A Novel Actinobacterial Cutinase Containing a Noncatalytic Polymer-Binding Domain.

The single putative cutinase-encoding gene from the genome of Kineococcus radiotolerans SRS30216 was cloned and expressed in Escherichia coli as a secreted fusion protein, designated YebF-KrCUT, where YebF is the extracellular carrier protein. The 294-amino-acid sequence of KrCUT is unique among currently characterized cutinases by having a C-terminal extension that consists of a short (Pro-Thr)-rich linker and a 55-amino-acid region resembling the substrate binding domain of poly(hydroxybutyrate) (PHB) depolymerases. Phylogenetically, KrCUT takes a unique position among known cutinases and cutinase-like proteins of bacterial and fungal origins. A modeled structure of KrCUT, although displaying a typical α/ß hydrolase fold, shows some unique loops close to the catalytic site. The 39-kDa YebF-KrCUT fusion protein and a truncated variant thereof were purified to electrophoretic homogeneity and functionally characterized. The melting temperatures (Tm) of KrCUT and its variant KrCUT206 devoid of the putative PHB-binding domain were established to be very similar, at 50 to 51°C. Cutinase activity was confirmed by the appearance of characteristic cutin components, C16 and C18 hydroxyl fatty acids, in the mass chromatograms following incubation of KrCUT with apple cutin as the substrate. KrCUT also efficiently degraded synthetic polyesters such as polycaprolactone and poly(1,3-propylene adipate). Although incapable of PHB depolymerization, KrCUT could efficiently bind PHB, confirming the predicted characteristic of the C-terminal region. KrCUT also potentiated the activity of pectate lyase in the degradation of pectin from hemp fibers. This synergistic effect is relevant to the enzyme retting process of natural fibers. IMPORTANCE To date, only a limited number of cutinases have been isolated and characterized from nature, the majority being sourced from phytopathogenic fungi and thermophilic bacteria. The significance of our research relates to the identification and characterization of a unique member of the microbial cutinases, named KrCUT, that was derived from the genome of the Gram-positive Kineococcus radiotolerans SRS30216, a highly radiation-resistant actinobacterium. Given the wide-ranging importance of cutinases in applications such as the degradation of natural and synthetic polymers, in the textile industry, in laundry detergents, and in biocatalysis (e.g., transesterification reactions), our results could foster new research leading to broader biotechnological impacts. This study also demonstrated that genome mining or prospecting is a viable means to discover novel biocatalysts as environmentally friendly and biotechnological tools.

Robotics for enzyme technology: innovations and technological perspectives.

The use of robotics in the life science sector has created a considerable and significant impact on a wide range of research areas, including enzyme technology due to their immense applications in enzyme and microbial engineering as an indispensable tool in high-throughput screening applications. Scientists are experiencing the advanced applications of various biological robots (nanobots), fabricated based on bottom-up or top-down approaches for making nanotechnology scaffolds. Nanobots and enzyme-powered nanomotors are particularly attractive because they are self-propelled vehicles, which consume biocompatible fuels. These smart nanostructures are widely used as drug delivery systems for the efficient treatment of various diseases. This review gives insights into the escalating necessity of robotics and nanobots and their ever-widening applications in enzyme technology, including biofuel production and biomedical applications. It also offers brief insights into high-throughput robotic platforms that are currently being used in enzyme screening applications for monitoring and control of microbial growth conditions. KEY POINTS: • Robotics and their applications in biotechnology are highlighted. • Robotics for high-throughput enzyme screening and microbial engineering are described. • Nanobots and enzyme-powered nanomotors as controllable drug delivery systems are reviewed.

Status of the application of exogenous enzyme technology for the development of natural plant resources.

Exogenous enzymes are extraneous enzymes that are not intrinsic to the subject. The exogenous enzyme industry has been rapidly developing recently. Successful application of recombinant DNA amplification, high-efficiency expression, and immobilization technology to genetically engineered bacteria provides a rich source of enzymes. Amylase, cellulase, protease, pectinase, glycosidase, tannase, and polyphenol oxidase are among the most widely used such enzymes. Currently, the application of exogenous enzyme technology in the development of natural plant resources mainly focuses on improving the taste and flavor of the product, enriching the active ingredient contents, deriving and transforming the structure of a chosen compound, and enhancing the biological activity and utilization of the functional ingredient. In this review, we discuss the application status of exogenous enzyme technology for the development of natural plant resources using typical natural active ingredients from plant, such as resveratrol, steviosides, catechins, mogrosides, and ginsenosides, as examples, to provide basis for further exploitation and utilization of exogenous enzyme technology.

Chicken Heads as a Promising By-Product for Preparation of Food Gelatins.

Every year, the poultry industry produces a large number of by-products such as chicken heads containing a considerable proportion of proteins, particularly collagen. To prepare gelatin is one of the possibilities to advantageously utilize these by-products as raw materials. The aim of the paper was to process chicken heads into gelatins. An innovative method for conditioning starting raw material was using the proteolytic enzyme. Three technological factors influencing the yield and properties of extracted gelatins were monitored including the amount of enzyme used in the conditioning of the raw material (0.4% and 1.6%), the time of the conditioning (18 and 48 h), and the first gelatin extraction time (1 and 4 h). The gelatin yield was between 20% and 36%. The gelatin gel strength ranged from 113 to 355 Bloom. The viscosity of the gelatin solution was determined between 1.4 and 9.5 mPa.s. The content of inorganic solids varied from 2.3% to 3.9% and the melting point of the gelatin gel was recorded between 34.5 and 42.2 °C. This study has shown that gelatin obtained from chicken heads has a promising potential with diverse possible applications in the food industry, pharmacy, and cosmetics.

Cross-linked enzyme aggregates of polyethylene terephthalate hydrolyse (PETase) from Ideonella sakaiensis for the improvement of plastic degradation.

Polyethylene terephthalate (PET) is one of the most produced plastics globally and its accumulation in the environment causes harm to the ecosystem. Polyethylene terephthalate hydrolyse (PETase) is an enzyme that can degrade PET into its monomers. However, free PETase lacks operational stabilities and is not reusable. In this study, development of cross-linked enzyme aggregate (CLEA) of PETase using amylopectin (Amy) as cross-linker was introduced to solve the limitations of free PETase. PETase-Amy-CLEA exhibited activity recovery of 81.9 % at its best immobilization condition. Furthermore, PETase-Amy-CLEA exhibited 1.37-, 2.75-, 2.28- and 1.36-fold higher half-lives than free PETase at 50 °C, 45 °C, 40 °C and 35 °C respectively. Moreover, PETase-Amy-CLEA showed broader pH stability from pH 5 to 10 and could be reused up to 5 cycles. PETase-Amy-CLEA retained >70 % of initial activity after 40 days of storage at 4 °C. In addition, lower Km of PETase-Amy-CLEA indicated better substrate affinity than free enzyme. PETase-Amy-CLEA corroded PET better and products yielded was 66.7 % higher than free PETase after 32 h of treatment. Hence, the enhanced operational stabilities, storage stability, reusability and plastic degradation ability are believed to make PETase-Amy-CLEA a promising biocatalyst in plastic degradation.

Integration of subcritical water extraction and treatment with xylanases and feruloyl esterases maximises release of feruloylated arabinoxylans from wheat bran.

Wheat bran is an abundant and low valued agricultural feedstock rich in valuable biomolecules as arabinoxylans (AX) and ferulic acid with important functional and biological properties. An integrated bioprocess combining subcritical water extraction (SWE) and enzymatic treatments has been developed for maximised recovery of feruloylated arabinoxylans and oligosaccharides from wheat bran. A minimal enzymatic cocktail was developed combining one xylanase from different glycosyl hydrolase families and a feruloyl esterase. The incorporation of xylanolytic enzymes in the integrated SWE bioprocess increased the AX yields up to 75%, higher than traditional alkaline extraction, and SWE or enzymatic treatment alone. The process isolated AX with tailored molecular structures in terms of substitution, molar mass, and ferulic acid, which can be used for structural biomedical applications, food ingredients and prebiotics. This study demonstrates the use of hydrothermal and enzyme technologies for upcycling agricultural side streams into functional bioproducts, contributing to a circular food system.

Upstream and Downstream Bioprocessing in Enzyme Technology.

The development of biotransformation must integrate upstream and downstream processes. Upstream bioprocessing will influence downstream bioprocessing. It is essential to consider this because downstream processes can constitute the highest cost in bioprocessing. This review comprehensively overviews the most critical aspects of upstream and downstream bioprocessing in enzymatic biocatalysis. The main upstream processes discussed are enzyme production, enzyme immobilization methodologies, solvent selection, and statistical optimization methodologies. The main downstream processes reviewed in this work are biocatalyst recovery and product separation and purification. The correct selection and combination of upstream and downstream methodologies will allow the development of a sustainable and highly productive system.

Tailored enzymes as next-generation food-packaging tools.

Enzymes have the potential for biotransformation in the food industry. Engineering tools can be used to develop tailored enzymes for food-packaging systems that perform well and retain their activity under adverse conditions. Consequently, novel tailored enzymes have been produced to improve or include new and useful characteristics for intelligent food-packaging systems. This review discusses the protein-engineering tools applied to create new functionality in food-packaging enzymes. The challenges in applications and anticipated directions for future developments are also highlighted. The development and discovery of tailored enzymes for smart food packaging is a promising way to ensure safe and high-quality food products.

Novel Thermophilic Bacterial Laccase for the Degradation of Aromatic Organic Pollutants.

We identified a putative laccase from the thermophilic bacterium Geobacillus yumthangensis. The putative laccase was produced recombinantly and its ability to catalyse the degradation of aromatic organic pollutants was investigated. The putative laccase exhibits broad pH and temperature stability, and, notably, it could catalyse the degradation of organic dyes as well as toxic pollutants including bisphenol A, guaiacol and phenol with a redox mediator. Our work further demonstrates the potential of using oxidative enzymes to break down toxic chemicals that possess major threats to human health and the environment.

Characterisation and optimisation of a novel laccase from Sulfitobacter indolifex for the decolourisation of organic dyes.

Laccases are multi­copper oxidases that possess the potential for industrial wastewater treatments. In this study, a putative laccase from Sulfitobacter indolifex was recombinantly produced and characterised. The enzyme was found to be stable and active at low to ambient temperature and across a range of pH conditions. The ability of the putative bacterial laccase to catalyse the decolourisation of seven common industrial dyes was also examined. Our results showed that the putative laccase could efficiently decolourise Indigo Carmine, Coomassie Brilliant Blue R-250, Congo Red, Malachite Green and Alizarin in the presence of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a redox mediator. Furthermore, the use of enzyme immobilisation technology to improve the operational stability and reusability of the putative laccase was also investigated. We found that immobilising the enzyme through the cross-linked enzyme aggregate method significantly improved its tolerance towards extreme pH as well as the presence of organic solvents. This work expands the arsenal of bacterial laccases available for the bioremediation of dye-containing wastewater.

Gene expression of microbial gelatinase activity for porcine gelatine identification.

In order to invent a porcine gelatine detection device using microbial resources, bacterial enzymes with a preference towards porcine gelatine and their candidate genes were evaluated. Five (n = 5) bacterial strains isolated from hot spring water and wet clay, Malaysia were screened for their gelatinase activity. The gelatinase enzyme was extracted and purified using ammonium sulphate precipitation prior to performing gelatinase assay on porcine, bovine and fish gelatine medium substrates. The G2 strain or Enterobacter aerogenes (Strain EA1) was selected for whole genome sequenced after showing a consistent trend of preference towards porcine gelatine. The gelatinase candidate gene gelEA1_9 was cloned and expressed. Based on one-way analysis of variance (ANOVA) with POST-HOC Duncan test (α = 0.05), the final product of gelEA1_9 was identified as a novel gelatinase. This gelatinase presented no significant difference in activity towards porcine gelatine. Hence, the present study demonstrated an enzyme-substrate interaction for porcine gelatine identification.

Immobilized laccase on zinc oxide nanoarray for catalytic degradation of tertiary butyl alcohol.

Laccase is an effective biocatalyst in bioremediation process; however, the application of the enzyme is limited due to its cost, recovery, and stability. In this study, we developed, characterized and evaluated the efficiency of immobilized laccase on zinc oxide nanostructure to catalyze biodegradation of TBA in comparison to the suspended enzyme. The results showed that both immobilized and suspended laccase were capable of catalyzing TBA biodegradation; however, the efficiency of the immobilized laccase on TBA removal was higher than that of the suspended enzyme. The repeatability testing revealed the potential of the immobilized laccase for repeatedly catalyzing TBA biodegradation with storage capacity. While the Vmax of immobilized enzyme was higher than suspended laccase (2.25 ± 0.542 mg TBA/h∙U vs. 1.47 ± 0.185 mg TBA/h∙U), the km of the immobilized enzyme was higher than the suspended laccase (67.9 ± 20.5 mg TBA/L vs. 33.5 ± 7.10 mg TBA/L). This suggests that the immobilized laccase is better in TBA removal, but has lower affinity with TBA than the suspended enzyme. Thus, immobilization of the enzyme can be applied to increase the efficiency and minimize the use of laccase for TBA remediation.

Microbial Keratinase: Next Generation Green Catalyst and Prospective Applications.

The search for novel renewable products over synthetics hallmarked this decade and those of the recent past. Most economies that are prospecting on biodiversity for improved bio-economy favor renewable resources over synthetics for the potential opportunity they hold. However, this field is still nascent as the bulk of the available resources are non-renewable based. Microbial metabolites, emphasis on secondary metabolites, are viable alternatives; nonetheless, vast microbial resources remain under-exploited; thus, the need for a continuum in the search for new products or bio-modifying existing products for novel functions through an efficient approach. Environmental distress syndrome has been identified as a factor that influences the emergence of genetic diversity in prokaryotes. Still, the process of how the change comes about is poorly understood. The emergence of new traits may present a high prospect for the industrially viable organism. Microbial enzymes have prominence in the bio-economic space, and proteases account for about sixty percent of all enzyme market. Microbial keratinases are versatile proteases which are continuously gaining momentum in biotechnology owing to their effective bio-conversion of recalcitrant keratin-rich wastes and sustainable implementation of cleaner production. Keratinase-assisted biodegradation of keratinous materials has revitalized the prospects for the utilization of cost-effective agro-industrial wastes, as readily available substrates, for the production of high-value products including amino acids and bioactive peptides. This review presented an overview of keratin structural complexity, the potential mechanism of keratin biodegradation, and the environmental impact of keratinous wastes. Equally, it discussed microbial keratinase; vis-à-vis sources, production, and functional properties with considerable emphasis on the ecological implication of microbial producers and catalytic tendency improvement strategies. Keratinase applications and prospective high-end use, including animal hide processing, detergent formulation, cosmetics, livestock feed, and organic fertilizer production, were also articulated.

Biochemical characterization of a stable azoreductase enzyme from Chromobacterium violaceum: Application in industrial effluent dye degradation.

The presence of dye, including azo functional group (NN) containing dyes, in industrial waste water is one of the major causes of water pollution. This report showcases the functional role of azoreductase from Chromobacterium violaceum (MTCC No: 2656) as a valuable enzyme for degradation of azo dyes. The enzyme was cloned, expressed, purified and biochemically characterized and further tested for degradation efficiency of azo group containing dyes like methyl red, amaranth and methyl orange. The degraded azo dye products (metabolites) resulted by the action of azoreductase enzyme had reduced toxicity on fibroblast cell lines (L929) as compared to raw and intact dye. Further, good stability of the enzyme makes it more suitable for various applications related to the degradation and decolourisation of effluent dyes.

Recent advances in the production strategies of microbial pectinases-A review.

Pectinases, member of the hydrolases family of enzymes, are one of the important enzymes of the biotechnological sector with 25% share in the global food and beverage enzyme market. They hold a leading position among the commercially produced industrial enzymes. These enzymes are sustainable and environmentally-friendly tool of nature with wide application perspective in many industrial processes, such as, retting and degumming of plant fibers, fermentation of tea and coffee, oil extraction, clarification of fruit juices and wine, valorization of industrial wastes, debasement of cellulosic biomass for biofuel production, wastewater remediation, desizing, scouring and bleaching of fabric, animal feed production, and protoplast fusion technology etc. However, like many other industrial enzymes, pectinases also face the constraint of low yield and productivity in its economized production. Therefore, pectinases have been the target of studies aiming to achieve the expression levels on a commercial scale. Among the natural sources of pectinases, microbial pectinases are employed frequently owing to its ease of production and unique physicochemical properties. This review mainly focuses on the production of pectinase enzymes along with different types of recent strategies used for their production optimization to get overexpression/production. The promise of genetic engineering approaches applied so far to get the higher production of these enzymes using simpler molecular devices and simple fermentation media is also covered in this review.

Patent Landscape on Structured Lipids Produced by Enzyme Technology.

BACKGROUND: Lipid modification results in several benefits for the food industry, biotechnology advances and human health. Customizing bioactive lipids is very appealing because it improves the product's nutritional quality. Lipases are sustainable biocatalysts that can be reused, show selectivity towards substrates and reactions occur in mild conditions. OBJECTIVES: We aimed at systematically searching for patents deposited worldwide, that approached the production of structured lipids by using lipases as biocatalysts. METHOD: A patent-search strategy was set up in Questel-Orbit and the search strategy adopted was based on the combination of specific keywords in the title/abstract of the documents, encompassing thoroughly the search scope. We revised all patents relating structured lipids produced by enzyme reactions and provided an overview of the main objectives of the patents describing it, as well as a view of the principal depositors, years of publication and principal countries of deposit, as a mean to access the technological landscape on the subject. RESULTS: Forty-four documents, published over the last 34 years, were retrieved. Nine main patents' objectives were found, and the two major groups are: SL with bioactive properties and/or with fatty acids (FA) esterified at specific triacylglycerol positions and SL analogous of natural lipids. China, Japan and USA were the three main patent depositors. CONCLUSION: Although the number of patents retrieved was relatively low, this review indicates that SL production aiming at improvements in nutritional/health and/or physical attributes for food enhancement is a new field, and technological interest and innovation have been increasing over the last ten years.

Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host.

l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)6 -tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni2+ -charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg-1 ) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17-fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017.

Biotechnological Applications of Marine Enzymes From Algae, Bacteria, Fungi, and Sponges.

Diversity is the hallmark of all life forms that inhabit the soil, air, water, and land. All these habitats pose their unique inherent challenges so as to breed the "fittest" creatures. Similarly, the biodiversity from the marine ecosystem has evolved unique properties due to challenging environment. These challenges include permafrost regions to hydrothermal vents, oceanic trenches to abyssal plains, fluctuating saline conditions, pH, temperature, light, atmospheric pressure, and the availability of nutrients. Oceans occupy 75% of the earth's surface and harbor most ancient and diverse forms of organisms (algae, bacteria, fungi, sponges, etc.), serving as an excellent source of natural bioactive molecules, novel therapeutic compounds, and enzymes. In this chapter, we introduce enzyme technology, its current state of the art, unique enzyme properties, and the biocatalytic potential of marine algal, bacterial, fungal, and sponge enzymes that have indeed boosted the Marine Biotechnology Industry. Researchers began exploring marine enzymes, and today they are preferred over the chemical catalysts for biotechnological applications and functions, encompassing various sectors, namely, domestic, industrial, commercial, and healthcare. Next, we summarize the plausible pros and cons: the challenges encountered in the process of discovery of the potent compounds and bioactive metabolites such as biocatalysts/enzymes of biomedical, therapeutic, biotechnological, and industrial significance. The field of Marine Enzyme Technology has recently assumed importance, and if it receives further boost, it could successfully substitute other chemical sources of enzymes useful for industrial and commercial purposes and may prove as a beneficial and ecofriendly option. With appropriate directions and encouragement, marine enzyme technology can sustain the rising demand for enzyme production while maintaining the ecological balance, provided any undesired exploitation of the marine ecosystem is avoided.

Application of enzyme technology in flavonoid ingredients of Chinese materia medica / 中草药

In recent years, enzyme technology is widely applied in the flavonoid ingredients of Chinese materia medica (CMM). Based on a large number of domestic and foreign literatures, the application of enzyme technology in the flavonoid ingredients is mainly embodied in three aspects: significantly improving the extraction rate of flavonoid ingredients by using alone or combined with other technology including enzyme-ultrasonic coupling technology and enzyme-microwave coupling technology, obtaining the effective ingredients with a better activity and higher bioavailability by changing the structure of some flavonoid ingredients, and increasing the active flavonoids content in the plants. Moreover, the immobilized enzyme technology is applied more and more. Enzyme technology, a new method in the study on flavonoid ingredients in CMM, can help to obtain more active flavonoids from the complex samples of CMM. Therefore, the enzyme technology has a good application prospect but also with some key problems to be further studied in future.