Hydrolysis Of Ester Bond Research Articles

Investigation of The Activity of Lipase Variants on Different 4-Nitrophenyl Esters by Spectrophotometric Assay

Microbial enzymes are important tools that are frequently used in the field of biotechnology. These microbial enzymes, which play a role in intracellular biological reactions, are used in many different industries. Lipase, proteases and amylases are important members of hydrolytic enzymes. Lipase enzyme, which has the most common usage area among hydrolytic enzymes, is an enzyme that catalyzes the hydrolysis of ester bonds between lipid molecules. The activity of lipase enzyme is commonly measured by spectrophotometric method. P-nitrophenol esters are commonly preferred for kinetic analysis. In the spectrophotometric analysis method, the colored product is measured as a result of the hydrolysis of p-nitrophenol ester substrates by the lipase enzyme. In this study, p-nitrophenyl acetate (Acetic acid 4-nitrophenyl ester), p-nitrophenyl butyrate (Butyric acid 4-nitrophenyl ester), p-nitrophenyl octanoate (Octanoic acid 4-nitrophenyl ester, 4 -Nitrophenyl caprylate), p-nitrophenyl dodecanoate (Dodecanoic acid 4-nitrophenyl ester), p-nitrophenyl palmitate (p-Nitrophenyl palmitate, Hexadecanoic acid 4-nitrophenyl ester) substrates were used. The products formed as a result of incubation of substrates with different carbon lengths with lipase enzyme periods were measured spectrophotometrically. Trials were carried out at 25°C between 5min-120min. As a result of the experiments carried out in four repetitions, it was determined that the activity of the lipase enzyme varies according to the length of the carbon chain of the substrates. Vmax values of wild lipase enzyme were calculated as 0.42 U/mg protein, 0.95 U/mg protein, 1.1 U/mg protein, 0.78 U/mg protein, 0.18 U/mg protein for pNP-acetate, pNP-buritate, pNP-octanoate, pNP-dodecanoate, pNP-palmitate, respectively. It was determined that the activity of lipase enzyme on p-nitrophenyl palmitate was very low. It was determined that the activity of wild lipase enzyme on the eight-carbon chain pNP-octanoate substrate was higher than the other substrates.

Dual-jet electrospun PDLGA/PCU nonwovens and their mechanical and hydrolytic degradation properties

A dual-jet electrospinning was used to mix a different hydrophilicity poly(carbonate urethanes) (PCUs) nanofibers with a biodegradable poly(D,L-lactide-co-glycolide) (PDLGA) copolyester microfibers. As a result, PDLGA/PCU partially degradable nonwovens consisting of an interlaced of both components fibers were obtained. In order to examine the hydrolytic degradation process of polyester fraction, as well as changes that occurred in the mechanical properties of the whole nonwovens, gel permeation chromatography, proton nuclear magnetic resonance spectroscopy, differential scanning calorimetry and scanning electron microscopy as well as static tensile test were performed. Obtained results showed that for the introduction of more hydrophobic PCU nanofibers (ChronoSil), the process of copolyester chain scission slowed down and the erosion mechanism proceeded in bulk. Unexpectedly, even greater deceleration of PDLGA fibers degradation was observed in case of more hydrophilic PCU (HydroThane), and erosion mechanism changed to surface. Enhancement the affinity of the whole nonwoven to the water, manifested by strong water uptake, facilitated the diffusion processes of both: water and acid degradation by-products, which limited autocatalysis reactions of the hydrolysis of ester bonds. On the other hand, strength tests showed the synergy in the mechanical characteristics of both components. Presented method allows influencing the mechanism and rate of polyester degradation without changing its chemical composition and physical properties, affecting only the physical interactions between the nonwoven and the degradation environment, and thus, on diffusion processes. Obtained partially degradable materials possessed also time prolonged functional properties, compared to the copolyester-only nonwoven itself, thus could be considered as promising for biomedical applications e.g. in drug release systems, implants or surgical meshes for supporting soft tissues.

Deterioration of Soil-biodegradable Mulch Films during Storage and Its Impact on Specialty Crop Production

Plastic mulch films contribute to improved crop yield and quality for vegetable and small fruit cropping systems. Although the single-season agronomic performance of conventional polyethylene mulches and soil-biodegradable mulches (BDMs) are similar, over time BDMs can begin to break down during storage and subsequently not provide season-long soil coverage. In this study, the changes in physicochemical properties of BDMs were investigated over 3 years of indoor storage (2015–18) under ideal environmental conditions in two laboratories. Mulches evaluated were black, 20–40 µm thick, suitable for annual vegetable production, and included three BDMs: two polybutylene adipate terephthalate (PBAT)-enriched mulches that are commercially available in North America, an experimental polylactic acid (PLA) and polyhydroxyalkanote-based film, and a conventional polyethylene mulch as a control. Tensile properties, specifically peak load and elongation at maximum tensile stress, decreased during storage, particularly for the PBAT-based BDMs, indicating a loss of strength. During year 3 of storage, the tensile properties declined extensively, suggesting embrittlement. The average molecular weight of PLA and PBAT slightly increased during year 1, perhaps due to release of monomers or oligomers, and then decreased extensively during years 2 and 3 due to hydrolysis of ester bonds (confirmed by Fourier transform infrared spectroscopic analysis). The structural integrity of BDMs was assessed during years 2 and 3 of the study (2017–18) in field trials at the locations where they were stored, Knoxville, TN, and Mount Vernon, WA, for vegetable production. The degradation of the BDMs during the cropping season was higher in 2018 compared with 2017, suggesting that degradation of mechanical and chemical properties while in storage may have contributed to rapid degradation of mulches in the field. In summary, BDMs undergo degradation even under ideal storage conditions and may perform best if deployed within 2 years of their receipt date. The farmer should verify that proper storage conditions have been used before receipt and that manufacturing date precedes the receipt date by no more than 6 months.

Three-dimensional structure of xylonolactonase from Caulobacter crescentus: A mononuclear iron enzyme of the 6-bladed β-propeller hydrolase family.

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d‐xylonolactone. We have determined crystal structures of Cc XylC in complex with d‐xylonolactone isomer analogues d‐xylopyranose and (r)‐(+)‐4‐hydroxy‐2‐pyrrolidinone at high resolution. Cc XylC has a 6‐bladed β‐propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe2+. The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side chains of three amino acid residues, Glu18, Asn146, and Asp196, which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe2+. The structure of Cc XylC was compared with eight other ester hydrolases of the β‐propeller hydrolase family. The previously published crystal structures of other β‐propeller hydrolases contain either Ca2+, Mg2+, or Zn2+ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe2+ as a catalytic metal. This could further expand our understanding of utilization of Fe2+ not only in oxidative enzymes but also in hydrolases.

Diversifying Arena of Drug Synthesis: In the Realm of Lipase Mediated Waves of Biocatalysis

Hydrolases, being most prominent enzymes used in industrial processes have left no stone unturned in fascinating the pharmaceutical industry. Lipases, being a part of acyl hydrolases are the ones that function similarly to esterases (except an interfacial action) wherein they generally catalyze the hydrolysis of ester bonds. Be it in terms of stereoselectivity or regioselectivity, lipases have manifested their promiscuous proficiency in rendering biocatalytic drug synthesis and intermediates thereof. Industrial utilization of lipases is prevalent since decades ago, but their distinctive catalytic competencies have rendered them suitable for maneuverability in various tides of biocatalytic industrial process development. Numbers of exquisite catalysts have been fabricated out of lipases using nanobiotechnology whereby enzyme reusability and robustness have been conferred to many of the organic synthesis procedures. This marks a considerable achievement of lipases in the second wave of biocatalysis. Furthermore, in the third wave an advent of genetic engineering has fostered an era of customized lipases for suitable needs. Be it stability or an enhanced efficacy, genetic engineering techniques have ushered an avenue for biocatalytic development of drugs and drug intermediates through greener processes using lipases. Even in the forthcoming concept of co-modular catalytic systems, lipases may be the frontiers because of their astonishing capability to act along with other enzymes. The concept may render feasibility in the development of cascade reactions in organic synthesis. An upcoming wave demands fulfilling the vision of tailored lipase whilst a far-flung exploration needs to be unveiled for various research impediments in rendering lipase as a custom fit biocatalyst in pharmaceutical industry.

Bacterial Degradation of Bacteriostatic Polyphenols, Tannins

Tannin degradation by bacteria has not been studied much as tannins are commonly known to be bacteriostatic due to enzyme inhibition, substrate deprivation, and the enzyme activity on the bacterial cell wall. However, about a handful of bacteria have been found to tolerate certain concentrations of tannin. This study focuses on isolating and identifying bacteria from decaying portions of tree bark for tannase production and effective catalysis of ester bond hydrolysis in tannins. Different concentrations of commercial tannic acid were used as the sole carbon source on mineral salt medium (MSM) agar plates, to test the maximum tolerable concentrations (MTCs) by the isolates. Tannin degradation was confirmed by a visual reading method and bacterial tannase activity and the biodegradation percentage were determined. One particular isolate was identified to have 50 g/L MTC of tannin, with a tannase activity of 51.61 U/mL that is optimum after 96 hours of incubation. The 16s rRNA sequencing results showed that the isolate belonged to Bacillus genus and the resulting bacterial strain isolate was found to be a new strain of Bacillus subtilis which was submitted to GenBank under the accession number MH330408.

Xylonolactonase from Caulobacter crescentus Is a Mononuclear Nonheme Iron Hydrolase

Caulobacter crescentus xylonolactonase (Cc XylC, EC 3.1.1.68) catalyzes an intramolecular ester bond hydrolysis over a nonenzymatic acid/base catalysis. Cc XylC is a member of the SMP30 protein family, whose members have previously been reported to be active in the presence of bivalent metal ions, such as Ca2+, Zn2+, and Mg2+. By native mass spectrometry, we studied the binding of several bivalent metal ions to Cc XylC and observed that it binds only one of them, namely, the Fe2+ cation, specifically and with a high affinity (Kd = 0.5 μM), pointing out that Cc XylC is a mononuclear iron protein. We propose that bivalent metal cations also promote the reaction nonenzymatically by stabilizing a short-lived bicyclic intermediate on the lactone isomerization reaction. An analysis of the reaction kinetics showed that Cc XylC complexed with Fe2+ can speed up the hydrolysis of d-xylono-1,4-lactone by 100-fold and that of d-glucono-1,5-lactone by 10-fold as compared to the nonenzymatic reaction. To our knowledge, this is the first discovery of a nonheme mononuclear iron-binding enzyme that catalyzes an ester bond hydrolysis reaction.

Biodegradation of phthalate esters by Pantoea dispersa BJQ0007 isolated from Baijiu

Baijiu is a famous Chinese alcoholic beverage and is produced by the co-fermentation of microorganisms with grains as the raw materials. Phthalate esters (PAEs) are toxic chemicals and frequently detected in Baijiu. Identification of microorganisms that could degrade PAEs was recognized as a possible way to solve the contamination. Pantoea spp. are one kind of dominant microorganisms for manufacturing Baijiu. Here, Pantoea dispersa BJQ0007 was isolated from Baijiu fermentation starter and showed the ability in degrading dimethyl phthalate, diethyl phthalate, dibutyl phthalate, diisobutyl phthalate, and di-(2-ethylhexyl) phthalate, with di-(2-ethylhexyl) phthalate as the preferred substrate. The whole genome was sequenced with a chromosome of 4,325,134 bp and three plasmids. The metabolic pathway of PAE degradation is proposed with an uncommon metabolic step by converting phthalate monoester to benzoate ester based on metabolic intermediate analysis and genome annotation. Two enzymes responsible for ester bond hydrolysis of PAEs were identified and belonged to enzyme families IV and not defined, respectively. The results revealed PAE degradation of Pantoea species and would promote the safety control of Baijiu products by offering bacterial and enzymatic resources.

Improving the Acid Resistance of Tannase TanBLp (AB379685) from Lactobacillus plantarum ATCC14917T by Site-Specific Mutagenesis.

Tannin acyl hydrolase referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannin to release gallic acid. The tannase TanBLp which cloned from Lactobacillus plantarum ATCC14917T has high activity in the pH range (7.0-9.0) at 40°C, it would be detrimental to the utilization at acidic environment. The catalytic sites and stability of TanBLp were analyzed using bioinformatics and site-specific mutagenesis. The results reiterated that the amino acid residues Ala164, Lys343, Glu357, Asp421 and His451 had played an important role in maintaining the activity. The optimum pH of mutants V75A, G77A, N94A, A164S and F243A were shifted from 8.0 to 6.0, and mutant V75A has the highest pH stability and activity at acidic conditions than other mutants, which was more suitable for industrial application to manufacture gallic acid. This study was of great significance to promote the industrialization and efficient utilization of tannase TanBLp.

Investigation of the pectin grafting with gallic acid and propyl gallate and their antioxidant activities, antibacterial activities and fresh keeping performance

In this paper, a method for the enzymatic modification of pectin, in which gallic acid (GA) and propyl gallate (PG) were grafted onto pectin molecules in an aqueous/organic two-phase system catalyzed by lipase, was proposed. The potential reaction mechanism was explored through UV–Vis, FTIR and 1H NMR spectroscopic methods and density functional theory. Results suggested that the lipase played a dual role during the modification by catalyzing the hydrolysis of methyl ester bonds of pectin in the aqueous phase and the esterification between the 4-OH of GA and PG and the –COOH of pectin in the organic phase. Moreover, the effects of GA and PG on the antioxidant and the antibacterial activities of pectin were evaluated, and results showed that the antioxidant and the antibacterial activities of modified pectin were better than those of native pectin. The effect of modified pectin on the quality of fresh bass (Lateolabrax maculatus) was further studied. Results suggested that, compared to control group, the total viable count, histamine level, malondialdehyde content and acid value of bass fillets treated with modified pectin were significantly reduced, whereas the sensory score was significantly increased.

The Role of Release Factors in the Hydrolysis of Ester Bond in Peptidyl-tRNA.

Class I release factors (RFs) recognize stop codons in the sequences of mRNAs and are required for the hydrolysis of peptidyl-tRNA in the ribosomal P site during the final step of protein synthesis in bacteria, resulting in the release of a complete polypeptide chain from the ribosome. A key role in this process belongs to the highly conserved GGQ motif in RFs. Mutations in this motif can reduce the hydrolysis rate or even completely inhibit the reaction. Previously, it was hypothesized that the amino acid residues of GGQ (especially glutamine) are essential for the proper coordination of the water molecule for subsequent hydrolysis of the ester bond. However, available structures of the 70S ribosome termination complex do not allow unambiguous identification of the exact orientation of the carbonyl group in peptidyl-tRNA relative to the GGQ, as well as of the position of the catalytic water molecule in the peptidyl transferase center (PTC). This mini-review summarizes key facts and hypotheses on the role of GGQ in the catalysis of peptide release, as well as suggests and discusses future experiments aimed to produce high-quality structural data for deciphering the precise mechanism of RF-mediated catalysis.

Determination of camostat and its metabolites in human plasma – Preservation of samples and quantification by a validated UHPLC-MS/MS method

ObjectivesCamostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. Design and methodsAn ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. ResultsBoth diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at −20 °C and −80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1–800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1–800 ng/mL. ConclusionsA methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.

Hydrolytic polysorbate 20 degradation – Sensitive detection of free fatty acids in biopharmaceuticals via UPLC-QDa analytics with isolator column

The enzymatic hydrolysis of polysorbates, e.g. induced by specific host cell proteins in biologics, is a known risk factor regarding the potential particle formation in the product over time. One of the root causes for this observation is an increase in free fatty acids (FA) within the formulation, which indicates the need for convenient monitoring of FA release. This study presents a novel UPLC-QDa based method to evaluate the content of the FAs esterified to polysorbate 20 (PS20) after hydrolysis. The presented method is label-free, i.e. independent of elaborate fluorophore-labeling and able to directly measure the ionized FAs. Furthermore, the method allows the determination of released FAs as percentage of ester bond hydrolysis and as absolute concentration expressed in ng/mL. Additionally, we describe for the first time in FA analytics the application of an isolator column, to remove trace levels of FAs present in the eluents to improve the sensitivity of the method. Lastly, the capabilities of the newly developed method are proven in case studies with three different monoclonal antibodies, which display characteristic FA release patterns in PS20-containing formulations. In summary, we developed a reliable, sensitive method for FA quantification in biologics, which could also be used as a predictive tool, considering FA solubility, regarding the formation of particles.

Fabrication of Antiswelling Loose Nanofiltration Membranes via a "Selective-Etching-Induced Reinforcing" Strategy for Bioseparation.

With diverse selectivity, higher permeance, and good antifouling property, loose polyamide nanofiltration (NF) membranes can be potentially deployed in various bioseparation applications. However, the loose NF membrane with a low crosslinking degree generally suffers from the alkali-induced pore swelling during chemical cleaning, resulting in degradation of separation performance with time. In this work, we conceive a novel strategy to tailor the separating layer through alkaline post-etching following the interfacial polymerization process, where piperazine and tannic acid (TA) were used as water-phase monomers, and trimesoyl chloride (TMC) and ferric acetylacetonate were employed as organic monomers in n-hexane. Thereinto, the polyester network formed by TA and TMC was selectively etched by alkaline treatment, thus obtaining a loose NF membrane, whose structure and performance could be facilely tailored by controlling the TA ratio and the etching pH. As a result, the well-designed loose NF membrane exhibited higher flux, better selectivity, and more stable separation performance in a long-term filtration of diluted cane molasses. Interestingly, the obtained loose NF membrane showed excellent antiswelling ability during alkaline cleaning because of network locking induced by Fe3+ chelation, decrease in the carboxyl proportion (more hydroxyl generation due to the ester bond hydrolysis), and enhanced interface interaction between the separation layer and the sublayer attributed to catechol adhesion effect. Therefore, such a "selective-etching-induced reinforcing" strategy could endow the polyamide NF membrane with both loose and antiswelling separation layer in a reliable and scalable way, which provides a new perspective for preparing highly selective and stable NF membrane for resource recovery.

Effect of chain length and branching on the in vitro metabolism of a series of parabens in human liver S9, human skin S9, and human plasma

Parabens are antimicrobial compounds used as preservatives in cosmetics, foods, and pharmaceuticals. Paraben exposure occurs through a variety of routes including dermal absorption, ingestion, and inhalation. Ester bond hydrolysis has been shown to be the predominant biotransformation for this chemical class. Here we evaluated a series of parabens of increasing alkyl chain length and branching in addition to the aryl side chain of phenyl paraben (PhP). We evaluated the parabens under full Michaelis-Menten (MM) parameters to obtain intrinsic clearance values and found different trends between human liver and skin, which correlate with the predominant esterase enzymes in those matrices, respectively. In liver, where carboxylesterase 1 (CES1) is the predominant esterase enzyme, the shorter chain parabens were more readily metabolized, while in skin, where carboxylesterase 2 (CES2) is the predominant esterase enzyme, the longer chain parabens were more readily metabolized. Alkyl chain branching reduced the hydrolysis rates relative to those for the straight chain compounds, while the addition of a phenyl group, as in PhP, showed an increase in hydrolysis, producing the highest observed hydrolysis rate for skin. These data summarize the structure-metabolism relationship for a series of parabens and contribute to the safety assessment of this class of compounds.

Reprocessable, biodegradable polyester-based solid-solid phase change materials networks from dynamic ionic crosslinking with high latent heat capability

Thermosetting polyethylene (PEG)-based solid-solid phase change materials networks (PCMNs) have high latent leat and form stability from the covalent crosslinking, yet they remain a great challenge in reprocessing and recycling of PCMNs without compromising a high latent heat above 100 J/g. Herein, the polyester-based solid-solid PCMNs with PEG as phase change component were synthesized via ionic crosslinking between the linear multiple carboxyl-functionalized phase change materials (CPCMs) polyester and zinc cations. The CPCMs were synthesized via polyesterification reaction between pyromellitic dianhydride (PMDA) and diol containing PEG and ethylene glycol, meanwhile forming multiple carboxyl along molecular chains. The dynamic ionic bonds and hydrolysable ester bonds endowed PCMNs with the reprocessability and biodegradability, respectively. PCMNs had the ΔH = 120 J/g with PEG weight fraction of ∼0.91 and concentration of carboxyl of ∼0.26 mmol/g. Also, PCMNs had good phase change ability, thermal stability and thermal reliability before and after thermal cycling and reprocessing. The robust approach enabled the combination of the reprocessing ability and high latent heat of PCMNs by ionic crosslinking.

N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors.

We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

Cold Active Lipases: Biocatalytic Tools for Greener Technology.

Lipases are enzymes that catalyze the ester bond hydrolysis in triglycerides with the release of fatty acids, mono- and diglycerides, and glycerol. The microbial lipases account for $400 million market size in 2017 and it is expected to reach $590 million by 2023. Many biotechnological processes are expedited at high temperatures and hence much research is dealt with thermostable enzymes. Cold active lipases are now gaining importance in the detergent, synthesis of chiral intermediates and frail/fragile compounds, and food and pharmaceutical industries. In addition, they consume less energy since they are active at low temperatures. These cold active lipases have not been commercially exploited so far compared to mesophilic and thermophilc lipases. Cold active lipases are distributed in microbes found at low temperatures. Only a few microbes were studied for the production of these enzymes. These cold-adapted enzymes show increased flexibility of their structures in response to freezing effect of the cold habitats. This review presents an update on cold-active lipases from microbial sources along with some structural features justifying high enzyme activity at low temperature. In addition, recent achievements on their use in various industries will also be discussed.

A Comparison on Biodegradation Behaviour of Polylactic Acid (PLA) Based Blown Films by Incorporating Thermoplasticized Starch (TPS) and Poly (Butylene Succinate-co-Adipate) (PBSA) Biopolymer in Soil

The comparative study of biodegradation of various blown films obtained from Poly (lactic) acid (PLA) has been studied via soil burial method. A total of 3-different types of films prepared from neat PLA and the reactive blends containing poly butylenes succinate-co-adipate (PBSA), and thermoplasticized starch (TPS); namely VPLA, PLA/PBSA, PLA/TPS respectively were the subjects of investigation. Several analytical techniques including weight loss method and analysis of mechanical properties were performed in each seven days interval until ninety days to elucidate the biodegradation in soil. The tensile modulus of VPLA and PLA/PBSA blown films were deteriorated to the tune of 60.32% and 71.28% respectively within 28 days, while PLA/TPS blown films recorded a significant reduction of 75.31% in the modulus value within 21 days of soil exposure compared to unexposed blown film samples. Similarly, blown films of PLA/TPS reported the highest rate of weight loss in the order of 40.06% in 90 days of soil burial with an estimated half-life of 103 days in soil environment compared to its counterparts. The depletion in both mechanical properties and weight of the film samples suggesting the occurrence of biodegradation in the real soil environment. Scanning electron microscopy (SEM) revealed the formation of coarse morphology for all three types of soil buried samples which trace of microbial action appeared on PLA/TPS films. Fourier transform infrared microscope (FTIR) showed the decrease in carbonyl index and variation in the intensities of carbonyl and hydroxyl peaks irrespective of the film samples after 90 days of soil exposure. Gel permeation chromatography (GPC) documented reduction in molecular weight and variation in polydispersity index (PDI) of post-exposed soil samples. The elemental analysis exhibited that the percentage of organic carbon and hydrogen content of all the films decreased while the oxygen percentage increased after soil burial due to the biodegradation of film specimens. Both Differential scanning calorimetry (DSC) and X-ray diffraction analysis (XRD) reported increase in crystallinity for soil exposed samples indicating the initiation of degradation kinetics preferably at the amorphous region of film composition. It has also been inferred that the biodegradation mechanism of VPLA and PLA/PBSA blown films predominantly driven by hydrolysis of ester bond. Contrastingly, the biodegradation kinetics in case of PLA/TPS film has been proceeded with microbial assimilation of TPS component which further thrived on hydrolysis of PLA component of the blown film system.

Crystal structure of fungal tannase from Aspergillus niger.

Tannases are serine esterases that were first discovered in fungi more than one and half centuries ago. They catalyze the hydrolysis of the gallolyl ester bonds in gallotannins to release gallic acid, which is an important intermediate in the chemical and pharmaceutical industries. Since their discovery, fungal tannases have found wide industrial applications, although there is scarce knowledge about these enzymes at the molecular level, including their catalytic and substrate-binding sites. While this lack of knowledge hinders engineering efforts to modify the enzymes, many tannases have been isolated from various fungal strains in a search for the desired enzymatic properties. Here, the first crystal structure of a fungal tannase, that from Aspergillus niger, is reported. The enzyme possesses a typical α/β-hydrolase-fold domain with a large inserted cap domain, which together form a bowl-shaped hemispherical shape with a surface concavity surrounded by N-linked glycans. Gallic acid is bound at the junction ofthe two domains within the concavity by forming two hydrogen-bonding networks with neighbouring residues. One is formed around the carboxyl group of the gallic acid and involves residues from the hydrolase-fold domain, including those from the catalytic triad, which consists of Ser206, His485 and Asp439. The other is formed around the three hydroxyl groups of the compound, with the involvement of residues mainly from the cap domain, including Gln238, Gln239, His242 and Ser441. Gallic acid is bound in a sandwich-like mode by forming a hydrophobic contact with Ile442. All of these residues are found to be highly conserved among fungal and yeast tannases.