Enzymatic properties and degradation characterization of a bis(2-hydroxyethyl) terephthalate hydrolase from Saccharothrix sp / 生物工程学报

The discovery of new enzymes for poly(ethylene terephthalate) (PET) degradation has been a hot topic of research globally. Bis-(2-hydroxyethyl) terephthalate (BHET) is an intermediate compound in the degradation of PET and competes with PET for the substrate binding site of the PET-degrading enzyme, thereby inhibiting further degradation of PET. Discovery of new BHET degradation enzymes may contribute to improving the degradation efficiency of PET. In this paper, we discovered a hydrolase gene sle (ID: CP064192.1, 5085270-5086049) from Saccharothrix luteola, which can hydrolyze BHET into mono-(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA). BHET hydrolase (Sle) was heterologously expressed in Escherichia coli using a recombinant plasmid, and the highest protein expression was achieved at a final concentration of 0.4 mmol/L of isopropyl-β-d-thiogalactoside (IPTG), an induction duration of 12 h and an induction temperature of 20 ℃. The recombinant Sle was purified by nickel affinity chromatography, anion exchange chromatography, and gel filtration chromatography, and its enzymatic properties were also characterized. The optimum temperature and pH of Sle were 35 ℃ and 8.0, and more than 80% of the enzyme activity could be maintained in the range of 25-35 ℃ and pH 7.0-9.0 and Co2+ could improve the enzyme activity. Sle belongs to the dienelactone hydrolase (DLH) superfamily and possesses the typical catalytic triad of the family, and the predicted catalytic sites are S129, D175, and H207. Finally, the enzyme was identified as a BHET degrading enzyme by high performance liquid chromatography (HPLC). This study provides a new enzyme resource for the efficient enzymatic degradation of PET plastics.

Expression, purification and characterization of a novel bis (hydroxyethyl) terephthalate hydrolase from Hydrogenobacter thermophilus / 生物工程学报

PET (polyethylene terephthalate) is one of the most important petrochemicals that is widely used in mineral water bottles, food and beverage packaging and textile industry. Because of its stability under environmental conditions, the massive amount of PET wastes caused serious environmental pollution. The use of enzymes to depolymerize PET wastes and upcycling is one of the important directions for plastics pollution control, among which the key is the depolymerization efficiency of PET by PET hydrolase. BHET (bis(hydroxyethyl) terephthalate) is the main intermediate of PET hydrolysis, its accumulation can hinder the degradation efficiency of PET hydrolase significantly, and the synergistic use of PET hydrolase and BHET hydrolase can improve the PET hydrolysis efficiency. In this study, a dienolactone hydrolase from Hydrogenobacter thermophilus which can degrade BHET (HtBHETase) was identified. After heterologous expression in Escherichia coli and purification, the enzymatic properties of HtBHETase were studied. HtBHETase shows higher catalytic activity towards esters with short carbon chains such as p-nitrophenol acetate. The optimal pH and temperature of the reaction with BHET were 5.0 and 55 ℃, respectively. HtBHETase exhibited excellent thermostability, and retained over 80% residual activity after treatment at 80 ℃ for 1 hour. These results indicate that HtBHETase has potential in biological PET depolymerization, which may facilitate the enzymatic degradation of PET.

Advances in poly(ethylene terephthalate) hydrolases / 生物工程学报

Plastics have brought invaluable convenience to human life since it was firstly synthesized in the last century. However, the stable polymer structure of plastics led to the continuous accumulation of plastic wastes, which poses serious threats to the ecological environment and human health. Poly(ethylene terephthalate) (PET) is the most widely produced polyester plastics. Recent researches on PET hydrolases have shown great potential of enzymatic degradation and recycling of plastics. Meanwhile, the biodegradation pathway of PET has become a reference model for the biodegradation of other plastics. This review summarizes the sources of PET hydrolases and their degradation capacity, degradation mechanism of PET by the most representative PET hydrolase-IsPETase, and recently reported highly efficient degrading enzymes through enzyme engineering. The advances of PET hydrolases may facilitate the research on the degradation mechanism of PET and further exploration and engineering of efficient PET degradation enzymes.

Computation-aided design of the flexible region of zearalenone hydrolase improves its thermal stability / 生物工程学报

The zearalenone hydrolase (ZHD101) derived from Clonostachys rosea can effectively degrade the mycotoxin zearalenone (ZEN) present in grain by-products and feed. However, the low thermal stability of ZHD101 hampers its applications. High throughput screening of variants using spectrophotometer is challenging because the reaction of hydrolyzing ZEN does not change absorbance. In this study, we used ZHD101 as a model enzyme to perform computation-aided design followed by experimental verification. By comparing the molecular dynamics simulation trajectories of ZHD101 at different temperatures, 32 flexible sites were selected. 608 saturated mutations were introduced into the 32 flexible sites virtually, from which 12 virtual mutants were screened according to the position specific score and enzyme conformation free energy calculation. Three of the mutants N156F, S194T and T259F showed an increase in thermal melting temperature (ΔTm>4 °C), and their enzyme activities were similar to or even higher than that of the wild type (relative enzyme activity 95.8%, 131.6% and 169.0%, respectively). Molecular dynamics simulation analysis showed that the possible mechanisms leading to the improved thermal stability were NH-π force, salt bridge rearrangement, and hole filling on the molecular surface. The three mutants were combined iteratively, and the combination of N156F/S194T showed the highest thermal stability (ΔTm=6.7 °C). This work demonstrated the feasibility of engineering the flexible region to improve enzyme performance by combining virtual computational mutations with experimental verification.

Expression and characterization of a novel halohydrin dehalogenase from Rhodospirillaceae bacterium / 生物工程学报

As a class of multifunctional biocatalysts, halohydrin dehalogenases are of great interest for the synthesis of chiral β-substituted alcohols and epoxides. There are less than 40 halohydrin dehalogenases with relatively clear catalytic functions, and most of them do not meet the requirements of scientific research and practical applications. Therefore, it is of great significance to excavate and identify more halohydrin dehalogenases. In the present study, a putative halohydrin dehalogenase (HHDH-Ra) from Rhodospirillaceae bacterium was expressed and its enzymatic properties were investigated. The HHDH-Ra gene was cloned into the expression host Escherichia coli BL21(DE3) and the target protein was shown to be soluble. Substrate specificity studies showed that HHDH-Ra possesses excellent specificity for 1,3-dichloro-2-propanol (1,3-DCP) and ethyl-4-chloro-3-hydroxybutyrate (CHBE). The optimum pH and temperature for HHDH-Ra with 1,3-DCP as the reaction substrate were 8.0 and 30 °C, respectively. HHDH-Ra was stable at pH 6.0-8.0 and maintained about 70% of its original activity after 100 h of treatment. The thermal stability results revealed that HHDH-Ra has a half-life of 60 h at 30 °C and 40 °C. When the temperature is increased to 50 °C, the enzyme still has a half-life of 20 h, which is much higher than that of the reported enzymes. To sum up, the novel halohydrin dehalogenase from Rhodospirillaceae bacterium possesses good temperature and pH stability as well as catalytic activity, and shows the potential to be used in the synthesis of chemical and pharmaceutical intermediates.

Cometabolic biodegradation of quizalofop-p-ethyl by Methylobacterium populi YC-XJ1 and identification of QPEH1 esterase

BACKGROUND: Quizalofop-p-ethyl (QPE), a unitary R configuration aromatic oxyphenoxypropionic acid ester (AOPP) herbicide, was widely used and had led to detrimental environmental effects. For finding the QPEdegrading bacteria and promoting the biodegradation of QPE, a series of studies were carried out. RESULTS: A QPE-degrading bacterial strain YC-XJ1 was isolated from desert soil and identified as Methylobacterium populi, which could degrade QPE with methanol by cometabolism. Ninety-seven percent of QPE (50 mg/L) could be degraded within 72 h under optimum biodegradation condition of 35°C and pH 8.0. The maximum degradation rate of QPE was 1.4 mg/L/h, and the strain YC-XJ1 exhibited some certain salinity tolerance. Two novel metabolites, 2-hydroxy-6-chloroquinoxaline and quinoxaline, were found by high-performance liquid chromatography/mass spectroscopy analysis. The metabolic pathway of QPE was predicted. The catalytic efficiency of strain YC-XJ1 toward different AOPPs herbicides in descending order was as follows: haloxyfop-pmethyl ≈ diclofop-methyl ≈ fluazifop-p-butyl N clodinafop-propargyl N cyhalofop-butyl N quizalofop-p-ethyl N fenoxaprop-p-ethyl N propaquizafop N quizalofop-p-tefuryl. The genome of strain YC-XJ1 was sequenced using a combination of PacBio RS II and Illumina platforms. According to the annotation result, one α/ß hydrolase gene was selected and named qpeh1, for which QPE-degrading function has obtained validation. Based on the phylogenetic analysis and multiple sequence alignment with other QPE-degrading esterases reported previously, the QPEH1 was clustered with esterase family V. CONCLUSION: M. populi YC-XJ1 could degrade QPE with a novel pathway, and the qpeh1 gene was identified as one of QPE-degrading esterase gene.

Endophytic fungi from medicinal plant Bauhinia forficata: Diversity and biotechnological potential

Bauhinia forficata is native to South America and used with relative success in the folk medicine in Brazil. The diversity, antibacterial activity, and extracellular hydrolytic enzymes of endophytic fungi associated with this plant were studied. Plant samples, which included leaves, sepals, stems, and seeds, were used. Ninety-five endophytic fungal were isolated (18 from leaves, 22 from sepals, 46 from stems, and nine from seeds), comprising 28 species. The most frequently isolated species were Acremonium curvulum (9.5%), Aspergillus ochraceus (7.37%), Gibberella fujikuroi (10.53%), Myrothecium verrucaria (10.53%) and Trichoderma piluliferum (7.37%). Diversity and species richness were higher in stem tissues, and Sorensen’s index of similarity between the tissues was low. Eleven fungi showed antibacterial activity. Aspergillus ochraceus, Gibberella baccata, Penicillium commune, and P. glabrum were those with the greatest antibacterial activity against Staphylococcus aureus and/or Streptococcus pyogenes. Thirteen species showed proteolytic activity, particularly Phoma putaminum. Fourteen species were cellulase positive, particularly the Penicillium species and Myrmecridium schulzeri. All isolates tested were xylanase positive and 10 showed lipolytic activity, especially Penicillium glabrum. It is clear that the endophytic fungi from B. forficata have potential for the production of bioactive compounds and may be a source of new therapeutic agents for the effective treatment of diseases in humans, other animals, and plants. To our knowledge, this is the first study of endophytic fungi from different tissues of B. forficata and their biotechnological potential.

Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels

Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.

Alkali production in the mouth and its relationship with certain patient's characteristics

Objectives To assess the relationships among alkali production, diet, oral health behaviors, and oral hygiene. Methods Data from 52 subjects including demographics, diet, and oral hygiene scores were analyzed against the level of arginine and urea enzymes in plaque and saliva samples. An oral habit survey was completed that included: use of tobacco (TB), alcohol (AH), sugary drinks (SD), and diet. Alkali production through arginine deiminase (ADS) and urease activities were measured in smooth-surface supragingival dental plaque and un stimulated saliva samples from all subjects. ADS and urease activities were measured by quantification of the ammonia generated from the incubation of plaque or saliva samples. Spearman correlations were used to compute all associations. Results Participants in the lowest SES (Socio-economic status) group had the habit of consuming sugary drinks the most and had the highest rate of tobacco use. Males consumed significantly more alcohol than females. No significant relationship was found between age or gender and alkali production. Higher rates of sugary drink consumption and tobacco use were significantly related to lower alkali production. Conclusion The study showed a relationship between alkali production and oral hygiene, diet, and certain oral health behaviors. Poor oral hygiene was significantly associated with age, lower SES, tobacco use, and alcohol, and sugary drinks consumption. Clinical relevance Certain oral health behaviors have an impact on oral hygiene and on alkali production; it is important to address these factors with patients as a strategy for caries control. .

The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.

Trypanosoma cruzi é altamente sensível ao estresse oxidativo causado por espécies reativas do oxigênio. Tripanotiona, o principal protetor do parasita contra o estresse oxidativo, é mantido reduzido pela tripanotiona redutase, pela presença deNADPH; a principal fonte da coenzima reduzida parece ser a via da pentose fosfato. As sete enzimas dessa via estão presentes nos quatro principais estágios do ciclo biológico do parasita; nós clonamos e expressamos as enzimas em Escherichia coli como proteínas ativas. Glucose 6-fosfato desidrogenase, que controla o fluxo da glucose da via em resposta à relação NADP/NADPH, é codificada por um número de genes por genoma haplóide e é induzida até 46-vezes por peróxido de hidrogênio em trypomastigotas metacíclicos. Os genes que codificam 6-fosfogluconolactonase, 6-fosfogluconato desidrogenase, transaldolase e transcetolase estão presentes no clone CL Brener como cópia única por genoma haplóide. 6-fosfogluconato desidrogenase é muito instável, mas foi estabilizada introduzindo duas pontes salinas por mutagênese sítio-dirigida. A Ribose-5-fosfato isomerase pertence ao Tipo B; genes que codificam enzimas Tipo A, presentes em mamíferos estão ausentes. A Ribulose-5-fosfato epimerase é codificada por dois genes. As enzimas da via têm um componente citosólico principal, embora várias delas tenham uma localização glicosomal secundária e também, localizações em menor número em outras organelas.

Hydrolytic enzymes in Paracoccidioides brasiliensis--ecological aspects

Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis. The yeast form of this pathogen is found in the animal host whereas the mycelial form is recovered from living and non-living organic material. The sole carbon source available in these habitats is represented by polysaccharides from the plant cell wall. Hydrolytic enzymes are necessary to convert these polymers into simple sugars for fungal metabolism. We report on the presence of ortholog genes of hydrolytic enzymes identified in the P. brasiliensis transcriptome and on hydrolytic activities in supernatants of induced P. brasiliensis cultures of mycelium and yeast cells. Enzymatic assays have shown cellulase and xylanase activities, both being higher in mycelium than in the yeast form. Amylase and chitinase activities were detected only in mycelium. Data so far reinforce the idea that mycelial P. brasiliensis is a saprobe.

Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification

Chromobacterium violaceum is a Gram-negative bacterium found in a wide variety of tropical and subtropical ecosystems. The complete genome sequence of C. violaceum ATCC 12472 is now available, and it has considerable biotechnological potential for various applications, such as environmental detoxification, as well as medical and agricultural use. We examined the biotechnological potential of C. violaceum for environmental detoxification. Three operons, comprising the ars operon, involved in arsenic resistance, the cyn operon, involved in cyanate detoxification, and the hcn operon, encoding a cyanase, responsible for biogenic production of cyanide, as well as an open reading frame, encoding an acid dehalogenase, were analyzed in detail. Probable catalytic mechanisms for the enzymes were determined, based on amino acid sequence comparisons and on published structural information for these types of proteins

Effect of ascorbic acid on stimulatory status of activated mouse peritoneal phagocytes.

Mouse peritoneal macrophages (MPM) when elicited by the antioxidant ascorbic acid have been found to be significantly stimulatory, exhibiting marked alteration at the cellular and enzyme levels. Alterations recorded were as follows--cellular yield per mouse, their protein content, lysosomal acid hydrolase levels and capability to phagocyte, all were significantly enhanced. The new stimulant was observed to produce no synergistic action on MPM when thioglycollate, BCG or endotoxin along with the same stimulated the latter. Levels of antioxidants like ascorbic acid and glutathione were found to be enhanced in elicited macrophages whereas superoxide dismutase levels varied when the three above stimulators were administered. However, the ascorbic acid elicited cells showed an increase in glutathione levels and a decrease in SOD levels but no change in total intracellular ascorbic acid levels. Further, though ascorbic acid interaction enhanced the phagocytic capability of MPM as compared to resident cells, no significant boosting of phagocytic process could be observed when each of three stimulators coupled with ascorbic acid was used for macrophage elicitation.

Alterations in kidney enzyme pattern in acute hypervitaminosis A

The relation of excessive doses of vitamin A with various kidney pathologies is well known however, information concerning the relation of kidney enzyme activity with acute hypervitaminosis A is rather scarce. In this study we describe the kidney enzymatic alterations observed in rats that received daily intramuscular injectiosn of 10.000, 30.000, 50.000 and 100.000IU of vitamin A palmitate (VA) during seven days (TREATED GROUPS). A comparison is made with the enzyme activity in healthy rats pairfed and treated with sodium palmitte by intramuscular injection (CONTROL GROUP). The treated rats showed a proportional increase (<0.05) in activity of acid maltase, transaminases or aminotransferase (GOT and GPT), alkaline phosphatase (ALP) and acid protease with all doses of VA administered, Amylase, lipase and arginase tend to decrease (p<0.05) in activity only with doses of 50.000 and 100.000 I.U. of VA. Several factors are responsible for these findings, such as kidney necrosis due to release of lysosomal acid hydrolases produced by hypervitaminosis A.

Distribution of digestive enzymes in gastrointestinal tract of young rhesus monkeys.

The distribution of different mucosal digestive enzymes along the gastrointestinal tract of young rhesus monkeys were studied by biochemical analysis of homogenized specimen from 13 different segments (i.e. stomach-fundus, body, antrum; small intestine-D1, D2, proximal, middle, distal; caecum; colon-ascending, transverse, descending and rectum), obtained from 6 monkeys of approximate age 8-12 months. The activity of all the enzymes was lowest in gastric mucosa rising through duodenum to peak in small intestine and declining towards the large bowel. There was no significant difference in levels of any of the enzymes between different parts of stomach or large bowel. This study suggests that like humans in rhesus monkeys maximum activity of digestive enzymes is present in small intestine and much less in gastric and large bowel mucosa.

Hydrolytic enzymes of rhesus placenta during Plasmodium cynomolgi infection: ultrastructural and biochemical studies.

Placenta in monkey demonstrated altered pathophysiology after P cynomolgi infection. The electronmicroscopic observations showed slight complete focal necrosis of the placental tissue, besides alterations in total protein, phosphatases and proteinases. These changes in cellular constituents of placenta during malaria infection may be responsible for malfunctioning of the organ and in turn, abnormal development of foetus.

Effect of depot medroxyprogesterone acetate and testosterone ananthate on the testis of albino rats: ultrastructural and biochemical studies.

Male albino rats were treated with depot medroxyprogesterone acetate (1 mg/animal/day) + testosterone ananthate (100 micrograms/100 g body weight/day) for 30 and 60 days. After 30 days of treatment, all the testicular enzymes like beta-glucuronidase, hyaluronidase, sorbitol dehydrogenase, lactate dehydrogenase, acid and alkaline phosphatase, registered non-significant decrease in their values. Fifty percent of the treated animals achieved sterility after 30 days of treatment. After 60 days of treatment the testis showed degenerative changes in Golgi phase and late spermatids. Changes in the Golgi phase spermatids were related with degeneration of the nuclear membrane. Changes in the late phase spermatids included mitochondrial hypertrophy of the midpieces, membrane lysis, absence of cristae and degeneration of annulus leading to detachment of tail. Cytoplasm of luminal area displayed hypertrophied mitochondria devoid of cristae, prominent appearance of Golgi bodies, intense lysosomal activity and ample vacuolation. Tail fragments of degenerated spermatids prevailed in luminal cytoplasm. Except for beta-glucuronidase which registered a significant decrease, levels of all the other testicular enzymes, viz. hyaluronidase, lactate dehydrogenase, sorbitol dehydrogenase, acid phosphatase and alkaline phosphatase were within their control limits. The ultrastructural and biochemical changes are correlated.