Current Biotechnology - Volume 4, Issue 2, 2015

Dihydrofolate reductase (DHFR) has emerged as a model enzyme to investigate the role of dynamics in catalysis. This review begins with a description of the timing of the protonation of the dihydrofolate substrate and the subsequent hydride transfer reaction. This is followed by a summary of the results obtained from bioinformatics, theoretical and experimental studies of coevolving DHFR residues. Some of these residues appear to be dynamically coupled to each other and to the reaction coordinate. Importantly, the coupling appears to expand across the entire enzyme as many of the coevolved amino acids are remote from the active site. The importance of introducing mutations during evolution in the correct evolutionary order is also discussed. These studies involved a combination of steady state and pre-steady state rate measurements, kinetic isotope effects, NMR spectroscopy and computational investigations of the wild type DHFR from E. coli and several mutants. The cumulative results implicate a role of motions across the entire protein in DHFR catalysis, which may serve as a guide in studies of other enzymatic systems.

Lipase B from Candida antarctica (CaLB) is one of the most largely employed biocatalysts for the synthesis of chiral fine chemicals. The successful application of this enzyme has also been promoted by advanced computational methods able to simulate enantiodiscrimination at molecular and energy level. Quantitative prediction of enantioselectivity remains a challenging task, affordable by means of sophisticated and rigorous QM/MM methods or by hybrid methods that combine molecular mechanics with experimental data and regression analysis. Most of the methods reported in the literature aim to predict CaLB enantiopreference and to understand the structural basis of enantiodiscrimination. Various experimental problems, such as resolution of alcohols, amines and carboxylic acids, solvent effect, entropic contribution of substrates, are expected to receive beneficial indications from novel advanced computational methods. However, the choice of the appropriate strategy is crucial for success in solving specific problems within a realistic time frame and with a convenient computational cost. In order to be competitive with experimental work, the rational and computational approach should be ideally within a high throughput scheme. Therefore, automation of computational procedures, software and scoring steps represents a new emerging and promising perspective to make the planning of biotransformation more effective and rational.

Oxidation reactions with oxygen and peroxides are difficult to control in the artificial environment of man-made chemistry. This makes oxizymes, i.e. oxidative enzymes that use oxygen or peroxide as co-substrates, very valuable targets for the chemical and pharmaceutical industries. Additionally, growing awareness of sustainability issues in society has encouraged the use of oxizymes in these industries. Some oxizymes generate hydrogen peroxide as a by-product. Hydrogen peroxide has antimicrobial effects and is therefore of interest for the food industries as well as other industries where microorganisms pose a danger to the consumer or patient. Hydrogen peroxide can also be used as a bleaching agent and thus applications of oxizymes in the textile industries as well as the pulp and paper industries are common practice. Oxizymes have recently been found to play an important role as auxiliary enzymes in the degradation of biomass. In this role, they support carbohydrate active enzymes in the degradation of cellulose and chitin, or assist in the deconstruction of lignin-derived polymers. They are therefore of importance for the biofuels industry which aims to create biofuels from renewable plant materials to replace petroleum-based fuels. Oxizymes have many more properties that make them useful for industrial applications. This review summarizes the technological advancements, which have made the use of enzymes in industry possible, as well as showcases different types of oxizymes currently used in different industries. Also, the challenges oxizymes face before their industrial applications can be fully developed, are discussed.

Hydroxynitrile lyase enzymes (HNLs) catalyze the stereoselective addition of HCN to carbonyl compounds to give valuable chiral hydroxynitriles. The discovery of new sources of HNL activity has been reported several times as the result of extensive screening of diverse plants for cyanogenic activity. Herein we report a two step-method that allows estimation of not only the native size of the active HNL enzyme but also its substrate specificity. Specifically, crude protein extracts from plant tissue are first subjected to blue native-PAGE. The resulting gel is then directly used for an activity assay in which the formation of hydrocyanic acid (HCN) is detected upon the cyanogenesis reaction of any cyanohydrin catalyzed by the enzyme of interest. The same gel may be used with different substrates, thus exploring the enzyme’s substrate scope already on the screening level. In combination with mass spectrometry, sequence information can be retrieved, which is demonstrated with the example of the so far unknown sequence of Prunus domestica HNL.

Thermotoga hypogea is a hyperthermophilic bacterium growing on carbohydrates and peptides, producing acetate, CO2 and H2 as major end products. Hydrogenase activity was detected in the cell-free extract of T. hypogea and a hydrogenase was purified to homogeneity. The purified enzyme was homotetrameric with a subunit of 65 kDa, and contained 16 atoms of Fe and 11.6 atoms of acid labile sulfur per mole subunit. The gene encoding the hydrogenase was sequenced, and four iron-sulfur clusters including the H-cluster were found to be present in the gene sequence, indicating it is an [FeFe]-hydrogenase. The enzyme was oxygen sensitive with a t1/2 of 3 min when exposed to air. It was thermostable with t1/2 values of approximately 40 and 15 min when incubated at 70°C and 85°C, respectively. The optimal temperature for the hydrogenase activity was about 85 °C. Both benzyl viologen (BV) and methyl viologen (MV) could serve as electron acceptors, and the apparent Vmax values were determined to be 1,142 and 607 μmol H2 oxidized min-1 mg-1, respectively. For H2 uptake activity, the apparent Km values for MV and BV were 0.17 and 0.24 mM, respectively. For H2 evolution activity, the apparent Km value for MV and apparent Vmax value were determined to be 1.1 mM and 192 μmol min-1 mg-1, respectively. Ferredoxin purified from T. hypogea served as electron carrier. The enzyme exhibited pH optima of 10.0 and 8.0 for the H2 uptake and evolution activities, respectively. The physiological function of the enzyme is proposed to catalyze the H2 evolution.

The numerical imperative of random mutagenesis and high-throughput screening has created an overwhelming pressure for single measurements of enzyme activity as a preliminary diagnostic of functional improvement even if this leads on to a more traditionally thorough kinetic analysis of each potentially interesting mutant. This paper argues, with examples, that it is therefore all the more important to apply kinetic thinking to the initial choice of conditions for the screening reaction and as far as possible match those conditions to those of the intended application. Without such planning there is a real risk of missing valuable mutants and of wasting time on many that do not fit the requirement. Key issues are choice of substrate concentration and direction of reaction and awareness of rate limiting steps in the mechanism.

Electric eel acetylcholinesterase (AChE) was immobilised on electrospun nylon 6: chitosan nanofibres using glutaraldehyde (GA) cross-linking. The immobilisation protocol was optimised and a GA solution of 5% (v/v) resulted in the immobilisation of 0.334 mg/cm2 of AChE onto the nanofibres. The immobilised enzyme was characterised with respect to pH and temperature and results compared to the free enzyme. The Vmax values obtained for the free and immobilised AChE enzymes were 0.345 and 0.287 µmol/min/mL, respectively, and the Km values for the free and immobilised AChE enzymes were 0.482 and 0.812 mM, respectively. Relative to free AChE, the immobilised enzyme showed considerable storage stability retaining ~50% of its activity when stored for 49 days at 4°C. Immobilised AChE also retained > 20% of its initial activity after 9 consecutive reuse cycles. When exposed to fixed concentrations of pesticides (carbofuran and demeton-S-methyl sulfone), immobilised AChE showed almost identical inhibition characteristics compared to the free enzyme. In conclusion, nylon-6:chitosan electrospun nanofibres were shown to be suitable supports for facile AChE immobilisation and the immobilised enzyme has potential for use in pesticide detection.

The Actinomadura R39 DD-peptidase is a very penicillin-sensitive enzyme. After a short time of contact with a liquid sample containing a beta-lactam antibiotic, the determination of the residual enzyme activity allows estimation of the concentration of the antibiotic in the sample. An alternative method utilizes the C-terminal domain of the Bacillus licheniformis penicillin sensor BlaR that is devoid of enzymatic activity. In this case, the protein is labelled with gold particles and the unreacted portion is captured by beta-lactam molecules covalently attached to a solid support. Both methods have been quite successful for the rapid estimation of beta-lactam antibiotics in milk.

Pleurotus ostreatus, using glucose as carbon source, is able to depolymerize a mixture of 2- naphthalene sulfonic acid polymers (NSAP), contained in a real petrochemical wastewater. On the contrary, its extracellular crude extracts, rich in laccases, are ineffective on NSAP degradation. Purified laccases and fungal extracellular extracts are able to depolymerize NSAP only in presence of specific synthetic or natural mediators. When P. ostreatus is grown on straw, the extracellular extracts are able to degrade the polymers, because they contain both high amounts of laccases and natural mediators. Because of the negligible cost of the straw this process appears very promising for enzymatic bioremediation of wastewater from rubber industry.

In recent years a lot of attention has been paid to biofuel production and the transition from first to second generation feedstocks, mainly from the perspective of reducing the use of fossil resources and avoiding competition with the food chain. These lignocellulosic materials can be used for fermentative production of other biochemicals, building blocks or biomaterials, making them attractive for a biobased economy. The many aspects that need to be considered for process design and industrial implementation for second generation sugar syrup, from technical, cost and sustainability perspective, are reviewed in this paper. The major classes of lignocellulosics, being cereal residues, hardwood and softwood, their compositions, and the main pretreatment methods, in view of efficiency in opening up the cell wall matrix and their potential to minimize the production of inhibitors, are compared. Still, due to naturally present potentially inhibitory sugars, as well as the inevitable production of some inhibitors in pretreatment, a detoxification step is likely necessary to prevent carry-over in the final product. In-depth studies have generated a very detailed view on enzymes required to saccharify the pretreated lignocellulosics efficiently, including the description of a new mechanism by the lytic polysaccharide monooxygenase. The enzyme classification, specificity, and their limitations, either substrate, enzyme or process related, are reviewed. Different ways to deal with minor impurities in the sugar syrup, from fermentation operational point of view, are deliberated upon. Finally, economic analysis in combination with life cycle assessment to value sustainability aspects, is discussed and shows the requirement for an integrated multidisciplinary approach to clear the way for production of universally fermentable sugar syrup from lignocellulosics.

Despite the higher activity possessed by bacterial β-amylases, plant β-amylases are widely used for industrial processes. It is crucial that targeted improvements must be made on bacterial β-amylases for enhanced production, thermal and operational stability for their commercial viability. In this study, Bacillus polymyxa BWB-01 was cultivated in submerged fermentation and influence of cultivation conditions (pH, temperature, carbon and nitrogen sources) on β-amylase production was investigated. β-amylase was purified in a three-step procedure before characterization. Maximum β-amylase production (75.88 U/ml) was obtained at pH 6.0 and 40 °C with soluble starch and malt extract as carbon and nitrogen sources. β-amylase was purified 5.64-fold with 19 % yield. The purified enzyme had optimum activity at 50 °C and pH 6.0, and exhibited remarkable stability over broad pH range (4.0 - 8.0) after 60 min incubation having residual activity of about 90% at pH 7.0, 6.0 and pH 5.0 with 50% residual activity at pH 4.0 and 8.0, respectively. The purified β-amylase uniquely retained 80% and 73% of its original activDespite the higher activity possessed by bacterial β-amylases, plant β-amylases are widely used for industrial processes. It is crucial that targeted improvements must be made on bacterial β- amylases for enhanced production, thermal and operational stability for their commercial viability. In this study, Bacillus polymyxa BWB-01 was cultivated in submerged fermentation and influence of cultivation conditions (pH, temperature, carbon and nitrogen sources) on β-amylase production was investigated. β-amylase was purified in a three-step procedure before characterization. Maximum β- amylase production (75.88 U/ml) was obtained at pH 6.0 and 40 °C with soluble starch and malt extract as carbon and nitrogen sources. β-amylase was purified 5.64-fold with 19% yield. The purified enzyme had optimum activity at 50 °C and pH 6.0, and exhibited remarkable stability over broad pH range (4.0 - 8.0) after 60 min incubation having residual activity of about 90% at pH 7.0, 6.0 and pH 5.0 with 50% residual activity at pH 4.0 and 8.0, respectively. The purified β-amylase uniquely retained 80% and 73% of its original activity after prolonged incubation (180 min) at pH 6.0 and 5.0, and still had residual activity of 65% after 300 min. The enzyme was moderately thermostable exhibiting 62% residual activity after 60 min at 50 °C and 54% at 60 °C after 30 min. β-amylase activity was enhanced by Mn2+ and Fe2+ but completely inhibited by Cu2+. B. polymyxa BWB-01 is a good producer of β-amylase with improved characteristics for application in food industry.ity after prolonged incubation (180 min) at pH 6.0 and 5.0, and still had residual activity of 65% after 300 min. The enzyme was moderately thermostable exhibiting 62% residual activity after 60 min at 50 °C and 54% at 60 °C after 30 min. β-amylase activity was enhanced by Mn2+ and Fe2+ but completely inhibited by Cu2+. B. polymyxa BWB-01 is a good producer of β-amylase with improved characteristics for application in food industry.

Catechol-O-methyltransferase (COMT) catalyzes the transfer of a methyl group from Sadenosyl- L-methionine to catechols. Because COMT has an important role in the metabolism of catecholamines, catechol estrogens, and drugs with a catechol moiety such as L-Dopa, measurement of COMT activity in several tissues is important. Furthermore, COMT inhibitors are used clinically in the treatment of Parkinson’s disease, and activation of COMT can control high blood pressure. Hence, high-throughput screening methods for COMT inhibitors and activators are needed. In this methodological article, two analytical methods for the measurement of COMT activity are described. One is very sensitive, and the other is a rapid assay. Norepinephrine, an endogenous compound was used as the substrate, and the enzymatic product (normetanephrine) was quantified with highperformance liquid chromatography-fluorescence or chemiluminescence detection.

Geobacillus kaustrophilus PW11, Geobacillus thermoleovorans PW13 and Geobacillus toebii PS4 were isolated form Tattapani hotspring of Himachal Pradesh, India and characterized for extracellular amylase activity. All the three Geobacillus spp. exhibited thermophilic amylase activity, which was predominantly extracellular. The activity was optimum at 90°C and pH 7.0. Interestingly, no amylase activity was observed at temperature less than 40°C, indicating its thermophilic nature. Moreover, pre incubation of enzyme at 100°C enhanced the amylase activity. The specific activity of amylase of PW11, PW13 and PS4 was 3898, 3597 and 3289 U mg-1, respectively at 90°C and pH 7.0. Starch, amylopectin and dextrin were observed to be the best substrates for amylase activity. Further, amylase activity of PW13 and PS4 remained unaffected in the presence of Triton X 100 (0.5%), but decreased by 40% in case of PW11 isolate. Among the metal ions tested, Mn2+, Co2+ and Fe2+ enhanced the amylase activity of PW11, PW13 and PS4 by 2-5 folds. While Hg2+ (1mM) strongly inhibited enzyme activity of PW13 and PS4, Cd2+ completely inhibited amylase activity of PW11. Amylase activity of PW13 and PS4 was enhanced by 2-2.5 folds in the presence of EDTA (5 mM). The thermophilic amylase activities of PW11, PW13 and PS4 are highly advantageous for downstream industrial applications.