**5.2 Substrates for inulinase production**

Media complexity and culture conditions influence the enzyme production critically. The morphogenesis and metabolic pathway involved in enzyme induction can be noticeably affected by altering the media components and the growth parameters. Therefore, this substitution may accelerate biocatalysis of substrate into desirable products.

Inulin, starch, sucrose, and inulin-rich plant extracts are been widely utilized as exclusive, cheap, and best carbon source for biosynthesis of inulinase by several microbes. This polyfructan along with naturally occurring inulin-rich material and mixed substrates contributes as potent inducers for inulinase production. This plant-derived abundant storage polysaccharide is also present in roots and tubers of Compositae and Gramineae plants and numerous invader weeds. The review mentions a wide substrate used for inulinase production mutant [9, 19]. Dahlia (*Dahlia pinnata*), rhizosphere of Jerusalem artichoke (*H. tuberosus*), chicory (*C. intybus*) roots, kuth (*Saussurea lappa*) roots, *Allium sativum,* and *Allium cepa* have broadly been exploited for this perseverance. Mature *C. intybus* root was found to be the best substrate for receiving maximum extracellular inulinase from *Fusarium oxysporum* [22].

#### **Figure 5.**

*Glance on varied inulinase producing microorganisms [55–78].*

## **5.3 Inulinase production**

Enzyme production is critically influenced by media complexity and culture conditions. Alterations in these two factors noticeably affect the morphogenesis and metabolic pathway involved in enzyme induction. It may accelerate biocatalysis of substrate into desirable products.

Inulinase enzymes are commercially produced consuming synthetic inulin and agroindustrial residues by submerged fermentation as well as by solid-state fermentation (SSF). Microorganisms, substrate, and cultivation method for inulinase production in certain studies reported in the literature [23, 24] are described later. The records show a resilient inclination to substitute high value synthetic inulin by agroindustrial substrates so as to make this enzyme production process cost effective. *Kluyveromyces* genus is reported to be the excellent inulinase producers [25]. Researchers explained that under optimum condition, the *Kluyveromyces marxianus* NRRL Y-7571 extracellular enzyme concentration extended to 391.9 U/g of dry fermented bagasse. Thus, due to the high availability and low rate sugarcane and corn industries, deposits (sugarcane bagasse, molasses, and corn steep liquor) can be economically attractive [26].

### **5.4 Factorial design**

The escalated microbial growth and enzyme yield throughout the fermentation need to be keenly monitored. This is well accomplished by optimizing the fermentation conditions. The single-dimensional traditional simple frequently employed optimization method encompasses fluctuation of one independent variable at given level and maintaining others constant. Since it lacks the possible interactions among factors, it is least preferred. Thus, an effectual experimental scheme like response surface method is adopted to operate optimal conditions for multivariable systems. It aids in appreciating interaction of parameters and recognizing optimal range for higher yield. It also includes variety of statistical techniques used for experimental design and model erection that measures and scrutinizes the optimum conditions. Effective optimization of fungal, bacterial, and yeast inulinase production consuming diverse substrates such as Jerusalem artichoke, sugarcane bagasse, and molasses in submerged or solid-state cultivation was stated in the literature [27]. Diagrammatic depiction of microorganisms and optimized experimental variables is accessible in **Figure 6** [13, 16, 28–33].

#### **5.5 Purification and properties of inulinases**

The nature, interaction, and additional specific properties can be well understood in case of pure enzymes than the crude ones. Enzyme purification thus serves as a crucial footstep. The efficacious purification is reliant on complexity, charge distribution, and physicochemical properties of enzyme. Size, polarity, ligand interactions, and solubility are few of the strategic factors that define the choice of purification techniques to be applied for purifying inulinase. Some common purification techniques hired are salt or solvent precipitation, ion exchange, affinity, hydrophobic interaction, gel exclusion chromatography, and ultrafiltration [34].

Implication of ammonium sulfate precipitation method followed with column chromatography, boosted *X. oryzae* endoinulinase recovery by 2.9-folds [35]. Thermostable endoinulinase from *Bacillus smithii* was purified by ammonium sulfate precipitation and ion exchange chromatography. The exoinulinase synthesized by *Arthrobacter* spp., *Arthrobacter globiformis*, *Bacillus stearothermophilus, Pseudomonas mucidolens,* and *Thermotoga maritima* was recovered and purified for further studies. Salt precipitation functioned better in bacterial inulinase

**249**

**Figure 6.**

*Bioconversion of Weedy Waste into Sugary Wealth DOI: http://dx.doi.org/10.5772/intechopen.91316*

purification, whereas organic solvent precipitation was preeminent for fungal inulinases. The extraordinary solubility of ammonium sulfate in water makes it more preferential for salt precipitation. This ammonium sulfate after cleavage gets converted into two ionic forms, thus sustaining its top most position in Hofmeister series. Structural integrity of protein is least exaggerated by this salt during the salting out progression. The increased probability of protein repression in organic solvent existence reduces its utility in enzyme purification. Maximum reports on use of ion exchange and gel exclusion chromatography followed by high selective affinity chromatography are noticed for biomolecule purification. The chemical structure and function of bacterial and fungal inulinase decide which purification techniques are to be employed for its purification. These techniques are reliable in convalescing interested protein in short time. The requisite factors like widely oscillating temperature and pH stability of inulinase, along with other vital characters, before being exploited for industrial applications need to be thoroughly inspected. Physical elements such as molecular weight (*M*r), Michaelis-Menten constant (*K*m), and maximal velocity (*V*max) are significantly imperative to characterize an enzyme. Heteromeric structure and any conformational variations are well enlightened by molecular weight studies of an enzyme. *K*m and *V*max values illuminate the enzyme kinetics and also emphasize on the specificity and affinity of inulinase for varied substrates. This affinity is designated by *K*m. *K*m is the substrate concentration that engages half of enzyme's active site. Lower *K*m illustrates higher affinity of

*Highlight on inulinase production by various microbes under specific fermentation conditions.*

The molecular masses of bacterial and fungal inulinases oscillate in the range from 28 to 450 kDa as denoted in **Figure 7** [36]. Most of the fungal inulinases have

enzyme toward specific substrate and vice versa.

**5.6 Structural peculiarities of purified inulinases**

#### *Bioconversion of Weedy Waste into Sugary Wealth DOI: http://dx.doi.org/10.5772/intechopen.91316*

**Figure 6.**

*Microorganisms*

**5.3 Inulinase production**

substrate into desirable products.

be economically attractive [26].

**5.5 Purification and properties of inulinases**

**5.4 Factorial design**

Enzyme production is critically influenced by media complexity and culture conditions. Alterations in these two factors noticeably affect the morphogenesis and metabolic pathway involved in enzyme induction. It may accelerate biocatalysis of

Inulinase enzymes are commercially produced consuming synthetic inulin and agroindustrial residues by submerged fermentation as well as by solid-state fermentation (SSF). Microorganisms, substrate, and cultivation method for inulinase production in certain studies reported in the literature [23, 24] are described later. The records show a resilient inclination to substitute high value synthetic inulin by agroindustrial substrates so as to make this enzyme production process cost effective. *Kluyveromyces* genus is reported to be the excellent inulinase producers [25]. Researchers explained that under optimum condition, the *Kluyveromyces marxianus* NRRL Y-7571 extracellular enzyme concentration extended to 391.9 U/g of dry fermented bagasse. Thus, due to the high availability and low rate sugarcane and corn industries, deposits (sugarcane bagasse, molasses, and corn steep liquor) can

The escalated microbial growth and enzyme yield throughout the fermentation need to be keenly monitored. This is well accomplished by optimizing the fermentation conditions. The single-dimensional traditional simple frequently employed optimization method encompasses fluctuation of one independent variable at given level and maintaining others constant. Since it lacks the possible interactions among factors, it is least preferred. Thus, an effectual experimental scheme like response surface method is adopted to operate optimal conditions for multivariable systems. It aids in appreciating interaction of parameters and recognizing optimal range for higher yield. It also includes variety of statistical techniques used for experimental design and model erection that measures and scrutinizes the optimum conditions. Effective optimization of fungal, bacterial, and yeast inulinase production consuming diverse substrates such as Jerusalem artichoke, sugarcane bagasse, and molasses in submerged or solid-state cultivation was stated in the literature [27]. Diagrammatic depiction of microorganisms and optimized experimental variables is accessible in **Figure 6** [13, 16, 28–33].

The nature, interaction, and additional specific properties can be well understood in case of pure enzymes than the crude ones. Enzyme purification thus serves as a crucial footstep. The efficacious purification is reliant on complexity, charge distribution, and physicochemical properties of enzyme. Size, polarity, ligand interactions, and solubility are few of the strategic factors that define the choice of purification techniques to be applied for purifying inulinase. Some common purification techniques hired are salt or solvent precipitation, ion exchange, affinity, hydrophobic interaction, gel exclusion chromatography, and ultrafiltration [34]. Implication of ammonium sulfate precipitation method followed with column

chromatography, boosted *X. oryzae* endoinulinase recovery by 2.9-folds [35]. Thermostable endoinulinase from *Bacillus smithii* was purified by ammonium sulfate precipitation and ion exchange chromatography. The exoinulinase synthesized by *Arthrobacter* spp., *Arthrobacter globiformis*, *Bacillus stearothermophilus, Pseudomonas mucidolens,* and *Thermotoga maritima* was recovered and purified for further studies. Salt precipitation functioned better in bacterial inulinase

**248**

*Highlight on inulinase production by various microbes under specific fermentation conditions.*

purification, whereas organic solvent precipitation was preeminent for fungal inulinases. The extraordinary solubility of ammonium sulfate in water makes it more preferential for salt precipitation. This ammonium sulfate after cleavage gets converted into two ionic forms, thus sustaining its top most position in Hofmeister series. Structural integrity of protein is least exaggerated by this salt during the salting out progression. The increased probability of protein repression in organic solvent existence reduces its utility in enzyme purification. Maximum reports on use of ion exchange and gel exclusion chromatography followed by high selective affinity chromatography are noticed for biomolecule purification. The chemical structure and function of bacterial and fungal inulinase decide which purification techniques are to be employed for its purification. These techniques are reliable in convalescing interested protein in short time. The requisite factors like widely oscillating temperature and pH stability of inulinase, along with other vital characters, before being exploited for industrial applications need to be thoroughly inspected.

Physical elements such as molecular weight (*M*r), Michaelis-Menten constant (*K*m), and maximal velocity (*V*max) are significantly imperative to characterize an enzyme. Heteromeric structure and any conformational variations are well enlightened by molecular weight studies of an enzyme. *K*m and *V*max values illuminate the enzyme kinetics and also emphasize on the specificity and affinity of inulinase for varied substrates. This affinity is designated by *K*m. *K*m is the substrate concentration that engages half of enzyme's active site. Lower *K*m illustrates higher affinity of enzyme toward specific substrate and vice versa.

#### **5.6 Structural peculiarities of purified inulinases**

The molecular masses of bacterial and fungal inulinases oscillate in the range from 28 to 450 kDa as denoted in **Figure 7** [36]. Most of the fungal inulinases have

#### **Figure 7.**

*Comparison of molecular masses of inulinase from numerous microbial sources obtained after SDS-PAGE electrophoresis.*

molecular weight exceeding 50.0 kDa. Three inulinases with molecular masses 42, 65, and 57 kDa were isolated and purified from *Kluyveromyces* species Y 85.

Characterization of fungal and bacterial endoinulinases is also investigated after its purification. The purified endoinulinase harvested from *Penicillium* sp. TN-88 has molecular mass of 68.0 kDa [37]. *Arthrobacter* sp. S37 also produced extracellular endoinulinase, which was purified and found to have approximately 75 kDa [38].

#### **5.7 Profitable approach of inulinase efficacy**

Owing to the scenarios in food, pharmaceutical and nutraceutical industries, microbial hydrolysis and bioconversion of inulin have established a new source of revenue to several workers [39].

Inulinase offers exciting perceptions in view of the budding need for the Ultrahigh-Fructose Syrup (UHFS) production from inulin. Approximate 95% pure fructose can be obtained by enzymatic hydrolysis of inulin in the presence of inulinase. Thereby, inulinase-producing microbes are been extensively exploited by numerous industries so as to get value-added UHFS from inulin-rich weeds.

Inulinase and inulinase producers along with superfluous microorganism amalgamation are prominently affianced for simultaneous saccharification and fermentation (SSF) of diverse substrates in ethanol production methods [39–41]. Ethanol is the greatest hired liquid biofuel either as a fuel or as a gasoline complement [42]. Agave, chicory, dahlia, Jerusalem artichoke tuber, and many other inulin-rich weeds aid as the finest raw resources for fuel ethanol production. Certain wild-type microbes were mutated to offer maximum yield. Various experimentations were performed on sugar-beet molasses and numerous plant extracts so as to be used as feedstock to gain ethanol.

Inulinases are furthermore broadly subjugated in commercialization of inulo [43], gluconic acid, sorbitol, pullulan, acetone-butanol [44], and other key products.

**251**

*Bioconversion of Weedy Waste into Sugary Wealth DOI: http://dx.doi.org/10.5772/intechopen.91316*

**6. Product formed after inulinolytic hydrolysis**

drinks and candy, cakes, and other food industries [45].

**6.1 Purification of fructose**

through microarrays [43].

review [47].

*6.2.1 In food industries*

**6.2 Commercial applications of fructose**

*6.2.2 Fortification of nominated fruit juice beverages*

considerably quality loss can be replaced with FOS and fructose.

The hydrolysis of inulin feedstock by inulinase yields astonishing amount of fructose in fermented broth. Carbohydrate, particularly fructose, is an indispensible chunk of the human diet. It owes exceptional properties and is nearly 1.5 times sweeter than sucrose, thus enhancing the palate and pleasure of several foodstuffs. It is recovered by passing through carpet bag filters containing activated charcoal

Beyond 30 proceeding years, pure crystalline fructose has stood at the heights in the market as a health supplement in food and beverage. Purity is the pivotal feature that draws a distinguishing sharp line between crystalline fructose and high fructose corn syrup (HFCS). Crystalline fructose products are characteristically 100% pure fructose, while HFCS comprehends nearly equivalent shares of fructose and glucose-like sucrose (table sugar). As pure crystalline fructose is bounteously sweeter than sugar, its minor amount is also adequate to accomplish the same level of sweetness. Thus, lower-sugar and trifling calorie foods typically contain pure crystalline fructose. Food genii company also favors pure crystalline fructose as it owns supplementary properties beyond sweetness, which marks it very lucrative in

The separation of FOS and fructose is frequently accomplished by reckonable chromatographic techniques. In dietetic products, optimal FOS separation is done by implementing glass-packed precoated silica gel with sodium acetate. Liquid chromatography (LC) with acetonitrile as a mobile phase is executed to purify nonstructural carbohydrates such as sugars and FOS with 3–19 degrees of polymerization. Auxiliary cost-effective methods exploiting activated charcoal fixed bed column with 80% degree of purification and 97.8% recovery of Fructose are superfluously proficient [43]. Purified fructose is assessed by diverse techniques such as NMR, MALDI-MS, MALDITOF, GC-MS, and ESI-MS [46]. The prebiotic fructose metabolism in microorganisms can be premeditated

Pure fructose along with FOSs is finely specified to exist in voluminous natural foods. Gigantic companies are manufacturing these extensively applicable healthy and calorie-free products via hydrolyzing inulin weeds by exploiting microbial inulinases. Few lucrative applications of fructose emphasized in the

Fructose serves as one of the key ingredients in food products such as energy and sports drinks, flavor boosted water, carbonated sodas and drinks, beverages, low-calorie food options, cereals, oatmeal, and yogurts and baked goods [3].

Investigation reveals that sucrose employed as fruit juice sweetener, with no

and is further crystalized using chilled solvents, ethanol specifically.

*Microorganisms*

75 kDa [38].

**Figure 7.**

*electrophoresis.*

weeds.

**5.7 Profitable approach of inulinase efficacy**

revenue to several workers [39].

feedstock to gain ethanol.

molecular weight exceeding 50.0 kDa. Three inulinases with molecular masses 42, 65, and 57 kDa were isolated and purified from *Kluyveromyces* species Y 85. Characterization of fungal and bacterial endoinulinases is also investigated after its purification. The purified endoinulinase harvested from *Penicillium* sp. TN-88 has molecular mass of 68.0 kDa [37]. *Arthrobacter* sp. S37 also produced extracellular endoinulinase, which was purified and found to have approximately

*Comparison of molecular masses of inulinase from numerous microbial sources obtained after SDS-PAGE* 

Owing to the scenarios in food, pharmaceutical and nutraceutical industries, microbial hydrolysis and bioconversion of inulin have established a new source of

Inulinase offers exciting perceptions in view of the budding need for the Ultrahigh-Fructose Syrup (UHFS) production from inulin. Approximate 95% pure fructose can be obtained by enzymatic hydrolysis of inulin in the presence of inulinase. Thereby, inulinase-producing microbes are been extensively exploited by numerous industries so as to get value-added UHFS from inulin-rich

Inulinase and inulinase producers along with superfluous microorganism amalgamation are prominently affianced for simultaneous saccharification and fermentation (SSF) of diverse substrates in ethanol production methods [39–41]. Ethanol is the greatest hired liquid biofuel either as a fuel or as a gasoline complement [42]. Agave, chicory, dahlia, Jerusalem artichoke tuber, and many other inulin-rich weeds aid as the finest raw resources for fuel ethanol production. Certain wild-type microbes were mutated to offer maximum yield. Various experimentations were performed on sugar-beet molasses and numerous plant extracts so as to be used as

Inulinases are furthermore broadly subjugated in commercialization of inulo [43], gluconic acid, sorbitol, pullulan, acetone-butanol [44], and other key products.

**250**
