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Chitinase B of "Microbulbifer degradans" 2-40 Contains Two Catalytic Domains with Different Chitinolytic Activities. Michael B. Howard, 2004.Chitinase B of "Microbulbifer degradans" 2-40 is a modular protein that is predicted to contain two glycoside hydrolase family18 [GH18] catalytic domains, two polyserine domains, and anacidic repeat domain . Each of the GH18 domains was shown tobe catalytically active against chitin . Activity assays revealthat the amino-terminal catalytic domain [GH18N] releases methylumbelliferonefrom 4'-methylumbelliferyl-N,N'-diacetylchitobiose 13.6-fold faster than the carboxy-terminal catalytic domain [GH18C] and releases chitobiose from the nonreducing end of chitooligosaccharides, therefore functioning as an exochitinase . GH18C releases methylumbelliferonefrom 4'-methylumbelliferyl-N,N',N"-triacetylchitotriose 2.7-foldfaster than GH18N and cleaves chitooligosaccharides at multiplebonds, consistent with endochitinolytic activity . Each domainwas maximally active from 30 to 37°C and from pH 7.2 to8.0 and was not affected by Mg2+, Mn2+, Ca2+, K+, EDTA, EGTA,or 1.0 M NaCl . The activity of each domain was moderately inhibitedby Ni2+, Sr2+, and Cu2+, while Hg2+ completely abolished activity.When the specific activities of various recombinant portionsof ChiB were calculated by using native chitin as a substrate,the polypeptide containing the endo-acting domain was twofoldmore active on native chitin than the other containing the exo-actingdomain . The presence of both domains in a single reaction increasedthe amount of reducing sugars released from native chitin to140% above the theoretical combined rate, indicating that the domains function cooperatively to degrade chitin . These data demonstrate that the GH18 domains of ChiB have different activities on the same substrate and function cooperatively to enhancechitin depolymerization. Thermus thermophilus as a Cell Factory for the Production of a Thermophilic Mn-Dependent Catalase Which Fails To Be Synthesized in an Active Form in Escherichia coli. Aurelio Hidalgo, 2004.Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H2O2-detoxifying systems . We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G) . When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins . Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used . Therefore, a requirement for a Thermus-specific activation factor was suggested . To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T . thermophilus under the control of the Pnar promoter . This promoter belongs to a respiratory nitrate reductase from of T . thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia . Upon induction in T . thermophilus HB8, a 20- to 30-fold increase in catalase specific activity was observed, whereas a 90- to 110-fold increase was detected when the laboratory strain T . thermophilus HB27::nar was used as the host . The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative . Therefore, our results validate the use of T . thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts . Effects of Multiple Deletions of Murein Hydrolases on Viability, Septum Cleavage, and Sensitivity to Large Toxic Molecules in Escherichia coli. Christoph Heidrich, 2002.The multiplicity of murein hydrolases found in most bacteria presents an obstacle to demonstrating the necessity of these potentially autolytic enzymes . Therefore, Escherichia coli mutants with deletions in multiple murein hydrolases, including lytic transglycosylases, amidases, and DD-endopeptidases, were constructed . Even a mutant from which seven different hydrolases were deleted was viable and grew at a normal rate . However, penicillin-induced lysis was retarded . Most of the mutants were affected in septum cleavage, which resulted in the formation of chains of cells . All three enzymes were shown to be capable of splitting the septum . Failure to cleave the septum resulted in an increase in outer membrane permeability, and thus the murein hydrolase mutants did not grow on MacConkey agar plates . In addition, the hydrolase mutants not only could be lysed by lysozyme in the absence of EDTA but also were sensitive to high-molecular-weight antibiotics, such as vancomycin and bacitracin, which are normally ineffective against E . coli. Arginine-Agmatine Antiporter in Extreme Acid Resistance in Escherichia coli. Ram Iyer, 2003.The process of arginine-dependent extreme acid resistance (XAR) is one of several decarboxylase-antiporter systems that protects Escherichia coli and possibly other enteric bacteria from exposure to the strong acid environment of the stomach . Arginine-dependent acid resistance depends on an intracellular proton-utilizing arginine  -decarboxylase and a membrane transport protein necessary for delivering arginine to and removing agmatine, its decarboxylation product, from the cytoplasm . The arginine system afforded significant protection to wild-type E . coli cells in our acid shock experiments . The gene coding for the transport protein is identified here as a putative membrane protein of unknown function, YjdE, which we now name adiC . Strains from which this gene is deleted fail to mount arginine-dependent XAR, and they cannot perform coupled transport of arginine and agmatine . Homologues of this gene are found in other bacteria in close proximity to homologues of the arginine decarboxylase in a gene arrangement pattern similar to that in E coli . Evidence for a lysine-dependent XAR system in E . coli is also presented . The protection by lysine, however, is milder than that by arginine .
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