At present time, most of the enzymes are derived from biological sources, including plants, animals, microalgae, algae, and other microorganisms. Microorganism enzymes, among the others, account for the biggest part of application and demand in industries. These enzymes from soil microorganisms are very essential not only for soil fertility but also for biotechnological significance. Soil enzymes from microalage, comprising Amylase, Carbonic anhydrase, Galactosidase, Laccase, L-Asparaginase, Lipase, Peroxidase, Phytase, Protease, and Superoxide dismutase attract a considerable amount of attention for themselves in various industries. Most of the enzymes mentioned above have a great potential for their application in biotechnology and agriculture. Majority of such enzymes are known for their versatility and countless applications in various fields; thus, performing research on these enzymes in the future would be auspicious. This review paper presents an overview of the potential of soil algae enzymes in fields like biotechnology, medicine and agriculture, and also provides areas for advancement in researches related to this field.
In the present study, Paenibacillus lautus strain BHU3 isolated from landfill soil was evaluated for the presence of potential endoglucanases which are the first candidate of cellulase enzyme system to act on cellulose. In-silico analysis revealed high potential thermostable endoglucanases which can efficiently interact with cellulose. The most potent and thermostable endoglucanase (locus tag id. CPZ30_18280) belonged to glycosyl family-5 and had interaction energy of − 12.981 kcal/mol for the best docked cluster containing three out of ten docking conformations, and Tm value of 73.3 °C. MD simulation of 100 ns proved highly stable binding interactions of CPZ30_18280 endoglucanase with cellulose with root mean square deviation (RMSD) values ranging from 0.15 to 0.30 nm. Consistent interactions with characteristic active site residues (tyrosine, tryptophan and aspartate) of glycosyl family-5 endoglucanases were found. Further, to enhance the production of endoglucanases, the fermentation conditions were optimized employing approaches like one factor at a time (OFAT) and response surface methodology (RSM). Maximum activity of endoglucanase was determined at 60 °C. The optimized conditions for enhanced production of endoglucanase (10.15 U/mL) were pH 6.63, yeast extract conc. 3.44 g/L, wheat bran 3.59%, and inoculum size 2.65%. Hence, P. lautus strain BHU3 has enormous potential to synthesize highly efficient thermostable endoglucanases under optimized regime using agro-wastes. Thus, it could find immense industrial applications including large scale cellulose conversion to bioethanol.
Metabolic biosensors are increasingly used in metabolic engineering and synthetic biology. In this study, using Saccharomyces cerevisiae as a model system, we developed a methodology to identify promoter elements that are responsive to glucaric acid. Through transcriptome analysis, it was found that multiple genes were upregulated when cells were exposed to high concentrations of glucaric acid. From the promoters of these candidate genes, the YCR012W promoter (P YCR012W) was observed to specifically respond to glucaric acid in a dose-dependent manner. To gain further insight into the binding site of glucaric acid-responsive activators, we truncated the promoter and revealed that the -564 to -464 bp regions of P YCR012W was essential for glucaric acid-responsive expression. To investigate the glucaric acid-responsive transcription factors, we predicted the transcription factor binding sites in the -564 to -464 bp region of P YCR012W and found that two transcription factors, Ash1p and Cbf1p, might be linked to glucaric acid responses. The strategies used in this study outline a method for the identification and development of metabolic biosensors.
Citrinin is a common mycotoxin produced by different fungi. Although considerable endeavors have been made to increase citrinin output to meet the requirements of research and industrial needs, few methods are effective, owing to potential safety concerns and technical limitations. In this study, a blue fungus that produces a yellow pigment was isolated from Ziziphus jujuba. The strain was then identified as Penicillium citrinum and named HR-087. Preparative liquid chromatography was used to extract the yellow pigment. Both LC–MS and NMR assays showed that it was citrinin. To enhance the citrinin production of this strain, the culture medium was optimized through orthogonal experiments, as well as batch and fed-batch process. The final titer and productivity of citrinin reached 9.62 g/L and 0.1 g/(L × h), respectively. The high titer achieved indicates the possibility of large-scale production of high purity citrinin for low-cost supply for both academic and diagnostic analysis standard.
Sucrose phosphorylase (SPase) can transfer the glucosyl group of sucrose to different compounds and has been widely used in industry. To overcome the low thermostability of the sucrose phosphorylase from Leuconostoc mesenteroides ATCC 12291 (LmSP), a method named PROSS was used to construct mutants with increased thermostability. All variants were screened by measuring their residual activities after heating at 50°C. Then, a single point mutant and a combined mutant with improved thermostability and activity were obtained. The half-lives of mutants at 50°C were approximately twice as high as those of the wild type. In addition, 2-O-α-d-glucosylglycerol (αGG) was synthesized by the wild type and the two improved variants, and the reaction conditions were optimized. Under the conditions of glycerol concentration of 3.2 mol/L, sucrose concentration of 1.2 mol/L, and enzyme concentration of 40 U/mL at 37°C for 60 h, the yield of αGG reached the maximum, and the sucrose conversion rate of the wild type, the mutant V23L and the combined mutant V23L/S424R were 62.3%, 70.7% and 76.3%, respectively. In this study, SPase mutants with higher activity and stability were obtained, and achieved high-level production of αGG.
The current nightmare for the whole world is COVID-19. The occurrence of concentrated pneumonia cases in Wuhan city, Hubei province of China, was first reported on December 30, 2019. SARS-CoV first disclosed in 2002 but had not outspread worldwide. After 18 years, in 2020, it reemerged and outspread worldwide as SARS-CoV-2 (COVID-19), as the most dangerous virus-creating disease in the world. Is it possible to create a favorable evolution within the short time (18 years)? If possible, then what are those properties or factors that are changed in SARS-CoV-2 to make it undefeated? What are the fundamental differences between SARS-CoV-2 and SARS? The study is one of the initiatives to find out all those queries. Here, four types of protein sequences from SARS-CoV-2 and SARS were retrieved from the database to study their physicochemical and structural properties. Results showed that charged residues are playing a pivotal role in SARS-CoV-2 evolution and contribute to the helix stabilization. The formation of the cyclic salt bridge and other intra-protein interactions specially network aromatic–aromatic interaction also play the crucial role in SAS-CoV-2. This comparative study will help to understand the evolution from SARS to SARS-CoV-2 and helpful in protein engineering.
The traditional homologous recombination (HR) gene-editing method faces problems such as low editing efficiency and absence of marker genes. CRISPR–Cas9-editing efficiency is high and has been widely used in bacteria and yeast. In comparison with CRISPR–Cas9, CRISPR–Cas12a has many outstanding advantages. Here, we report an Acidaminococcus sp. BV3L6 (As) Cas12a-based genome-editing method used for Starmerella bombicola. To demonstrate the high efficiency of the CCMGE system, we verified a counter-selectable marker in S. bombicola, orotidine 5’-phosphate decarboxylase (100% for URA3). We also tested the common gene UDP-glucosyltransferase (100% for UGTA) using a 300 bp donor containing hygromycin expression cassette. This toolkit was further extended to simultaneously edit two genes (18% for UGTA and leu) and three genes (13.8% for UGTA, leu and URA3). The system greatly reduces the screening time for such multi-site editing. Based on the CCMGE system, the PHA (polyhydroxyalkanoate)-producing strain was constructed by increasing the copy number of the PHA synthase (PHAC). The PHA content and DCW reached 11.8% and 30.1 g/L, respectively. The yield of PHA was about three times higher than that of the single-copy strain using the same fermentation method.
The ribosome-binding site (RBS) in the 5′ untranslated region is recognized by 16S rRNA to start translation and is an essential element of the gene expression system. RBSs have been widely applied in regulating gene expression in various scenarios, including Gram-negative or Gram-positive bacteria. Here, we first rationally designed and constructed an RBS mutant library containing 66 RBSs. The strength of these RBSs in E. coli and C. glutamicum was characterized individually. The RBS strength spanned about 200 and 15 times in the two species, respectively. The strength of RBSs in C. glutamicum was generally lower than that of in E. coli. A total of 18 RBSs showed similar strength (within twofold differences) between the species in our study, and the correlation analysis of the strength of RBSs between E. coli and C. glutamicum (R 2 = 0.7483) revealed that these RBSs can be used across species. The sequence analysis revealed that the RBS region with two Ts stated was beneficial for RBS to function cross-species. The RBS characterized here can be used to precisely regulate gene expression in both hosts, and the characteristics of cross-species RBSs provide basic information for RBS rational design.
d-allulose, the epimer at C-3 position of d-fructose, is a low-calorie functional rare sugar, which is regarded as one of the most potential sweeteners. At present, the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase (DAE). However, industrial applications of DAE are still limited by its poor thermostability. Herein, directed evolution was applied to improve the thermostability of DAE from Clostridium cellulolyticum H10 (CcDAE). Two optimal mutants D281G and C289R, exhibiting 13.80-fold and 13.88-fold t 1/2 values as that of wild type at 65 ℃, respectively, were obtained. To further enhance the thermostability, the triple mutant A107P/D281G/C289R was constructed after combination of mutants D281G, C289R, and previously identified thermostability-enhanced mutant A107P. The T m and optimal temperature of triple mutant were increased by 14.39 ℃ and 5 ℃, respectively, compared to the wild type, meanwhile, the half-life of triple mutant was 58.85-fold as that of wild type at 65 ℃. Furthermore, the conversion rate of triple mutant was increased from 24.76% of wild type to 27.53% using 300 g/L d-fructose as substrate at 70 ℃. The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.
Cyclodextrin (CD) is produced by the catalysis of starch or starch derivatives by cyclodextrin glucosyltransferase (CGTase), and its yield is mainly limited by the product and reaction specificity of CGTase. In this study, we use CGTase derived from Bacillus stearothermophilus NO2, exhibiting high expression levels and good stability for molecular modification. The N353A mutant effectively decreases the hydrolysis activity, and the ratio of the k cat values (cyclization to hydrolysis activity) is 86.46, which is threefold that of the wild type. The E142P mutant effectively enhances α-CD specificity, which increases the ratio of k cat values (α-CD to β-CD formation) from 2.18 of the wild-type to 2.42. The N353A/E142P mutant weakens the hydrolysis side reaction and enhances α-CD specificity, and the proportion of α-CD products is 53.67%, which is 15.62% higher than that of the wild-type. This research focuses on CGTase reaction and product specificities, which suggest a novel method for the industrial production of α-CD.
l-Threonine transaldolase could catalyze the transaldolation of l-threonine and aldehyde to generate β-hydroxy-α-amino acids with high diastereoselectivity. A novel l-threonine transaldolase (PmLTTA) was identified from Pseudomonas sp. through genome mining. PmLTTA exhibited high activity in the synthesis of l-threo-phenylserine from l-threonine and benzaldehyde, with specific activity of 5.48 U mg–1. However, the application of PmLTTA was impeded by the low conversion ratio and variable diastereoselectivity, which were caused by the toxicity of aldehydes and kinetic/thermodynamic controls in the transaldolation reaction. To solve these issues, alcohol dehydrogenase was used to remove the by-product acetaldehyde, and then carboxylic acid reductase was introduced to alleviate the inhibition of benzaldehyde and toxicity of DMSO. Finally, a multi-enzyme cascade reaction, comprising of PmLTTA, carboxylic acid reductase, alcohol dehydrogenase and glucose dehydrogenase, was constructed to prepare l-threo-phenylserine from cheap benzoic acid, in which alleviated inhibition of aldehydes and desirable diastereoselectivity were achieved. Under the optimized conditions, the conversion ratio of 57.1% and de value of 95.3% were reached. This study provides an efficient and green approach for the synthesis of chiral l-threo-phenylserine from industrial byproduct, and provides guidance for the development of cascade reactions influenced by the toxic intermediates and complicated kinetic/thermodynamic controls.
Due to abundant availability, biofuel production from bamboo residues is gaining popularity. Bamboo biomass and its residues are widely available in the north-eastern part of India, which can be sustainably exploited for green energy production. The aim of the present study was to evaluate the pretreatments of dewaxed bamboo residues with dilute alkali (NaOH), and dilute acid, and the effect of pretreatment on biomass hydrolysis was performed with a commercial enzyme (Zytex). Results showed that maximum fermentable sugars were 490 mg/g obtained from 10% biomass with 0.6% (w/v) of alkali, and acid pretreated biomass produced 320 mg/g of sugars from 2% (w/v) acid with 10% biomass. Changes in biomass structure during the pretreatment process are correlated with FTIR, SEM, and component analysis for lignin, cellulose, and hemicelluloses. Fermentation studies of the hydrolysate showed that the yield of ethanol was 77% of the theoretical maximum at 36 h. Results indicate the scope of utilization of bamboo residues as substrates for biofuels, and alkaline pretreatment is an effective pretreatment process for bioethanol production.
The present research work highlights the biosurfactant application toward enhanced pyrene degradation by Pseudomonas sp. Lipopeptide-type biosurfactant was produced from Free Air CO2 Enriched (FACE) soil bacterium Bacillus sp. SS105. The different concentrations of biosurfactant at different time intervals were optimized for maximum pyrene degradation and quantified through analytical methods such as the scanning process of UV–Vis spectrophotometer and GC–MS analysis. In the absence of biosurfactant, Pseudomonas sp. could degrade only 8% of pyrene at the concentration of 5 mg per 100 ml, while the degradation rate enhanced by 95% in presence of biosurfactant (5 mg/100 ml of culture). This result demonstrated that biosurfactant produced from Bacillus sp. SS105 has potential to enhance the bioavailability and biodegradation of pyrene by Pseudomonas sp.
As the world is facing a Covid-19 pandemic, this virus teaches a lesson about the importance of on-site disinfection. On-site disinfection/sterilization with real-time monitoring of biomedical waste generated from the medical facilities is mandatory to prevent hospital-acquired infection (HAI). In this study, the life cycle assessment of two technologies, i.e., microwave (radiation-based) and autoclave (steam-based) were performed to summarize the inside-out evaluation of both technologies in terms of efficiency, efficacy, and cost-effectiveness. The results of disinfection efficacy indicated a log 10 reduction (almost 100%) in the vegetative load of microorganisms compared to the control, showing a similar level of disinfection efficacy of both strategies. Additionally, both technologies were compared on several parameters, and it was discovered that the autoclave uses more time and resources than the microwave. The total cost of an autoclave to the government is approximately double that of a microwave, while the operational cost of an autoclave is more than double that of a microwave. The findings from this study indicate that MACS may be used as a dry technique of biomedical disinfection, and its portability, tunability, and compactness make it a suitable alternative for biomedical disinfection and sterilization.
Osmoporation is a novel encapsulation approach for bioactive compounds based on the osmoresistance mechanisms of microbial cells. To the best of our knowledge, this is the first study investigating the production of fisetin-enriched yoghurt using Lactobacillus acidophilus-based bio-capsules via osmoporation as the starter culture. Results showed that the milk acidification with fisetin-loaded L. acidophilus progressed at a slower pace due to complex mechanisms induced by osmoporation and internalized fisetin. Milk fermentation using fisetin bio-capsules reached a maximum acidification rate of 0.18 pH units/h after 23 h and pH 4.6 was achieved after 32 h. Besides, the antioxidant activity of yoghurts produced with fisetin bio-capsules did not change during cold storage, while the antioxidant activity of yoghurt produced with non-encapsulated fisetin was reduced by 2.5-fold after 28 days. Overall, this study shows that fisetin osmoporation using L. acidophilus is a versatile encapsulation bioprocess that enables the delivery of preserved phytoactives into fermented foods like yoghurt. This strategy has the potential to be extended to other applications in the dairy industry using lactic acid bacteria as both the encapsulation matrix and fermentation agent.