**2.4 Steroids**

Microorganisms are able to hydroxylate steroids in different positions C-1 to C-21. These represent the most widespread type of steroid bioconversion carried *Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

#### **Figure 6.**

of **76** using white-rot fungus *Coriolus versicolor* afforded eight polyhydroxylated steroids, 7β-hydroxydiosgenin (**81**), (25R)-spirost-5-en-3β,7β,21-triol (**82**), (25R) spirost-5-en-3β,7β,12β-triol (**83**), (25R)-spirost-5-en-3β,7α,15α,21-tetraol (**84**), (25R)-spirost-5-en-3β,7β,12β,21-tetraol (**85**), (25R)-spirost-5-en-3β,7α,12β,21 tetraol (**86**), and (25R)-spirost-5-en-3β,7β,11α,21-tetraol (**87**). The 3β-hydroxyl group and double bond in the B-ring of 76 were found to be important structural

*Biotransformation products of oleanolic acid (***62***), echnocystic acid (***68***) and betulinic acid (***63***).*

*AD androst-4-ene-3,17-dione; ADD androsta1,4-diene-3,17-dione; 16-AD androst-4-ene-3,16-dione.*

*A. nidulans* ++ ++ ++ ++ *C elegans* ++ ++

*Rhizopus* sp. ++ ++ ++

*The ability of different fungi to transform diosgenin (***76***).*

**Fungi Diosgenona (**77**) AD (**79**) ADD (**80**) Progesterone (**102**) 16-AD**

*F. solani* ++ ++

Microbial transformation of **76** using *Cunninghamella blakesleeana* AS 3.970 afforded polyhydroxylated derivatives, such as (25R)-spirost-5-en-3β,7α,12β-triol (**88**), (25R)-spirost-5-en-3β,7α,12β,15α,21-pentaol (**89**), (25R)-spirost-5-en-3β,7α,12β,18-tetraol (**90**), (25R)-spirost-5-en-3β,7α,12β,15α-tetraol (**91**), (25R) spirost-5-en-3β,7α,11α,21-tetraol (**92**), (25R)-spirost-5-en-3β,7β,15α,21-tetraol (**93**), and (25R)-spirost-5-en-3β,7β,12β,18-tetraol (**94**) [47], specifically, the hydroxylation, ketonization, and methoxylation by *Cunninghamella blakesleeana*, *C. elegans, Helicostylum piriforme*, and *Streptomyces virginiae*, at C-7, C-9, C-11, C-12, and C-25 positions of **76**. Biotransformation of **76** by *Syncephalastrum racemosum* afforded (25R)-spirost-5-en-3β,7α,9α-triol (**95,** 1%), (25R)-spirost-5-en-3β,9α,12α-triol-7 one (**96**, 2%), (25R)-spirost-5-en-3β,9α-diol-7,12-dione (**97**, 1.5%), (25R)-spirost-4 en-9α,12β,14α-triol-3-one (**98**, 0.66%), and (25S)-spirost-4-en-9α,14α,25β-triol-3 one (**99**, 0.66%) [48]. *C. echinulata* (CGMCC3.2716) metabolized **76** to afford **81** (0.9%), **83** (7.7%), (25R)-spirost-5-en-3β,7β-diol-11-one (**100**, 7, 1.5%), and (25R)-

Microorganisms are able to hydroxylate steroids in different positions C-1 to C-21. These represent the most widespread type of steroid bioconversion carried

determinants for their activity [46].

**2.4 Steroids**

**282**

**Figure 5.**

*Microorganisms*

**Table 3.**

spirost-5-en-3β,7β,11α-triol (**101**, 6.2%) (**Figure 6**) [49].

*Biotransformation products of diosgenin (***76***).*

out by fungi. The commercialized microbial process in the steroid field was in the production of 11α-hydroxyprogesterone. This process was realized for the first time by Peterson and Murray (1952), which patented this process of 11α-hydroxylation of progesterone (**102**) by *Rhizopus* species [50]. Microbial hydroxylation of **102** by *A. griseola* produced two hydroxylated pregnane identified as 6β,14α-dihydroxyprogesterone (**103**) and 7α,14α-dihydroxyprogesterone (**104**). *R. pusillus* produced 6β,11α-dihydroxyprogesterone (**105**) with excellent yield (65.5%) and 7α,14αdihydroxyprogesterone (**106**) (**Figure 7**) [51].

Industry, which is carried by different microorganisms, such as different species of *Curvularia* spp., *Cunninghamella* spp. and fungi *Trichoderma hamatum*, *Cochliobolus lunatus*. Structural transformation of steroidal compounds through microorganisms has emerged as an important application in the steroidal drug industry. Microbial conversions of steroids generally involve dehydrogenation, esterification, halogenation, isomerization, methoxylation, and side-chain modification of steroidal skeleton. Recently, *Mucor circinelloides lusitanicus* transformed 5-en-3β-ol steroids (**108** and **109**) into di- and trihydroxy products. The compound

**Figure 7.** *Biotransformation products of progesterone (***102***).*

microorganisms to reduce 17-keto- to 17β-hydroxysteroids was evidenced for a wide variety of substrates and microorganisms of different taxonomy: bacteria, fungi,

drugs

14α *Mucor* sp. [56] 15β *Bacillus* sp. [56, 62] 16α *Streptomyces* sp. [63, 64]

*Some examples of steroid hydroxylation reactions promoted by microorganisms and their applications.*

C-7β *Mortierella* sp. Obtaining drugs for prostate cancer [56, 57]

11α *Aspergillus* sp.*, Rhizopus* sp. Obtaining of anti-inflammatory,

**Microorganisms Applications Reference**

dexamethasone

Production of bile acids and drugs for neuropsychiatry and immunology

Obtaining anti-inflammatory drugs, like hydrocortisone, prednisone acetate,

immunosuppressive, anti-allergic drugs, and production of contraceptive [55, 56]

[56–60]

[56, 61]

and yeast [54, 56, 57, 66].

**Figure 10.**

**sites**

**Table 4.**

**285**

**Hydroxylation**

*The ability of different microorganism to transform progesterone (***102***).*

*Nigrospora* sp.*, Acremonium* sp*., Phycomyces* sp.

C-7α *Fusarium* sp*.*, *Gibberella* sp.,

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

11β *Curvularia* sp*., Absidia* sp*.,*

sp.

*Cunninghamella* sp*., Trichoderma* sp*., Cochliobolus*

#### **Figure 8.**

*Biotransformation products of 5-en-3β-ol steroids.*

**108** yielded 3β,7α,11α-trihydroxypregna-5-en-20-one (**110**, 46.4%), and **109** afforded **111** (3β,7α-dihydroxyandrost-5-en-17-one, 43.6%) (**Figure 8**) [52].

Microbial transformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (**112**) is by four filamentous fungi, *Cunninghamella elegans* (**113**–**119**), *Macrophomina phaseolina* (**115**, **117**, **120**–**122**), *Rhizopus stolonifer* (**113**, **123**), and *Gibberella fujikuroi* (**115**–**117**, **123**). These metabolites were obtained as a result of biohydroxylation of **112** at C-6β, 7β, 11α, 14α, 15β, 16β, and 17α positions (**Figure 9**) [53].

The 11α-, 11β-, 15α, and 16α-hydroxylations are currently established processes in the steroid industry mainly for the production of adrenal cortex hormones and their analogues. 11α-, 11β-, and 16α-hydroxylations are usually performed using *Rhizopus* spp. or *Aspergillus* spp., *Curvularia* spp. or *Cunninghamella* spp. and *Streptomyces* spp., respectively (**Figure 10**) (**Table 4**) [54].

Boldenone (**124**) is an important steroid hormone drug which is the derivative of testosterone. Biotransformation of **124** by *Arthrobacter simplex* and recombinant *Pichia pastoris* with 17β-hydroxysteroid dehydrogenase from *Saccharomyces cerevisiae* produces BD (**124**) from androst-4-ene-3,17-dione (**79**, AD) efficiently [65]. Many microorganisms such as *Mucor racemosus*, *Nostoc muscorum*, and *Arthrobacter oxydans* can utilize androst-1,4-diene-3,17-dione (**80**, ADD) as substrate to produce testosterone through 17β-carbonyl reduction reactions (**Table 5**). The ability of

**Figure 9.** *Biotransformation products of (20S)-20-hydroxymethylpregna-1, 4-dien-3-one (***112***).*

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

#### **Figure 10.**

**108** yielded 3β,7α,11α-trihydroxypregna-5-en-20-one (**110**, 46.4%), and **109** afforded **111** (3β,7α-dihydroxyandrost-5-en-17-one, 43.6%) (**Figure 8**) [52]. Microbial transformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one

*Macrophomina phaseolina* (**115**, **117**, **120**–**122**), *Rhizopus stolonifer* (**113**, **123**), and

result of biohydroxylation of **112** at C-6β, 7β, 11α, 14α, 15β, 16β, and 17α positions

The 11α-, 11β-, 15α, and 16α-hydroxylations are currently established processes in the steroid industry mainly for the production of adrenal cortex hormones and their analogues. 11α-, 11β-, and 16α-hydroxylations are usually performed using *Rhizopus* spp. or *Aspergillus* spp., *Curvularia* spp. or *Cunninghamella* spp. and *Strep-*

Boldenone (**124**) is an important steroid hormone drug which is the derivative of testosterone. Biotransformation of **124** by *Arthrobacter simplex* and recombinant

*cerevisiae* produces BD (**124**) from androst-4-ene-3,17-dione (**79**, AD) efficiently [65]. Many microorganisms such as *Mucor racemosus*, *Nostoc muscorum*, and *Arthrobacter oxydans* can utilize androst-1,4-diene-3,17-dione (**80**, ADD) as substrate to produce testosterone through 17β-carbonyl reduction reactions (**Table 5**). The ability of

*Pichia pastoris* with 17β-hydroxysteroid dehydrogenase from *Saccharomyces*

*Biotransformation products of (20S)-20-hydroxymethylpregna-1, 4-dien-3-one (***112***).*

(**112**) is by four filamentous fungi, *Cunninghamella elegans* (**113**–**119**),

*tomyces* spp., respectively (**Figure 10**) (**Table 4**) [54].

*Biotransformation products of 5-en-3β-ol steroids.*

(**Figure 9**) [53].

**Figure 9.**

**284**

**Figure 8.**

*Microorganisms*

*Gibberella fujikuroi* (**115**–**117**, **123**). These metabolites were obtained as a

*The ability of different microorganism to transform progesterone (***102***).*


#### **Table 4.**

*Some examples of steroid hydroxylation reactions promoted by microorganisms and their applications.*

microorganisms to reduce 17-keto- to 17β-hydroxysteroids was evidenced for a wide variety of substrates and microorganisms of different taxonomy: bacteria, fungi, and yeast [54, 56, 57, 66].


[54, 55, 70–73, 76, 77, 80, 81], testosterone [54, 55, 74–76, 81], cortexolone (**126**)

*various microorganisms. (d) Reduction and hydroxylation of prednisone (***126***) microorganisms.*

*The ability of different fungi to transform DHEA (***125***), testosterone, cortexolone (***126***) and prednisone (***127***). (a) Hydroxilation of 3β-hydroxy-5-androsten-17-one (DHEA) by various microorganisms. (b) Reduction of C-17 and hydroxilation of testosterone by various microorganisms. (c) Hydroxylation of cortexolone (***123***) by*

Sclareolide (**128**) is a natural product isolated from several plant species which displays phytotoxic and cytotoxic activities against several human tumor cells lines. This compound has also been used as starting material for the synthesis of various bioactive products. Regarding the biotransformation of the **128** with different microorganisms, mono- (**130**, **131**, **135**, **140**–**142**) and dihydroxylation (**132**–**134**, **136**, **139**, **143**, **146**), oxidation (**129**, **144**), hydroxylation/oxidation (**145**),

epimerization (**137**), and cyclization (**138**) products have been obtained [83]. The microbial transformation of **128** by *Curvularia lunata* yielded 3-ketoesclareolide (**129**), 1β-hydroxysclareolide (**130**), 3β-hydroxysclareolide (**131**), 1α,3β-dihydroxysclareolide (**133**), and 1β,3β-dihydroxysclareolide (**134**) [84]. The incubation of **128**

*blakesleeana* metabolized **128** to afford **129**, **135**, **134**, and **138**–**140**. Biotransforma-

hydroxysclareolide (**142**) [86]. Fermentation of **148** with *A. niger* using a nutrient-

As most important phytochemicals in food, the dietary flavonoids exert a wide range of benefits for human health. Recent researches have explored diverse biological and pharmacological activities of natural flavonoids—antioxidant activity, anti-inflammatory activity, anti-Alzheimer's disease, antibacterial activity, antifungal activity, anti-HIV activity, anticoagulant activity, antileishmanial activity, and

with *Cunninghamella elegans* afforded **129**, **131**, **133,** and **135**–**137** [85]. *C*

tion of **128** with *C. echinulata* yielded 5-hydroxysclareolide (**141**) and 7β-

rich culture medium yielded **141** and **144**–**146** (**Figure 12**) [83].

[78, 79], and prednisone (**127**) (**Figure 11a**–**d**) [54, 55, 82].

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

**2.5 Diterpene**

**Figure 11.**

**2.6 Flavonoids**

**287**

#### **Table 5.**

*Reduction of the C-17 carbonyl group of steroids by (17βHSDs) different microorganisms.*

The oxidation of 17β-hydroxyl group was observed along with hydroxylation of steroids at C5 (*Penicillium crustosum, P. chrysogenum*), C6 (*Bacillus stearothermophilus, B. obtusa, P. blakesleeanus*), C7 (α/β) (*A. coerulea*, *Botrytis cinerea*, *B. obtusa*, *P. blakesleeanus*, *Rhizopus stolonifer*), C10 (*Absidia glauca*), C11 (α/β) (*A. coerulea*, *B. obtusa*, *Cephalosporium aphidicola*, *R. stolonifer*), C12 (*A. glauca*, *B. obtusa*), C14 (*Bacillus* sp.), and C15 (*A. glauca*, *Aspergillus fumigatus*, *B. obtusa*) [54, 56, 57, 67–69]. The biotransformation of **79** with different microorganisms is shown. Compound **79** is an endogenous weak androgen steroid hormone and intermediate in the biosynthesis of estrone and of testosterone from dehydroepiandrosterone (DHEA) [70]. DHEA is an endogenous steroid hormone. It functions as a metabolic intermediate in the biosynthesis of the androgen and estrogen sex steroids. Various microorganisms have had the ability to biotransform steroidal compounds such as AD (**79**) [54], DHEA (**125**)

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

#### **Figure 11.**

*The ability of different fungi to transform DHEA (***125***), testosterone, cortexolone (***126***) and prednisone (***127***). (a) Hydroxilation of 3β-hydroxy-5-androsten-17-one (DHEA) by various microorganisms. (b) Reduction of C-17 and hydroxilation of testosterone by various microorganisms. (c) Hydroxylation of cortexolone (***123***) by various microorganisms. (d) Reduction and hydroxylation of prednisone (***126***) microorganisms.*

[54, 55, 70–73, 76, 77, 80, 81], testosterone [54, 55, 74–76, 81], cortexolone (**126**) [78, 79], and prednisone (**127**) (**Figure 11a**–**d**) [54, 55, 82].

#### **2.5 Diterpene**

Sclareolide (**128**) is a natural product isolated from several plant species which displays phytotoxic and cytotoxic activities against several human tumor cells lines. This compound has also been used as starting material for the synthesis of various bioactive products. Regarding the biotransformation of the **128** with different microorganisms, mono- (**130**, **131**, **135**, **140**–**142**) and dihydroxylation (**132**–**134**, **136**, **139**, **143**, **146**), oxidation (**129**, **144**), hydroxylation/oxidation (**145**), epimerization (**137**), and cyclization (**138**) products have been obtained [83]. The microbial transformation of **128** by *Curvularia lunata* yielded 3-ketoesclareolide (**129**), 1β-hydroxysclareolide (**130**), 3β-hydroxysclareolide (**131**), 1α,3β-dihydroxysclareolide (**133**), and 1β,3β-dihydroxysclareolide (**134**) [84]. The incubation of **128** with *Cunninghamella elegans* afforded **129**, **131**, **133,** and **135**–**137** [85]. *C blakesleeana* metabolized **128** to afford **129**, **135**, **134**, and **138**–**140**. Biotransformation of **128** with *C. echinulata* yielded 5-hydroxysclareolide (**141**) and 7βhydroxysclareolide (**142**) [86]. Fermentation of **148** with *A. niger* using a nutrientrich culture medium yielded **141** and **144**–**146** (**Figure 12**) [83].

#### **2.6 Flavonoids**

As most important phytochemicals in food, the dietary flavonoids exert a wide range of benefits for human health. Recent researches have explored diverse biological and pharmacological activities of natural flavonoids—antioxidant activity, anti-inflammatory activity, anti-Alzheimer's disease, antibacterial activity, antifungal activity, anti-HIV activity, anticoagulant activity, antileishmanial activity, and

The oxidation of 17β-hydroxyl group was observed along with hydroxylation of

steroids at C5 (*Penicillium crustosum, P. chrysogenum*), C6 (*Bacillus stearothermophilus, B. obtusa, P. blakesleeanus*), C7 (α/β) (*A. coerulea*, *Botrytis cinerea*, *B. obtusa*, *P. blakesleeanus*, *Rhizopus stolonifer*), C10 (*Absidia glauca*), C11 (α/β) (*A. coerulea*, *B. obtusa*, *Cephalosporium aphidicola*, *R. stolonifer*), C12 (*A. glauca*, *B. obtusa*), C14 (*Bacillus* sp.), and C15 (*A. glauca*, *Aspergillus fumigatus*, *B. obtusa*) [54, 56, 57, 67–69]. The biotransformation of **79** with different microorganisms is shown. Compound **79** is an endogenous weak androgen steroid hormone and intermediate in the biosynthesis of estrone and of testosterone from dehydroepiandrosterone (DHEA) [70]. DHEA is an endogenous steroid hormone. It functions as a metabolic intermediate in the biosynthesis of the androgen and estrogen sex steroids. Various microorganisms have had the ability to biotransform steroidal compounds such as AD (**79**) [54], DHEA (**125**)

*Reduction of the C-17 carbonyl group of steroids by (17βHSDs) different microorganisms.*

*P.*

*membranaefaciens Torulopsis* spp. *Hortaea werneckii Phaeotheca triangularis P. herbarum P. ostreatus Rhodotorula aurantiaca R. mucilaginosa*

**Fungi Yeast Bacteria**

*Candida albicans C. pelliculosa C. pseudotropicalis C. robusta C. tropicalis C. utilis Cryptococcus albidus C. laurentii C. tsukubaensis Debaryomyces hansenii D. kloeckeri D. nicotianae D. subglobosus D. vini Hansenula anomala H. califórnica H. schnegii H. suaveolens Kloeckera jensenii Saccharomyces carlsbergensis S. cerevisiae S. fragilis S. lactis S. oviformis S. turbidans S. validus Pichia farinosa*

*B. stearothermophilus Bacteroides fragilis Brevibacterium sterolicum Clostridium paraputrificum Comamonas testosteroni Lactobacillus bulgaricus Mycobacterium* spp. *B. stearothermophilus Bacteroides fragilis Brevibacterium sterolicum Clostridium paraputrificum Comamonas testosteroni (syn. Pseudomonas testosteroni*) *Lactobacillus bulgaricus Pediococcus cerevisiae Sarcina lutea Staphylococcus aureus Streptomyces globisporus S. sphaeroides S. viridochromogenes S. hydrogenans S. lavendulae*

*Fusarium culmorum F. oxysporum* var. *cubense F. solani Mucor piriformis M. spinosus P. chrysogenum P. crustosum P. blakesleeanus R. stolonifer Septomyxa affinis T. piriforme Trichoderma viride Zygodesmus* sp.

*Actinomucor elegans Agaricus silvaticus A. pantherina A. spissa Armillaria mellea Corticium centrifugum Fusarium spp. Gibberella saubinetti Mucor* spp. *Penicillium* spp. *Aphanocladium album Aspergillus chevalieri*

*Microorganisms*

*A. flavus A. oryzae A. tamarii B. obtusa C. aphidicola Ceratocystis paradoxa*

*C. lunatus*

*lecaniicorni*

**Table 5.**

**286**

*Colletotrichum musae C. radicicola*

*Exophiala jeanselmei* var.

**Figure 12.** *Biotransformation products of sclareolide (***128***).*

anti-obesity activity [87–91]. Microbial biotransformation strategies for production of flavonoids have attracted considerable interest because they allow yielding novel flavonoids, which do not exist in nature.

The microorganisms tend to hydroxylate flavanones at the C-5, 6, and 4<sup>0</sup> positions; however, for prenylated flavanones, dihydroxylation often takes place on the Δ4(5) double bond on the prenyl group (the side chain of A ring), although cyclization of the prenyl group to dihydrofurane derivatives is rather common biotransformation pathway of prenylated flavonoids. Prenylated flavanones are a unique class of naturally occurring flavonoids characterized by the presence of a prenylated side chain (prenyl, geranyl) in the flavonoid skeleton [95]. The prenyl chain generally refers to the 3,3-dimethylallyl substituent (3,3-DMA), geranyl and lavandulyl. It is proposed that the prenyl-moiety makes the backbone compound more lipophilic, which leads to its high affinity with cell membranes. The prenylation brings the flavonoids with enhancement of antibacterial, anti-inflammatory, antioxidant, cytotoxicity, larvicidal, as well as estrogenic activities. **Figure 16** demonstrated

*The main reactions during biotransformation of chalcone whit microorganisms.*

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

*Biotransformation products obtained from biotransformation of chalcones* **147-152** *with A. niger.*

**Figure 14.**

**289**

**Figure 13.**

The main reactions during microbial biotransformation are hydroxylation, dehydroxylation, O-methylation, O-demethylation, glycosylation, deglycosylation, dehydrogenation, hydrogenation, C ring cleavage of the benzo-γ-pyrone system, cyclization, and carbonyl reduction. *Cunninghamella*, *Penicillium*, and *Aspergillus* strains are very popular to biotransform flavonoids, and they can perform almost all the reactions with excellent yields (**Figure 13**). Isoflavones are usually hydroxylated at the C-3<sup>0</sup> position of the B ring by microorganisms. Chalcones **147-152** were regioselectively cyclized to flavanones (**Figure 14**). Hydrogenation of flavonoids was only reported on transformation of chalcones to dihydrochalcones (**Figure 14**) [92, 93].

*Aspergillus niger* is one of the most applied microorganisms in the flavonoids' biotransformation; for example, *A. niger* can transfer flavanone to flavan-4-ol, 2<sup>0</sup> hydroxydihydrochalcone, flavone, 3-hydroxyflavone, 6-hydroxyflavanone, and 4<sup>0</sup> hydroxyflavanone. The hydroxylation of flavones by microbes usually happens on the ortho position of the hydroxyl group on the A ring and C-4<sup>0</sup> position of the B ring, and microbes commonly hydroxylate flavonols at the C-8 position. Natural flavonoids, such as naringenin (**166**), hesperetin (**167**), chrysin (**168**), apigenin (**169**), and luteolin (**170**) were subjected to microbiological transformations by *Rhodotorula glutinis* (KCh 735). Yeast was able to regioselectively C-8 hydroxylate **167**, **168, 169**, and **170** to generate **171** (17%), **172** (31%), **173** (12.9%), and **174** (25%), respectively. Naringenin (**166**) was transformed to carthamidin (**175**) and isocarthamidin (**176**) in a ratio of 1:19, respectively (**Figure 15**) [94].

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

#### **Figure 13.**

anti-obesity activity [87–91]. Microbial biotransformation strategies for production of flavonoids have attracted considerable interest because they allow yielding novel

The main reactions during microbial biotransformation are hydroxylation, dehydroxylation, O-methylation, O-demethylation, glycosylation, deglycosylation, dehydrogenation, hydrogenation, C ring cleavage of the benzo-γ-pyrone system, cyclization, and carbonyl reduction. *Cunninghamella*, *Penicillium*, and *Aspergillus* strains are very popular to biotransform flavonoids, and they can perform almost all the reactions with excellent yields (**Figure 13**). Isoflavones are usually hydroxylated at the C-3<sup>0</sup> position of the B ring by microorganisms. Chalcones **147-152** were regioselectively cyclized to flavanones (**Figure 14**). Hydrogenation of flavonoids

*Aspergillus niger* is one of the most applied microorganisms in the flavonoids' biotransformation; for example, *A. niger* can transfer flavanone to flavan-4-ol, 2<sup>0</sup>

hydroxydihydrochalcone, flavone, 3-hydroxyflavone, 6-hydroxyflavanone, and 4<sup>0</sup>

hydroxyflavanone. The hydroxylation of flavones by microbes usually happens on the ortho position of the hydroxyl group on the A ring and C-4<sup>0</sup> position of the B ring, and microbes commonly hydroxylate flavonols at the C-8 position. Natural flavonoids, such as naringenin (**166**), hesperetin (**167**), chrysin (**168**), apigenin (**169**), and luteolin (**170**) were subjected to microbiological transformations by *Rhodotorula glutinis* (KCh 735). Yeast was able to regioselectively C-8 hydroxylate **167**, **168, 169**, and **170** to generate **171** (17%), **172** (31%), **173** (12.9%), and **174** (25%), respectively. Naringenin (**166**) was transformed to carthamidin (**175**) and



was only reported on transformation of chalcones to dihydrochalcones

isocarthamidin (**176**) in a ratio of 1:19, respectively (**Figure 15**) [94].

flavonoids, which do not exist in nature.

*Biotransformation products of sclareolide (***128***).*

(**Figure 14**) [92, 93].

**288**

**Figure 12.**

*Microorganisms*

*The main reactions during biotransformation of chalcone whit microorganisms.*

The microorganisms tend to hydroxylate flavanones at the C-5, 6, and 4<sup>0</sup> positions; however, for prenylated flavanones, dihydroxylation often takes place on the Δ4(5) double bond on the prenyl group (the side chain of A ring), although cyclization of the prenyl group to dihydrofurane derivatives is rather common biotransformation pathway of prenylated flavonoids. Prenylated flavanones are a unique class of naturally occurring flavonoids characterized by the presence of a prenylated side chain (prenyl, geranyl) in the flavonoid skeleton [95]. The prenyl chain generally refers to the 3,3-dimethylallyl substituent (3,3-DMA), geranyl and lavandulyl. It is proposed that the prenyl-moiety makes the backbone compound more lipophilic, which leads to its high affinity with cell membranes. The prenylation brings the flavonoids with enhancement of antibacterial, anti-inflammatory, antioxidant, cytotoxicity, larvicidal, as well as estrogenic activities. **Figure 16** demonstrated

#### **Figure 14.** *Biotransformation products obtained from biotransformation of chalcones* **147-152** *with A. niger.*

#### **Figure 15.**

*Biotransformation products of flavanone (***166***,* **167***) and flavone (***168***–***170***).*

sugar moiety to chalcones, flavanones, and isoflavanones with high regioselectivity. Therefore, it is possible to use *Beauveria* and *Absidia* for the microbial transformation

Bavachinin (**182**) is one kind compound of flavanones and isolated from the aerial parts and dried fruits of *Psoralea corylifolia*, and bavachinin displays a broad range of biological activities, such as antioxidant, antibacterial, antifungal, antiinflammatory, antitumor, anti-pyretic, and analgesic properties [100, 101]. Bavachinin (**182**) was subject to biotransformation by cultured cells of *A. flavus* (ATCC 30899); *C. elegans* (CICC 40250) afforded the same product **183** [(S)-6- ((R)-2,3-dihydroxy-3-methylbutyl)-2-(4-hydroxyphenyl)-7-methoxychromen-4 one]. On the other hand, one major product **184** [(2S,4R)-2-(4-hydroxyphenyl)-7 methoxy-6-(3-methylbut-2-en-1-yl)-chromen-4-ol] was obtained by *P. raistrickii* (ATCC 10490) by the reduction at the position of ketone group of the C-ring

The biotransformation of xanthohumol (**185**), a prenylated chalcone isolated from hops by selected fungi, *Absidia coerulea* (AM93), *Rhizopus nigricans* (UPF701), *Mortierella mutabilis* (AM404), and *Beauveria bassiana* (AM446), was investigated. The incubation of *A. coerulea* with **185** resulted in the isolation of xanthohumol 4<sup>0</sup>

O-β-D-glucopyranoside (**186**, 29%). This metabolite was also produced by *R. nigricans* (**186**, 14.2%). Biotransformation of **185** with *B. bassiana* and *M. mutabilis* yielded xanthohumol 7-O-β-D(4‴-O-methyl)-glucopyranoside (**187**, 23%) and isoxanthohumol 7-O-β-glucopyranoside (**188**, 49%), respectively (**Figure 19**) [103]. The compounds **188** (9.3%) and **186** (12%) were also observed as products of **185** transformation by *Cunninghamella elegans* [104]. Another way to obtain **188** is by the transformation of isoxanthohumol (**189**, 61.6%) with *Absidia glauca*; although


of simple or prenylated flavonoids by glycosidation reactions [97, 99].

(**Figure 18**) [102].

**291**

**Figure 17.**

**Figure 18.**

*Biotransformation products of prenylnaringenin (***178***).*

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

*Biotransformation products of bavachinina (***182***).*

**Figure 16.** *Microbial biotransformation of kurarinone (***177***) using C. echinulate and C. militaris.*

the microbial biotransformation of kurarinone (**177**) using *C. echinulata* and *C. militaris* [96, 97].

Incubation of *Absidia coerulea* (AM93) with prenylnaringenin (**178**) led to metabolite **179** (8-prenylnaringenin 7-O-β-D-glucopyranoside, 49.3%), while *B. bassiana* transformed **<sup>178</sup>** into **<sup>180</sup>** (8-prenylnaringenin 7-O-β-D-4″-O-methylglucopyranoside, 32.9%); the metabolites **179** and **180** originated in Sabouraud medium. In the absence of glucose in the culture of *A. coerulea*, the sulfation of substrate **178** (8-prenylnaringenin-7-sulfate, **181**, 31.1%) occurs, while *B. bassiana* into the same product (**180**). The capacity of some fungi—*Cunninghamella elegans*, *Streptomyces fulvissimus*, *Mucor ramannianus*, and *B. bassiana*—in the sulfation of certain phenolic compounds has been reported (**Figure 17**) [98].

Regioselective glycosylation of biologically active flavonoid aglycones catalyzed by microorganisms is an interesting and desired reaction, which significantly increases the water solubility of the compound and, therefore, may improve bioavailability of flavonoids. *Absidia glauca* AM177, *A. coerulea* AM93, *Rhizopus nigricans* UPF701, *Beauveria bassiana* AM278, and *B. bassiana* AM446 are able to conjugate

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

**Figure 17.** *Biotransformation products of prenylnaringenin (***178***).*

**Figure 18.** *Biotransformation products of bavachinina (***182***).*

sugar moiety to chalcones, flavanones, and isoflavanones with high regioselectivity. Therefore, it is possible to use *Beauveria* and *Absidia* for the microbial transformation of simple or prenylated flavonoids by glycosidation reactions [97, 99].

Bavachinin (**182**) is one kind compound of flavanones and isolated from the aerial parts and dried fruits of *Psoralea corylifolia*, and bavachinin displays a broad range of biological activities, such as antioxidant, antibacterial, antifungal, antiinflammatory, antitumor, anti-pyretic, and analgesic properties [100, 101]. Bavachinin (**182**) was subject to biotransformation by cultured cells of *A. flavus* (ATCC 30899); *C. elegans* (CICC 40250) afforded the same product **183** [(S)-6- ((R)-2,3-dihydroxy-3-methylbutyl)-2-(4-hydroxyphenyl)-7-methoxychromen-4 one]. On the other hand, one major product **184** [(2S,4R)-2-(4-hydroxyphenyl)-7 methoxy-6-(3-methylbut-2-en-1-yl)-chromen-4-ol] was obtained by *P. raistrickii* (ATCC 10490) by the reduction at the position of ketone group of the C-ring (**Figure 18**) [102].

The biotransformation of xanthohumol (**185**), a prenylated chalcone isolated from hops by selected fungi, *Absidia coerulea* (AM93), *Rhizopus nigricans* (UPF701), *Mortierella mutabilis* (AM404), and *Beauveria bassiana* (AM446), was investigated. The incubation of *A. coerulea* with **185** resulted in the isolation of xanthohumol 4<sup>0</sup> - O-β-D-glucopyranoside (**186**, 29%). This metabolite was also produced by *R. nigricans* (**186**, 14.2%). Biotransformation of **185** with *B. bassiana* and *M. mutabilis* yielded xanthohumol 7-O-β-D(4‴-O-methyl)-glucopyranoside (**187**, 23%) and isoxanthohumol 7-O-β-glucopyranoside (**188**, 49%), respectively (**Figure 19**) [103]. The compounds **188** (9.3%) and **186** (12%) were also observed as products of **185** transformation by *Cunninghamella elegans* [104]. Another way to obtain **188** is by the transformation of isoxanthohumol (**189**, 61.6%) with *Absidia glauca*; although

the microbial biotransformation of kurarinone (**177**) using *C. echinulata* and *C.*

Incubation of *Absidia coerulea* (AM93) with prenylnaringenin (**178**) led to metabolite **179** (8-prenylnaringenin 7-O-β-D-glucopyranoside, 49.3%), while *B. bassiana* transformed **<sup>178</sup>** into **<sup>180</sup>** (8-prenylnaringenin 7-O-β-D-4″-O-methylglucopyranoside, 32.9%); the metabolites **179** and **180** originated in Sabouraud medium. In the absence of glucose in the culture of *A. coerulea*, the sulfation of substrate **178** (8-prenylnaringenin-7-sulfate, **181**, 31.1%) occurs, while *B. bassiana* into the same product (**180**). The capacity of some fungi—*Cunninghamella elegans*, *Streptomyces fulvissimus*, *Mucor ramannianus*, and *B. bassiana*—in the sulfation of

Regioselective glycosylation of biologically active flavonoid aglycones catalyzed

by microorganisms is an interesting and desired reaction, which significantly increases the water solubility of the compound and, therefore, may improve bioavailability of flavonoids. *Absidia glauca* AM177, *A. coerulea* AM93, *Rhizopus nigricans* UPF701, *Beauveria bassiana* AM278, and *B. bassiana* AM446 are able to conjugate

certain phenolic compounds has been reported (**Figure 17**) [98].

*Microbial biotransformation of kurarinone (***177***) using C. echinulate and C. militaris.*

*Biotransformation products of flavanone (***166***,* **167***) and flavone (***168***–***170***).*

*militaris* [96, 97].

**Figure 16.**

**290**

**Figure 15.**

*Microorganisms*

significantly decreased the DPPH free radical scavenging potential; however, further dehydrogenation Δ2(3) to 2,3-dehydrosilbyn-7-sulfate (**206**) drastically enhanced the DPPH free radical scavenging potential activity [109] (**Figure 21**).

Enzymes are the most proficient catalysts, offering much more competitive processes than chemical catalysts. A number of enzyme-based processes have been commercialized for producing several valuable products. During the 1980s and 1990s, engineering of enzymes based on structural information allowed extension of their substrate ranges, enabling the synthesis of unusual intermediates. Accordingly, the use of enzymes has been expanded to the manufacture of pharmaceutical

(biocatalysts) are highly enantio-, chemo-, and regioselective in a wide range of reaction conditions. Selectivity is extremely desirable in the synthesis of different synthesis products, since it offers advantages such as minimizing the side reactions that do not require protection and deprotection steps, which allows for shorter synthesis. Biocatalysis provides a technology that is environmentally safer, and it effectively reduces the level of waste and even eliminates the waste generation rather than remediation and disposal of wastes at the end of the process. In addition

**2.7 Enzymes isolated from microorganisms and their application**

**Figure 20.**

**Figure 21.**

**293**

*Biotransformation products of sylbin (***204***).*

*Biotransformation products of isoxanthohumol (***189***).*

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

intermediates and fine chemicals [110]. Microorganisms and enzymes

**Figure 19.** *Biotransformation products of xanthohumol (***185***).*

the efficiency of this process was high (61.6% yield), it required the chemical isomerization of **185** to **189**, prior to biotransformation [105].

<sup>2</sup>″-(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″:3<sup>0</sup> ,4<sup>0</sup> ]-4,2<sup>0</sup> -dihydroxy-6<sup>0</sup> methoxychalcone (**190**), mixture of diastereoisomers of (2S, 2″S) and (2S, 2″R) 2″- (2″-hydroxyisopropyl)-dihydrofurano-[4″,5″:7,8]-4<sup>0</sup> -hydroxy-5-methoxyflavanone (**191**), and (Z)-2″-(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″-6,7]-3<sup>0</sup> ,4<sup>0</sup> dihydroxy-4-methoxyaurone (**192**) were obtained by transformation of **185** in *Aspergillus ochraceus* (AM 465) culture (**Figure 19**) [106].

Incubation of xanthohumol (**185**) both with *Fusarium avenaceum* (AM11) and *F. oxysporum* (AM727) gave a single metabolite 2″-(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″:3<sup>0</sup> ,4<sup>0</sup> ]-4<sup>0</sup> ,2-dihydroxy-6<sup>0</sup> -methoxy-α,β-dihydrochalcone (**193)**, which turned out to be the product of the prenyl group cyclization and α,β-double bond reduction. *F. tricinctum* reduced α,β-double bond of **185** to give 4,2<sup>0</sup> ,4<sup>0</sup> -trihydroxy-6<sup>0</sup> -methoxy-3-prenyl-α,β-dihydrochalcone (**194**). *Penicillium albidum* (AM79) oxidized **185** at the double bond of prenyl group to xanthohumol H (**195**) [107]. The culture of the yeast, *Rhodotorula marina* (AM 77), converted **185** and 4 methoxychalcone (**196**) to α,β-dihydroxanthohumol (**197**) and 4-methoxydihydrochalcone (**198**) with the yields of 18% and 20%, respectively [108]. *Penicillium albidum* (AM79) dihydroxylated the Δ2″(3″) double bond of xanthohumol to produce 3<sup>0</sup> -[3″-hydroxy-3″-methylbutyl]-4,2<sup>0</sup> ,4<sup>0</sup> -trihydroxy-6<sup>0</sup> -methoxychalcone (199, 22.84%) (**Figure 19**).

*B. bassiana* AM278 and *Absidia glauca* AM177 converted isoxanthohumol (**189**) into glucoside derivatives (**200**, **201**), whereas *Fusarium equiseti* AM15 transformed it into (2R)-2-(2-hydroxyisopropyl)-dihydrofurano-[2,3:7,8]-4-hydroxy5 methoxyflavanone (**202**) (**Figure 20**) [95, 106].

*C. echinulata* (ATCC 9244) sulfated silybin (**203**) to silybin-7-sulfate (**204**) and 2,3-dehydrosylibin-7-sulfate (**205**). Sulfonation at the C-7 position of silybin

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

**Figure 20.** *Biotransformation products of isoxanthohumol (***189***).*

significantly decreased the DPPH free radical scavenging potential; however, further dehydrogenation Δ2(3) to 2,3-dehydrosilbyn-7-sulfate (**206**) drastically enhanced the DPPH free radical scavenging potential activity [109] (**Figure 21**).

### **2.7 Enzymes isolated from microorganisms and their application**

Enzymes are the most proficient catalysts, offering much more competitive processes than chemical catalysts. A number of enzyme-based processes have been commercialized for producing several valuable products. During the 1980s and 1990s, engineering of enzymes based on structural information allowed extension of their substrate ranges, enabling the synthesis of unusual intermediates. Accordingly, the use of enzymes has been expanded to the manufacture of pharmaceutical intermediates and fine chemicals [110]. Microorganisms and enzymes (biocatalysts) are highly enantio-, chemo-, and regioselective in a wide range of reaction conditions. Selectivity is extremely desirable in the synthesis of different synthesis products, since it offers advantages such as minimizing the side reactions that do not require protection and deprotection steps, which allows for shorter synthesis. Biocatalysis provides a technology that is environmentally safer, and it effectively reduces the level of waste and even eliminates the waste generation rather than remediation and disposal of wastes at the end of the process. In addition

**Figure 21.** *Biotransformation products of sylbin (***204***).*

the efficiency of this process was high (61.6% yield), it required the chemical

dihydroxy-4-methoxyaurone (**192**) were obtained by transformation of **185** in

*oxysporum* (AM727) gave a single metabolite 2″-(2″-hydroxyisopropyl)-dihy-

bond reduction. *F. tricinctum* reduced α,β-double bond of **185** to give 4,2<sup>0</sup>

it into (2R)-2-(2-hydroxyisopropyl)-dihydrofurano-[2,3:7,8]-4-hydroxy5-

2,3-dehydrosylibin-7-sulfate (**205**). Sulfonation at the C-7 position of silybin

which turned out to be the product of the prenyl group cyclization and α,β-double


,4<sup>0</sup>

*B. bassiana* AM278 and *Absidia glauca* AM177 converted isoxanthohumol (**189**) into glucoside derivatives (**200**, **201**), whereas *Fusarium equiseti* AM15 transformed

*C. echinulata* (ATCC 9244) sulfated silybin (**203**) to silybin-7-sulfate (**204**) and


(**191**), and (Z)-2″-(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″-6,7]-3<sup>0</sup>

,2-dihydroxy-6<sup>0</sup>

methoxychalcone (**190**), mixture of diastereoisomers of (2S, 2″S) and (2S, 2″R) 2″-

Incubation of xanthohumol (**185**) both with *Fusarium avenaceum* (AM11) and *F.*

,4<sup>0</sup> ]-4,2<sup>0</sup>




,4<sup>0</sup> -

> ,4<sup>0</sup> -trihy-



isomerization of **185** to **189**, prior to biotransformation [105]. <sup>2</sup>″-(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″:3<sup>0</sup>

(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″:7,8]-4<sup>0</sup>

,4<sup>0</sup> ]-4<sup>0</sup>

*Biotransformation products of xanthohumol (***185***).*


methoxyflavanone (**202**) (**Figure 20**) [95, 106].

drofurano-[4″,5″:3<sup>0</sup>

22.84%) (**Figure 19**).

droxy-6<sup>0</sup>

**Figure 19.**

*Microorganisms*

duce 3<sup>0</sup>

**292**

*Aspergillus ochraceus* (AM 465) culture (**Figure 19**) [106].

to, biocatalysts have many attractive features in the context of green chemistry and sustainable development. Various enzymes used in different industrial processes have been described in the literature. **Table 6** indicates some enzymes, their source, and some applications [111–113].

sustainable developments in a variety of industrial applications as they show important environmental benefits due to their biodegradability, specific stability under extreme conditions, improved use of raw materials, and decreased amount of waste products. Although major advances have been made in the last decade, our knowledge of the physiology, metabolism, enzymology, and genetics of this fascinating group of extremophilic microorganisms and their related enzymes is still limited

The outstanding properties of thermozymes are suited to industries that employ

**Source Enzyme Activity Bioprocess/industry Reference**

oxidation by oxidative pentose phosphate cycle

Carboxylesterase Carboxyl ester hydrolysis Agriculture, food, and

Degradation of palm oil Treatment of palm oil-

Pharmaceutical industry

Enhanced production of biohydrogen

containing wastewater

Biofuel, cosmetics, or perfume production, leather and pulp industries

Biomedical, pharmaceutical, food, and environmental

from lignocellulose

Biofuel production [126]

Leather industry [123]

pharmaceutical industries

[117]

[119]

[120]

[121]

[122]

[124]

[125]

elevated temperatures, such as the pulp and paper, food, brewing, and feed processing industries. Thermophiles are often highly resistant to harsh conditions such as chemical denaturing agents, wide pH ranges, and/or nonaqueous solvents. Examples of such enzymes are cellulases, xylanases, pectinases, chitinases, amylases, pullulanases, proteases, lipases, glucose isomerases, alcohol dehydrogenases, and esterases. Thermophilic enzymes have played important roles not only at the industrial level but also in pharmaceutical applications requiring use of specific

aldolases for the synthesis of enantiopure compounds (**Table 7**) [118].

Aldolase Stereoselective C-C bond formation

Hydrogenase Final stage of glucose

substrates

Protease Degradation of hair waste from tannery

Chitinase Hydrolysis of β-(1, 4)-

Pullulanase Hydrolysis of α-(1, 6)-

*Extremophile microorganisms and some applications of their enzymes.*

glycosidic bonds in chitin

Endoxylanase β-(1,4)-xylan cleavage Biofuel production

glucosidic linkages

[114–116].

*Sulfolobus solfataricus S. acidocaldarius Thermoproteus texas Hyperthermus butylicus*

*Pyrococcus furiosus*

*Geobacillus thermoleovorans*

Microbial community from solid-state fermentation reactor

*Sulfolobus tokodaii*

*Acidothermus cellulolyticus*

*Thermotoga neapolitana*

**Table 7.**

**295**

*Bacillus pumilus* Acidic

thermostable lipase

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

*Geobacillus* sp. Lipase Hydrolysis of diver's lipid
