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L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC 90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine, food industry, and diagnostics. Among various organisms expressing L-ASNases, thermophiles and hyperthermophiles produce enzymes with superior performances-stable and heat resistant thermo-ASNases. This review is an attempt to take a broader view on the thermo-ASNases. Here we discuss the position of thermo-ASNases in the large family of L-ASNases, their role in the heat-tolerance cellular system of thermophiles and hyperthermophiles, and molecular aspects of their thermoactivity and thermostability. Different types of thermo-ASNases exhibit specific L-asparaginase activity and additional secondary activities. All products of these enzymatic reactions are associated with diverse metabolic pathways and are important for mitigating heat stress. Thermo-ASNases are quite distinct from typical mesophilic L-ASNases based on structural properties, kinetic and activity profiles. Here we attempt to summarize the current understanding of the molecular mechanisms of thermo-ASNases' thermoactivity and thermostability, from amino acid composition to structural-functional relationships. Research of these enzymes has fundamental and biotechnological significance. Thermo-ASNases and their improved variants, cloned and expressed in mesophilic hosts, can form a large pool of enzymes with valuable characteristics for biotechnological application.

L-asparaginase (L-ASNase) is a biotechnologically relevant enzyme for the pharmaceutical, biosensor and food industries. Efforts to discover new promising L-ASNases for different fields of biotechnology have turned this group of enzymes into a growing family with amazing diversity. Here, we report that thermophile Melioribacter roseus from Ignavibacteriae of the Bacteroidetes/Chlorobi group possesses two L-ASNases-bacterial type II (MrAII) and plant-type (MrAIII). The current study is focused on a novel L-ASNase MrAII that was expressed in Escherichia coli , purified and characterized. The enzyme is optimally active at 70 °C and pH 9.3, with a high L-asparaginase activity of 1530 U/mg and L-glutaminase activity ~19% of the activity compared with L-asparagine. The kinetic parameters K M and V max for the enzyme were 1.4 mM and 5573 µM/min, respectively. The change in MrAII activity was not significant in the presence of 10 mM Ni 2+ , Mg 2+ or EDTA, but increased with the addition of Cu 2+ and Ca 2+ by 56% and 77%, respectively, and was completely inhibited by Zn 2+ , Fe 3+ or urea solutions 2-8 M. MrAII displays differential cytotoxic activity: cancer cell lines K562, Jurkat, LnCap, and SCOV-3 were more sensitive to MrAII treatment, compared with normal cells. MrAII represents the first described enzyme of a large group of uncharacterized counterparts from the Chlorobi-Ignavibacteriae-Bacteroidetes clade.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli , purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters K M and V max for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni 2+ , Cu 2+ , Mg 2+ , Zn 2+ and Ca 2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe 3+ . The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production., Competing Interests: The authors have declared that no competing interests exist.

L-asparaginase is extensively used in the treatment of acute lymphoblastic leukemia and several other lymphoproliferative diseases. In addition to its biomedical application, L-asparaginase is also of prospective use in food industry to reduce the formation of acrylamide, which is classified as probably neurotoxic and carcinogenic to human, and in biosensors for determination of L-asparagine level in medicine and food chemistry. The importance of L-asparaginases in different fields, disadvantages of commercial ferments, and the fact that they are widespread in nature stimuli the search for biobetter L-asparaginases from new producing microorganisms. In this regard, extremofile microorganisms exhibit unique physiological properties such as thermal stability, adaptability to extreme cold conditions, salt and pH tolerance and so provide one of the most valuable sources for novel L-asparaginases. The present review summarizes the recent results on studying the structural, functional, physicochemical and kinetic properties, stability of extremophilic L-asparaginases in comparison with their mesophilic homologues.

Cephalosporin acid synthetase (CASA) is responsible for specific to synthesis of cephalosporin-acids, its expression in Escherichia coli cells is accompanied by accumulation of unprocessed insoluble precursor. In order to optimize conditions of recombinant CASA production we have studied the effects of several parameters of strain cultivation, including growth media composition, temperature, and inoculation dose. Also plasmids for production of CASA variants with the signal sequence of Erwinia carotovora L-asparaginase (ansCASA) and "leaderless" CASA were created in search of more efficient expression constructs. Removal of the N-terminal secretion signal sequence reduced the production of functionally active CASA more than 10-fold and inhibited strain growth. Insertion of the L-asparaginase signal sequence increased the specific enzyme activity in the resultant recombinant strain. The ansCASA producing strain was used to develop the method of immobilization of the recombinant enzyme on an epoxy-activated macroporous acrylic support. The resultant biocatalyst performed effective synthesis of cefazolin from 3-[(5-methyl-1,3,4-thiadiazol-2-il)-thiomethyl]-7- aminocephalosporanic acid (MMTD-7-ACA) and methyl ester of 1(H)-tetrazolilacetic acid (МETzAA), under mild conditions a transformation level of MMTD-7-ACA to cefazolin of 95% is reached.

An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of β-lactam antibiotics of the cephalosporin acids class (cefazolin, cefalotin, cefezole etc.). A comparison of the previously determined genomic sequence of E. coli VKPM B-10182 with a genome of the parent E. coli strain ATCC 9637 was performed. Multiple mutations indicating the long selection history of the strain were detected, including mutations in the genes of RNase and β-lactamases that could enhance the level of enzyme synthesis and reduce the degree of degradation of the synthesized cephalosporin acids. The CASA gene--a direct homolog of the penicillin G-acylase gene--was identified by bioinformatics methods. The homology of the gene was confirmed by gene cloning and the expression and determination of its enzymatic activity in the reaction of cefazolin synthesis. The CASA gene was isolated and cloned into the original expression vector, resulting in an effective E. coli BL2l(DE3) pMD0107 strain producing CASA.

Inorganic polyphosphate performs many regulatory functions in living cells. The yeast exopolyphosphatase PPN1 is an enzyme with multiple cellular localization and probably variable functions. The Saccharomyces cerevisiae strain with overexpressed PPN1 was constructed for large-scale production of the enzyme and for studying the effect of overproduction on polyphosphate metabolism. The ΔPPN1 strain was transformed by the vector containing this gene under a strong constitutive promoter of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae. Exopolyphosphatase activity in the transformant increased 28- and 11-fold compared to the ΔPPN1 and parent strains, respectively. The content of acid-soluble polyphosphate decreased ∼6-fold and the content of acid-insoluble polyphosphate decreased ∼2.5-fold in the cells of the transformant compared to the ΔPPN1 strain. The recombinant enzyme was purified. The substrate specificity, cation requirement, and inhibition by heparin were found to be similar to native PPN1. The molecular mass of a subunit (∼33 kD) and the amino acid sequence of the recombinant enzyme were the same as in mature PPN1. The recombinant enzyme was localized mainly in the cytoplasm (40%) and vacuoles (20%). The overproducer strain had no growths defects under phosphate deficiency or phosphate excess. In contrast to the parent strains accumulating polyphosphate, the transformant accumulated orthophosphate under phosphate surplus., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Vectors for the expression of the CefT transporter of the MFS family in Acremonium chrysogenum--a producer of beta-lactam antibiotic cephalosporin C--and in Saccharomyces cerevisiae as a fusion with the cyan fluorescent protein (CFP) have been created. The subcellular localization of the CefT-CFP hybrid protein in yeast cells has been investigated. It was shown that the CefT-CFP hybrid protein is capable of complementation of the qdr3, tpo 1, and tpo3 genes encoding for orthologous MFS transporters of Saccharomycetes, making the corresponding strains resistant to spermidine, ethidium bromide, and hygromycin B. High-yield strain VKM F-4081D of A. chrysogenum, expressing the cefT-cfp fusion, was obtained by an agrobacteria conjugated transfer. It was also shown that the constitutive expression of cefT in A. chrysogenum VKM F-4081D led to a change in the biosynthetic profiles of cephalosporin C and its precursors. This resulted in a 25-35% decrease in the finite product accumulated in the cultural liquid with a simultaneous increase in the concentration of its intermediators.

Using pulse electrophoresis in controlled homogenous electric field we conducted molecular karyotyping of highly-productive and laboratory strains of Acremonium chrysogenum generating antibiotic cephalosporin C (cefC). Differences in size of several chromosomes of highly active strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the highly active strain was not associated with alteration of localization and copy number ofcephalosporin C biosynthesis and transport genes. A cluster of "early" cefC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the "late genes", on chromosome II (2.3 Mb). Both clusters are presented as a single copy perA. chrysogenum genome in the wild-type and in CB26/8 producer strains. Based on comparative analysis of laboratory and industrial cefC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.