Enzyme Source Research Articles

Purification and Properties of a Plasmin-like Marine Protease from Clamworm (Perinereis aibuhitensis).

Marine organisms are a rich source of enzymes that exhibit excellent biological activity and a wide range of applications. However, there has been limited research on the proteases found in marine mudflat organisms. Based on this background, the marine fibrinolytic enzyme FELP, which was isolated and purified from clamworm (Perinereis aibuhitensis), has exhibited excellent fibrinolytic activity. We demonstrated the FELP with a purification of 10.61-fold by precipitation with ammonium sulfate, ion-exchange chromatography, and gel-filtration chromatography. SDS-PAGE, fibrin plate method, and LC-MS/MS indicated that the molecular weight of FELP is 28.9 kDa and identified FELP as a fibrinolytic enzyme-like protease. FELP displayed the maximum fibrinolytic activity at pH 9 (407 ± 16 mm2) and 50 °C (724 ± 27 mm2) and had excellent stability at pH 7-11 (50%) or 30-60 °C (60%), respectively. The three-dimensional structure of some amino acid residues of FELP was predicted with the SWISS-MODEL. The fibrinolytic and fibrinogenolytic assays showed that the enzyme possessed direct fibrinolytic activity and indirect fibrinolysis via the activation of plasminogen; it could preferentially degrade Aα-chains of fibrinogen, followed by Bβ- and γ-chains. Overall, the fibrinolytic enzyme was successfully purified from Perinereis aibuhitensis, a marine Annelida (phylum), with favorable stability that has strong fibrinolysis activity in vitro. Therefore, FELP appears to be a potent fibrinolytic enzyme with an application that deserves further investigation.

Fucanases Related to the GH107 Family from Members of the PVC Superphylum

The glycoside hydrolase 107 (GH107) family includes fucanase enzymes from only two bacterial phyla, Bacteroidota and Pseudomonadota. The goal of this work was to explore the diversity of putative fucanase enzymes related to this family in organisms of the PVC superphylum (Planctomycetota, Verrucomicrobiota, Chlamydiota), in order to expand our knowledge of the fucoidan-degrading potential in this ecologically and biotechnologically relevant group. Using hidden Markov model- and peptide-based annotation tools, 26 GH107 homolog sequences were identified in metagenome and genome datasets. The sequences formed two distinct clusters in a phylogenetic analysis, only one including members of the GH107 family. The endo-acting fucoidan degrading activity was confirmed in an enzyme included in the most divergent cluster. The fucanase, which probably originated in an uncultured planctomycete from the sampled subantarctic sediments, was cloned and expressed in Escherichia coli. The enzyme catalyzed the rapid hydrolysis of internal glycosidic bonds of fucoidan from Macrocystis pyrifera, a macroalgae species abundant at the site. It was active in a wide range of temperatures (5–45 °C), salinities (9.5–861 mM NaCl), and pH values (4.5–9), mainly producing sulfated α-(1,3)-linked fuco-oligosaccharides of various lengths. The PVC superphylum represents a promising source of fucanase enzymes with various biotechnological applications.

Immunomodulating Enzymes from Streptococcus pyogenes-In Pathogenesis, as Biotechnological Tools, and as Biological Drugs.

Streptococcus pyogenes, or Group A Streptococcus, is an exclusively human pathogen that causes a wide variety of diseases ranging from mild throat and skin infections to severe invasive disease. The pathogenesis of S. pyogenes infection has been extensively studied, but the pathophysiology, especially of the more severe infections, is still somewhat elusive. One key feature of S. pyogenes is the expression of secreted, surface-associated, and intracellular enzymes that directly or indirectly affect both the innate and adaptive host immune systems. Undoubtedly, S. pyogenes is one of the major bacterial sources for immunomodulating enzymes. Major targets for these enzymes are immunoglobulins that are destroyed or modified through proteolysis or glycan hydrolysis. Furthermore, several enzymes degrade components of the complement system and a group of DNAses degrade host DNA in neutrophil extracellular traps. Additional types of enzymes interfere with cellular inflammatory and innate immunity responses. In this review, we attempt to give a broad overview of the functions of these enzymes and their roles in pathogenesis. For those enzymes where experimentally determined structures exist, the structural aspects of the enzymatic activity are further discussed. Lastly, we also discuss the emerging use of some of the enzymes as biotechnological tools as well as biological drugs and vaccines.

Purification and characterization of an α-l-arabinofuranosidase, α-l-AFase, for hydrolyzed ginsenoside Rc from Bacillus subtilis

Natural ginsenoside needs to be converted into rare ginsenoside before it can be readily absorbed into the bloodstream for action. In this study, an α-l-arabinofuranosidase (α-l-AFase) gene Bsafs2 was cloned from Bacillus subtilis (B. subtilis). Bsafs2 was ligated to the expression vector pET28a(+), and the expression vector was constructed and transformed into Escherichia coli (E. coli) BL21 heterologous recombinant expression to obtain α-l-AFase. α-l-AFase can hydrolyze at the C20 site of Ginsenoside Rc to obtain rare ginsenoside Rd. Studies on the enzymatic property showed that α-l-AFase had good tolerance to ethanol, glucose, and l-arabinose. The optimum temperature of α-l-AFase was 40 °C and pH = 5.5. Kinetic parameters Km of α-l-AFase for pNPαAraf and Ginsenoside Rc were 1.93 and 8.9 mmol/L, the Vmax were 26 and 154 μmol/min/mg, the Kcat were 24.14 and 1.48 S-1, respectively. This study provides the enzyme source for the biotransformation of Ginsenoside Rc.

Measurement of Mucinase Activity.

Mucinase consists of some proteases, glycosidases, sulfatases, and sialidases. It is not practical to measure individual enzyme activities when measuring mucinase activity. In this method, mucinase activity is measured using porcine gastric mucin as a substrate and feces as an enzyme source. This description includes fecal pellet preparation, reaction procedure of mucinase, measurement of reducing sugars liberated during the procedure, and determination of nitrogen content in the fecal preparations.

Penicillium citrinum CFAM 521 Isolated From the Amazon Region: A Novel Source of a Fibrinolytic Enzyme.

Fibrinolytic agents are essential in treating thrombosis, playing a critical role in improving survival rates in cardiovascular diseases. Microbial fibrinolytic proteases have emerged as promising alternatives due to their affordability, specificity, lower toxicity, and reduced side effects. Consequently, the search for microorganisms capable of producing these enzymes has gained significant economic importance in the pharmaceutical industry. This study reports and characterizes a novel fibrinolytic enzyme produced by Penicillium citrinum CFAM 521, a strain isolated from the Amazon region. The enzyme was purified using a polyethylene glycol (PEG)-phosphate salt aqueous two-phase system (ATPS). The effects of PEG molecular weight, PEG concentration, and phosphate concentration on the protease partition coefficient (K) were evaluated through a 22 full factorial design. The enzyme exhibited both fibrinolytic and fibrinogenolytic activities. After partitioning in a two-phase system with 10% (w/w) PEG and 15% (w/w) sodium phosphate, the fibrinolytic proteases were predominantly retained in the salt-rich bottom phase (K = 0.33). The enzyme has a molecular weight of 34 kDa, with optimal pH and temperature at 9°C and 37°C, respectively. Inhibitory analysis confirmed that it is a serine protease, and its activity was enhanced by the addition of Mn2+. Notably, the enzyme exhibited no hemolytic activity. Therefore, P. citrinum CFAM 521 represents a novel source of fibrinolytic enzymes, highlighting its potential as an alternative for the development of thrombolytic agents.

Cellulolytic enzymes in Microbulbifer sp. Strain GL-2, a marine fish intestinal bacterium, with emphasis on endo-1,4-β-glucanases Cel5A and Cel8.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two β-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-β-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-β-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

<i>Nocardiopsis synnemataformans</i> NBRM9, an extremophilic actinomycete producing extremozyme cellulase, using lignocellulosic agro-wastes and its biotechnological applications

<abstract> <p>Actinomycetes are an attractive source of lignocellulose-degrading enzymes. The search for actinomycetes producing extremozyme cellulase using cheap lignocellulosic waste remains a priority goal of enzyme research. In this context, the extremophilic actinomycete NBRM9 showed promising cellulolytic activity in solid and liquid assays. This actinomycete was identified as <italic>Nocardiopsis synnemataformans</italic> based on its phenotypic characteristics alongside phylogenetic analyses of 16S rRNA gene sequencing (OQ380604.1). Using bean straw as the best agro-waste, the production of cellulase from this strain was statistically optimized using a response surface methodology, with the maximum activity (13.20 U/mL) achieved at an incubation temperature of 40 °C, a pH of 9, an incubation time of 7 days, and a 2% substrate concentration. The partially purified cellulase (PPC) showed promising activity and stability over a wide range of temperatures (20–90 °C), pH values (3–11), and NaCl concentrations (1–19%), with optimal activity at 50 °C, pH 9.0, and 10% salinity. Under these conditions, the enzyme retained >95% of its activity, thus indicating its extremozyme nature. The kinetics of cellulase showed that it has a V<sub>max</sub> of 20.19 ± 1.88 U/mL and a Km of 0.25 ± 0.07 mM. The immobilized PPC had a relative activity of 69.58 ± 0.13%. In the in vitro microtiter assay, the PPC was found to have a concentration-dependent anti-biofilm activity (up to 85.15 ± 1.60%). Additionally, the fermentative conversion of the hydrolyzed bean straw by <italic>Saccharomyces cerevisiae</italic> (KM504287.1) amounted to 65.80 ± 0.52% of the theoretical ethanol yield. Overall, for the first time, the present work reports the production of extremozymatic (thermo, alkali-, and halo-stable) cellulase from <italic>N. synnemataformans</italic> NBRM9. Therefore, this strain is recommended for use as a biotool in many lignocellulosic-based applications operating under harsh conditions.</p> </abstract>

Application of microbial enzymes in medicine and industry: current status and future perspectives.

Microbes are a major source of enzymes due to their ability to be mass-cultivated and genetically modified. Compared with plant and animal enzymes, microbial enzymes are more stable and active. Enzymes are generally classified into six classes based on their reaction, substrate specificity and mechanism of action. In addition to their application in medicine for treating diseases, these compounds are used as anti-inflammatory, thrombolytic and digestive agents. However, challenges such as immunogenicity, tissue specificity and short in vivo half-life make clinical trials complex. Enzymes are metabolic catalysts in industry and their production and extraction must be optimized to preserve profitability due to rising demand. The present review highlights the increasing importance of bacterial enzymes in industry and medicine and explores methods for their production, extraction and purification.

Isolation, Molecular Characterization, Optimization and Purification of Amylase Enzyme from Locally Isolated Bacillus Species in Different Regions of Munshiganj, Bangladesh

Amylase is a kind of enzyme that facilitates the breakdown of carbohydrates in the body. This enzyme has many applications in different industries. Generally, in pharmaceutical sector it is used for the treatment of pancreatic disorder, pancreatic enzyme replacement therapy (PERT) and as a digestive aid. In this study, the bacterial strain was isolated from soil samples collected from potato dumpsites in different areas of Munshiganj, Bangladesh. After subculturing on the nutrient agar plate, 31 colonies were obtained, of which 9 isolates were found as amylase producer on starch agar medium. Of these, 2 isolates (MC-04 and MC-15) were selected based on the starch hydrolysis clear zone ratio. The isolate MC-04 showed crude enzyme activity of 2.82 IU/ml and specific enzyme activity of 3.42 IU/mg, and isolate MC-15 showed crude enzyme activity of 3.16 IU/ml and specific enzyme activity of 3.77 IU/mg. The best isolate, MC-15, was then identified by morphology, biochemical and molecular characterization and confirmed by 16S-rRNA gene sequencing. After 16S-rRNA sequencing, the isolate (MC-15) was identified as Bacillus subtilis. This amylase production of this strain had also been optimized under certain conditions such as different incubation periods, pH, temperatures and different carbon sources. We found that the best incubation period was 48 h, the optimum pH-7.0, the optimum temperature at 40°C and 2% starch was considered as the best source of carbon. Finally, the crude amylase enzyme was purified by precipitation with ammonium sulfate, dialysis, and single-step gel filtration chromatography. The enzymatic activity of the purified amylase was found to be 8.91 IU/ml, that was 2.82-fold greater enzymatic activity than the raw enzyme. The experiments confirmed that Bacillus subtilis may be a good source of amylase enzyme for industrial application in Bangladesh. Dhaka Univ. J. Pharm. Sci. 22(2): 147-154, 2023 (December)

Isolation and Molecular identification of Endophytic fungi from Michelia champaca Linn. and screening for its Extracellular Enzyme Production

Fungal endophytes are the group of microorganisms that occupy parts and colonize living, internal tissues of plants without causing any immediate, negative effect or noticeable injury to the host. In this study, fungal endophytes were targeted for isolation from young and healthy leaves of Michelia champaca Linn. from Guwahati region, Assam. The colony morphology was screened for observation of spores/ conidia or fruiting bodies under light compound microscope after staining. The endophyte was screened for the presence of various enzymatic activities like Amylolytic, Lipolytic, Cellulolytic, Proteolytic and catalase activity. Results showed that the isolated endophyte was a potential source of amylolytic, cellulolytic and Lipolytic activities whereas it showed negative result for catalase and proteolytic activities. It was further characterized using 1, 5.8S ribosomal RNA gene ITS sequencing and phylogenetic analysis. The molecular characterization identified it as Cladosporium pseudocladosporioides. Hence, this study showed that fungal isolate from M. champaca could be a potential source for enzymes with biological activities.

Proteolytic Activity of Bacillus Strains Isolated from Soil of Rice Agrocenosis

Microorganisms are the most common sources of commercial enzymes due to their physiological and biochemical properties, facile culture conditions, and ease of cell manipulation. Among microbial enzymes, proteases are ubiquitous in nature and have been found in all living forms encompassing the eukaryotes like plants, animals, fungi, and protists as well as the prokaryotic domains of bacteria and archaea. Proteases are the most important for the industry and constitute approximately 60% of the total industrial enzyme market. Among the bacteria, the genus Bacillus has a very prominent place in terms of the commercial production of proteases. Earlier from the water and bottom sediments of the Black Sea, we have isolated a number of producers of proteolytic enzymes from Bacillus species. The aim of this work was to investigate the ability of representatives of a number of soil bacilli species to synthesize enzymes that hydrolyze such protein substrates as elastin, fibrin, fibrinogen, and keratin. Methods. The objects of the study were 8 cultures (KS 1 — KS 8) isolated from the soil of the rice agrocenosis. Cultures were grown under conditions of deep cultivation at 28 °С, with a mixing speed of for the nutrient medium of 230 rpm for 4 days. Methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic, and keratinase) activity in the culture liquid supernatant were used. Disulfide reductase activity was measured spectrophotometrically at 412 nm by evaluating the yellow sulfide formed during the reduction of 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). Results. The study of the spectrum of proteolytic activities of 8 freshly isolated strains showed that only KS 6 under experimental conditions did not show the ability to hydrolyze any of the studied substrates (casein, elastin, fibrin, fibrinogen, and keratin). Strains KS 1, KS 2, KS 7, and KS 8 showed higher levels of activity compared to other strains studied. The most interesting for further research are: І) strain KS 1, which showed the highest fibrinolytic activity, ІІ) strain KS 2 as the most effective producer with elastase and fibrinogenolytic activity, III) KS 7 and KS 8, which simultaneously showed the highest rates as keratinase (7 U/mL and 9 U/mL) and sulfate reductase (33 μmol/min and 31 μmol/min) activity, respectively. Conclusions. According to the catalytic properties, a number of representatives of Bacillus, isolated from the soil of the rice agrocenosis may be promising for further research as an enzyme producer with proteolytic activity.

Applications of Microbial Enzymes: The Need of an Hour

A growing need for sustainable solutions is one of the primary drivers of the demand for industrial enzymes. One of the most significant and beneficial sources of many enzymes has been and still is the microbial world. Numerous industrial procedures, such as chemical synthesis used to create chemicals and pharmaceuticals, have a number of drawbacks: Lack of enantiomeric specificity for chiral synthesis, low pH, high pressure, high temperature, and low catalytic efficiency. Enzyme research and interest are still advancing, which helps industrial biocatalysis succeed even more. There should be a lot of intriguing discoveries in the field of biotransformation over the coming years. The value of biotechnologically and industrially significant microbial enzymes is the main topic of this study, which comprises 44 papers, including research studies and review articles. Also, it offers novel insights into the micro-organisms that manufacture these enzymes as well as the procedures employed for their purification and separation.

High potential low-cost crude enzymes from Algerian fungal strains for plant polysaccharides hydrolysis

Fungi secrete large enzyme sets, such as glycosyl hydrolases (GH) capable of targeting plant polymers, making them a valuable enzymes source. In this study, three fungal strains were isolated from the rhizosphere area and identified as Trichoderma harzianum, Aspergillus tubingensis and Aspergillus tamarii by molecular analysis. The strains were able to grow on mono-, di- and polysaccharides as sole carbon sources, and even on D-galactose and cellulose which is rare for Aspergillus species. Besides, GH activities such as cellobiohydrolase, β-1,4-D-galactosidase, β-mannosidase glucoamylase and β-1,4-D-galactosidase were revealed in secretome of the strains cultivated on wheat bran with optimum obtained between 4th and 7th day. Furthermore, hydrolysis of wheat bran with T. harzianum, A. tubingensis and A. tamarii culture supernatant resulted in the release of 4.6, 5.5 and6.8 g/L of glucose, respectively. The yield increased two-fold by applying a mix of T. harzianum and A. tubingensis enzymes extracts at a ratio 1:1. In addition, the combination of the crude enzymes with commercial cellulase enhanced 3 times the glucose content. The results present enzyme extracts with high yield in saccharification of complex substrates with a potential application in bioethanol production.

Characterization of trypsin enzyme extracted from intestines of yellowfin tuna (Thunnus albacares) and bigeye tuna (Thunnus obesus) from Indonesian seawater

Recently, there has been a major concern about fisheries waste produced by tuna processing factories. There are many ways to handle the waste from tuna processing factories, specifically the tuna viscera which contains the stomach, pyloric caeca, kidneys, and intestines. The tuna viscera have been known as a source of digestive enzymes, one of them is a protease, including trypsin. The objective of this research was to analyze the enzymatic characteristic of trypsin extracted from two different tuna species. In this study, the trypsin extracted from the intestines of yellowfin and bigeye tuna was characterized by analyzing its enzyme activity as well as optimum temperature and pH, protein concentration, kinetic studies, and molecular weight. The results reveal that the enzyme activity and protein concentration observed in bigeye tuna (Thunnus obesus) were significantly higher than in yellowfin tuna (Thunnus albacares). In addition, trypsin bigeye and yellowfin tuna had the optimum temperature and pH from both crude extracts within the range of 50-60˚C and 7-8, respectively. Moreover, the molecular weights of trypsin enzymes extracted from yellowfin (T. albacares) and bigeye tuna (T. obesus) were 29 kDa.

Andean soil-derived lignocellulolytic bacterial consortium as a source of novel taxa and putative plastic-active enzymes

An easy and straightforward way to engineer microbial environmental communities is by setting up liquid enrichment cultures containing a specific substrate as the sole source of carbon. Here, we analyzed twenty single-contig high-quality metagenome-assembled genomes (MAGs) retrieved from a microbial consortium (T6) that was selected by the dilution-to-stimulation approach using Andean soil as inoculum and lignocellulose as a selection pressure. Based on genomic metrics (e.g., average nucleotide and amino acid identities) and phylogenomic analyses, 15 out of 20 MAGs were found to represent novel bacterial species, with one of those (MAG_26) belonging to a novel genus closely related to Caenibius spp. (Sphingomonadaceae). Following the rules and requirements of the SeqCode, we propose the name Andeanibacterium colombiense gen. nov., sp. nov. for this taxon. A subsequent functional annotation of all MAGs revealed that MAG_7 (Pseudobacter hemicellulosilyticus sp. nov.) contains 20, 19 and 16 predicted genes from carbohydrate-active enzymes families GH43, GH2 and GH92, respectively. Its lignocellulolytic gene profile resembles that of MAG_2 (the most abundant member) and MAG_3858, both of which belong to the Sphingobacteriaceae family. Using a database that contains experimentally verified plastic-active enzymes (PAZymes), twenty-seven putative bacterial polyethylene terephthalate (PET)-active enzymes (i.e., alpha/beta-fold hydrolases) were detected in all MAGs. A maximum of five putative PETases were found in MAG_3858, and two PETases were found to be encoded by A. colombiense. In conclusion, we demonstrate that lignocellulose-enriched liquid cultures coupled with genome-resolved metagenomics are suitable approaches to unveil the hidden bacterial diversity and its polymer-degrading potential in Andean soil ecosystems.

Isolation, Production and Characterization of Pectinase Enzyme from Fungal Sources: A Review

Pectin, a plant cell wall polysaccharide, is broken down by the de-esterification, hydrolysis and trans-elimination procedure using the pectinase enzyme. They are also known as pectic enzymes and include the commercial pectinases pectolyase, polygalacturonase, and pectozyme, which have all undergone extensive research. This study aimed to know the procedure of isolation, screening and characterization of pectinase-producing fungi from different food sources and their application in juice extraction, wine clarification, coffee extraction, tea, vegetable oil extraction, cocoa, etc. Pectinase enzymes are obtained naturally by a variety of insects, fungi, yeasts, plants, bacteria, and microorganisms; moreover, animal or human cells cannot make pectinase enzymes. The present review discusses the production, classification, application and microbial sources of the pectinase enzyme.

Cow's milk as an inflammatory factor, an exploratory study on the social network Instagram

Introduction: Cow's milk is a source of nutrients, including water, protein, carbohydrates, fat, vitamins, minerals and enzymes. However, it has been the target of criticism that raises doubts about its impact on human health, with cow's milk consumption being associated with inflammatory processes. Objective: To map and analyze the information about cow's milk as a pro-inflammatory factor disseminated by health professionals on the social networking platform Instagram®. Method: An exploratory study was carried out between 03/07/2023 and 03/11/2023 on the Instagram® social network. Brazilian health profiles were selected, such as doctors, nutritionists, physiotherapists, psychologists and nurses, regardless of gender, who had at least one image or video publication related to milk as an inflammatory factor. The publications were analyzed in terms of communicative practice, interaction with users, content and the quality of the information disseminated. Results: Publications in video format showed higher engagement when compared to those in image format. Among the opinions on cow's milk, 9 profiles suggested an association with inflammation and a negative impact on health, while 11 profiles supported its consumption. 60% of the posts analyzed had no scientific articles to back up their views. Conclusion: The social network Instagram® has proved to be an important strategy for disseminating health-related content and citizen participation. However, it is necessary to consider the limits and challenges of its use, especially the spread of unscientific information.

Genome-wide analysis of lipolytic enzymes and characterization of a high-tolerant carboxylesterase from Sorangium cellulosum.

Microorganisms are important sources of lipolytic enzymes with characteristics for wide promising usages in the specific industrial biotechnology. The cellulolytic myxobacterium Sorangium cellulosum is rich of lipolytic enzymes in the genome, but little has been investigated. Here, we discerned 406 potential lipolytic enzymes in 13 sequenced S. cellulosum genomes. These lipolytic enzymes belonged to 12 families, and most are novel with low identities (14-37%) to those reported. We characterized a new carboxylesterase, LipB, from the alkaline-adaptive So0157-2. This enzyme, belonging to family VIII, hydrolyzed glyceryl tributyrate and p-nitrophenyl esters with short chain fatty acids (≤C12), and exhibited the highest activity against p-nitrophenyl butyrate. It retained over 50% of the activities in a broad temperature range (from 20°C to 60°C), alkaline conditions (pH 8.0-9.5), and the enzymatic activity was stable with methanol, ethanol and isopropanol, and stimulated significantly in the presence of 5 mM Ni2+. LipB also exhibited β-lactamase activity on nitrocefin, but not ampicillin, cefotaxime and imipenem. The bioinformatic analysis and specific enzymatic characteristics indicate that S. cellulosum is a promising resource to explore lipolytic enzymes for industrial adaptations.

Enzymatic synthesis of prebiotic galactooligosaccharides from galactose derived from gum arabic.

A novel enzymatic process was established for galactooligosaccharides (GOS) synthesis by using plant-derived galactose as substrate, without producing any byproducts. The galactose was prepared from the acid hydrolysate of gum arabic. The yeast Kluyveromyces lactis producing β-galactosidase capable of catalyzing GOS synthesis from galactose was screened out. The synthesis conditions using the yeast cells as enzyme source were optimized by both single-factor experiment and response surface methodology, with the highest GOS yield reached 45%. The composition of reaction mixture contained only GOS and unreacted galactose, which could be easily separated by the cation exchange resin column. The structures of major GOS products were identified as Gal-β-D-(1→6)-Gal, Gal-β-D-(1→3)-Gal, and Gal-β-D-(1→6)-Gal-β-D-(1→6)-Gal by MS and NMR spectra. Moreover, the β-galactosidase-containing cells can be recycled for at least 30 batches of GOS synthesis at 35°C, with the enzyme activity remaining above 60%.