Recent bioinspired efforts of designing novel nanoenzyme-based electrocatalysts are driven by the urgency of making bioelectrofuels more affordable and efficient. Unlike natural enzymes, nanoenzyme-modified electrodes with large surface areas enclose numerous biomimicking active sites to facilitate enhanced microbial growth followed by increased reactant-to-bioelectrofuel conversion.
Bungarus candidus venom exhibited high hyaluronidase, acetylcholinesterase and phospholipase A activities; low proteinase, 5'-nucleotidase, alkaline phosphomonoesterase and phosphodiesterase activities and moderately high L-amino acid oxidase activity. SP-Sephadex C-50 ion exchange chromatographic fractionation of the venom and Sephadex G-50 chromatography of the major lethal venom fractions indicate that the venom contains at least two highly lethal, basic phospholipases A with LD50 (i.v.) values of 0.02 micrograms/g (F6A) and 0.18 micrograms/g (F4A), respectively; as well as two polypeptide toxins with LD50 (i.v.) values of 0.17 micrograms/g and 0.83 micrograms/g, respectively. The major lethal toxin is the basic lethal phospholipase A, F6A, which accounts for approximately 13% of the venom protein and has a mol. wt of 21,000.
Sumatran pit viper (Trimeresurus sumatranus sumatranus) venom was fractionated by DEAE-Sephacel ion exchange chromatography into seven fractions. Fractions 4, 5 and 6 were lethal to mice and exhibited strong hemorrhagic activity, as well as some enzymatic activities. Fraction 6 also exhibited potent anticoagulant and thrombin-like activities. Analysis of the biological and enzymatic properties of the three lethal fractions suggests that the major lethal component of fractions 4 and 5 may be the hemorrhagic principle, and that the lethality of fraction 6 may be due to the hemorrhagic principle and/or the anticoagulant principle.
Some enzymatic activities and toxic properties of four samples of Ophiophagus hannah (king cobra) venom were investigated. There is little intraspecific variation in enzyme contents, protein composition and toxic properties of the venom. The venom does not exhibit hemolytic or edema-inducing activity but is characterized by an exceptionally high alkaline phosphomonoesterase activity. DEAE-Sephacel ion exchange chromatography and Sephadex G-75 gel filtration chromatography of the venom indicate that the major lethal toxins are the low mol.wt, non-enzymatic basic proteins. Venom fractions exhibiting high enzymatic activities apparently do not play an important role in the lethality in mice of Ophiophagus hannah venom.
Melioidosis has been recognized as an important cause of sepsis in the tropics. The disease caused by an environmental saprophyte Burkholderia pseudomallei, affects mostly adults with underlying immunocompromised conditions. In this study, the enzymatic profiles of 91 clinical and 9 environmental isolates of B. pseudomallei were evaluated using the APIZYM system, in addition to assessment of protease, phospholipase C and sialidase activities using agar plate methods and other assays. The activity of 10 enzymes - alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase were detected in >75% of the clinical isolates. The majority of B. pseudomallei isolates in this study exhibited protease and phospholipase activities. No sialidase activity was detected. Five Burkholderia thailandensis isolates had similar APIZYM profiles as B. pseudomallei clinical isolates except for the lower detection rate for N-acetyl-β-glucosaminidase. The subtle differences in the number of enzymes secreted and the levels of enzymatic activities of phenotypically identical clinical and environmental strains of B. pseudomallei give weight to the fact that the causative agent of melioidodis originates from the environment.
The present study explores the utilisation of a new raw material from lignocellulose biomass, Meranti wood sawdust (MWS) for high commercial value xylooligosaccharides (XOS) production using immobilised xylanase. The xylanase was immobilised by a combination of entrapment and covalent binding techniques. The hemicellulosic xylan from MWS was extracted using a standard chlorite delignification method. The production of total and derivatives of XOS from the degradation of the hemicellulosic xylan of MWS were compared to the production from the commercial xylan from Beechwood. The utilisation of the extracted xylan from MWS yielded 0.36 mg/mL of total XOS after 60 h of hydrolysis. During the hydrolysis reaction, the immobilised xylanase released a lower degree of polymerisation (DP) of XOS, mainly X2 and X3, which were the major products of xylan degradation by xylanase enzymes. The production of XOS with a lower DP from MWS demonstrated the biotechnological potential of the MWS in the future. The XOS production retained about 70% of its initial XOS production during the second cycle. This is also the first report on the utilisation of MWS wastes in enzymatic hydrolysis using immobilised xylanase for XOS production.
LML-type structured lipids are one type of medium- and long-chain triacylglycerols. LML was synthesized using immobilized Talaromyces thermophilus lipase (TTL)-catalyzed interesterification of tricaprylin and ethyl linoleate. The resin AB-8 was chosen, and the lipase/support ratio was determined to be 60 mg/g. Subsequently, the immobilized TTL with strict sn-1,3 regiospecificity was applied to synthesize LML. Under the optimized conditions (60 °C, reaction time 6 h, enzyme loading of 6% of the total weight of substrates, substrate of molar ratio of ethyl linoleate to tricaprylin of 6:1), Triacylglycerols with two long- and one medium-chain FAs (DL-TAG) content as high as 52.86 mol% was obtained. Scale-up reaction further verified the industrial potential of the established process. The final product contained 85.24 mol% DL-TAG of which 97 mol% was LML after purification. The final product obtained with the high LML content would have substantial potential to be used as functional oils.
Biocarriers are pivotal in enhancing the reusability of biocatalyst that would otherwise be less economical for industrial application. Ever since the induction of enzymatic technology, varied materials have been assessed for their biocompatibility with enzymes of distinct functionalities. Herein, cellulase was immobilized onto polymethacrylate particles (ICP) as the biocarrier grafted with ethylenediamine (EDA) and glutaraldehyde (GA). Carboxymethyl cellulose (CMC) was used as a model substrate for activity assay. Enzyme immobilization loading was determined by quantifying the dry weight differential of ICP (pre-& post-immobilization). Cellulase was successfully demonstrated to be anchored upon ICP and validated by FTIR spectra analysis. The optimal condition for cellulase immobilization was determined to be pH 6 at 20 °C. The maximum CMCase activity was achieved at pH 5 and 50 °C. Residual activity of ∼50% was retained after three iterations and dipped to ∼18% on following cycle. Also, ICP displayed superior pH adaptability as compared to free cellulase. The specific activity of ICP was 65.14 ± 1.11% relative to similar amount of free cellulase.
The present study reports the effects of three commercial immobilized lipases namely Novozyme 435 from Candida antarctica lipase B (CALB), Lipozyme TL IM from Thermomyces lanuginosus and Lipozyme RM IM from Rhizomucor miehei on the production of trimethylolpropane (TMP) ester from high oleic palm methyl ester (HO-PME) and TMP. The TMP ester is a promising base oil for biolubricants that are easily biodegradable and non-toxic to humans and the environment. Enzymatic catalysts are insensitive to free fatty acid (FFA) content, hence able to mitigate the side reactions and consequently reduce product separation cost. The potential of these enzymes to produce TMP ester in a solvent-free medium was screened at various reaction time (8, 23, 30 and 48 h), operating pressure (0.1, 0.3 and 1.0 mbar) and enzyme dosage (1, 3, 5 and 10% w/w). The reaction was conducted at a constant temperature of 70 °C and a molar ratio of 3.9:1 (HO-PME: TMP). Novozyme 435 produced the highest yield of TMP ester of 95.68 ± 3.60% under the following conditions: 23 h reaction time, 0.1 mbar operating pressure and 5% w/w of enzyme dosage. The key lubrication properties of the produced TMP ester are viscosity index (208 ± 2), pour point (- 30 ± - 2 °C), cloud point (- 15 ± - 2 °C), onset thermal degradation temperature (427.8 °C), and oxidation stability, RPVOT (42 ± 4 min). The properties of the TMP ester produced from the enzymatic transesterification are comparable to other vegetable oil-based biolubricants produced by chemical transesterification.
One of the potentials of carrier-free cross-linked enzyme aggregates (CLEA) immobilization is the ability to be separated and reuse. Yet, it might be impeded by the poor mechanical stability resulting low recyclability. CLEA of CGTase from Bacillus lehensis G1 (CGTase G1-CLEA) using chitosan (CS) as a cross-linker demonstrated high activity recovery however, displayed poor reusability. Therefore, the relationship between mechanical strength and reusability is studied by enhancing the CS mechanical properties and applying a new co-aggregation approach. Herein, CS was chemically cross-linked with glutaraldehyde (GA) and GA was introduced as a co-aggregant (coGA). CGTase G1-CLEA developed using an improved synthesized chitosan-glutaraldehyde (CSGA) cross-linker and a new coGA technique showed to increase its mechanical stability which retained 63.4% and 52.2%, respectively compared to using CS that remained 33.1% of their initial activity after stirred at 500 rpm. The addition of GA impacted the morphology and interaction consequently stabilizing the CLEAs durability in production of cyclodextrins. As a result, the reusability of CGTase G1-CLEA with CSGA and coGA increased by 56.6% and 42.8%, respectively compared to previous CLEA after 5 cycles for 2 h of reaction. This verifies that the mechanical strength of immobilized enzyme influences the improvement of its operational stability.
Recurrent outbreaks of muscular sarcocystosis among tourists visiting islands in Malaysia have focused international attention on sarcocystosis, a disease once considered rare in humans. Sarcocystis species require two hosts, definitive and intermediate, to complete their life cycle. Humans can serve as definitive hosts, with intestinal sarcocystosis for two species acquired from eating undercooked meat: Sarcocystis hominis, from beef, and Sarcocystis suihominis, from pork. Symptoms such as nausea, stomachache, and diarrhea vary widely depending on the number of cysts ingested but appear more severe with pork than with beef. Humans serve as intermediate hosts for Sarcocystis nesbitti, a species with a reptilian definitive host, and possibly other unidentified species, acquired by ingesting sporocysts from feces-contaminated food or water and the environment; infections have an early phase of development in vascular endothelium, with illness that is difficult to diagnose; clinical signs include fever, headache, and myalgia. Subsequent development of intramuscular cysts is characterized by myositis. Presumptive diagnosis based on travel history to tropical regions, elevated serum enzyme levels, and eosinophilia is confirmed by finding sarcocysts in muscle biopsy specimens. There is no vaccine or confirmed effective antiparasitic drug for muscular sarcocystosis, but anti-inflammatory drugs may reduce symptoms. Prevention strategies are also discussed.
CeO2 nanoparticles (NPs) have shown promising approaches as therapeutic agents in biology and medical sciences. The physicochemical properties of CeO2-NPs, such as size, agglomeration status in liquid, and surface charge, play important roles in the ultimate interactions of the NP with target cells. Recently, CeO2-NPs have been synthesized through several bio-directed methods applying natural and organic matrices as stabilizing agents in order to prepare biocompatible CeO2-NPs, thereby solving the challenges regarding safety, and providing the appropriate situation for their effective use in biomedicine. This review discusses the different green strategies for CeO2-NPs synthesis, their advantages and challenges that are to be overcome. In addition, this review focuses on recent progress in the potential application of CeO2-NPs in biological and medical fields. Exploiting biocompatible CeO2-NPs may improve outcomes profoundly with the promise of effective neurodegenerative therapy and multiple applications in nanobiotechnology.
α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg) of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups) on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0-9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 °C. Immobilized TASKA retained 50% of its initial activity after 5-12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports.
Cellulose nanomaterial with rod-like structure and highly crystalline order, usually formed by elimination of the amorphous region from cellulose during acid hydrolysis. Cellulose nanomaterial with the property of biocompatibility and nontoxicity can be used for enzyme immobilization. In this work, urease enzyme was used as a model enzyme to study the surface modification of cellulose nanomaterial and its potential for biosensor application. The cellulose nanocrystal (CNC) surface was modified using 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation to introduce the carboxyl group at C6 primary alcohol. The success of enzyme immobilization and surface modification was confirmed using chemical tests and measured using UV-Visible spectrophotometer. The immobilization strategy was then applied for biosensor application for urea detection. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques were used for electroanalytical characterization of the urea biosensor.
A new biosensor for the analysis of nitrite in food was developed based on hemoglobin (Hb) covalently immobilized on the succinimide functionalized poly(n-butyl acrylate)-graphene [poly(nBA)-rGO] composite film deposited on a carbon-paste screen-printed electrode (SPE). The immobilized Hb on the poly(nBA)-rGO conducting matrix exhibited electrocatalytic ability for the reduction of nitrite with significant enhancement in the reduction peak at −0.6 V versus Ag/AgCl reference electrode. Thus, direct determination of nitrite can be achieved by monitoring the cathodic peak current signal of the proposed polyacrylic-graphene hybrid film-based voltammetric nitrite biosensor. The nitrite biosensor exhibited a reproducible dynamic linear response range from 0.05⁻5 mg L−1 nitrite and a detection limit of 0.03 mg L−1. No significant interference was observed by potential interfering ions such as Ca2+, Na⁺, K⁺, NH₄⁺, Mg2+, and NO₃− ions. Analysis of nitrite in both raw and processed edible bird’s nest (EBN) samples demonstrated recovery of close to 100%. The covalent immobilization of Hb on poly(nBA)-rGO composite film has improved the performance of the electrochemical nitrite biosensor in terms of broader detection range, lower detection limit, and prolonged biosensor stability.
The modelling of splicing systems is simulated by the process of cleaving and recombining DNA molecules with the presence of a ligase and restriction enzymes which are biologically called as endodeoxyribonucleases. The molecules resulting from DNA splicing systems are known as splicing languages. Palindrome is a sequence of strings that reads the same forward and backward. In this research, the splicing languages resulting from DNA splicing systems with one non-palindromic restriction enzyme are determined using the notation from Head splicing system. The generalisations of splicing languages for DNA splicing systems involving a cutting site and two non-overlapping cutting sites of one non-palindromic restriction enzyme are presented in the first and second theorems, respectively, which are proved using direct and induction methods. The result from the first theorem shows a trivial string which is the initial DNA molecule; while the second theorem determines a splicing language consisting of a set of resulting DNA molecules from the respective DNA splicing system.
Conjugated linoleic acid (CLA)-rich triacylglycerols (TAG) have received significant attention owing to their health promoting properties. In this study, CLA-rich TAG were successfully synthesized by an immobilized mutant lipase (MAS1-H108A)-catalyzed esterification of CLA-rich fatty acids and glycerol under vacuum. MAS1-H108A was first immobilized onto ECR1030 resin. Results showed that the lipase/support ratio of 41 mg/g was suitable for the immobilization and the thermostability of immobilized MAS1-H108A was greatly enhanced. Subsequently, the immobilized MAS1-H108A was employed for the synthesis of CLA-rich TAG and 95.21% TAG with 69.19% CLA was obtained under the optimized conditions. The TAG content (95.21%) obtained by immobilized MAS1-H108A is the reported highest value thus far, which was significantly higher than that (9.26%) obtained by Novozym 435 under the same conditions. Although the TAG content comparable to the results obtained in this study could also be obtained by Novozym 435, the used enzyme amount is approximately 5-fold of the immobilized MAS1-H108A. Additionally, the immobilized MAS1-H108A exhibited excellent recyclability during esterification retaining 95.11% of its initial activity after 10 batches. Overall, such immobilized mutant lipase with superior esterification activity and recyclability has the potential to be used in oils and fats industry.
Psychrophiles are cold-living microorganisms synthesizing enzymes that are permanently active at almost near-zero temperatures. Psychrozymes are supposed to be structurally more flexible than their homologous proteins. This structural flexibility enables these proteins to undergo conformational changes during catalysis and improve catalytic efficiency at low temperatures. The outstanding characteristics of the psychrophilic enzymes have attracted the attention of the scientific community to utilize them in a wide variety of industrial and pharmaceutical applications. In this review, we first highlight the current knowledge of the cold-adaptation mechanisms of the psychrophiles. In the sequel, we describe the potential applications of the enzymes in different biotechnological processes specifically, in the production of industrial and pharmaceutical products. KEY POINTS: • Methods that organisms have evolved to survive and proliferate at cold environments. • The economic benefits due to their high activity at low and moderate temperatures. • Applications of the psychrophiles in biotechnological and pharmaceutical industry.