From the Discovery of Extremozymes to an Enzymatic Product: Roadmap Based on Their Applications

With the advent of the industrial revolution, the use of toxic compounds has grown exponentially, leading to a considerable pollution of the environment. Consequently, the development of more environmentally conscious technologies is an urgent need. Industrial biocatalysis appears as one potential solution, where a higher demand for more robust enzymes aims to replace toxic chemical catalysts. To date, most of the commercially available enzymes are of mesophilic origin, displaying optimal activity in narrow ranges of temperature and pH (i.e., between 20°C and 45°C, neutral pH), limiting their actual application under industrial reaction settings, where they usually underperform, requiring larger quantities to compensate loss of activity. In order to obtain novel biocatalysts better suited for industrial conditions, an efficient solution is to take advantage of nature by searching and discovering enzymes from extremophiles. These microorganisms and their macromolecules have already adapted to thrive in environments that present extreme physicochemical conditions. Hence, extremophilic enzymes stand out for showing higher activity, stability, and robustness than their mesophilic counterparts, being able to carry out reactions at nonstandard conditions. In this brief research report we describe three examples to illustrate a stepwise strategy for the development and production of commercial extremozymes, including a catalase from an Antarctic psychrotolerant microorganism, a laccase from a thermoalkaliphilic bacterium isolated from a hot spring and an amine-transaminase from a thermophilic bacterium isolated from a geothermal site in Antarctica. We will also explore some of their interesting biotechnological applications and comparisons with commercial enzymes.

Enhanced Optimization of Bioethanol Production from Palm Waste Using the Taguchi Method

In the present study, palm fiber (PF) and palm fronds (PFN) were selected as local agricultural wastes for the extraction of different biopolymers (cellulose, hemicelluloses, and lignin) by alkaline sodium hydroxide (PF, 2.37% NaOH at 86.5 °C for 1.6 h; PFN, 6% NaOH at 90 °C for 1 h) and bioethanol production. The processes of extraction were optimized by the experimental design method of Taguchi. The total carbohydrates of PF and PFN obtained were 24.4% and 31.0%, respectively. In addition, the untreated palm fiber (UPF), untreated palm frond (UPFN), cellulose palm fibers (CPF), and cellulose palm fronds (CPFN) were subjected to enzymatic hydrolysis processes using crude enzymes and commercial enzymes at 48 °C and pH 5.5. The results indicate that the maximum reducing sugars used were CPF 229.90, CPFN 243.69, UPF 120.19, and UPFN 100.00 (mg/g), which were obtained at a crude enzyme loading. CPF and CPFN hydrolysates were then successfully converted into bioethanol by a separate enzymatic hydrolysis and fermentation by Saccharomyces cerevisiae. Anaerobic cultivation of the hydrolysates with S.cerevisiae resulted in 0.222 g/g and 0.213 g/g bioethanol in the case of CPF and CPFN, respectively. Optimization processes could be an innovative approach to the sustainable development of bioethanol production.

Screening and identification of thermophilic cellulolytic bacteria isolated from sawdust compost

Composting process mainly depends on the metabolic pathways of the microorganism and involves the activity of different enzymes. Thermophilic cellulase-producing bacteria isolated from sawdust compost were tested for formation of a visible zone around the colonies on the agar plates medium containing carboxymethyl cellulose at 50ºC. Screening of carboxymethyl cellulase producing isolates was further realized on the basis in liquid medium by DNS method. Among 29 isolates investigated, V1 and V11 strains exhibited maximum enzyme activity of 1.9 and 2.3 U/mL, respectively. These isolates were selected for morphological, physiological and biochemical studies and 16S rRNA gene analysis. They were found a Gram-positive, rod-shaped spore forming cells, which were identified as Bacillus megaterium (V1) and Bacillus subtilis (V11) based on cell morphology, nucleotide homology and phylogenetic analysis. The optimal temperature for activity of endoglucanases (CMCase) ranged from 35–45°C (strain V1) and 40– 50ºC (strain V11). Our findings showed that Bacillus megaterium (V1) and Bacillus subtilis (V11) cellulase demonstrate thermophilic characteristics within wide range of temperature and meets the requirements for commercial enzymes.

Enzymatic Pretreatment of Byproducts from Soapstock Splitting and Glycerol Processing for Improvement of Biogas Production

This study investigated acid splitting wastewater (ASW) and interphase (IF) from soapstock splitting, as well as matter organic non glycerol (MONG) from glycerol processing, as potential substrates for biogas production. Batch and semicontinuous thermophilic anaerobic digestion experiments were conducted, and the substrates were preliminary treated using commercial enzymes kindly delivered by Novozymes A/C. The greatest enhancement in the batch digestion efficiency was achieved when three preparations; EversaTransform, NovoShape, and Lecitase were applied in the hydrolysis stage, which resulted in the maximum methane yields of 937 NL/kg VS and 915 NL/kg VS obtained from IF and MONG, respectively. The co-digestion of 68% ASW, 16% IF, and 16% MONG (wet weight basis) performed at an organic loading rate (OLR) of 1.5 kg VS/m3/day provided an average methane yield of 515 NLCH4/kg VSadded and a volatile solid reduction of nearly 95%. A relatively high concentration of sulfates in the feed did not significantly affect the digestion performance but resulted in an increased hydrogen sulfide concentration in the biogas with the peak of 4000 ppm.

Effect of Several Pretreatments on the Lactic Acid Production from Exhausted Sugar Beet Pulp

Exhausted sugar beet pulp (ESBP), a by-product of the sugar industry, has been used as a substrate to produce lactic acid (LA). Due to the fact that ESBP contains a high percentage of pectin and hemicellulose, different pretreatments were studied to solubilize them and to facilitate the access to cellulose in the subsequent enzymatic hydrolysis. Several pretreatments were studied, specifically biological, oxidant with alkaline hydrogen peroxide (AHP), and thermochemical with acid (0.25, 0.5, or 1% w/v of H2SO4). Pretreated ESBP was enzymatically hydrolysed and fermented with the strain Lactiplantibacillus plantarum for LA production. The hydrolysis was carried out with the commercial enzymes Celluclast®, pectinase, and xylanase, for 48 h. After that, the hydrolysate was supplemented with yeast extract and calcium carbonate before the bacteria inoculation. Results showed that all the pretreatments caused a modification of the fibre composition of ESBP. In most cases, the cellulose content increased, rising from 25% to 68% when ESBP was pretreated thermochemically at 1% w/v H2SO4. The production of LA was enhanced when ESBP was pretreated thermochemically. However, it was reduced when biological and AHP pretreatments were applied. In conclusion, thermochemical pretreatment with 1% w/v H2SO4 had a positive impact on the production of LA, increasing its concentration from 27 g/L to 50 g/L.

Protoplast isolation from Dictyopteris pacifica and Scytosiphon lomentaria, using a simple commercial enzyme preparation

Background Protoplasts (i.e., naked plant cells) can be used for in vitro manipulations and genetic improvement in cultivars with economic value. During the last decade, protoplast research in economic brown algae has been scarce, and it is usually hampered by the use of non-commercial enzymes or crude extracts for isolating protoplasts. Dictyopteris pacifica is part of a brown algal genus well known by its wide chemical diversity and biological properties. Scytosiphon lomentaria is an edible brown seaweed with antioxidant, antitumor, and antiviral properties. So far, there are no protoplast isolation protocols using commercial enzymes for these two economic brown algae. In this study, we obtained protoplasts from cultured samples of D. pacifica and S. lomentaria using commercially available enzymes. Additionally, we investigated the effects of Driselase inclusion and Ca-chelation pre-treatment on protoplast yields in order to optimize the conditions for protoplast preparations. Results Protoplasts were isolated from Dictyopteris pacifica and Scytosiphon lomentaria using the commercially available Cellulase Onozuka RS (1%) and Alginate lyase (4 U mL−1), and short incubation time (4 h). Driselase did not show significant effects on protoplast production in both species. Ca-chelation pre-treatment only increased the number of protoplasts in D. pacifica. Under optimal conditions, the protoplast yields from D. pacifica and S. lomentaria were 4.83 ± 2.08 and 74.64 ± 32.49 × 106 protoplasts g−1 fresh weight, respectively. The values obtained for S. lomentaria were 2–3 orders of magnitude higher than previously reported. Conclusions Our results show that high protoplast yields can be obtained from D. pacifica and S. lomentaria using a simple mixture of commercial enzymes (Cellulase RS and Alginate lyase) and short incubation time (4 h). This work also represents the first report of protoplast isolation in D. pacifica. The method proposed here can help to expand protoplast technology in more brown algal species.

Characterization of a Novel Keratinase With Surfactant Stability in a Wide Range of pH Activity From a Native Isolate, Bacillus Mojavensis FUM125

Keratinases are enzymes with the most diverse sources and applications. Different forms of keratinase have been applied in environment and variety of industries, highlighting the necessity for novel potential keratinases, which could be applicable in variety of industries. Accordingly, the present study aimed to identify and characterize a novel keratinase producing bacterium with high potential in variety of industries. In the present study, the native isolate of Bacillus sp. FUM125 was isolated, identified and optimized for the keratinolytic activity. The keratinase was purified and characterized using biochemical assays. The Bacillus sp. FUM125 isolate was identified as Bacillus mojavensis R-OH-1 with 99.8% similarity. The isolate showed the maximum keratinolytic activity at pH of 8.5 after 24-hour incubation at 37°C (2.1-fold enzyme production). According to the biochemical analysis, the keratinase belonged to a serine protease family, whit 33.5 kDa molecular weight and was stable in a wide range of pH and temperature with maximum keratinolytic activity at 60°C and pH 8. Among the metal ions, K+, Ca2+, Na2+ and Mg2+ increased the enzyme activity. The activity was increased by the reducing agents of DTT and beta-mercaptoethanol. Based on the substrate profile findings, the enzyme was active in various soluble and insoluble substrates. The enzyme showed a half-life of 98 min in the optimal temperature and the ratio of keratinolytic:caseinolytic to be 0.95. Our enzyme with higher temperature and pH stability compared to existing commercial enzymes can be considered as a potential candidate for use in various industries.

Extremophilic Oxidoreductases for the Industry: Five Successful Examples With Promising Projections

In a global context where the development of more environmentally conscious technologies is an urgent need, the demand for enzymes for industrial processes is on the rise. Compared to conventional chemical catalysts, the implementation of biocatalysis presents important benefits including higher selectivity, increased sustainability, reduction in operating costs and low toxicity, which translate into cleaner production processes, lower environmental impact as well as increasing the safety of the operating staff. Most of the currently available commercial enzymes are of mesophilic origin, displaying optimal activity in narrow ranges of conditions, which limits their actual application under industrial settings. For this reason, enzymes from extremophilic microorganisms stand out for their specific characteristics, showing higher stability, activity and robustness than their mesophilic counterparts. Their unique structural adaptations allow them to resist denaturation at high temperatures and salinity, remain active at low temperatures, function at extremely acidic or alkaline pHs and high pressure, and participate in reactions in organic solvents and unconventional media. Because of the increased interest to replace chemical catalysts, the global enzymes market is continuously growing, with hydrolases being the most prominent type of enzymes, holding approximately two-third share, followed by oxidoreductases. The latter enzymes catalyze electron transfer reactions and are one of the most abundant classes of enzymes within cells. They hold a significant industrial potential, especially those from extremophiles, as their applications are multifold. In this article we aim to review the properties and potential applications of five different types of extremophilic oxidoreductases: laccases, hydrogenases, glutamate dehydrogenases (GDHs), catalases and superoxide dismutases (SODs). This selection is based on the extensive experience of our research group working with these particular enzymes, from the discovery up to the development of commercial products available for the research market.

Effect of different commercial enzymes on the clotting of milk and certain properties of curd

More researches published data about the milk curd properties, evaluated the importance in the cheese making, but an analysis of importance of these properties in practical applications is usually lacking. We investigate the milk curd behaviour using different enzyme preparations at the cutting of curd. We focused on the well measurable properties as clotting time, viscosity of curd, texture properties an whey separation rate of cur at cutting time. Approximately five minutes difference was determined between the clotting times. Investigated the curd properties we found significant differences between the hardness on samples clotted with CHY MAX® M 1000 and NATUREN® Premium 145 enzymes. Other properties did not show significant differences, but in some case differences were remarkable. Discovered differences e.g. approx. 5% whey separation rate difference and the different trends of adhesive force and adhesiveness confirm that such studies should be carried out. Summarized effect of different enzymes can alter the cheese making technology in the practice, significantly. Considering every aspect, in our investigation the CHY MAX® M 1000 enzyme seemed the best.