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Frances Jurnak - One of the best experts on this subject based on the ideXlab platform.

  • characterization and implications of ca2 binding to Pectate Lyase c
    Journal of Biological Chemistry, 2003
    Co-Authors: Steven R. Herron, Robert D. Scavetta, Michael Garrett, Margaret Legner, Frances Jurnak
    Abstract:

    Ca(2+) is essential for in vitro activity of Erwinia chrysanthemi Pectate Lyase C (PelC). Crystallographic analyses of 11 PelC-Ca(2+) complexes, formed at pH 4.5, 9.5, and 11.2 under varying Ca(2+) concentrations, have been solved and refined at a resolution of 2.2 A. The Ca(2+) site represents a new motif for Ca(2+), consisting primarily of beta-turns and beta-strands. The principal differences between PelC and the PelC-Ca(2+) structures at all pH values are the side-chain conformations of Asp-129 and Glu-166 as well as the occupancies of four water molecules. According to calculations of pK(a) values, the presence of Ca(2+) and associated structural changes lower the pK(a) of Arg-218, the amino acid responsible for proton abstraction during catalysis. The Ca(2+) affinity for PelC is weak, as the K(d) was estimated to be 0.132 (+/-0.004) mm at pH 9.5, 1.09 (+/-0.29) mm at pH 11.2, and 5.84 (+/-0.41) mm at pH 4.5 from x-ray diffraction studies and 0.133 (+/-0.045) mm at pH 9.5 from intrinsic tryptophan fluorescence measurements. Given the pH dependence of Ca(2+) affinity, PelC activity at pH 4.5 has been reexamined. At saturating Ca(2+) concentrations, PelC activity increases 10-fold at pH 4.5 but is less than 1% of maximal activity at pH 9.5. Taken together, the studies suggest that the primary Ca(2+) ion in PelC has multiple functions.

  • Structure of a Plant Cell Wall Fragment Complexed to Pectate Lyase C
    The Plant Cell, 1999
    Co-Authors: Robert D. Scavetta, Steven R. Herron, Arland T. Hotchkiss, Nobuhiro Kita, Noel T. Keen, Jacques A. E. Benen, Harry C. M. Kester, Jaap Visser, Frances Jurnak
    Abstract:

    The three-dimensional structure of a complex between the Pectate Lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, α-D-Gal p A-([1→4]-α-D-Gal p A) 3 -(1→4)-D-Gal p A, is composed of five galacturonopyranose units (D-Gal p A) linked by α-(1→4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the Pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the Pectate component in plant cell walls. The conformation of the Pectate fragment is a mix of 2 1 and 3 1 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca 2+ ions found in the complex. The observed protein–oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the Pectate Lyase family of proteins. Because the R218K PelC–galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the Pectate Lyase superfamily, is the amino acid that initiates proton abstraction during the β elimination cleavage of polygalacturonic acid.

  • sequence profile of the parallel β helix in the Pectate Lyase superfamily
    Journal of Structural Biology, 1998
    Co-Authors: Susan E. Heffron, Gregory R Moe, Volker Sieber, J Mengaud, Pascale Cossart, Jacqueline Vitali, Frances Jurnak
    Abstract:

    The parallel β helix structure found in the Pectate Lyase superfamily has been analyzed in detail. A comparative analysis of known structures has revealed a unique sequence profile, with a strong positional preference for specific amino acids oriented toward the interior of the parallel β helix. Using the unique sequence profile, search patterns have been constructed and applied to the sequence databases to identify a subset of proteins that are likely to fold into the parallel β helix. Of the 19 families identified, 39% are known to be carbohydrate-binding proteins, and 50% belong to a broad category of proteins with sequences containing leucine-rich repeats (LRRs). The most striking result is the sequence match between the search pattern and four contiguous segments of internalin A, a surface protein from the bacterial pathogenListeria monocytogenes.A plausible model of the repetitive LRR sequences of internalin A has been constructed and favorable 3D–1D profile scores have been calculated. Moreover, spectroscopic features characteristic of the parallel β helix topology in the Pectate Lyases are present in the circular dichroic spectrum of internalin A. Altogether, the data support the hypothesis that sequence search patterns can be used to identify proteins, including a subset of LRR proteins, that are likely to fold into the parallel β helix.

  • the refined three dimensional structure of Pectate Lyase e from erwinia chrysanthemi at 2 2 a resolution
    Plant Physiology, 1996
    Co-Authors: Susan E. Lietzke, Marilyn D Yoder, Robert D. Scavetta, Frances Jurnak
    Abstract:

    The crystal structure of Pectate Lyase E (PelE; EC 4.2.2.2) from the enterobacteria Erwinia chrysanthemi has been refined by molecular dynamics techniques to a resolution of 2.2 A and an R factor (an agreement factor between observed structure factor amplitudes) of 16.1%. The final model consists of all 355 amino acids and 157 water molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.721[deg] for bond angles. The structure of PelE bound to a lanthanum ion, which inhibits the enzymatic activity, has also been refined and compared to the metal-free protein. In addition, the structures of Pectate Lyase C (PelC) in the presence and absence of a lutetium ion have been refined further using an improved algorithm for identifying waters and other solvent molecules. The two putative active site regions of PelE have been compared to those in the refined structure of PelC. The analysis of the atomic details of PelE and PelC in the presence and absence of lanthanide ions provides insight into the enzymatic mechanism of Pectate Lyases.

  • Functional Implications of Structure-Based Sequence Alignment of Proteins in the Extracellular Pectate Lyase Superfamily
    Plant Physiology, 1995
    Co-Authors: Bernard Henrissat, Marilyn D Yoder, Susan E. Lietzke, Susan E. Heffron, Frances Jurnak
    Abstract:

    Pectate Lyases are plant virulence factors that degrade the Pectate component of the plant cell wall. The enzymes share considerable sequence homology with plant pollen and style proteins, suggesting a shared structural topology and possibly functional relationships as well. The three-dimensional structures of two Erwinia chrysanthemi Pectate Lyases, C and E, have been superimposed and the structurally conserved amino acids have been identified. There are 232 amino acids that superimpose with a root-mean-square deviation of 3 A or less. These amino acids have been used to correct the primary sequence alignment derived from evolution-based techniques. Subsequently, multiple alignment techniques have allowed the realignment of other extracellular Pectate Lyases as well as all sequence homologs, including pectin Lyases and the plant pollen and style proteins. The new multiple sequence alignment reveals amino acids likely to participate in the parallel beta helix motif, those involved in binding Ca2+, and those invariant amino acids with potential catalytic properties. The latter amino acids cluster in two well-separated regions on the Pectate Lyase structures, suggesting two distinct enzymatic functions for extracellular Pectate Lyases and their sequence homologs.

Krishanu Chakrabarti - One of the best experts on this subject based on the ideXlab platform.

  • degumming of ramie fiber and the production of reducing sugars from waste peels using nanoparticle supplemented Pectate Lyase
    Bioresource Technology, 2013
    Co-Authors: Arka Mukhopadhyay, Dhrubajyoti Chattopadhyay, Nalok Dutta, Krishanu Chakrabarti
    Abstract:

    Abstract Banana, citrus and potato peels were subjected to treatment with hydroxyapatite nanoparticle (NP) supplemented purified Pectate Lyase (NP-PL), isolated from Bacillus megaterium AK2 to produce reducing sugar (RS). At both 50 and 90 °C production of RS by NP-PL was almost twofold greater than that by untreated Pectate Lyase (PL) from each of the three peels. The optimal production of RS from banana and citrus peels were after 24 and 6 h of incubation while it was 24 and 4 h for potato peels at 50 and 90 °C, respectively, on NP-PL treatment. NP-PL could degum raw, decorticated ramie fibers as well as enhance fiber tenacity and fineness. The weight loss of the fibers were 24% and 31% better (compared to PL treatment) after 24 and 48 h of processing. These findings have potential implications for the bio-ethanol, bio-fuel and textile industries.

  • improvement of thermostability and activity of Pectate Lyase in the presence of hydroxyapatite nanoparticles
    Bioresource Technology, 2012
    Co-Authors: Arka Mukhopadhyay, Dhrubajyoti Chattopadhyay, Anjan Kumar Dasgupta, Krishanu Chakrabarti
    Abstract:

    Abstract The activity and half-life of Pectate Lyase (PL) from Bacillus megaterium were nine- and 60-fold, respectively, higher at 90 °C in the presence of hydroxyapatite nanoparticles (NP-PLs) than in the presence of 1 mM CaCl 2 . Thermodynamic analysis of the nanoparticle-induced stability revealed an enhanced entropy–enthalpy compensation by the NP-PLs since a reciprocal linearity of the enthalpy–entropy change to 90 °C was observed. Without nanoparticles, the linearity range was 70 °C. Such compensation reflected the maintenance of the native structure of proteins. The remarkable enhancement of activity and stability of the NP-PL system at high temperatures may be utilized commercially e.g. in the food industry or the processing of natural fibers that may require a thermotolerant enzyme.

  • arg 235 is an essential catalytic residue of bacillus pumilus dks1 Pectate Lyase to degum ramie fibre
    Biodegradation, 2011
    Co-Authors: Snehasish Basu, Dhrubajyoti Chattopadhyay, Abhrajyoti Ghosh, Amit Bera, Arunava Roy, Krishanu Chakrabarti
    Abstract:

    After 24 h of incubation with only purified Pectate Lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of Pectate Lyase the pel gene encoding a Pectate Lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of 35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5–9.0. Both Ca2+ and Mn2+ ions were required for activity on Na-Pectate salt substrates, while the enzyme was strongly inhibited by Zn2+ and EDTA. The deduced nucleotide sequence of the DKS1 Pectate Lyase (EU652988) showed 90% homology to Pectate Lyases from Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1 Pectate Lyase.

  • degumming and characterization of ramie fibre using Pectate Lyase from immobilized bacillus pumilus dks1
    Letters in Applied Microbiology, 2009
    Co-Authors: Snehasish Basu, Manabendra N Saha, Dhrubajyoti Chattopadhyay, Krishanu Chakrabarti
    Abstract:

    Aims: The present study was aimed at finding the optimal conditions for the production of Pectate Lyase using immobilized Bacillus pumilus DKS1 cells in calcium-alginate (Ca-alginate) beads and determining the efficient degumming of ramie fibre. Methods and Results: The active cells of B. pumilus DKS1 were immobilized in Ca-alginate and used for the production of Pectate Lyase. The production of enzyme increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 38AE 5Um l )1 at 18 g l )1 . This was about 1AE5-fold higher than that obtained by free cells. Degummed fibre using immobilized cells showed better tenacity than that prepared by using nonimmobilized cells. Conclusions: The Ca-alginate entrapment is a promising immobilization method of B. pumilus DKS1 for semicontinuous enzyme production. Enzyme production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation. Fibre degumming by using immobilized cells produced better quality fibre. Significance and Impact of the Study: This is the first report of degumming of fibre using enzyme from immobilized B. pumilus cells as per our knowledge. High-quality degummed fibre could be prepared with relatively inexpensive inputs for use in the textile and paper industry.

  • thermodynamic characterization of a highly thermoactive extracellular Pectate Lyase from a new isolate bacillus pumilus dks1
    Bioresource Technology, 2008
    Co-Authors: Snehasish Basu, Manabendra N Saha, Dhrubajyoti Chattopadhyay, Abhrajyoti Ghosh, Amit Bera, Krishanu Chakrabarti
    Abstract:

    Abstract An extracellular Pectate Lyase (EC 4.2.2.2) was purified from the culture filtrate of a newly isolated Bacillus pumilus DKS1 grown in pectin containing medium. Using ion-exchange and gel filtration chromatography, this enzyme was purified and found to have a molecular weight of around 35 kDa. The purified enzyme exhibited maximal activity at a temperature of 75 °C and pH 8.5. The presence of 1 mM calcium and manganese enhanced Pectate Lyase activity and was strongly inhibited by zinc, nickel and EDTA. The thermal inactivation studies revealed an entropy–enthalpy compensation pattern below a critical temperature. The alkaliphilicity and high thermostability of this Pectate Lyase may have potential implications in fibre degumming.

Jose A Mercado - One of the best experts on this subject based on the ideXlab platform.

  • the nanostructural characterization of strawberry pectins in Pectate Lyase or polygalacturonase silenced fruits elucidates their role in softening
    Carbohydrate Polymers, 2015
    Co-Authors: Sara Pose, Miguel A Quesada, Andrew R Kirby, Candelas Paniagua, Keith W Waldron, Victor J Morris, Jose A Mercado
    Abstract:

    To ascertain the role of pectin disassembly in fruit softening, chelated- (CSP) and sodium carbonate-soluble (SSP) pectins from plants with a Pectate Lyase, FaplC, or a polygalacturonase, FaPG1, downregulated by antisense transformation were characterized at the nanostructural level. Fruits from transgenic plants were firmer than the control, although FaPG1 suppression had a greater effect on firmness. Size exclusion chromatography showed that the average molecular masses of both transgenic pectins were higher than that of the control. Atomic force microscopy analysis of pectins confirmed the higher degree of polymerization as result of pectinase silencing. The mean length values for CSP chains increased from 84 nm in the control to 95.5 and 101 nm, in antisense FaplC and antisense FaPG1 samples, respectively. Similarly, SSP polyuronides were longer in transgenic fruits (61, 67.5 and 71 nm, in the control, antisense FaplC and antisense FaPG1 samples, respectively). Transgenic pectins showed a more complex structure, with a higher percentage of branched chains than the control, especially in the case of FaPG1 silenced fruits. Supramolecular pectin aggregates, supposedly formed by homogalacturonan and rhamnogalacturonan I, were more frequently observed in antisense FaPG1 samples. The larger modifications in the nanostructure of pectins in FaPG1 silenced fruits when compared with antisense Pectate Lyase plants correlate with the higher impact of polygalacturonase silencing on reducing strawberry fruit softening.

  • antisense inhibition of a Pectate Lyase gene supports a role for pectin depolymerization in strawberry fruit softening
    Journal of Experimental Botany, 2008
    Co-Authors: Nieves Santiagodomenech, Juan Munozblanco, Jose A Mercado, Silvia Jimenezbemudez, Antonio J Matas, Jocelyn K C Rose, Miguel A Quesada
    Abstract:

    Cell wall disassembly in softening fruits is a complex process involving the cumulative action of many families of wall-modifying proteins on interconnected polysaccharide matrices. One strategy to elucidate the in vivo substrates of specific enzymes and their relative importance and contribution to wall modification is to suppress their expression in transgenic fruit. It has been reported previously that inhibiting the expression of Pectate Lyase genes by antisense technology in strawberry (Fragaria x ananassa Duch.) fruit resulted in prolonged fruit firmness. This suggested that pectin depolymerization might make a more important contribution to strawberry fruit softening than is often stated. In this present study, three independent transgenic lines were identified exhibiting a greater than 90% reduction in Pectate Lyase transcript abundance. Analyses of sequential cell wall extracts from the transgenic and control fruit collectively showed clear quantitative and qualitative differences in the extractability and molecular masses of populations of pectin polymers. Wall extracts from transgenic fruits showed a reduction in pectin solubility and decreased depolymerization of more tightly bound polyuronides. Additional patterns of differential extraction of other wall-associated pectin subclasses were apparent, particularly in the sodium carbonate- and chelator-soluble polymers. In addition, microscopic studies revealed that the typical ripening-associated loss of cell-cell adhesion was substantially reduced in the transgenic fruits. These results indicate that Pectate Lyase plays an important degradative role in the primary wall and middle lamella in ripening strawberry fruit, and should be included in synergistic models of cell wall disassembly.

  • antisense inhibition of Pectate Lyase gene expression in strawberry fruit characteristics of fruits processed into jam
    Journal of Food Engineering, 2007
    Co-Authors: Rafael Sesmero, Miguel A Quesada, Jose A Mercado
    Abstract:

    We have analyzed several quality parameters of strawberry jam prepared from transgenic fruits with reduced expression of a Pectate Lyase gene. Two independent lines showing a reduction in Pectate Lyase mRNA transcript level of 90% (Apel 14) and 99% (Apel 23) have been studied. At harvest, ripen fruits from these two lines were significantly firmer than control. Soluble solid content was similar in all genotypes. Control and transgenic fruits were processed into jam and the textural properties of jam berries and medium were analyzed separately using a back extrusion test. Transgenic fruits resisted the cooking process better than control, as reflected by the higher weight of berries in these jams. The back extrusion test of jam berries showed that cooked fruits from both transgenic lines were firmer than control, and the firmness values correlated positively with the degree of Pectate Lyase silencing. By contrast, jam mediums of these lines were similar in firmness but slightly less viscous than control. Overall, the results obtained indicate that inhibition of Pectate Lyase gene expression can improve several quality traits of strawberry jam, such as texture and content of whole berries.

  • manipulation of strawberry fruit softening by antisense expression of a Pectate Lyase gene
    Plant Physiology, 2002
    Co-Authors: Silvia Jimenezbermudez, Juan Munozblanco, Miguel A Quesada, Jose Redondonevado, Jose Luis Caballero, J M Lopezaranda, Victoriano Valpuesta, Fernando Pliegoalfaro, Jose A Mercado
    Abstract:

    Strawberry (Fragaria × ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry Pectate Lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the Pectate Lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that Pectate Lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.

Juan Gao - One of the best experts on this subject based on the ideXlab platform.

  • a novel pl9 Pectate Lyase from paenibacillus polymyxa kf 1 cloning expression and its application in pectin degradation
    International Journal of Molecular Sciences, 2019
    Co-Authors: Ye Yuan, Yan Zhao, Xinyu Zhang, Yifa Zhou, Juan Gao, Han Zhang
    Abstract:

    Pectate Lyases play an important role in pectin degradation, and therefore are highly useful in the food and textile industries. Here, we report on the cloning of an alkaline Pectate Lyase gene (pppel9a) from Paenibacillus polymyxa KF-1. The full-length gene (1350 bp) encodes for a 449-residue protein that belongs to the polysaccharide Lyase family 9 (PL9). Recombinant PpPel9a produced in Escherichia coli was purified to electrophoretic homogeneity in a single step using Ni2+-NTA affinity chromatography. The enzyme activity of PpPel9a (apparent molecular weight of 45.3 kDa) was found to be optimal at pH 10.0 and 40 °C, with substrate preference for homogalacturonan type (HG) pectins vis-a-vis rhamnogalacturonan-I (RG-I) type pectins. Using HG-type pectins as substrate, PpPel9a showed greater activity with de-esterified HGs. In addition, PpPel9a was active against water-soluble pectins isolated from different plants. Using this Lyase, we degraded citrus pectin, purified fractions using Diethylaminoethyl (DEAE)-sepharose column chromatography, and characterized the main fraction MCP-0.3. High-performance gel permeation chromatography (HPGPC) analysis showed that the molecular mass of citrus pectin (~230.2 kDa) was reduced to ~24 kDa upon degradation. Ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS) and monosaccharide composition analyses demonstrated that PpPel9a worked as an endo-Pectate Lyase, which acted primarily on the HG domain of citrus pectin. In vitro testing showed that the degradation product MCP-0.3 significantly promotes the growth of Lactobacillus plantarum and L. rhamnosus. In this regard, the enzyme has potential in the preparation of pharmacologically active pectin products.

  • Screening of a Novel Polysaccharide Lyase Family 10 Pectate Lyase from Paenibacillus polymyxa KF-1: Cloning, Expression and Characterization
    MDPI AG, 2018
    Co-Authors: Yan Zhao, Ye Yuan, Xinyu Zhang, Yifa Zhou, Juan Gao
    Abstract:

    Pectate Lyase (EC 4.2.2.2) catalyzes the cleavage of α-1,4-glycosidic bonds of pectin polymers, and it has potential uses in the textile industry. In this study, a novel Pectate Lyase belonging to polysaccharide Lyase family 10 was screened from the secreted enzyme extract of Paenibacillus polymyxa KF-1 and identified by liquid chromatography-MS/MS. The gene was cloned from P. polymyxa KF-1 genomic DNA and expressed in Escherichia coli. The recombinant enzyme PpPel10a had a predicted Mr of 45.2 kDa and pI of 9.41. Using polygalacturonic acid (PGA) as substrate, the optimal conditions for PpPel10a reaction were determined to be 50 °C and pH 9.0, respectively. The Km, vmax and kcat values of PpPel10a with PGA as substrate were 0.12 g/L, 289 μmol/min/mg, and 202.3 s−1, respectively. Recombinant PpPel10a degraded citrus pectin, producing unsaturated mono- and oligogalacturonic acids. PpPel10a reduced the viscosity of PGA, and weight loss of ramie (Boehmeria nivea) fibers was observed after treatment with the enzyme alone (22.5%) or the enzyme in combination with alkali (26.3%). This enzyme has potential for use in plant fiber processing

Jiangning Song - One of the best experts on this subject based on the ideXlab platform.

  • process optimization of high level extracellular production of alkaline Pectate Lyase in recombinant escherichia coli bl21 de3
    Biochemical Engineering Journal, 2015
    Co-Authors: Huilin Wang, Jiangning Song
    Abstract:

    Abstract Eco-effective high-level production of alkaline Pectate Lyase (PL) is a necessary prerequisite for the application of this enzyme in large-scale industries, such as bioscouring. For this purpose, in this work, a multi-step glycerol feeding strategy based on specific growth rate for high cell density cultivation of recombinant Escherichia coli was first established, which could effectively control cell growth and acetate yield. Next, the effects of induction time, temperature, and inducer concentration on cell growth and extracellular secretory production of recombinant PL were investigated. When induced by IPTG (using either low IPTG concentration at 25 °C or high IPTG concentration at 30 °C), two different extracellular secretion strategies were found to be appropriate for extracellular PL production. On the other hand, when cultivations were induced by mild continuous lactose feeding strategy, the extracellular and total PL activities reached 4478 and 5337 U/mL, respectively, representing the highest PL yield reported to date.