High Hydrolytic Activity Research Articles

Lipase immobilization on alumina membranes using a traditional and a nature-inspired method for active degradation of oil fouling

This study compared the traditional method of silanization using 3-aminopropyltriethoxysilane (APTES) with the innovative technique of polydopamine coating (PDA) for lipase immobilization on α-alumina membranes. Three lipases were tested, and Eversa Transform 2.0 (ET2) was chosen to be immobilized in the membrane due to its high hydrolytic activity towards soybean oil. Polydopamine deposition with and without glutaraldehyde activation presented the highest membrane hydrolytic activities (1553 ± 209 and 1845 ± 283 U·m−2, respectively). The membrane without activation maintained 41.1 ± 1.7 % of the free enzyme activity while the one with glutaraldehyde preserved only 23.9 ± 1.0 %. When tested in oil–water separation using a soybean oil emulsion, the membranes with ET2 immobilized by PDA showed water permeance 2-fold higher than the nonactive membranes (300 ± 12 and 156 ± 4 L·h−1·m−2·bar, respectively), showing good fouling-degrading capacity. They also presented self-cleaning properties when activated through the use of proper pH and temperature. The water permeance recovery for the control membrane was 34% while the membranes with immobilized ET2 by PDA and APTES showed recoveries of 69% and 49%, respectively. Permeance recovery for ET2 immobilized by silanization was similar to the PDA-coated membrane with no enzyme (45%).

Screening and characterization of novel lipase producing Bacillus species from agricultural soil with high hydrolytic activity against PBAT poly (butylene adipate co terephthalate) co-polyesters

Screening and characterization of novel lipase producing Bacillus species from agricultural soil with high hydrolytic activity against PBAT poly (butylene adipate co terephthalate) co-polyesters

Influence of Textural Properties of Divinylbenzene Copolymers on the Immobilization of Lipase B from Candida antarctica

Previous studies have investigated the preparation of heterogeneous biocatalysts based on the immobilization of lipases on distinct types of supports. However, few works have investigated the influence of the textural properties of these supports on the immobilization parameters. Thus, the present article reports the preparation of copolymers based on divinylbenzene by aqueous suspension polymerizations, using different amounts of porogenic agents to prepare particles with distinct textural properties. The particles were used for immobilization of lipase from Candida antarctica fraction B and the performance of the biocatalysts was evaluated in hydrolysis reactions, using p-nitrophenyl laurate as substrate. The use of Sty/DVB particles resulted in higher immobilization yields (89.5% and 99.2%) and higher hydrolytic activities, when compared to the Sty/VBC/DVB particles. Particularly, the increase of the pore diameters of the particles resulted in higher immobilization yields. Also, the hydrolytic activities depended simultaneously on the average pore diameter, porosity, and specific area (the most influential variable) of the supports. Thus, it was observed that the distinct morphological features of the polymer support can exert significant and conflicting effects on the final biocatalyst performance, since the specific surface area is normally expected to decrease with the increase of the average pore size.

Yeast Multi-Enzymatic Systems for Improving Colour Extraction, Technological Parameters and Antioxidant Activity of Wine.

Wine yeasts are a heterogeneous microbial group with high enzymatic potential that makes them a useful tool in winemaking. With a better understanding of their oenological properties, selection procedures can be optimised to obtain more efficient strains. The present study aims to isolate and select yeasts from wine grape surface by studying their production of enzymes that hydrolyse plant cell wall polymers and by linking them to different technological parameters and antioxidant activity of wines. Yeasts that are able to produce carbohydrolases and related enzymes of oenological importance were firstly selected on plates and subsequently identified. Then, a secondary selection of yeasts was carried out according to technological effects of their extracellular enzyme extracts on short macerations. In this way, the colour extraction, total polyphenol content, clarification, filterability and antioxidant activity were studied. This approach makes it possible to correlate the microorganism capacity to produce cell wall-depolymerizing enzymes with their technological effects. From 366 isolates, 96 strains (26.2%) showed at least one of the polysaccharidase activities and 55 strains (57.3%) of them exhibited activities of multiple enzymes that degrade plant cell wall polymers. Sixteen strains were selected and identified as Aureobasidium, Candida, Debaryomyces, Hanseniaspora, Metschnikowia, Pichia, Saccharomyces and Torulaspora. Pectinolytic enzymes had the highest hydrolytic activity. Aureobasidium pullulans had a broader enzyme blend and higher activity, dominated by pectinases and followed by xylanases and cellulases. Moreover, the Torulaspora delbrueckii m7-2 strain produced high amounts of polysaccharidase and this was strain-dependent. Strains that produced enzyme extracts with a wide range of activities that were also the highest, also had the best chromatic and technological properties. Cluster analysis confirmed that A. pullulans R-22, m11-2, m86-1 and m86-2 and T. delbrueckii m7-2 could be correlated with a better effect on filterability, clarification and extraction of bioactive compounds, encouraging future studies regarding their application in winemaking. The study of yeast multi-enzymatic systems impacting the grape maceration process enables a proper selection criterion for wine yeasts to improve colour extraction, technological parameters and antioxidant activity of Malbec wine. This work shows that A. pullulans and T. delbruekii have a high enzymatic potential for oenological purposes.

The dynamics of indigenous epiphytic bacterial and fungal communities of barley grains through the commercial malting process in Western Canada

Microbial activity is present at every step of the malting process. It is, therefore, critical to manage the grain-associated microbial communities for the production of high-quality malts. This study characterized barley and malt epiphytic microbiota by metabarcoding the internal transcribed spacer (ITS) 2 region and the 16S rRNA gene V1–V4 metabarcodes, respectively. We elucidated the changes in the diversity and the compositional and functional changes of the grain-associated microbiota and inferred the impact of such changes on malting efficiency and premature yeast flocculation (PYF) of the commercial malt end product. Through the malting process, the fungal diversity decreased while bacterial community diversity increased. Lactic acid bacteria (LAB) and some mycotoxin-producing fungi (e.g. Fusarium spp.) were found to be significantly enriched in malts. Most potential fungal pathogens, however, did not change in abundance through the malting process. Fungi (e.g. Aureobasidium, Candida) and bacteria (e.g. LAB, Arthrobacter, Brachybacterium) with the potential to generate organic acids or exhibit high hydrolytic enzymatic activity for degrading the endosperm cell walls and storage proteins were detected in greater abundance in kilned malt, suggesting their contribution to malting efficiency. Bacterial and fungal operational taxonomic units (OTUs) associated with PYF-positive malt were mainly identified as Aureobasidium, Candida, and Leuconostoc, while Pleosporaceae, Steptococcus, and Leucobacter were associated with PYF-negative malt. The ecological networks of the field and steeped barley samples were found to be larger and denser, while that of the malt microbiome was smaller and less connected. A decrease in the proportion of negative interactions through the malting process suggested that malting destabilized the microbial networks. In summary, this study profiled the microbiota of commercial malting barley and malt samples in western Canada; the findings expanded our knowledge in the microbiology of malting while providing potential insights regarding the management of microbial-associated problems, such as PYF, in commercial malting.

Screening and efficient production of engineered lipase B from Candida Antarctica for eco-friendly recycling of waste polycaprolactone

Waste plastics cause serious environmental pollution. Plastic recycling is crucial to combat this global problem. Enzymatic plastic depolymerization is a promising approach for plastic recycling owing to its environmentally friendly nature. Polycaprolactone (PCL), a widely used biodegradable plastic, has been constantly dumped into land and sea. In this study, we screened and produced a PCL-depolymerizing enzyme with high catalytic activity for PCL recycling. Lipase B from Candida Antarctica (CalB) is capable of depolymerizing PCL. Among the three CalB variants with high hydrolytic or synthetic activity reported previously by our group, we found that CalB-658 (V221K and A281I mutations) exhibited higher PCL-depolymerizing activity than the wild-type CalB using a PCL emulsion agar plate. We produced this CalB variant using Pichia pastoris as the recombinant secretory expression host. During the fed-batch fermentation of the recombinant P. pastoris using a 5 L bioreactor, CalB-658 was produced extracellularly at a concentration of 844.3 mg/L with a lipase activity of 13,753.3 U/L. The PCL-depolymerizing activity of CalB-658 was compared with that of the wild-type CalB. During the depolymerization of 60 mg PCL films for 32 h, the wild-type CalB and CalB-658 degraded 54.2% and 97.3% of the PCL film, respectively. Moreover, CalB-658 produced 5.0-fold more 6‑hydroxy hexanoic acid (65 g/L), the monomer of PCL, compared to the wild-type CalB (13 g/L), during a 40 h depolymerization reaction of PCL powder, indicating that CalB-658 exhibited higher PCL-depolymerizing activity compared to the wild-type CalB. This result was confirmed by scanning electron microscopy. Therefore, we have successfully developed a screening and extracellular production system for a PCL-depolymerizing enzyme with high catalytic activity that can produce large quantities of the enzyme with a relatively simple purification step.

Site-directed mutation of β-galactosidase from Streptococcus thermophilus for galactooligosaccharide-enriched yogurt making

β-Galactosidase is one of the most important enzymes used in dairy processing. It converts lactose into glucose and galactose, and also catalyzes galactose to form galactooligosaccharides (GOS), so-called prebiotics. However, most of the β-galactosidases from the starter cultures have low transgalactosylation activities, the process that results in galactose accumulation in yogurt. Here, a site-directed mutation strategy was attempted, to genetically modify β-galactosidase from Streptococcus thermophilus. Out of 28 Strep. thermophilus strains, a β-galactosidase gene named bgaQ, encoded for high β-galactosidase hydrolysis activity (BgaQ), was cloned from the strain Strep. thermophilus SDMCC050237. It was 3,081 bp in size, with 1,027 deduced amino acid residuals, which belonged to the GH2 family. After replacing the Tyr801 and Pro802 around the active sites of BgaQ with His801 and Gly802, the GOS synthesis of the generated mutant protein BgaQ-8012 increased from 20.5% to 26.7% at 5% lactose, and no hydrolysis activity altered obviously. Subsequently, the purified BgaQ or BgaQ-8012 was added to sterilized milk inoculated with 2 starters from Strep. thermophilus SDMCC050237 and Lactobacillus delbrueckii ssp. bulgaricus ATCC11842. The GOS yields with added BgaQ or BgaQ-8012 increased to 5.8 and 8.3 g/L, respectively, compared with a yield of 3.7 g/L without enzymes added. Meanwhile, the addition of the BgaQ or BgaQ-8012 reduced the lactose content by 49.3% and 54.4% in the fermented yogurt and shortened the curd time. Therefore, this study provided a site-directed mutation strategy for improvement of the transgalactosylation activity of β-galactosidase from Strep. thermophilus for GOS-enriched yogurt making.

The precursors of C8 alcohols from soybean: Purification, distribution and hydrolysis properties of glycosidically bound volatiles.

C8 alcohols, such as 1-octen-3-ol and 3-octanol, have been variously described as mushroom, musty or earthy and are common volatile compounds of soymilk. C8 alcohols were the major volatiles formed during soybean soaking, and about 95% of the total C8 alcohols were originated from the enzymatic hydrolysis of glycosides of alcohols in soybean hypocotyls. In addition to 1-octen-3-yl β-primeveroside, a new glycoside was purified from the methanol extracts of soybean hypocotyls and were confirmed as 3-octanyl β-primeverosides by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The crude glycosidase extracted from soybean hypocotyls showed much higher hydrolysis activities toward C8 alcohol β-primeverosides than that of almond β-glycosidase. Besides, increasing nonenzymatic hydrolysis was observed when the purified β-primeverosides were heated at pH values lower than 6. The nonenzymatic hydrolysis activation energies (Ea ) were 30.41kJ/mol and 38.08kJ/mol for 1-octen-3-yl β-primeveroside and 3-octanyl β-primeveroside, respectively. In addition to C8 alcohols, nonenzymatic hydrolysis also resulted in the formation of 2,3-octanedione, 3-hydroxy-2-octanone and 3-octanone. The nonenzymatic hydrolysis of β-primeverosides in the soy milk was also studied under the same conditions as those in the model systems. The degree of hydrolysis was similar but the molar yields of the volatile components were lower than those obtained in the model system. PRACTICAL APPLICATION: The results indicated that a different strategy or technology is required to suppress the formation of C8 alcohols than that applied to off-flavor compounds formed de novo during processing of soybean seeds.

Influence of enzymative catalysis on technological parameters of apple juice production

The article provides data on the investigation of the effect of enzymatic catalysis on the technological parameters of apple juice production. Apples are distinguished by the presence of a heterogeneous high-molecular complex of biopolymers, which complicates their processing: it prevents juice output, hinders its clarification, filterability of wine materials and negatively affects the colloidal stability of wines. Enzymatic catalysis based on the action of microbial preparations, contributes in many respects to the successful solution of the problems of intensifying technological processes of fruit and berry raw materials processing and improving the quality of obtained products. Enzyme preparations of complex action, causing deep hydrolysis of high-molecular components of the peel and pulp of fruits, provide a more complete extraction of the liquid fraction, better clarification and filterability of juices. The foothill zone of Adygea has special soil and climatic conditions that affect the formation of physical and chemical characteristics of raw materials. Considering that apple tree occupies the first place in terms of planting area among fruit crops in the Republic of Adygea, an urgent task is to study the effect of enzymatic catalysis on technological properties of juices obtained during the processing of apples grown in these soil and climatic conditions. Enzyme preparations of complex action, recommended for the processing of fruit juices and wine materials, made in France and Germany, have been selected as objects of the research. The results of the studies on the use of enzyme preparations in the processing of apple juices, taking into account their influence on the juice yield and such technological parameters as the speed and quality of clarification, filterability and dynamics of alcoholic fermentation, have been presented. The effect of enzymatic catalysis on the mass concentrations of polysaccharides, starch, total nitrogen and phenolic substances has been studied. A higher hydrolytic activity of the Extrazyme enzyme preparation has been established, when added to the pulp; it contributes to the deep transformation of high-molecular substances and, as a result, to optimize the technological parameters of juice production

Preparation of a novel solid acid bearing sulfur-containing active groups and evaluation of its activity for cellulose hydrolysis

A novel metal-free solid acid was prepared by co‑carbonation followed sulfonation, using cellulose and sodium thiosulfate as raw materials. The effects of sulfur-doping on the structure and catalytic activity of the solid acid were discussed. The results reveal that S content of the solid acid was up to 23.53 wt%, in which S was present in the form of sulfoxide, sulfone, thiophene and disulfide groups in addition to sulfonic acid group. The solid acid exhibited high adsorption capacity and catalytic activity in cellulose hydrolysis compared with carbon-based solid acid, which is initially attributed to the appearance of sulfoxide and thiophene. Sulfoxide and thiophene can enhance the adsorption capacity of the solid acid through hydrogen bonding and CH-π interaction respectively, leading to improvement in hydrolytic activity of the solid acid.

Identification and molecular characterization of propionylcholinesterase, a novel pseudocholinesterase in rice

ABSTRACT Cholinesterase is consisting of acetylcholinesterase (AChE) and pseudocholinesterase in vertebrates and invertebrates. AChE gene has been identified in several plant species, while pseudocholinesterase gene has not yet been found in any plant species. In this study, we report that the AChE gene paralog encodes propionylcholinesterase (PChE), a pseudocholinesterase in rice. PChE was found to be located adjacent to AChE (Os07g0586200) on rice chromosome 7 and designated as Os07g0586100. Phylogenetic tree analysis showed a close relationship between rice AChE and PChE. PChE-overexpressing rice had higher hydrolytic activity toward propionylthiocholine than acetylthiocholine and showed extremely low activity against butyrylthiocholine. Therefore, the PChE gene product was characterized as a propionylcholinesterase, a pseudocholinesterase. The rice PChE displayed lower sensitivity to the cholinesterase inhibitor, neostigmine bromide, than electric eel, maize, and rice AChEs. The recombinant PChE functions as a 171 kDa homotetramer. PChE was expressed during the later developmental stage, and it was found be localized in the extracellular spaces of the rice leaf tissue. These results suggest that the rice plant possesses PChE, which functions in the extracellular spaces at a later developmental stage. To the best of our knowledge, this study provides the first direct evidence and molecular characterization of PChE in plants.

Ni0.25Co0.75O nanowire array supported on Cu@CuO foam, an inexpensive and durable catalyst for hydrogen generation from ammonia borane

The development of highly active and stable catalysts is desirable for hydrogen generation from ammonia borane (AB). In this study, Ni0.25Co0.75O nanowire array supported on Cu@CuO foam (Ni0.25Co0.75O/Cu@CuO) has been successfully fabricated. The Ni0.25Co0.75O/Cu@CuO catalyst exhibited high performance towards AB hydrolysis with a high hydrogen generation rate (HGR) of 4.348 Lhydrogen min−1 gcat.−1. More importantly, the activity of Ni0.25Co0.75O/Cu@CuO has only decreased slightly after twelve cycles. This work provides a simple method to fabricate an inexpensive film catalyst with high activity and good stability for ammonia borane hydrolysis.

Anaerobic hydrolysis of complex substrates in full-scale aerobic granular sludge: enzymatic activity determined in different sludge fractions

Complex substrates, like proteins, carbohydrates, and lipids, are major components of domestic wastewater, and yet their degradation in biofilm-based wastewater treatment technologies, such as aerobic granular sludge (AGS), is not well understood. Hydrolysis is considered the rate-limiting step in the bioconversion of complex substrates, and as such, it will impact the utilization of a large wastewater COD (chemical oxygen demand) fraction by the biofilms or granules. To study the hydrolysis of complex substrates within these types of biomass, this paper investigates the anaerobic activity of major hydrolytic enzymes in the different sludge fractions of a full-scale AGS reactor. Chromogenic substrates were used under fully mixed anaerobic conditions to determine lipase, protease, α-glucosidase, and β-glucosidase activities in large granules (>1 mm in diameter), small granules (0.2–1 mm), flocculent sludge (0.045–0.2 mm), and bulk liquid. Furthermore, composition and hydrolytic activity of influent wastewater samples were determined. Our results showed an overcapacity of the sludge to hydrolyze wastewater soluble and colloidal polymeric substrates. The highest specific hydrolytic activity was associated with the flocculent sludge fraction (1.5–7.5 times that of large and smaller granules), in agreement with its large available surface area. However, the biomass in the full-scale reactor consisted of 84% large granules, making the large granules account for 55–68% of the total hydrolytic activity potential in the reactor. These observations shine a new light on the contribution of large granules to the conversion of polymeric COD and suggest that large granules can hydrolyze a significant amount of this influent fraction. The anaerobic removal of polymeric soluble and colloidal substrates could clarify the stable granule formation that is observed in full-scale installations, even when those are fed with complex wastewaters.Key points• Large and small granules contain >70% of the hydrolysis potential in an AGS reactor.• Flocculent sludge has high hydrolytic activity but constitutes <10% VS in AGS.• AGS has an overcapacity to hydrolyze complex substrates in domestic wastewater.Graphical abstract

Cellulolytic and mannanolytic aerobic bacteria isolated from Buffalo rumen (Bubalus babalis) and its potency to degrade fiber in palm kernel meal

Abstract. Sari SLA, Triyanto T, Zuprifal Z, Prijambada ID. 2021. Cellulolytic and mannanolytic aerobic bacteria isolated from Buffalo rumen (Bubalus babalis) and its potency to degrade fiber in palm kernel meal. Biodiversitas 22: 2829-2837. Palm kernel meal (PKM) is potential to be used as feed, but its high fiber content causes PKM meal difficult to be digested by monogastric animals. Ruminants are especially effective in digesting plant fibers because of the presence of microbes in their rumens. Based on those facts, this research was conducted to obtain mannanolytic and cellulolytic bacteria from buffalo rumens (Bubalus babalis, Linnaeus, 1758), which can degrade fibers in PKM. Bacteria were isolated from buffalo rumen by using PKM- isolation media. Screening of hydrolytic activities was done based on clear zone formation on screening media. A total of five bacterial isolates with the highest hydrolytic activities were then assayed quantitatively for their abilities to degrade mannan and cellulose, then identified based on 16S rRNA gene sequences. This research successfully isolated 34 bacterial isolates. The screening result demonstrated that all isolates could hydrolyze mannan, cellulose and polysaccharide in PKM. Isolate BR25 showed the highest hydrolytic ability on PKM and mannan screening media with clear zone diameter/colony diameter ratio (dz/dc ratio) of 2.99 and 3.53, respectively. Isolate BR31 showed the highest cellulolytic ability with dz/dc ratio value of 2.22. Five isolates with the highest hydrolytic activity, i.e. BR14, BR16, BR23, BR25, and BR30 showed the ability to grow on submerged media which contain locust bean gum (LBG) and microcrystalline cellulose (MCC) respectively, as single carbon source and isolate BR25 showed the highest ability to degrade mannan and cellulose. Based on the gene sequence of 16S rRNA, isolates BR14, BR16, BR23, BR25, and BR30 were identified to be closely related to Exiguobacterium acetylicum, Bacillus cereus, Klebsiella quasipneumoniae, Paenibacillus polymyxa, and Acinetobacter baumannii with 98.57-100% level of similarity.

The role of microorganisms in biodegradation of chitosan/tannic acid materials

High utilization of thermoplastic polymers with low degradation rates as packaging materials generates a large amount of waste. Therefore, it should be replaced by natural polymers that can be degraded by microorganisms. In this paper, chitosan (CTS)/tannic acid (TA) materials in the weight ratios of 80CTS/20TA and 50CTS/50TA were prepared as potential packaging materials. The results showed that these materials were similarly degraded in soil and compost. However, in comparison to 50CTS/50TA, 80CTS/20TA was slightly better degraded in soil. After 14 days of biodegradation, the chemical structure of materials was changed resulting from adhesion of the microorganisms. The smallest changes were observed on 80CTS/20TA film. Bacterial species were collected and identified from materials after the degradation process. Microorganisms with the highest hydrolytic activity were chosen for the degradation study. Biodegradation and hydrolytic activity were observed only in a few strains, which indicate difficulties in material degradation. Soil bacteria degraded the films better than bacteria isolated from the compost. This study showed also that consortia of bacteria added to soil and compost had a positive effect on the biodegradation of the tested materials and increased the biodegradation of these materials in the studied environments.

Identification and characterization of extracellular enzymes secreted by Aspergillus spp. involved in lipolysis and lipid-antioxidation during katsuobushi fermentation and ripening

A mild-flavored soup stock made from katsuobushi is an important element of traditional Japanese cuisine and is the basic seasoning responsible for the taste. Fermented and ripened katsuobushi, known as karebushi, is manufactured by simmering skipjack tuna that is then smoke-dried, fermented, and ripened in a repeated molding process by five dominant Aspergillus species. Here, our aim was to characterize and identify the lipolytic enzymes secreted by the dominant Aspergillus species, especially A. chevalieri and A. pseudoglaucus, which are involved in hydrolyzing lipids during the molding process. The crude enzyme preparations from the five Aspergillus spp. cultivated on katsuobushi solid medium hydrolyzed triglycerides in fish oil, and more saturated and unsaturated fatty acids (C16:0, C16:1, C18:0, C18:1) were produced than major polyunsaturated fatty acids (C20:5, C22:6). On the basis of ion exchange chromatograms, the composition of the lipolytic enzymes was different in the five species. There was at least one active fraction with high hydrolytic activity toward fish oil in four of the Aspergillus spp., but not A. sydowii; the lipolytic enzyme secreted by A. sydowii had quite high activity toward the artificial substrate p-nitrophenyl butyrate, but low activity toward the natural oil. The lipolytic fractions from A. chevalieri and A. pseudoglaucus were further purified by hydrophobic interaction chromatography then gel-filtration chromatography; LC-MS-MS Mascot analysis identified a variety of lipolytic enzymes, including cutinase, esterase, phospholipase, and carboxyl esterase in the lipolytic fractions from these species. The identified enzymes had 30%–70% identity to previously reported or manually annotated lipases or esterases from taxa other than Aspergillus. The different lipolytic enzymes likely acted on triglycerides in the katsuobushi fish oil. Furthermore, catalase B and Cu/Zn superoxide dismutase, which limit oxidative damage of lipids, were also identified. These antioxidant enzymes may prevent lipid oxidation and rancidity as the lipolytic enzymes hydrolyze lipids during the long fermentation and ripening process. Umami and richness tastes tended to increase in extracts from culture of protease- and peptidase-producing A. sydowii. Our results will aid in the selection and application of desirable strains of Aspergillus species as starter cultures to improve the storage and quality of fermented and ripened karebushi.

Metaproteomic Discovery and Characterization of a Novel Lipolytic Enzyme From an Indian Hot Spring.

Lipolytic enzymes are produced by animals, plants and microorganisms. With their chemo-, regio-, and enantio-specific characteristics, lipolytic enzymes are important biocatalysts useful in several industrial applications. They are widely used in the processing of fats and oils, detergents, food processing, paper and cosmetics production. In this work, we used a new functional metaproteomics approach to screen sediment samples of the Indian Bakreshwar hot spring for novel thermo- and solvent-stable lipolytic enzymes. We were able to identify an enzyme showing favorable characteristics. DS-007 showed high hydrolytic activity with substrates with shorter chain length (<C8) with the maximum activity observed against p-nitrophenyl butyrate (C4). For substrates with a chain length >C10, significantly less hydrolytic activity was observed. A preference for short chain acyl groups is characteristic for esterases, suggesting that DS-007 is an esterase. Consistent with the high temperature at its site of isolation, DS-007 showed a temperature optimum at 55°C and retained 80% activity even after prolonged exposure to temperatures as high as 60°C. The enzyme showed optimum activity at pH 9.5, with more than 50% of its optimum activity between pH 8.0 and pH 9.5. DS-007 also exhibited tolerance toward organic solvents at a concentration of 1% (v/v). One percent of methanol increased the activity of DS-007 by 40% in comparison to the optimum conditions without solvent. In the presence of 10% methanol, DMSO or isopropanol DS-007 still showed around 50% activity. This data indicates that DS-007 is a temperature- and solvent-stable thermophilic enzyme with reasonable activity even at lower temperatures as well as a catalyst that can be used at a broad range of pH values with an optimum in the alkaline range, showing the adaptation to the habitat’s temperature and alkaline pH.

Enhancing PET hydrolytic enzyme activity by fusion of the cellulose–binding domain of cellobiohydrolase I from Trichoderma reesei

The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (IsPETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETaseS121E/D186H/R280A (IsPETaseEHA) variant. We found that fusing a cellulose–binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETaseEHA showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETaseEHA_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 ℃ and 40 ℃, respectively. The catalytic activity of IsPETaseEHA_CBM was increased by 86 % when the protein concentration was increased from 2.5 μg/mL to 20 μg/mL. These findings suggest that the fusion of polymer-binding module to IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.

Solid-state fermentation production and characterization of an alkaline lipase from a newly isolated Burkholderia gladioli strain

The newly isolated Burkholderia gladioli BRM58833 strain was shown to secrete an alkaline lipase highly active and stable in organic solvents. Lipase production was optimized through the cultivation of the strain by solid-state fermentation in wheat bran. The lipase extraction conditions were also optimized. The low-cost extract obtained has shown a high hydrolytic activity of 1096.7 ± 39.3 U·gds−1 (units per gram of dry solids) against pNPP and 374.2 ± 20.4 U·gds−1 against triolein. Proteomic analysis revealed the optimized extract is composed of two esterases and three true lipases, showing a preference for long-chain substrates. The highest activity was obtained at 50 °C and pH 9. However, the extract maintained more than 50% of its maximum activity between pH 8.0 and 10.0 and throughout the whole temperature range evaluated (32–70 °C). The enzymes were inhibited by SDS, EDTA, ZnSO4 and FeCl3 and activated by FeSO4, MgCl2 and BaCl2. The lipases conserved their activity when incubated in solvents as acetonitrile, diethyl ether, n-heptane n-hexane, toluene, methanol and t-butanol. The resistance of these lipases to solvents and expressive thermostability when compared to other lipases, reveal their potential both in hydrolysis reactions and in synthesis of esters.

Implications for the PET decomposition mechanism through similarity and dissimilarity between PETases from Rhizobacter gummiphilus and Ideonella sakaiensis

The development of a superb polyethylene terephthalate (PET) hydrolyzing enzyme requires an accurate understanding of the PET decomposition mechanism. However, studies on PET degrading enzymes, including the PET hydrolase from Ideonella sakaiensis (IsPETase), have not provided sufficient knowledge of the molecular mechanisms for the hardly accessible substrate. Here, we report a novel PET hydrolase from Rhizobacter gummiphilus (RgPETase), which has a hydrolyzing activity similar to IsPETase toward microcrystalline PET but distinct behavior toward low crystallinity PET film. Structural analysis of RgPETase reveals that the enzyme shares the key structural features of IsPETase for high PET hydrolysis activity but has distinguished structures at the surface-exposed regions. RgPETase shows a unique conformation of the wobbling tryptophan containing loop (WW-loop) and change of the electrostatic surface charge on the loop dramatically affects the PET-degrading activity. We further show that effect of the electrostatic surface charge to the activity varies depending on locations. This work provides valuable information underlying the uncovered PET decomposition mechanism.