Active Extracellular Enzyme Research Articles

Phytoplankton-derived polysaccharides and microbial peptidoglycans are key nutrients for deep-sea microbes in the Mariana Trench

BackgroundThe deep sea represents the largest marine ecosystem, driving global-scale biogeochemical cycles. Microorganisms are the most abundant biological entities and play a vital role in the cycling of organic matter in such ecosystems. The primary food source for abyssal biota is the sedimentation of particulate organic polymers. However, our knowledge of the specific biopolymers available to deep-sea microbes remains largely incomplete. One crucial rate-limiting step in organic matter cycling is the depolymerization of particulate organic polymers facilitated by extracellular enzymes (EEs). Therefore, the investigation of active EEs and the microbes responsible for their production is a top priority to better understand the key nutrient sources for deep-sea microbes.ResultsIn this study, we conducted analyses of extracellular enzymatic activities (EEAs), metagenomics, and metatranscriptomics from seawater samples of 50–9305 m from the Mariana Trench. While a diverse array of microbial groups was identified throughout the water column, only a few exhibited high levels of transcriptional activities. Notably, microbial populations actively transcribing EE genes involved in biopolymer processing in the abyssopelagic (4700 m) and hadopelagic zones (9305 m) were primarily associated with the class Actinobacteria. These microbes actively transcribed genes coding for enzymes such as cutinase, laccase, and xyloglucanase which are capable of degrading phytoplankton polysaccharides as well as GH23 peptidoglycan lyases and M23 peptidases which have the capacity to break down peptidoglycan. Consequently, corresponding enzyme activities including glycosidases, esterase, and peptidases can be detected in the deep ocean. Furthermore, cell-specific EEAs increased at 9305 m compared to 4700 m, indicating extracellular enzymes play a more significant role in nutrient cycling in the deeper regions of the Mariana Trench.ConclusionsTranscriptomic analyses have shed light on the predominant microbial population actively participating in organic matter cycling in the deep-sea environment of the Mariana Trench. The categories of active EEs suggest that the complex phytoplankton polysaccharides (e.g., cutin, lignin, and hemicellulose) and microbial peptidoglycans serve as the primary nutrient sources available to deep-sea microbes. The high cell-specific EEA observed in the hadal zone underscores the robust polymer-degrading capacities of hadal microbes even in the face of the challenging conditions they encounter in this extreme environment. These findings provide valuable new insights into the sources of nutrition, the key microbes, and the EEs crucial for biopolymer degradation in the deep seawater of the Mariana Trench.6w3UcpMmGbcStWK5zJqVACVideo

New Malolactic Bacteria Strains Isolated from Wine Microbiota: Characterization and Technological Properties

Malolactic fermentation (MLF) or biological decrease of wine acidity is defined as the enzymatic bioconversion of malic acid in lactic acid, a process performed by lactic acid bacteria (LAB). The procedures for the isolation of new indigenous LAB strains from the red wines produced in Copou Iasi wine center (NE of Romania) undergoing spontaneous malolactic fermentation, resulted in the obtaining of 67 catalase-negative and Gram-positive LAB strains. After testing in the malolactic fermentative process, application of specific screening procedures and identification (API 50 CH), two bacterial strains belonging to the species Oenococcus oeni (strain 13-7) and Lactobacillus plantarum (strain R1-1) with high yield of malolactic bioconversion, non-producing biogenic amines, and with active extracellular enzymes related to wine aroma, were retained and characterized. Tested in synthetic medium (MRS-TJ) for 10 days, the new isolated LAB strains metabolized over 98% of the malic acid at ethanol concentrations between 10 and 14 % (v/v), low pH (>3.0), total SO2 doses up to 70 mg/L and temperatures between 15 and 35 °C, showing high potential for future use in the winemaking process as bacterial starter cultures, in order to obtain high quality wines with increased typicity.

Vertical variation in Vibrio community composition in Sansha Yongle Blue Hole and its ability to degrade macromolecules

With the advantages of wide distribution, fast growth, and broad metabolic spectrum to organic carbon compounds, Vibrio may play an important role in organic carbon cycling. However, the ecological roles of Vibrio in many marine environments have not been explored. Here, the world’s deepest ‘blue hole’, the Sansha Yongle Blue Hole (SYBH) in the South China Sea, which is a geographically semi-enclosed environment featuring unique chemical characters, was investigated. The abundance, diversity and carbon source utilization capability of Vibrio were studied by quantification and high-throughput sequencing of Vibrio specific 16S rRNA genes and cultivation methods. The abundance of Vibrio in water column of the SYBH ranged from 3.78 × 104 to 7.35 × 106 16S rRNA gene copies L−1. Free-living Vibrio was more abundant than particle-associated Vibrio (~ 1.20 × 106 versus~ 2.68 × 105 gene copies L−1), indicating that Vibrio prefers a free-living life style. The Vibrio assemblages showed clear vertical stratification and could be divided into three groups: aerobic-transition, middle anaerobic and bottom anaerobic zones. Dissolved oxygen (DO), temperature, pH and salinity were the main environmental factors affecting the abundance and community composition. Cultivated Vibrio demonstrated a degrading capability to various macromolecular substrates, including starch, Tween 20/40/80, DNA, gelatin, alginate, casein, chitin, lecithin, κ-carrageenan, mannan, xylan and hyaluronic acid. This suggests that Vibrio could produce a variety of highly active extracellular enzymes. Our study provides new insights into the distribution pattern and possible role in carbon cycle of Vibrio in the unique environment of a ‘blue hole’.

Efficient production of recombinant tannase in Aspergillus oryzae using an improved glucoamylase gene promoter

A tannase-encoding gene, AotanB, from Aspergillus oryzae RIB40 was overexpressed in A.oryzae AOK11 niaD-deficient mutant derived from an industrial strain under the control of an improved glucoamylase gene promoter PglaA142. The recombinant tannase, designated as rAoTanBO, was produced efficiently as an active extracellular enzyme. Purified rAoTanBO showed a smeared band with a molecular mass of approximately 80-100kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rAoTanBO had a molecular mass of 65kDa, after treatment with endo-β-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30-35°C and pH 6.0. The tannase activity of purified rAoTanBO towards natural and artificial substrates was 2-8 folds higher than that of the recombinant enzyme produced by Pichia pastoris, designated as rAoTanBP. N-terminus of the mature rAoTanBP had six more amino acids than the N-terminus of the mature rAoTanBO. Kinetic analyses showed that rAoTanBO had higher catalytic efficiency (kcat/Km) than rAoTanBP. rAoTanBO was stable up to 60°C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP.

Development of a Cold-Adapted Pseudoalteromonas Expression System for the Pseudoalteromonas Proteins Intractable for the Escherichia coli System.

Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psychrophilic strain Pseudoalteromonas sp. SM20429 was established. Based on the shuttle vector, three reporter vectors were constructed to compare the strength of the cloned promoters at low temperature. The promoter of xylanase gene from Pseudoalteromonas sp. BSi20429 was chosen due to its high activity at 10–15°C. An expression vector pEV containing the chosen promoter, multiple cloning sites and a His tag was constructed for protein expression and purification. With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h. Recombinant pseudoalterin purified from the culture by Ni affinity chromatography had identical N-terminal sequence, similar molecular mass and substrate specificity as the native pseudoalterin. In addition, another two cold-adapted enzymes were also successfully expressed by this system. Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system.

Isolation and Screening of Cold Active Extracellular Enzymes Producing Psychrotrophic Bacteria from Soil of Jammu City

found to be present in garden soil samples (33.33%) and bacterial load was 13.5×105 Cfu/ gm. Only eleven isolates among 40 were grown at 4°C. The isolated psychrotrophic bacteria were belong to the genera of Bacillus, Paenibacillus, Pseudomonas, Staphylococcus, Arthrobacter, E coli and Micrococcus. All the above isolates of bacteria were tested for the production of cold active extracellular enzymes like proteases, cellulases, amylases, pectinases, and lipases. Enzymatic performance was evaluated at 4, 15 and 30°C by plate assay method. Lipase activities at both 4 and 15°C were detected as maximum than others by 27.5% and 85% of the bacterial isolates respectively. The potential of these psychrotrophic bacteria as a resource of enzymes have found their way into biotechnological and industrial applications.

A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

Expression and purification of aggregation-prone and disulfide-containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high-value target proteins. Here, we present a novel gene-fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag-Acidic-Target Tag) results in near-complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one-step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization.

Isolation, Purification, and Properties of α-Amylase from Bacillus subtilis-7A

Cultivation ofBacillus subtilis-7A on waste from alcohol production yielded an active extracellular enzyme α-amylase with MW 75 kDa. The enzyme was isolated from the culture medium by 60% saturated ammonium sulfate and purified until homogeneous by gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. The optimum temperatures for the complex and purified enzyme are 30 and 50°C, respectively. The optimum activity for both preparations occurred at pH 6.5. The substrate specificity of the isolated preparations was studied.

Molecular basis for the association of group IIA phospholipase A(2) and decorin in human atherosclerotic lesions.

Group IIA secretory nonpancreatic phospholipase A(2) (snpPLA(2)) is associated with collagen fibers in the extracellular matrix of human atherosclerotic plaques. Decorin, a small proteoglycan (PG) carrying chondroitin/dermatan sulfate glycosaminoglycans (GAGs), forms part of the collagen network in human arteries. To explore whether snpPLA(2) may be associated with collagen fibers via interaction with decorin, we performed (1) immunohistochemistry to compare the relative in vivo localization of snpPLA(2) and decorin in human atherosclerotic tissue and (2) in vitro experiments to study the interaction between snpPLA(2) and decorin. In atherosclerotic lesions, decorin was detected within the snpPLA(2)-positive part of the intima close to the media. Electrophoretic mobility shift assay showed that snpPLA(2) binds to decorin synthesized by human fibroblasts. Native and GAG-depleted decorin enhanced the association of snpPLA(2) to collagen types I and VI in a solid-phase binding assay. Furthermore, snpPLA(2) bound efficiently to a recombinant decorin core protein fragment B/E (Asp45-Lys359). This binding was competed with soluble decorin and inhibited at NaCl concentrations >150 mmol/L. The decorin core protein fragment B/E competed better than dermatan sulfate for binding of snpPLA(2) to decorin-coated microtiter wells. The enzymatic activity of snpPLA(2) increased 2- to 3-fold in the presence of decorin or GAG-depleted decorin. The results show that snpPLA(2) binds preferentially to the decorin protein core rather than to the GAG chain and that this interaction enhances snpPLA(2) activity. As a consequence, this active extracellular enzyme may contribute to the pathogenesis of atherosclerosis by modifying lipoproteins and releasing inflammatory lipid mediators at places of lipoprotein retention in the arterial wall.

Secretion processing and activation of Erwinia chrysanthemi proteases

E chrysanthemi, a phytopathogenic enterobacterium, secretes several enzymes into the medium such as pectinases cellulases and proteases. It also produces 3 distinct and antigenically related extracellular proteases. The proteases secretion pathway seems to be distinct from that of the other extracellular enzymes since pleiotropic mutants impaired in cellulase and pectinase are unimpaired in protease secretion. E chrysanthemi proteases B and C secretion occurs without an N-terminal signal peptide and is dependent upon specific secretion functions which are encoded by genes adjacent to the protease structural genes. This secretion pathway might be analogous to the α-hemolysin secretion pathway in E coli. Protection against intracellular proteolytic activity is achieved by 2 distinct mechanisms: the proteases are synthesized as inactive precursors with an N-terminal extension of 15 aminoacids (protease B) and 17 aminoacids (protease C) absent in the mature active extracellular enzymes; an intracellular specific inhibitor is produced by osme E chrysanthemi strains.

Role of Planococcus citreus in the spoilage of Penaeus shrimp

An extensive survey of marine products revealed that only products containing shrimp contained P. citreus P. citreus isolates accounted for 13–24% of the microbial flora of Penaeus shrimp during extended ice storage. After 10 days of shrimp ice storage, Plano-coccus and Pseudomonas were the predominant microorganisms present in Penaeus shrimp. All isolates recovered exhibited good growth over a range of pH 7–10, 5–35 °C and 5–120 g NaCl/1. Proteolysis studies revealed that P. citreus isolates were able to hydrolyze gelatin, cottonseed, soy and more important to the fisheries microbiologists, shrimp protein. P. citreus grew well at ice and refrigerated temperatures (5 °C) and all isolates tested produced an active extracellular enzyme system capable of degrading shrimp protein at this temperature. P. citreus counts increased in the inoculated shrimp stored at 5 °C from 3.0 x 103 bacteria/gram to 1.5 × 108 bacteria /gram at the 16th day. The potential of P. citreus as a “spoiler” of raw shrimp was shown by the rapid increase in total volatile nitrogen/amino acid nitrogen (TVN/AA-N) ratio, pH and trimethyl-amine nitrogen (TMN) content as well as the reduction in percent total extractable protein (TEP) of the inoculated samples. The extracellular proteolytic enzyme of P. citreus was active at refrigeration or iced temperatures; however, maximum activity was exhibited at 35 °C and pH 8. The importance of the extracellular protease enzyme for shrimp quality is discussed.