**Chia-Hung Kuo 1,2,\* , Chun-Yung Huang 1,\* , Chwen-Jen Shieh 3,\* and Cheng-Di Dong 4,\***


Enzymes, also known as biocatalysts, are proteins produced by living cells and found in a wide range of species, including animals, plants, and microorganisms. Due to their specificity, enzymes are often widely used to catalyze various chemical reactions as proficient biocatalysts in many applications. Since an enzyme is a protein, it easily becomes denatured and loses its activity under unfavorable conditions of the surrounding environment. Therefore, in order to prolong the activity and increase enzyme stability, immobilization technology is commonly used for improving the overall efficiency of enzyme catalysis. Immobilized enzymes can maintain high efficiency, specificity, and mild reaction characteristics to overcome the shortcomings of free enzymes [1]. Immobilized enzymes have many advantages, such as exhibiting high storage stability [2], easy separation and recovery [3], reusability [4], continuous operation in a bioreactor [5,6], and high stability. The commonly used methods for immobilizing enzymes on carriers are adsorption, cross-linking, covalent binding, and entrapment [7]. Among these methods, adsorption is the simplest and most common way to immobilize enzymes. The adsorption method mainly works through van der Waals interactions, hydrophobic interactions, or electrostatic interactions between the carrier and the enzyme. Therefore, adsorption can maintain the maximum activity of enzymes since there is no or significantly less configurational change in the enzyme's structure.

Remonatto et al. [8] investigated the immobilization process of lipases by physical adsorption using clay supports, including diatomite, vermiculite, montmorillonite KSF (MKSF), and kaolinite. The results showed the lipase immobilized on MKSF support had an improvement in the catalytic performance that presented a 69.47% immobilization yield and higher hydrolytic activity (270.7 U g−<sup>1</sup> ). The Vmax value of immobilized lipase was 13 times greater than the free one, indicating that the lipase activity was improved. Lipases are versatile enzymes that have several industrial applications [9]. The lipases immobilized on MKSF demonstrate high temperature and pH stability, making them suitable for industrial applications. More specifically, inorganic clays are low-cost supports with high adsorption capacity, environmentally friendly properties, and renewable abundance.

The biocementation of soil has received significant attention as an environmentally friendly alternative to chemical stabilization methods. An enzyme called urease is required to catalyze the chemical reaction that generates calcium carbonate precipitates from urea hydrolysis [10,11]. Pinto Vilar and Ikuma [12] reported the adsorption of a specific target protein, urease, as part of a complex, natural protein extract on soils through different surface chemistries with emphasis on the retained activity of the adsorbed urease. The adsorption and retention of urease activity is a critical first step for successful biocementation,

**Citation:** Kuo, C.-H.; Huang, C.-Y.; Shieh, C.-J.; Dong, C.-D. Enzymes and Biocatalysis. *Catalysts* **2022**, *12*, 0. https://doi.org/

Received: 26 August 2022 Accepted: 30 August 2022 Published: 2 September 2022

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which is a catalytic method used for soil stabilization. The results showed that the mass of proteins adsorbed was similar across soils with different surface chemistries (i.e., sand only, iron-coated sand, and hydrophobic sand). Urease was preferentially adsorbed in soils with hydrophobic contents greater than 20% (*w*/*w*) compared to total proteins contained in crude bacterial protein extracts. A comparison of the urease adsorption into silica sand and soil mixtures, as well as iron-coated sand, showed that it was much lower than the total protein adsorption. There was preferential adsorption of urease within the crude protein extract onto hydrophobic surfaces, which in turn yielded higher urease activity. These results suggest that soil surface manipulations may significantly enhance enzymatic activity, leading to improved outcomes for biocementation.

The packed-bed bioreactor is a simple piece of equipment that includes a pump and a column packed with immobilized enzymes and is commonly used for continuous production [13,14]. In the industry, the packed-bed bioreactor is commonly used to maximize the efficiency of enzyme reactions since it is energy-efficient, reduces reaction volumes, and is convenient to operate continuously [15]. Ethyl esters of omega 3 fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) ethyl esters, are active pharmaceutical ingredients used to reduce triglyceride levels in the treatment of hyperlipidemia [16]. The first time DHA and EPA ethyl esters were produced continuously from DHA + EPA concentrate and ethyl acetate in an ultrasonic packed-bed bioreactor was reported by Kuo et al. [17]. The mass transfer kinetic model was used to evaluate the efficiency of the ultrasound. The results showed that the ultrasonic packed-bed bioreactor had a higher external mass transfer coefficient compared to the packed-bed bioreactor. With ultrasonication, the highest conversion of 99% was obtained at a flow rate of 1 mL min−<sup>1</sup> and substrate concentration of 100 mM. When increasing the substrate concentration and flow rate to 500 mM and 5 mL min−<sup>1</sup> , the conversion remained at 93%. The effect of ultrasonication was also evaluated by the kinetic model using an ordered mechanism in the batch reaction. There was an 8.9 times greater V0 max/K<sup>2</sup> value in the ultrasonic bath, indicating that ultrasonication greatly enhanced lipase-catalyzed transesterification efficiency. For long-term operation, the ultrasonic packed-bed bioreactor that performed under the highest conversion conditions showed that the enzyme remained stable for at least 5 days and maintained a conversion of 98%. The ultrasonic packed-bed bioreactor is a continuously operating system that, in terms of enzyme usage and production cost, is superior to batch production. Additionally, it has a good chance of being used in industrial mass production or production capacity in the future.

Enzymes from microorganisms play a crucial role as metabolic catalysts, and they can be used in numerous industries and applications. Recent developments in the discovery of stable enzymes have expanded their uses to encompass organic synthesis and the production of specialized chemicals, pharmaceutical intermediates, and agrochemicals [18]. In order to improve enzyme performance, microbial screening or gene cloning is generally used [19–21]. It is also common for enzyme studies to use a particular medium for microbiological fermentation in order to produce enzymes.

Dakhmouche Djekrif et al. [22] demonstrated the ability of a yeast strain *Clavispora lusitaniae* ABS7, isolated from wheat grains that produced significant amounts of amylopullulanase on by-products of milk manufacturing (whey). The chromatographic profile on Sephacryl S-200 revealed two α-amylase and pullulanase-specific activities eluted together with the protein peak. The elution on DEAE-cellulose confirmed the presence of both activities in the same fraction. The α-amylase and pullulanase were purified with purification rates of 50.45 and 44.59 and yields of 23.88% and 21.11%, respectively. The purified enzyme showed a single band on the SDS-PAGE gel with an estimated molecular weight of 75 KDa. The coexistence of the two pullulanase and amylase activities was analyzed from the band, which suggested that the *C. lusitaniae* ABS7 strain had a bifunctional amylolytic enzyme with two active sites: one for α-amylase and the other for pullulanase, thus allowing simultaneous hydrolysis of α-1,4 and α-1,6 glycosidic bonds. The thin layer chromatography also confirmed that the enzyme digested starch to maltose and glucose and pullulan to maltotriose, maltose, and glucose. These two activities are probably localized in two distinct active sites of a type II amylopullulanase with saccharifying power. The α-amylase and pullulanase had pH optima at 9 and temperature optima at 75 ◦C and 80 ◦C, respectively. After heat treatments for 3 h at 75 ◦C, 2 h at 100 ◦C, and 3 h at 100 ◦C, α-amylase retained 88%, 51.76%, and 38.6% activity, respectively. The pullulanase maintained 91% and 42% activity after incubations at 80 ◦C and 100 ◦C for 3 h, respectively. The results showed the excellent thermostability of *C. lusitaniae* ABS7 amylopullulanase type II. The effect of various metal ions and chemical reagents on the α-amylase and pullulanase activities were also studied. Because of its excellent stability and compatibility with commercial laundry detergents, the alkalothermophilic amylopullulanase of *C. lusitaniae* ABS7 is suitable for industrial use, especially in detergents.

Sequential fermentation (SF) is usually used in the fermentation process of rice wine [23,24], which combines solid-state (SSF) and submerged (SmF) fermentation. De Oliveira et al. [25] developed an SF method for producing protease from *Aspergillus tamarii* URM4634 using wheat bran as a substrate. A 2<sup>3</sup> full factorial design was used to examine the effects of glucose concentration, medium volume, and inoculum size on protease production. Moreover, glucose concentration and medium volume were optimized using a 2 2 central composite rotational design to achieve a maximum protease activity of 180.17 U mL−<sup>1</sup> . The protease activity produced by the SF method was increased approximately 9-fold over the conventional SmF method. A temperature of 50 ◦C and pH of 7 were found to be optimal for *A. tamarii* URM4634 protease. In the kinetic and thermodynamic study, the enzyme exhibited values of the Michaelis constant (Km), maximum rate (Vmax), and turnover number (kcat) of 16.26 mg mL−<sup>1</sup> , 147.06 mg mL−<sup>1</sup> min−<sup>1</sup> , and 195.37 s−<sup>1</sup> , respectively. The activation energy (E\*a) of 40.38 kJ mol−<sup>1</sup> was estimated for azocasein hydrolysis. Protease thermostability was performed in the temperature range of 50 to 80 ◦C. The results indicated that the protease was thermostable at 50 ◦C, which is a commonly used temperature in many industrial processes, as evidenced by a half-life of 231.05 min and a decimal-reduction time of 767.53 min.

Nitrile hydratase is a biocatalytic enzyme that is commonly used to synthesize acrylamide from acrylonitrile [26,27]. Grill et al. [28] investigated a new nitrile hydratase, *Gordonia hydrophobica* nitrile hydratase (GhNHase), which was produced in *Escherichia coli* and applied as a cell-free extract (CFE). An enzymatic dynamic kinetic resolution with GhNHase was employed to prepare the precursor of API levetiracetam, 2-(pyrrolidine-1-yl) butanamide, via enantioselective. The GhNHase was used to convert (RS)-2-(pyrrolidine-1 yl) butanenitrile to (S)-2-(pyrrolidine-1-yl) butanamide. The effect of GhNHase, substrate concentration, and reaction temperature on the product titer and enantiomeric excess was explored. The results showed that the substrate concentration, reaction pH, and amount of biocatalyst were critically important factors for achieving (S)-configured amide conversions. Among APIs with amide groups, nitrile is often used as the precursor to introduce the amide group. GhNHase showed full conversion of the nitriles, including benzonitrile, 3-cyanopyridine, and pyrazine-2-carbonitrile, indicating that substrates with cyano groups directly attached to aromatic systems might be preferred.

Cyanide hydratases (CynHs) (EC 4.2.1.66) belong to the nitrilase family and catalyze the hydration of cyanide to formamide. Sedova et al. [29] used a new CynH from *Exidia glandulosa* (protein KZV92691.1, namely, NitEg), which was overproduced in *Escherichia coli*. The cell-free extract had a specific activity of 280 U mg protein−<sup>1</sup> , which was increased to 697 U mg protein−<sup>1</sup> after purification via cobalt ion affinity chromatography. The purified NitEg (4.0 µg protein mL−<sup>1</sup> ) could convert 25 mM free cyanide (fCN) to formamide in 60 min. The NitEg exhibited values of the Michaelis constant (Km), maximum rate (Vmax), and turnover number (kcat) of 22.2 mM, 1335 U mg−<sup>1</sup> , and 927 s−<sup>1</sup> , respectively. Enzyme performance is often represented by the Vmax/K<sup>m</sup> parameter [30]. The Vmax/K<sup>m</sup> ratio (U mg−<sup>1</sup> mM−<sup>1</sup> ) was 60 in NitEg, which was similar to *Aspergillus niger* CynH (62) but higher than *Gloeocercospora sorghi* CynH (49), indicating the high performance of NitEg. The optimal activity of NitEg occurred at 40–45 ◦C and a pH range of approximately 6–9.

A simulated electroplating effluent was composed of 100 mM fCN and 1 mM AgNO<sup>3</sup> or 1 mM CuSO4. The results showed that the NitEg at a concentration of 14 µg enzyme mL−<sup>1</sup> was sufficient to remove more than 97% fCN in the simulated electroplating effluent after 1 h. The NitEg demonstrated excellent performance in fCN solutions of up to 100 mM concentrations under alkaline conditions [29].

Phenylalanine ammonia lyase (PAL) is an important enzyme involved in the phenylpropanoid pathway, which catalyzes the biosynthesis of polyphenolic compounds such as phenylpropanoids, flavonoids, and lignin in plants. Hsieh et al. [31] investigated the expression of BoPAL4, a Bamboo PAL protein, in *Escherichia coli* Top10. BoPAL4 contained a 2106 bp open-reading frame and encoded a 701- amino acids polypeptide. L-Phe, L-Tyr, and L-3,4-dihydroxy phenylalanine (L-DOPA) were used as substrates to examine PAL, tyrosine ammonia-lyase (TAL), and L-DOPA ammonia-lyase (DAL) activities of BoPAL4. The optimal reaction pH and temperature for BoPAL4 on three substrates were at 9.0, 8.5, 9.0, and 50, 60, 40 ◦C, respectively. In addition, the kcat value of 1.87 s−<sup>1</sup> and K<sup>m</sup> value of 640 µM indicated that the Phe-123 to His mutation of BoPAL4 had a higher L-Phe binding affinity than wild-type BoPAL4. BoPAL4 can catalyze L-Phe, L-Tyr, and L-DOPA to yield trans-cinnamic acid, p-coumaric acid, and caffeic acid, respectively. Trans-cinnamic acid derivatives have been shown to have beneficial effects on human health and may therefore enhance the utility of BoPAL4.

Trypsin is a serine protease that hydrolyzes peptides and proteins on the carboxyl side of lysines and arginines. The robustness and high enzymatic activity of trypsin make it to be use in diverse products and many biotechnological applications [32,33]. The robustness of trypsin is based on three intrinsic disulfide bridges and is also influenced by the direct presence of Ca2+-ions in the calcium-binding loop. Simon et al. [34] inserted an additional fourth disulfide bridge by substitution of Glu70 and Glu80 to Cys70 and Cys80, named aTn. The results found that disulfide bonds eliminated the enzyme's dependence on Ca2+-ions. The disulfide bond in aTn prevents autolysis in absence of Ca2+-ions situation. In addition, the aTn did not seem to denature completely even at 90 ◦C indicated the disulfide bridge also contributed to increase thermal stability. The nonstructural antigen protein 3 of the hepatitis C virus (HCV NS3), commonly used for ELISA diagnosis of HCV, has protease and helicase activities. Huang et al. [35] developed a clone with a special design to produce a truncated NS3 recombinant protein (without protease domain) and overexpressed in the *E. coli* expression system. As the temperature shifted from 37 to 25 ◦C, the yield of the soluble fraction of HCV NS3 was increased from 4.15 to 11.1 mg L −1 . In terms of solubility, purity, antigenic efficacy, and stability, low-temperature (25 ◦C) protein expression exhibits superior performance than high-temperature expression at 37 ◦C. The comparison of recombinant HCV ELISA with two commercial kits, Abbott HCV EIA 2.0 and Ortho HCV EIA 3.0, was also carried out and thoroughly discussed. It was demonstrated that truncated NS3 produced at 25 ◦C showed a better discriminating ability than the proteins produced at 37 ◦C and was competitive with commercial kits, suggesting that it might have potential for HCV ELISA diagnosis.

Velázquez-De Lucio et al. [36] discussed exogenous enzymes as zootechnical additives in animal feed. Exogenous enzymes have been shown to increase the bioavailability and digestibility of nutrients as well as eliminate some anti-nutritional factors from agroindustrial and agroforestry wastes used as animal feed. A general classification of enzymes depends on the substrate on which they act; commercially, in animal nutrition, enzymes are divided into three categories based on their purpose: carbohydrases, proteases, and phytase. The enzymes used as zootechnical additives in animal feed, such as those for poultry, swine, ruminant, fish, and dogs, were also discussed. Additionally, the production methods of various enzymes for animal feeding are also introduced. A description of enzymes used for animal nutrition, their mode of action, production, and new sources of production are presented in this review, along with studies on different animal models.

Neubauer et al. [37] described the application of a Hidden Markov Model (HMM) for the identification of novel flavin-dependent halogenases in metagenomes. They chose

metagenomic data since they contain many genomes, even from uncultivable organisms, which expands the scope for identification. With this HMM, 254 complete and partial putative flavin-dependent halogenase genes in 11 metagenomic data sets were identified. In another study, the HMM strategy was used to screen the bacterial associates of the *Botryococcus braunii* consortia (PRJEB21978) [38] and identify several putative flavin-dependent halogenase genes. Two of these novel halogenase genes were found in one gene cluster of the *Botryococcus braunii* symbiont *Sphingomonas* sp. In vitro activity tests revealed that both heterologously expressed enzymes are active flavin-dependent halogenases able to halogenate indole, indole derivatives, and phenol derivatives while preferring bromination over chlorination.

Metagenomics is the study of directly obtaining all the genetic material in the environment. Metagenomics can be used as an innovative strategy to study these unculturable microorganisms by using DNA extracted from environmental samples. Sousa et al. [39] discussed the most relevant information reported in the last decade about the application of metagenomics for the discovery of promising biocatalysts in soil and water. Metagenomic studies are classified into the categories of raw resources, human-manipulated resources, and unspecified resources. A compilation of metagenomic data obtained from different environments was provided. The metagenomic studies conducted for enzyme discovery demonstrated that a considerable number of promising environments are yet to be explored. The authors analyzed and discussed the research on metagenomics from a global perspective. The data and discussion offered in this review will be fascinating for a large community of researchers working on metagenomic studies to examine microbial functionality.

In conclusion, this Special Issue shows that enzymes and biocatalysis have found numerous applications in various fields. These applications include the production of APIs, wastewater treatment, transformation of health ingredients, laundry detergent applications, fortification of feed, and biocementation. Moreover, there are studies focusing on the immobilization and production procedures of enzymes. Furthermore, an extensive review of metagenomics studies related to water and soil is included.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

