Industrial Biocatalysts Research Articles

Applicability of Recombinant Laccases From the White-Rot Fungus Obba rivulosa for Mediator-Promoted Oxidation of Biorefinery Lignin at Low pH.

Utilization of lignin-rich side streams has been a focus of intensive studies recently. Combining biocatalytic methods with chemical treatments is a promising approach for sustainable modification of lignocellulosic waste streams. Laccases are catalysts in lignin biodegradation with proven applicability in industrial scale. Laccases directly oxidize lignin phenolic components, and their functional range can be expanded using low-molecular-weight compounds as mediators to include non-phenolic lignin structures. In this work, we studied in detail recombinant laccases from the selectively lignin-degrading white-rot fungus Obba rivulosa for their properties and evaluated their potential as industrial biocatalysts for the modification of wood lignin and lignin-like compounds. We screened and optimized various laccase mediator systems (LMSs) using lignin model compounds and applied the optimized reaction conditions to biorefinery-sourced technical lignin. In the presence of both N–OH-type and phenolic mediators, the O. rivulosa laccases were shown to selectively oxidize lignin in acidic reaction conditions, where a cosolvent is needed to enhance lignin solubility. In comparison to catalytic iron(III)–(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation systems, the syringyl-type lignin units were preferred in mediated biocatalytic oxidation systems.

Study of individual domains' functionality in fused lipolytic biocatalysts based on Geobacillus lipases and esterases

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes.

Structural and Functional Analysis of the Only Two Pyridoxal 5′-Phosphate-Dependent Fold Type IV Transaminases in Bacillus altitudinis W3

Aminotransferases are employed as industrial biocatalysts to produce chiral amines with high enantioselectivity and yield. BpTA-1 and BpTA-2 are the only two pyridoxal 5′-phosphate-dependent fold type IV transaminase enzymes in Bacillus altitudinis W3. Herein, we compared the structures and biochemical characteristics of BpTA-1 and BpTA-2 using bioinformatic analysis, circular dichroism spectroscopy, atomic force microscopy and other approaches. BpTA-1 and BpTA-2 are similar overall; both form homodimers and utilize a catalytic lysine. However, there are distinct differences in the substrate cofactor-binding pocket, molecular weight and the proportion of the secondary structure. Both enzymes have the same stereoselectivity but different enzymatic properties. BpTA-2 is more active under partial alkaline and ambient temperature conditions and BpTA-1 is more sensitive to pH and temperature. BpTA-2 as novel enzyme not only fills the building blocks of transaminase but also has broader industrial application potential for (R)-α-phenethylamines than BpTA-1. Structure-function relationships were explored to assess similarities and differences. The findings lay the foundation for modifying these enzymes via protein engineering to enhance their industrial application potential.

Modelling of enzyme kinetics: cellulose enzymatic hydrolysis case

Abstract Enzymes as industrial biocatalysts offer numerous advantages over traditional chemical processes resulting on improvements in process economy and environmental sustainability. Because enzymes are extensively used in different industrial areas, the enzyme kinetics is an important factor for industry as it is able to estimate the extent of substrate conversion under known conditions and evaluate reactor performance. Furthermore, kinetic modelling is useful in the analysis, prediction, and optimization of an enzymatic process. Thus, kinetic modelling is a powerful tool for biochemical reaction engineering. In addition to the aforementioned, in the industrial technology, modelling together with simulation play a key role because they help to understand how a system behaves under specific conditions, and thus they allow saving on costs and lead times. Enzymatic conversion of renewable cellulosic biomass into biofuels is at the heart of advanced bioethanol production. In the production of bioethanol from cellulosic biomass, enzymatic hydrolysis of cellulose to fermentable sugars accounts for a large portion (∼30%) of the total production costs. Therefore, a thorough understanding of enzymatic hydrolysis is necessary to create a robust model which helps designing optimal conditions and economical system. Nevertheless, it is a challenging task because cellulose is a highly complex substrate and its enzymatic hydrolysis is heterogeneous in nature, and thus the whole process of cellulose conversion to glucose involves more steps than classical enzyme kinetics. This chapter describes the bases of enzyme kinetic modelling, focussing on Michaelis-Menten kinetics, and presents the models classification based on the fundamental approach and methodology used. Furthermore, the modelling of cellulose enzymatic hydrolysis is described, also reviewing some model examples developed for cellulose hydrolysis over the years. Finally, the application of enzyme kinetics modelling in food, pharmaceutical and bioethanol industry is presented.

A novel cold-adapted phospho-beta-galactosidase from Bacillus velezensis and its potential application for lactose hydrolysis in milk

A phospho-β-galactosidase gene (BsGal1332) was cloned from Bacillus velezensis and successfully expressed in Escherichia coli BL21(DE3). The active BsGal1332 was identified to be a homodimer with a combined molecular mass of approximately 113 kDa, and it belonged to the glycoside hydrolase family 1. The BsGal1332 displayed relative strict substrate specificity for galactosyl compounds compared with the other phospho-β-galactosidases. The purified BsGal1332 showed the maximum activity at pH 8.0 and 50 °C for 2-nitrophenyl-β-d-galactopyranoside (oNPGal) and at 40 °C for lactose. BsGal1332 was slightly activated by K+ and Na+, but not strongly affected by Ca2+, and was stable at pH 6.0–7.0 and 40 °C or below it. The activity of BsGal1332 decreased quickly after incubation at 50 °C or higher temperature, suggesting it was a cold-adapted enzyme. Moreover, BsGal1332 could hydrolyze lactose and oNPGal with Km values of 23.68 and 2.36 mM and kcat values of 117.55 and 155.61 s−1 at 4 °C, respectively. Additionally, 1 U of the BsGal1332 could thus be capable of hydrolyzing about 38% of the lactose in 1 mL of milk after incubating at 4 °C for 4 h. Taken together, these properties of BsGal1332 made it a new promising industrial biocatalyst for efficient lactose hydrolysis in milk.

Topology engineering via protein catenane construction to strengthen an industrial biocatalyst

Protein topology engineering has emerged as a new dimension to alter protein stability and function. Inspired by the art of nature, where backbone cyclization is frequently adopted to enhance the stability of natural peptide products and thermostable enzymes; herein, we report protein topology engineering of an industrial thermolabile gamma lactamase via catenation. Two different protein catenanes were successfully constructed via SpyTag/SpyCatcher modules and two different peptide dimer domains. The designed protein catenanes were functionally synthesized in Escherichia coli. A comparison of their biochemical properties revealed that protein topology played a key role in the stability of gamma lactamase. Protein catenation enhanced both the thermo- and proteolytic stabilities of gamma lactamase. Gamma lactamase was stabilized by ∼8 °C in one of the catenated forms. Moreover, Cat1-MhIHL-V54L and Cat2-MhIHL-V54L displayed 1.8- and 2.4-fold higher enzyme efficiencies (Kcat/Km), respectively, than the unattenuated enzyme. Therefore, our results proved that protein catenane construction could be a general strategy to strengthen industrial biocatalysts by mechanisms distinct from those of the conventional direct evolution schemes, whereby our results offer wide applications in the fine chemical industry.

Characterization of alkylguaiacol-degrading cytochromes P450 for the biocatalytic valorization of lignin

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA, encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC, encoding the meta-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL, encoding a CYP255A2 family P450 and an ortho-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O-demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities (kcat/KM): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA's binding affinities for the different guaiacols and was the inverse of GcoAEP4's specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin.

Harnessing the sponge microbiome for industrial biocatalysts.

Within the marine sphere, host-associated microbiomes are receiving growing attention as prolific sources of novel biocatalysts. Given the known biocatalytic potential of poriferan microbial inhabitants, this review focuses on enzymes from the sponge microbiome, with special attention on their relevant properties and the wide range of their potential biotechnological applications within various industries. Cultivable bacterial and filamentous fungal isolates account for the majority of the enzymatic sources. Hydrolases, mainly glycoside hydrolases and carboxylesterases, are the predominant reported group of enzymes, with varying degrees of tolerance to alkaline pH and growing salt concentrations being common. Prospective areas for the application of these microbial enzymes include biorefinery, detergent, food and effluent treatment industries. Finally, alternative strategies to identify novel biocatalysts from the sponge microbiome are addressed, with an emphasis on modern -omics-based approaches that are currently available in the enzyme research arena. By providing this current overview of the field, we hope to not only increase the appetite of researchers to instigate forthcoming studies but also to stress how basic and applied research can pave the way for new biocatalysts from these symbiotic microbial communities in a productive fashion. KEY POINTS: • The sponge microbiome is a burgeoning source of industrial biocatalysts. • Sponge microbial enzymes have useful habitat-related traits for several industries. • Strategies are provided for the future discovery of microbial enzymes from sponges.

Protease—A Versatile and Ecofriendly Biocatalyst with Multi-Industrial Applications: An Updated Review

Proteases are important industrial biocatalysts that constitute the largest group of enzymes acting as proteinases, peptidases, and amidases with a broad range of industrial applications. In this review, particular attention has been given to comprehensively scrutinize the proteases. After the succinct introduction, classification of proteases as exopeptidases (amino and carboxy proteases) and endopeptidases (serine, aspartic, cysteine, and metalloproteases), sources of alkaline, acidic and neutral protease like animal, plant and microbial sources along with the multi-industrial applications have been dissertated. Now a day’s, mostly proteases, which are present in the market, are produced from microbial sources because of the fast production rate and the limited requirement of cultivation. In addition to this, a critique on the applications of proteases in food, detergent, leather, pharmaceutical, cosmetics, silk degumming, silver recovery, chemical industry, and wastewater treatment industries is also concisely addressed. Finally, protein engineering and immobilization strategies to improve the catalytic properties of protease are thoroughly vetted. The quest for novel sources of protease enzyme has been encouraged to fulfill their ever-increasing demands for industrial exploitation.

Supplementation of watermelon peels as an enhancer of lipase and esterase production by Yarrowia lipolytica in solid-state fermentation and their potential use as biocatalysts in poly(ethylene terephthalate) (PET) depolymerization reactions

Yarrowia lipolytica is a yeast that presents high biotechnological potential due to its ability to produce many metabolites, among them lipases and esterases, which are important industrial biocatalysts. Since Brazil is an agroindustrial country, it generates an enormous diversity of residues or byproducts that can be used as a platform for biomolecules production. This work aims to evaluate lipase and esterase production by Y. lipolytica via solid-state fermentation using soybean bran and soybean bran supplemented with watermelon peels in different contents, and subsequent use of the enzyme extracts for poly(ethylene terephthalate) (PET) hydrolysis. Supplementation of watermelon peels in the lowest content led to an improvement of lipase activity in almost 31%, reaching 75.22 U g−1. Esterase productivity was 1.5-fold higher when 20 wt% of watermelon peels were added to the media culture. Timecourse evaluation of enzymes production showed a maximum lipase activity in 14 h and similar esterase activity in 14 and 20 h of fermentation. Proteases production were also intensified in supplemented samples. Enzymes produced with 5 wt% watermelon peels supplementation led to higher terephthalic acid concentration (up to 42.02 µmol L−1) during PET depolymerization. Results suggest a great potential of enzyme production in low cost fermentative media to act as biocatalysts in PET hydrolysis reactions.

Cloning, expression and characterization of a new endo-β-N-acetylglucosaminidase from Streptomyces alfalfae

Endo-β-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-β-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×10⁶ U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.

Resonance Raman spectroscopic studies of peroxo and hydroperoxo intermediates in lauric acid (LA)-bound cytochrome P450 119

Cytochromes P450 bind and cleave dioxygen to generate a potent intermediate compound I, capable of hydroxylating inert hydrocarbon substrates. Cytochrome P450 119, a bacterial cytochrome P450 that serves as a good model system for the study of the intermediate states in the P450 catalytic cycle. CYP119 is found in high temperature and sulfur rich environments. Though the natural substrate and redox partner are still unknown, a potential application of such thermophilic P450s is utilizing them as biocatalysts in biotechnological industry; e.g., the synthesis of organic compounds otherwise requiring hostile environments like extremes of pH or temperature. In the present work the oxygenated complex of this enzyme bound to lauric acid, a surrogate substrate known to have a good binding affinity, was studied by a combination of cryoradiolysis and resonance Raman spectroscopy, to trap and characterize active site structures of the key fleeting enzymatic intermediates, including the peroxo and hydroperoxo species.

Industrial Biocatalysis: an Insight into Trends and Future Directions

Industrial biocatalysts are gaining popularity each day for the purpose of eco-friendliness, cost-effectiveness and long-term sustainability. However, finding a suitable biocatalyst for any particular process fulfilling these criteria is still a challenging task and has to be taken care of for upscaling of the process. Emerging technologies are being adopted for the betterment of the process involving biocatalysis in terms of process economics and efficiency in terms of yield and productivity. Various recent measures for finding suitable novel industrial biocatalysts and their improvement in properties using emerging techniques have been reviewed in this manuscript. Novel techniques focussing on biocatalytic-related areas in the recent times and their future prospects have been summarized. Isolation of novel biocatalysts for industrial processes is still being considered as the deciding factor for efficient industrial production using enzymes. Metagenomics and classical ways of finding these catalysts for isolation and screening have not gone out of fashion. Immobilization on novel matrices and co-immobilization on novel matrices to overcome mass transfer limitation and for reducing the cost are very common in recent studies. Metabolic and genome mining for improvement and directed evolutions are also being used for generation of new-age catalysts improvement. Enzyme cascade, in silico studies and computational biology are upcoming areas related to biocatalysis. Importance of industrial biocatalysis is limited by non-availability of efficient catalysts in terms of productivity, yield, cost-effectiveness and eco-friendliness. These are being removed gradually by finding the suitable candidate for catalysing these reactions. Classical and metagenomic isolation and screening of these efficient catalysts are presently in vogue but genome mining is the upcoming trend. Directed evolution, genome engineering and metabolic engineering are second steps considered for desired improvements in properties. Process optimization using various non-conventional solvents, immobilization on novel matrices, co-immobilization and multi-enzyme cascades are being done for improving economic efficiency. Further need for process sustainability and economic consideration are forcing researchers to take the route of in silico biology and high-level computational methods.

Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation.

The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The P. polymyxa levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the P. polymyxa levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges.IMPORTANCE Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa, it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.

Engineering a Highly Active Sucrose Isomerase for Enhanced Product Specificity by Using a "Battleship" Strategy.

The sucrose isomerase SmuA from Serratia plymuthica efficiently catalyses the isomerisation of sucrose into isomaltulose, an artificial sweetener used in the food industry. However, the formation of a hygroscopic by‐product, trehalulose, necessitates additional separation to obtain a crystalline product. Therefore, we have improved the product specificity of SmuA by first introducing a few exploratory amino acid exchanges around the active site and investigating their influence. Then, we devised a second set of mutations, either at promising positions from the preceding cycle, but with a different side chain, or at alternative positions in the vicinity. After seven iterative cycles involving just 55 point mutations, we obtained the triple mutant Y219L/D398G/V465E which showed 2.3 times less trehalulose production but still had high catalytic efficiency (k cat/K M=11.8 mM−1 s−1). Not only does this mutant SmuA appear attractive as an industrial biocatalyst, but our semirational protein‐engineering strategy, which resembles the battleship board game, should be of interest for other challenging enzyme optimization endeavours.

A high-throughput screening assay for the directed evolution-guided discovery of halohydrin dehalogenase mutants for epoxide ring-opening reaction

Halohydrin dehalogenases (HHDHs) are valuable biocatalysts involved in the synthesis of β-substituted alcohols via their nucleophile-mediated ring-opening activity. To use directed evolution to unleash the latent potential of HHDHs for the synthesis of β-substituted alcohols, we report a high-throughput assay for screening HHDHs mutagenesis libraries. The assay is performed in a 96-well microtiter plate format using a cell-free extract or whole-cells in the presence of the desired nucleophile. The developed method relies upon the color change of bromothymol blue, due to the pH change caused by HHDH-catalyzed ring-opening of the epoxide substrate. The assay was validated using gas chromatography and subsequently applied to high-throughput screening of halohydrin dehalogenase HheC mutagenesis library. Active mutants were found for the tested substrates. Due to its simplicity and flexibility towards the use of nucleophiles and epoxides, the method is an attractive alternative to the existing assays for HHDH epoxide ring-opening reaction and could be helpful in the rapid discovery of industrial biocatalysts.

Visualized characterization of bacterial penicillin G acylase for the hydrolysis of β-lactams using an activatable NIR fluorescent probe

Penicillin G acylase (PGA) identified from bacteria, is used as industrial biocatalyst for the hydrolysis of penicillins along with the production of 6-aminopenicillanic acid, a key intermediate of the semi-synthetic β-lactam antibiotics. Due to wide usage of PGA, some novel homology bioactive enzymes are investigated in various bacteria, and visualized analysis technique would be necessary and helpful for the development and utilization of PGA function in living systems. In the present work, an activated off-on near-infrared (NIR) fluorescent probe (DDAP) deduced from a novel fluorophore has been designed and developed for sensitively and selectively detecting endogenous PGA in real-time. The hydrolysis mechanism has been elucidated using in silico docking analysis, which confirmed DDAP as the specific substrate of PGA. Using DDAP as the fluorescent probe, the endogenous bacterial PGA was imaged in living systems successfully, which could be applied to identify bacteria with PGA expression from complex microorganism samples. Furthermore, using DDAP as the staining agent, the endogenous PGA in various bacteria has been profiled visually on native-PAGE gel, and the PGA activity in bacterial lysates could be effectively determined. The PAGE-gel for PGA analysis stained by DDAP was more sensitive than generally used silver and Coomassie Blue dyes. Finally, a series of β-lactams were hydrolyzed by recombinant PGA to further confirm its hydrolase property, corresponding to the hydrolysis of DDAP.

Isolation of naringinase producing soil bacteria from <em>Psidium guajava</em> L. and <em>Terminalia chebula</em> Retz and its enzymatic activity

In commercial citrus juice, bitterness causes serious challenges in fruit juice manufacturing industries. The prime cause of bitterness is due to the presence of naringin compound. It is noted that microbial enzyme retains some specific catalytic reactions of physicochemical and biological properties possessing high degree of industrial and medical applications. The microbial enzyme naringinase can be immobilized for industrial use to reduce the cost of debittering of juice. Since environment friendly industrial biocatalysts are economically more viable, so the focus on the present study is debittering of juice at low cost without using chemicals which alter the nutrient composition. In the present study, four strains of naringin degrading bacteria were isolated from the soil of Psidium guajava L. and Terminalia chebula Retz in Dibrugarh University Botanical Garden and were investigated for naringinase activity of soil microbes and their growth conditions at different parameters. The turbidity of cell culture and concentration of proteins in the culture media have been utilized for the optimization of various growth conditions, like temperature and pH for microbial growth. The optimal growth of the four isolates was observed in a media of pH 6 and selected for further study. All the four isolates showed good extracellular naringinase activity. Among the four isolates, oval and rod shaped gram positive bacteria showed the highest specific activity (405.31 U/mg) and lowest activity was shown by rod shaped gram negative bacteria (231.77 U/mg). Furthermore, rod shaped isolate exhibited maximum growth and highest protein content among the four isolates. These results suggested that in addition to the naringinase enzyme, some other proteins were also produced by the isolates. These proteins might have some significance in supporting the growth of the microorganisms.

Engineering lytic polysaccharide monooxygenases (LPMOs).

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze the hydroxylation of glycosidic bonds found in the most abundant and recalcitrant polysaccharides on Earth. Since their discovery in 2010, these enzymes have received extensive attention in both fundamental and applied research due to their remarkable oxidative power and synergistic interplay with hydrolytic enzymes. The harsh and unnatural conditions used in industrial enzymatic saccharification processes and the sensitivity of LPMOs for damage induced by reactive oxygen species call for enzyme engineering to develop LPMOs to become robust industrial biocatalysts. Other engineering targets include improved catalytic activity, adjusted substrate specificity and the introduction of completely new activities. Reaching these targets not only requires appropriate methods for measuring enzyme activity, but also requires in-depth knowledge of the active site and the reaction mechanism, which is yet to be achieved in the LPMO field. Here we describe what has been done in the LPMO engineering field so far. Furthermore, we address the difficulties involved in properly assessing LPMO functionality, which are due to common side reactions taking place in LPMO reactions and which complicate screening methods.

Baker's Yeast Catalyzed Multicomponent Reactions: A New Hope?

AbstractBaker's yeast was primarily used as a leavening agent in the food processing industry and later as biocatalyst in the brewing industry. Ready availability and low cost has made baker's yeast a primary research object among the scientific community. It is well known to reduce carbonyl groups. Several other simple single step transformations have also been carried out using baker's yeast. Multicomponent reactions are a close fit to the concept of “ideal” synthesis and, in recent years, they have become a rapid platform for the drug discovery process. Due to the promiscuous nature of enzymes, they were not explored as potential biocatalyst for multicomponent reactions. In this review, the application of baker's yeast in multicomponent reactions has been discussed.