Microorganisms as Biocatalysts and Enzyme Sources

Microbial-catalyzed biotransformations have considerable potential for the generation of an enormous variety of structurally diversified organic compounds , especially natural products with complex structures like triterpenoids, flavonoids, steroids, steroidal saponins, and sesquiterpenoids. They offer efficient and eco-nomical ways to produce semisynthetic analogues and novel lead molecules. Microorganisms such as bacteria and fungi could catalyze chemo -, regio -, and stereo specific hydroxylations of diverse substrates that are extremely difficult to produce by chemical routes. During recent years, considerable research has been performed on the microbial transformation of bioactive compounds, in order to obtain biologically active molecules with diverse structural features. In green chemistry, biotransformations are an important chemical methodology toward more sustainable industrial processes.


Introduction
Microbial transformation is regarded as an enzymatic reaction by using the metabolic activities of microorganisms to modify the chemical structures of bioactive substrates for finding the new chemical derivatives with the potent bioactivities and physical-chemical characteristics. It has a number of advantages over chemical synthesis such as higher stereo-and regioselectivity but is also enantiospecific, allowing the production of chiral products from racemic mixtures. The conditions for biotransformations are mild, and in the majority of cases, they do not require the protection of pre-existing functional groups. Furthermore, some reactions that do not occur when using chemical approaches are easily carried out by microbial transformation. Microbial factories show advantages, for instance, growing rapidly and ease of large-scale production [1][2][3].
The use of microorganisms may be a highly efficient method of production of these compounds. The reactions involved in biotransformation of organic compounds by whole cells of various microorganisms include oxidation, reduction, hydroxylation, esterification, methylation, demethylation, isomerization, hydrolysis, glycosylation, and hydrogenation [4,5].
Biotransformation may be carried out with isolated enzyme systems or with intact organism. Although isolated enzyme systems may be more specific and activities, such as antibacterial, anti-allergic, antioxidative, anti-inflammation, and antitumor activities [18].
Ginsenoside Rb1 (61) is the most predominant protopanaxadiol-type ginsenoside in Panax species (ginseng). Several microbial transformations of this substrate (Ginsenoside Rb1) have been accomplished with an ample and varied group of microorganisms, all of these having β-glucosidase activities.  Deglycosylation appears to be the major transformation pathway, and the intermediate and the final hydrolysis products of 61 depended on the microorganisms used. The biotransformation of various triterpenes, such as 61-64, has been described in the literature. For each triterpenoid, the transforming microorganism together with the type and site of the reaction catalyzed is given in Table (72). In this way there were two types of reactions in the biotransformation of triterpenic acids 58, 64, and 68: hydroxylation and glycosylation [41]. Biotransformation of 58 by C. muscae yielded nine hydroxylated and glycosylated metabolites. The specific hydroxylation (7β, 15α, and 21β) was main reaction type. In addition, the selective glycosylation at C-28 was another main reaction type. It was also observed that the 3β-OH group was selectively dehydrogenated into carbonyl group [42].

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
Applications Reference
Production of bile acids and drugs for neuropsychiatry and immunology [55,56] C-7β Mortierella sp.

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 anti-obesity activity [87][88][89][90][91]. Microbial biotransformation strategies for production of flavonoids have attracted considerable interest because they allow yielding novel flavonoids, which do not exist in nature.
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 0hydroxydihydrochalcone, flavone, 3-hydroxyflavone, 6-hydroxyflavanone, and 4 0hydroxyflavanone.  The microorganisms tend to hydroxylate flavanones at the C-5, 6, and 4 0 positions; however, for prenylated flavanones, dihydroxylation often takes place on the Δ 4(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 microbial biotransformation of kurarinone (177) using C. echinulata and C. militaris [96,97]. 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  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].

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  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][112][113].

Extremophiles
A very interesting research area in biology and biotechnology is the of extremophile microorganisms. Extremophiles can be divided into group according to (i) temperature tolerance, (ii) salt concentration, (iii) pH range, or (iv) pressure conditions. Enzymes from extremophilic microorganisms offer versatile tools for  Table 6. Enzymes, source, and some applications.
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 [114][115][116].
The outstanding properties of thermozymes are suited to industries that employ 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 (

Conclusion
Due to microorganisms' abundant multienzyme systems, microbial transformation possesses advantages against chemosynthesis of environmental friendliness, mild reaction conditions, and high stereo-, regio, and chemo-selectivities as well as in improving conversion rates and reducing cost. Thus, microbial transformation technique is being increasingly used to structurally modify natural and synthetic compounds.
The hydrolytic and reductive capabilities of microorganisms have been known and are currently used in preparative and industrial reactions. Various classes of bioactive organic compounds have been subjected to enzymatic transformation to obtain more active and less toxic substances or to elucidate their metabolic pathways.
For example, biotransformation-derived steroids are used for a wide range of pharmacotherapeutic purposes, such as anti-inflammatory, immunosuppressive, progestational, diuretic, anabolic, as neurosteroids, and as contraceptive. Researchers continue to discover more useful steroid compounds and to isolate microorganisms that can perform the structural transformations desired. New technologies such as genomics, metanogenomics, gene shuffling, and DNA evolution provide valuable tools for improving or adapting enzyme properties to the desired requirements.
An alternative may be extremophilic microorganisms such as biocatalysts for countless future industrial applications that are more environmentally friendly.