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.

The Growth and Morphological Characterization Of Tropical Thermophilic Bacterium Parageobacillus caldoxylosilyticus ER4B

Parageobacillus caldoxylosilyticus is a rod-shaped thermophilic bacterium that can grow optimally at high temperatures. The thermophilicity of the bacterium is expected to be largely accounted for by the production of thermostable enzymes which has valuable applications in many fields. However, the species is poorly studied, hence, the growth conditions at high temperatures remained unclear until today. Therefore, this study aimed to determine the growth characterization of P. caldoxylosilyticus, including growth media preferences, optimal growth temperature, as well as minimum and maximum growth temperature. P. caldoxylosilyticus strain ER4B isolated from oil palm empty fruit bunch compost was used in this study. The bacterial strain was first identified using 16S rRNA sequencing, and the subsequent BLAST result showed that it is closest to P. caldoxylosilyticus strain UTM6. It is found that ER4B grew best in LB as compared to R2A, TSB, and NB medium. Further temperature tests determined the optimum growth temperature of the strain to be at 64°C Besides, the bacterium forms mucoid circular punctiform colonies that are yellowish in color on an agar plate, and the colony is usually 2 mm to 4 mm in diameter. The microscopic analysis also revealed that strain ER4B is a Gram-positive rod-shaped bacterium that has a length ranging from 3 µm to 6 µm, with a diameter of around 0.5 µm.

Brevibacillus marinus sp. nov., a thermophilic bacterium isolated from deep sea sediment in the South China Sea

A novel thermophilic bacterium, designated SCSIO 07484T, was isolated from marine sediment sampled in the South China Sea. Growth occurred at 30–60 °C, pH 6.0–8.0 and in the presence of 0–3 % (w/v) NaCl. Cells of strain SCSIO 07484T were rod-shaped and flagellum-forming. No soluble pigment was observed. The phylogenetic analysis of the 16S rRNA gene sequences indicated that SCSIO 07484T belonged to the family Paenibacillaceae and clustered with members of the genus Brevibacillus in the phylogenetic trees with less than 96.2 % similarities. The cell wall contained meso-diaminopimelic acid. Whole-cell hydrolysates contained arabinose, glucose and ribose. The predominant menaquinone was MK-7. Major fatty acids were iso-C16 : 0, iso-C15 : 0, C16 : 0 and iso-C14 : 0. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine were its diagnostic polar lipids. The whole genome size of strain SCSIO 07484T was 4 079 826 bp with a DNA G+C content of 56.2 mol%, including one circular chromosome of 3 978392 bp and one plasmid of 101434 bp. Based on the polyphasic analysis of strain SCSIO 07484T, it is considered to represent a novel species of the genus Brevibacillus , for which the name Brevibacillus marinus sp. nov. is proposed with the type strain SCSIO 07484T (=DSM 106769T=CGMCC 1.15814T).

Not All That Glitters Is Gold: The Paradox of CO-dependent Hydrogenogenesis in Parageobacillus thermoglucosidasius

The thermophilic bacterium Parageobacillus thermoglucosidasius has recently gained interest due to its ability to catalyze the water gas shift reaction, where the oxidation of carbon monoxide (CO) is linked to the evolution of hydrogen (H2) gas. This phenotype is largely predictable based on the presence of a genomic region coding for a carbon monoxide dehydrogenase (CODH—Coo) and hydrogen evolving hydrogenase (Phc). In this work, seven previously uncharacterized strains were cultivated under 50% CO and 50% air atmosphere. Despite the presence of the coo—phc genes in all seven strains, only one strain, Kp1013, oxidizes CO and yields H2. The genomes of the H2 producing strains contain unique genomic regions that code for proteins involved in nickel transport and the detoxification of catechol, a by-product of a siderophore-mediated iron acquisition system. Combined, the presence of these genomic regions could potentially drive biological water gas shift (WGS) reaction in P. thermoglucosidasius.

Biohydrogen production from food processing wastewater by a newly isolated thermophilic bacterium

The aim of this work was a comparative study of biohydrogen production from cheese whey and confectionary wastewater by a newly isolated thermophilic microbial strain Thermoanaerobacterium thermosaccharolyticum SP-H2. Experimental results showed that the fermentative hydrogen was successfully produced with the highest hydrogen yield of 3.9 mL H2/mL cheese whey or 80 mL H2/g chemical oxygen demand. The profile of soluble metabolite products showed that hydrogen generation by a new isolate was mainly acetate-type fermentation in the case of confectionary wastewater and mixed ethanol-acetate-lactate type fermentation in the case of cheese whey. The more optimal metabolic pathway of confectionary wastewater fermentation was confirmed by the better kinetic characteristics according to the Gompertz model.

The protective effect of manganese superoxide dismutase from thermophilic bacterium HB27 on hydrochloric acid-induced chemical cystitis in rats

Purpose To evaluate the effects of manganese superoxide dismutase (Mn-SOD) from thermophilic bacterium HB27 (name as Tt-SOD) on chemical cystitis. Methods Control and experimental rats were infused by intravesical saline or hydrochloric acid (HCl) on the first day of the experiments. Saline, sodium hyaluronate (SH) or Tt-SOD were infused intravesically once a day for three consequent days. On the fifth day, the rats were weighted and sacrificed following a pain threshold test. The bladder was harvested for histological and biochemical analyses. Results Tt-SOD could reduce the bladder index, infiltration of inflammatory cells in tissues, serum inflammatory factors and SOD levels, mRNA expression of inflammatory factors in tissues, and increase perineal mechanical pain threshold and serum MDA and ROS levels in HCl-induced chemical cystitis. Furthermore, Tt-SOD alleviated inflammation and oxidative stress by the negative regulation of the NF-κB p65 and p38 MAPK signaling pathway. Conclusions Intravesical instillation of Tt-SOD provides protective effects against HCl-induced cystitis.

A Survey of Spontaneous Antibiotic-Resistant Mutants of the Halophilic, Thermophilic Bacterium Rhodothermus marinus

Rhodothermus marinus is a halophilic extreme thermophile, with potential as a model organism for studies of the structural basis of antibiotic resistance. In order to facilitate genetic studies of this organism, we have surveyed the antibiotic sensitivity spectrum of R. marinus and identified spontaneous antibiotic-resistant mutants. R. marinus is naturally insensitive to aminoglycosides, aminocylitols and tuberactinomycins that target the 30S ribosomal subunit, but is sensitive to all 50S ribosomal subunit-targeting antibiotics examined, including macrolides, lincosamides, streptogramin B, chloramphenicol, and thiostrepton. It is also sensitive to kirromycin and fusidic acid, which target protein synthesis factors. It is sensitive to rifampicin (RNA polymerase inhibitor) and to the fluoroquinolones ofloxacin and ciprofloxacin (DNA gyrase inhibitors), but insensitive to nalidixic acid. Drug-resistant mutants were identified using rifampicin, thiostrepton, erythromycin, spiramycin, tylosin, lincomycin, and chloramphenicol. The majority of these were found to have mutations that are similar or identical to those previously found in other species, while several novel mutations were identified. This study provides potential selectable markers for genetic manipulations and demonstrates the feasibility of using R. marinus as a model system for studies of ribosome and RNA polymerase structure, function, and evolution.