The use of chemicals around the globe in different industries has increased tremendously, affecting the health of people. The modern world intends to replace these noxious chemicals with environmental friendly products for the betterment of life on the planet. Establishing enzymatic processes in spite of chemical processes has been a prime objective of scientists. Various enzymes, specifically microbial proteases, are the most essentially used in different corporate sectors, such as textile, detergent, leather, feed, waste, and others. Proteases with respect to physiological and commercial roles hold a pivotal position. As they are performing synthetic and degradative functions, proteases are found ubiquitously, such as in plants, animals, and microbes. Among different producers of proteases, Bacillus sp. are mostly commercially exploited microbes for proteases. Proteases are successfully considered as an alternative to chemicals and an eco-friendly indicator for nature or the surroundings. The evolutionary relationship among acidic, neutral, and alkaline proteases has been analyzed based on their protein sequences, but there remains a lack of information that regulates the diversity in their specificity. Researchers are looking for microbial proteases as they can tolerate harsh conditions, ways to prevent autoproteolytic activity, stability in optimum pH, and substrate specificity. The current review focuses on the comparison among different proteases and the current problems faced during production and application at the industrial level. Deciphering these issues would enable us to promote microbial proteases economically and commercially around the world.

/pdf/microbial-proteases-applications-o4nt9ikd6n.pdf

Microbial Proteases Applications.

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25 oC makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

/pdf/discovery-molecular-mechanisms-and-industrial-applications-40im449bq9.pdf

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes.

Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.

https://www.pnas.org/content/pnas/113/9/2502.full.pdf

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species.

Due to its clear inherited backgrounds as well as simple and diverse genetic manipulation systems, Bacillus subtilis is the key Gram-positive model bacterium for studies on physiology and metabolism. Furthermore, due to its highly efficient protein secretion system and adaptable metabolism, it has been widely used as a cell factory for microbial production of chemicals, enzymes, and antimicrobial materials for industry, agriculture, and medicine. In this mini-review, we first summarize the basic genetic manipulation tools and expression systems for this bacterium, including traditional methods and novel engineering systems. Secondly, we briefly introduce its applications in the production of chemicals and enzymes, and summarize its advantages, mainly focusing on some noteworthy products and recent progress in the engineering of B. subtilis. Finally, this review also covers applications such as microbial additives and antimicrobials, as well as biofilm systems and spore formation. We hope to provide an overview for novice researchers in this area, offering them a better understanding of B. subtilis and its applications.

/pdf/bacillus-subtilis-a-universal-cell-factory-for-industry-5eaaefvivf.pdf

Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine.

A large variety of microorganisms produce potent biocatalysts as enzymes which vary in their microbial sources, chemical properties, and mechanisms. Usually, microbial enzymes catalyze the reactions of hydrolysis, oxidation, or reduction. Microbial enzymes have different active site motifs targeting diversified substrates. They may catalyze the reaction by completely different mechanisms even when they belong to the same class. Microbial enzymes are mainly produced by submerged fermentation and solid state fermentation. The enormous diversity of microbial enzymes makes them an interesting group of products for application in many areas such as agricultural, chemical industry, food processing industry, textile industry, pharmaceuticals, wood processing industry, analytical applications, cosmetics, and environmental pollution control, such as bioremediation and biodegradation. In this chapter, classification and microbial resources, microbial production, and industrial applications of a group of industrially used microbial enzymes, such as carbohydrases, proteases and lipases, are described.

Microbial Enzymes of Use in Industry

Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001

Bacillus subtilis and its closely related species are important strains for industry, agriculture, and medicine However, it is difficult to perform genetic manipulations using the endogenous recombination machinery In many bacteria, phage recombineering systems have been employed to improve recombineering frequencies To date, an efficient phage recombineering system for B subtilis has not been reported Here, we, for the first time, identified that GP35 from the native phage SPP1 exhibited a high recombination activity in B subtilis On this basis, we developed a high-efficiency GP35-meditated recombineering system Taking single-stranded DNA (ssDNA) as a recombineering substrate, ten recombinases from diverse sources were investigated in B subtilis W168 GP35 showed the highest recombineering frequency (171 ± 015 × 10−1) Besides targeting the purine nucleoside phosphorylase gene (deoD), we also demonstrated the utility of GP35 and Beta from Escherichia coli lambda phage by deleting the alpha-amylase gene (amyE) and uracil phosphoribosyltransferase gene (upp) In all three genetic loci, GP35 exhibited a higher frequency than Beta Moreover, a phylogenetic tree comparing the kinship of different recombinase hosts with B subtilis was constructed, and the relationship between the recombineering frequency and the kinship of the host was further analyzed The results suggested that closer kinship to B subtilis resulted in higher frequency in B subtilis In conclusion, the recombinase from native phage or prophage can significantly promote the genetic recombineering frequency in its host, providing an effective genetic tool for constructing genetically engineered strains and investigating bacterial physiology

A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35

Objective To explore the composition of the ocular microbiome in normal subjects and patients with Meibomian gland dysfunction (MGD). Subjects and methods Seventy subjects (140 eyes) were enrolled in our study. Signs of dry eye were evaluated and bacterial species in the conjunctival sac (CS) and Meibomian gland (MG) secretions were then identified by 16S rRNA gene sequencing. Additionally, 17 subjects (34 eyes) were further evaluated to determine differences in the microbiomes in the surface and deep layers of MG using a segmental secretion analysis. Results The positive bacterial isolation rate was markedly higher in MG secretions than in the CS. The bacterial composition of the control and mild group was simple, whereas the composition of bacteria was more complex as the severity of MGD increased. The positive bacterial isolation rate and number of bacterial types were significantly higher in the severe MGD group than those in the control, mild and moderate MGD groups. Corynebacterium macginleyi was only detected in the severe MGD group, with an isolation rate of up to 26.3%. Furthermore, a new grading system for bacterial severity of MGD was proposed and the severity of MGD appeared to be positively correlated with a higher grade of bacterial severity. The segmental secretion analysis showed severe MGD had a significantly higher incidence of bacterial discordance rate. Conclusion The severity of MGD was positively correlated with a higher isolation rate, a greater number of bacterial species, and a higher grade of bacterial severity, which implied that MGD might be correlated with bacterial changes. This study provided some basis for the indications of antibiotic in clinical practice.

/pdf/pathogens-in-the-meibomian-gland-and-conjunctival-sac-1y713ql17k.pdf

Pathogens in the Meibomian gland and conjunctival sac: microbiome of normal subjects and patients with Meibomian gland dysfunction.

http://www.im.ac.cn/UserFiles/File/2011/2011.03/AprB-Biochimie%20final%20version%20on%20%20web.pdf

Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family.

PDZ domain-containing proteases, also known as HtrA family proteases, play important roles in bacterial cells by modulating disease pathogenesis and cell-envelope stress responses. These proteases have diverse functions through proteolysis- and nonproteolysis-dependent modes. Here, we report that the genome of the causative agent of rice bacterial blight, Xanthomonas oryzae pv. oryzae, encodes seven PDZ domain-containing proteins. Systematic inactivation of their encoding genes revealed that PXO_01122 and PXO_04290 (prc) are involved in virulence. prc encodes a putative HtrA family protease that localizes in the bacterial periplasm. Mutation of prc also resulted in susceptibility to multiple environmental stresses, including H2O2, sodium dodecylsulfate, and osmolarity stresses. Comparative subproteomic analyses showed that the amounts of 34 periplasmic proteins were lower in the prc mutant than in wild-type. These proteins were associated with proteolysis, biosynthesis of macromolecules, carbohydrate or energy metabolism, signal transduction, and protein translocation or folding. We provide in vivo and in vitro evidence demonstrating that Prc stabilizes and directly binds to one of these proteins, DppP, a dipeptidyl peptidase contributing to full virulence. Taken together, our results suggest that Prc contributes to bacterial virulence by acting as a periplasmic modulator of cell-envelope stress responses.

https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-08-13-0234-R

The Periplasmic PDZ Domain-Containing Protein Prc Modulates Full Virulence, Envelops Stress Responses, and Directly Interacts with Dipeptidyl Peptidase of Xanthomonas oryzae pv. oryzae

Jie Wu

Papers

Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001

A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35

Pathogens in the Meibomian gland and conjunctival sac: microbiome of normal subjects and patients with Meibomian gland dysfunction.

Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family.

The Periplasmic PDZ Domain-Containing Protein Prc Modulates Full Virulence, Envelops Stress Responses, and Directly Interacts with Dipeptidyl Peptidase of Xanthomonas oryzae pv. oryzae