Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates

Biocatalysis has been increasingly used for pharmaceutical synthesis in an effort to make manufacturing processes greener and more sustainable. Biocatalysts that possess excellent activity, specificity, thermostability and solvent-tolerance are highly sought after to meet the requirements of practical applications. Generating biocatalysts with these specific properties can be achieved by either discovery of novel biocatalysts or protein engineering. Meanwhile, chemoenzymatic routes have also been designed and developed for pharmaceutical synthesis on an industrial scale. This review discusses the recent discoveries, engineering, and applications of biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. Key classes of biocatalysts include reductases, oxidases, hydrolases, lyases, isomerases, and transaminases.     

Production of soluble and active microbial transglutaminase in Escherichia coli for site‐specific antibody drug conjugation

Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro‐domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine‐scan of the mTGase pro‐domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro‐domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro‐domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30–75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site‐specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.     

Dedifferentiation of gland cells in hydra and further development of interstitial cells arising from them

Summary Asexual, non-budding hydras were treated in the 175000 solution of E 39 solubile (Bayer). They were feeding, growing and budding for six days. The interstitial cells found in the ectoderm at the moment of treatment differentiated into cnidoblasts during that period. The cells that happened to be in the gastroderm at that time, differentiated into interstitial cells which were not able to cross the mesoglea due to the E 39 activity. In the gastroderm a great number of cnids appeared.These observations support the hypothesis that in normal hydra the interstitial cells formed by the dedifferentiation of gland cells pass into the ectoderm. There they are transformed into cnidoblasts which pass into the mesoglea. Prom the gastroderm the cnidoblasts come into the ectoderm of the tentacles through endodermal cells and the coelenteric fluid.

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Zusammenfassung Asexuelle, knospenlose Hydren wurden mit einer Lösung von Bayer E 39 (175000), solubile behandelt. Einige Tiere wurden nach 4, 6, 10 und 24 Std fixiert, die übrigen nach 24 Std in das Aquariumwasser zurückversetzt. Diese Hydren wurden in Zeitabschnitten von 24 Std innerhalb von 8 Tagen fixiert.Im Verlauf der ersten 6 Tage fressen und wachsen alle Hydren und bilden Knospen. Am 7. Tag (bei einigen am 8. Tag) beginnt die Depression: Die Nahrungsaufnahme hört auf, die Polypen kontrahieren sich allmählich und ihre Tentakel verkürzen sich. Bis zum 16. Tag sind alle Hydren eingegangen. In den ersten Stunden nach der Behandlung (bis zu 10 Std) sind imEktoderm fast gar keine Veränderungen zu bemerken, imGastroderm kommt es jedoch zu Veränderungen in der Größe, Form und im Bau der zymogenen Zellen. Nach dem 1. Tag verschwinden die interstitiellen Zellen allmählich, aber die Cnidoblasten und die Cniden verbleiben vollzählig bis zum 7. Tag, dann aber nimmt die Zahl der Cnidoblasten ab. In geringerer Zahl bleiben jedoch die Cniden bis zum Lebensende erhalten. Die zymogenen Zellen dedifferenzieren sich zu interstitiellen Zellen, einige unter ihnen verwandeln sich in kleine kugelförmige, ausgesprochen basophile Zellen, welche sich an der Mesogloea ansammeln.Am Anfang, wenn E 39 noch nicht zu voller Wirkung gekommen ist, durchwandern interstitielle Zellen die Mesogloea und gliedern sich in das Ektoderm ein. Dieser Zellübergang nimmt allmählich ab, hört aber niemals vollkommen auf. Während dieser Zeit häufen sich immer mehr reife Cniden im Gastroderm an. Die Autoren sind der Meinung, daß es sich hierbei um Cniden handelt, die sich einerseits aus jenen Cnidoblasten entwickelt haben, die am Anfang der Behandlung aus dem Ektoderm in das Gastroderm eingewandert sind, andererseits von interstitiellen Zellen abstammen, die durch Dedifferenzierung aus zymogenen Zellen entstanden und im Gastroderm übriggeblieben sind. Sie entwickelten sich dort, weil sie infolge der Wirkung von E 39 weder durch die Mesogloea hindurchtreten, noch in die Tentakel einwandern konnten. Diese Prozesse werden in der Arbeit ausführlich diskutiert.Auf Grund dieser Beobachtungen nehmen die Autoren an, daß auch in der normalen Hydra die aus zymogenen Zellen entstandenen interstitiellen Zellen in das Ektoderm einwandern, wo sie sich teilen und zu Cnidoblasten ausdifferenzieren.Ein Teil dieser Cnidoblasten wandert durch die Mesogloea in das Gastroderm ein, aber aus dem Gastroderm mittels entodermaler Zellen und gastraler Flüssigkeit wieder aus und gelangt schließlich in das Ektoderm der Tentakel.

     

Das Gastroderm im Prozeß der Regeneration der Hydra

Zusammenfassung Es wurden Amputationen an Hydren in der hypostomalen Region durchgeführt. Das Material wurde in verschiedenen Zeitabschnitten fixiert (von 5 min bis 19 Std). Die Heilung der Wunde findet unter Mitwirkung aller entodermalen Zellen statt. Der Prozeß der Regeneration geht von zymogenen Zellen aus, welche sich zu Neoblasten entdifferenzieren. Diese wandeln sich durch Differentiation in ektodermale Zellelemente um. Das alte Ektoderm hat an der Bildung des jungen Ektoderms im Gebiete der Wunde keinen Anteil.Der Wandel des Entoderms im Verlaufe der Heilung der Wunde und der Regeneration wurde mit Hilfe histochemischer Methode zur Darstellung der sauren Phosphatase verfolgt. Es wurde die Annahme geäußert, daß die zymogenen Zellen auch bei der normalen Hydra das Ektoderm mit neuen Zellen versorgen.

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The gastroderm in the process of the regeneration of hydra

Summary The amputations of the hydra in the hypostomal region have been performed. The material has been fixed in various time intervals after the amputation (from 5 min to 19 hours).The healing of the wound is done with the participation of all endodermal cells, the regeneration process with the help of zymogenic cells which dedifferenciate into neoblasts, and the neoblasts develop into ectodermal cell elements by differentiation. The old ectoderm does not take part in forming the young ectoderm in the wound area.The participation of endoderm in healing the wound and in the regeneration has been stated by the method for the acid phosphatase. It is supposed that the zymogenic cells also in normal hydra supply the ectoderm with new cells.

     

Rho GTPases and spermatogenesis

Rho GTPases, such as Rho, Rac and Cdc42, are known to regulate many cellular processes including cell movement and cell adhesion. While the cellular events of germ cell movement are crucial to spermatogenesis since developing germ cells must migrate progressively from the basal to the adluminal compartment but remain attached to the seminiferous epithelium, the physiological significance of Rho GTPases in spermatogenesis remains largely unexplored. This paper reviews some recent findings on Rho GTPases in the field with emphasis on the studies in the testis, upon which future studies can be designed to delineate the role of Rho GTPases in spermatogenesis.     

甲壳质脱乙酰基酶的研究概况及进展

甲壳质脱乙酰基酶（ｃｈｉｔｉｎｄｅａｃｅｔｙｌａｓｅ）最初是从真菌毛霉（Ｍｕｃｏｒ．ｒｏｕｘｉ）分离纯化的一种乙酰基转移酶。这种酶可以催化脱去甲壳质分子中Ｎ－乙酰葡糖胺链上的乙酰基,而使之变成壳多糖［１］。除几种真菌外,在昆虫中也发现了这种酶的存在［２］。真菌的甲壳质脱乙酰基酶主要参与真菌细胞壁的形成［３］,还与真菌自溶的过程中的细胞壁裂解有关［４］。最近又发现它参与植物和病原微生物的相互作...     

The role of the Robertsonian rearrangements in the origin of the XX/XY1Y2 sex chromosome system and in the chromosomal differentiation in Harttia species (Siluriformes, Loricariidae)

Harttia is a genus of the subfamily Loricariinae that posses a broad chromosomal variation. In addition to interspecific karyotype diversity within this group, a multiple sex chromosome system, XX/XY₁Y₂, has been described for Harttia carvalhoi. Thus, this study aimed to determine the role of chromosomal rearrangements in karyotype differentiation in Harttia by classical and molecular cytogenetic procedures. The results show that Robertsonian rearrangements have a prominent role in the chromosomal diversification of the species analysed, which initially leads to hypothesize a diploid number reduction in Harttia torrenticola and H. carvalhoi. The metacentric chromosome 1, shared between H. torrenticola and H. carvalhoi, could have originated from centric fusions from the ancestral karyotype. A centric fission event associated with the first metacentric pair allowed for the origination of a multiple sex chromosome system XX/XY₁Y₂, specific to H. carvalhoi. This study highlights the relevance of Robertsonian rearrangements in karyotypic differentiation of the species studied and demonstrates that the occurrence of a centric fission, as opposed to a previously hypothesised chromosome fusion, is directly implicated in the origin of the sex chromosome system of H. carvalhoi.