate ability of fungi to co-produce all inulinases from a cocktail, could be useful as a new, easy approach to FOS production by fungal enzymes without their separation and purification, contributing to cheaper and faster production processes.

Exo-inulinase production from Aspergillus fumigatus NFCCI 2426: purification, characterization, and immobilization for continuous fructose production.

Aspergillus fumigatus was found to produce thermostable exo-inulinase (EC 3.8.1.80; 38 U/ml) on inulin-rich infusions. Exo-inulinase (14.6 U/mg) was immobilized on glutaraldehyde activated Ca-alginate beads for continuous generation of fructose by hydrolyzing sucrose, chicory, and dandelion substrates. Immobilization of enzyme was confirmed by microscopic and spectroscopic techniques. The exo-inulinase was purified using ion-exchange (1.30-folds) and size-exclusion chromatography (2.71-folds). The purified exo-inulinase showed 64 kDa band on gel and was optimally active at 60 °C and pH 6.0. Kinetic constants, Km and Vmax of purified exo-inulinase, were 5.88 mM and 1.66 µM/min, respectively, and its relative activity was found to be enhanced (125.8%) in the presence of calcium ion. Immobilized preparation was utilized for continuous generation of fructose from chicory juice (26 to 70%) and dandelion root extracts (16 to 24%) by recycling upto five cycles, respectively. In comparison to other sweeteners, such as sucrose, fructose is considered as a healthy alternative. The present study demonstrated the use of immobilized exo-inulinase in continuous generation of fructose from some underutilized plant sources that can be used in food industry. PRACTICAL APPLICATION: Thermostable exo-inulinase produced by A. fumigatus was immobilized on calcium alginate matrix and was employed for continuous hydrolysis of chicory juice and dandelion root extract for generation of fructose syrup.

The Important Roles Played in Substrate Binding of Aromatic Amino Acids in Exo-Inulinase From Kluyveromyces cicerisporus CBS 4857.

Inulinase is a member of the glycoside hydrolase family 32 (GH32). It catalyzes the randomly hydrolyzation of 2,1-ß-D-fructosidic linkages in inulin and plays a role in the production of high-fructose syrup. In this study, detailed roles of the conserved residues W79, F113, M117, R181, C239, and W334 of the exo-inulinase from Kluyveromyces cicerisporus CBS4857 (KcINU1) in substrate binding and stabilization were evaluated by in silico analysis and site-directed mutagenesis. These residues belong to the conserved WG, FSGSMV, RDP, ECP, and WQY regions of the GH32 and are located around the catalytic pocket of KcINU1. Zymogram assay showed relatively weaker band for F113W and similar band for M117A compared to the wild-type enzyme toward inulin and sucrose, whereas all other variants showed no observable stain on the native polyacrylamide gel electrophoresis. These results were further confirmed with the dinitrosalicylic acid colorimetric method. It showed that the residual activities of F113W toward inulin and sucrose were 33.8 ± 3.3% and 96.2 ± 5.5%, respectively, and that of M117A were 103.8 ± 1.3% and 166.5 ± 12%, respectively. Results from fluorescence spectra indicated that there is a significant conformational change that happened in F113W compared to the wild-type enzyme, while M117A exhibited limited impact although the quenching effect was increased.

Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability.

BACKGROUND: Inulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. In the present study, a recombinant exo-inulinase (rKcINU1), derived from Kluyveromyces cicerisporus CBS4857, was proven as an N-linked glycoprotein, and the removal of N-linked glycan chains led to reduced activity. RESULTS: Five N-glycosylation sites with variable high mannose-type oligosaccharides (Man3-9GlcNAc2) were confirmed in the rKcINU1. The structural modeling showed that all five glycosylation sites (Asn-362, Asn-370, Asn-399, Asn-467 and Asn-526) were located at the C-terminus ß-sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site N-glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five N-glycosylation sites removed was constructed, which resulted in the loss of all enzyme activity. Interestingly, the N362Q led to an 18% increase in the specific activity against inulin, while a significant decrease in thermostability (2.91 °C decrease in T m ) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity. CONCLUSION: The increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the N-linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent N-glycans to bring rigidity. Meanwhile, the N-glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the ß-sandwich domain configuration. This may have facilitated the capture and transfer of substrates to the enzyme active cavity, in a manner quite similar to that of carbohydrate binding modules (CBMs), i.e. the chains endowed the ß-sandwich domain with the functions of CBM. This study discovered a unique C-terminal sequence which is more favorable to glycosylation, thereby casting a novel view for glycoengineering of enzymes from fungi via redesigning the amino acid sequence at the C-terminal domain, so as to optimize the enzymatic properties.

Enhanced inulinase production by Fusarium solani JALPK from invasive weed using response surface methodology.

The present study is the first report of utilizing Tithonia rotundifolia weed as a substrate for inulinase production from Fusarium solani JALPK. It also deals with the statistical optimization of culture conditions to enhance the enzyme yield. Amongst the 11 variables screened by Plackett- Burman design, Inulin in combination with Agave sisalana extract, Tithonia rotundifolia extract and NaNO3 had a significant influence on inulinase production and their concentrations were further optimized employing Box Behnken design. An enhancement of inulinase production from 970 EU/mL to 3261.011 EU/mL was gained after media optimization. Amongst the screened carbon sources Tithonia rotundifolia was found to be very effective in stimulating elevated inulinase synthesis. The Tithonia rotundifolia weed extract was treated with inulinase from Fusarium solani JALPK to form fructose which was estimated spectrophotometrically. This liberated fructose was also confirmed by osazone formation test and FTIR. HPTLC analysis of product revealed the exoinulinase nature of the enzyme produced by Fusarium solani JALPK since fructose was the only end product after hydrolysis of inulin rich weed in fermented broth. Thus the elevated extracellular inulinase yielding novel property of Fusarium solani JALPK (KY914560) contributes in considering it as a potential candidate with food, pharmaceutical and bioremediation applications.

Purification and characterization of two isoforms of exoinulinase from Penicillium oxalicum BGPUP-4 for the preparation of high fructose syrup from inulin.

Two exoinulinases, Exo-I and Exo-II from the culture broth of Penicillium oxalicum BGPUP-4 was purified using three-step purification method i.e., isopropanol precipitation, Q-Sepharose and Sephadex G-100 column chromatography. The molecular weight of Exo-I and Exo-II was determined to be 64.85 kDa and 32.54 kDa, respectively using MALDI-TOF. Exo-I and Exo-II showed high specificity for inulin and their respective Vmax/Km ratio was 3.74 and 7.20. Besides, both the inulinases also displayed specificity for lactose, sucrose and raffinose. Exo-I and Exo-II were stable at a pH range of 4.0-8.0 with pH optima 5.0. Optimal temperature for both the inulinases was 55 °C, and both the isoforms retained approximately 50% of their activity up to 70 °C. Ag+, Hg2+, Ba2+, Cu2+ and Ca2+ ions shown stimulatory effect on inulinases activity, while Fe2+, Mn2+, Co2+and EDTA completely inhibited enzyme activity. Purified enzyme was successfully used for the preparation of high fructose syrup from inulin.

Purification and Characterization of Exo-Inulinase from Paenibacillus sp. d9 Strain.

This study intended to purify and characterise exo-inulinase of diesel-degrading Paenibacillus sp. D9. The whole genome sequencing of Paenibacillus sp. D9 revealed to possess the sacC gene that is encoded as exo-inulinase/levanase. This isolate was capable of producing a maximum of 50.9 IU/mL of exo-inulinase activity within 3 days at 30 °C, 200 rpm and pH of 7.0 on minimal salt medium agar supplemented with 1% (w/v) inulin. An exo-inulinase of 58.5 kDa was purified using ammonium sulphate precipitation, HiTrap QFF column and MMC column chromatographies with a specific activity of 4333 IU/mg, 7.1% recovery and a 4.3-fold increase in purity. The purified D9 exo-inulinase had temperature and pH optimum at 40 °C and pH 4.0, respectively, with the Michaelis constant of 5.5 mM and a maximal velocity of 476.2 IU/mg, respectively. Catalytic constant, k cat was calculated to be 42.6 s-1 with a catalytic efficiency (k cat /K m ) of 7.6 s-1 mM-1. The presence of Ca2+ enhanced the activity of D9 exo-inulinase while Hg2+ completely inhibited the activity, other compounds such as Fe3+ and Cu2+ had an inhibitory effect. The results of amino acid alignment and the complete degradation of inulin into fructose by the purified enzyme confirmed that inulinase from Paenibacillus sp. D9 is an exo-form. The phylogenetic tree based on the protein sequences indicates that bacterial exo-inulinases possess a common ancestry.

Immobilization of thermostable exo-inulinase from mutant thermophilic Aspergillus tamarii-U4 using kaolin clay and its application in inulin hydrolysis.

In this study, attempts were made to immobilize purified exo-inulinase from mutant thermophic Aspergillus tamarii-U4 onto Kaolinite clay by covalent bonding cross-linked with glutaraldehyde with an immobilization yield of 66% achieved. The free and immobilized inulinases were then characterized and characterization of the enzymes revealed that temperature and pH optima for the activity of the free and immobilized enzymes were both 65 °C and pH 4.5 respectively. The free inulinase completely lost its activity after incubation at 65 °C for 6 h while the immobilized inulinase retained 16.4% of its activity under the same condition of temperature and incubation time. The estimated kinetic parameters Km and Vmax for the free inulinase as estimated from Lineweaver-Burk plots were 0.39 mM and 4.21 µmol/min for the free inulinase and 0.37 mM and 4.01 µmol/min for the immobilized inulinase respectively. Inulin at 2.5% (w/v) and a flow rate of 0.1 mL was completely hydrolysed for 10 days at 60 °C in a continuous packed bed column and the operational stability of the system revealed that the half-life of the immobilized inulinase was 51 days. These properties make the immobilized exo-inulinase from Aspergillus tamarii-U4 a potential candidate for the production of fructose from inulin hydrolysis.

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 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/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.

The importance of the non-active site and non-periodical structure located histidine residue respect to the structure and function of exo-inulinase.

Here, we have studied the role of a histidine residue with the lowest solvent accessibility among other histidine residues at the end of a short connecting structure (189AELH192) of the catalytic domain of the exo-inulinase through creation of H192A mutant. Site-directed mutagenesis method was applied to create the mutant enzyme. Molecular dynamics (MD) simulations, spectroscopic, calorimetric and kinetics analysis were used to study the structural and functional consequences of His192 substitution. Accordingly, the thermo-stabilities and catalytic performance were decreased upon H192A mutation. In silico and experimental approaches evidently confirm that His192 residue of exo-inulinase possesses structural and functional importance regardless of the lack of direct interaction with the substrate or involvement in the catalytic activity of exo-inulinase.