Gewinnung und Charakterisierung von Proteasen aus <i>Thermoactinomyces vulgaris</i>. II. Selektion von leistungsfähigen Stämmen

Klingenberg, P.; Zickler, F.; Leuchtenberger, A.; Ruttloff, H.

doi:10.1002/jobm.19790190104

Cited by 13 publications

(1 citation statement)

References 6 publications

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“…2). The proposed signal peptide would thus be 29 amino acids in length. The sequence pattern A-V-A-/-L-S-P occurs at the proposed processing site, and the hydrophobicity profile (data not shown) has a good fit to the compilation of known signal peptides (53,65,66).…”

Section: Resultsmentioning

confidence: 99%

The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis

Hofemeister¹,

König²,

Hoang³

et al. 1994

Appl Environ Microbiol

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We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic a-amylase (AmyTV1). A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacilus subtilis. The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T. vulgaris 94-2A culture. The amino acid sequence was aligned with several known oa-amylase sequences. We found 83% homology with the 48-kDa a-amylase part of the Bacilus polymyxa Pj-a-amylase polyprotein and 50%o homology with Taka amylase A of AspergiUlus oryzae but only 45% homology with another T. vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47. The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B. subtilis. Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1. The expression levels in B. subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacilus amyloliquefaciens a-amylase gene. A number of thermostable enzymes have been isolated from Thermoactinomyces vulganis strains. Among these were thermitase (4, 13, 14, 28, 29), a protease from the subtilisin family (2, 5, 36), and a variety of oa-amylases (pullulanases). The a-amylases of strains R47 and 42, like fungal glucoamylases (1, 15, 49, 54), hydrolyze starch and pullulan (1, 54). The a-amylase of T. vulgaris 94-2A, however, utilizes only starch and glycogen as substrates, not pullulan. a-Amylase 1 of T. vulganis 94-2A (AmyTV1) is a protein of 53 kDa and was previously shown to exhibit striking homology to Taka amylase A of Aspergillus oryzae for a short N-terminal sequence (22, 60, 67). Smaller peptides of 33 and 18 kDa have been shown to be products of limited AmyTV1 proteolysis (21). The AmyTV1 amylase is unusual because of its temperature optimum at 62.5°C in a low pH range (4.8 to 6), its relatively short half-life of about 5 min at 70°C, and the production of maltose and maltotriose in the hydrolysate, which lacks glucose (47). In order to overcome problems of poor growth and product yield in T. vulgaris 94-2A, we cloned the gene encoding the AmyTV1 amylase and performed expression studies with Bacillus subtilis. The transcription start site(s) of the gene in B. subtilis was studied after deletion of the 5'-flanking region and after its replacement with the promoter of the B. amyloliquefaciens cx-amylase gene. MATERIALS AND METHODS Bacterial strains, plasmids, and phages. The T. vulgaris strain used was originally isolated and described by Klingenberg et al. (29, 47). The strain 94-2A was selected for higher enzyme production (29). The EMBL3 lambda phage (12) was used for preparation of a T. vulgaris DNA bank in Escherichia

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Section: Resultsmentioning

confidence: 99%

The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis

Hofemeister¹,

König²,

Hoang³

et al. 1994

Appl Environ Microbiol

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Gewinnung und Charakterisierung von Proteasen aus Thermoactinomyces vulgaris. VI. Mitt. Zur Herstellung eines Trockenpraparates im Labor‐ und kleintechnischen Mnßstab unter besonderer Berücksichtigung des Öleinflusses

Leuchtenberger

Täufel

Ruttloff

1983

Acta Biotechnologica

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A dried proteolytic enzyme preparation “Thermitase” is produced of the cultural solution of Thermoactinomyces vulgaris. The procedure comprises concentration, sedimentation, ethanol and sodiumsulfate precipitation and drying in laboratorial and semitechnical scale. The elimination of rape seed oil which impairs the salt sedimentation was specially taken into account. A certain sedimentation procedure using caoline or a aqueous solution of CaCl2 and Na2HPO4 or filtration was elaborated to eliminate the lipid components in the concentrated solution. The method is convenient to get good yields of the thermostable protease.

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Wirkung von Ölen und Fettsäuren auf Wachstum und Enzymbildung bei Thermoactinomyces vulgaris. IV. Einfluß von Art und Menge des Öles sowie des Zeitpunktes der Ölzugabe

Leuchtenberger¹,

Ruttloff²

1985

Acta Biotechnologica

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The effect of different quantities and kinds of vegetable and animal oils and the importance of the time of the oil addition on growth and protease synthesis by T. vulgaris was investigated. The used oils stimulate the protease production if they are added in a suitable concentration. However, the stimulation effect of each oil is different. Culture inoculation with spores gives the best results, when oil was added to the medium 1 up to 2 hours after beginning of the fermentation. The enzyme activity is equal to or lower than the control, when the oil addition was carried out before or 3 hours after starting the fermentation.

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Gewinnung und Charakterisierung von Proteasen aus Thermoactinomyces vulgaris. II. Selektion von leistungsfähigen Stämmen

Cited by 13 publications

References 6 publications

The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis

The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis

Gewinnung und Charakterisierung von Proteasen aus Thermoactinomyces vulgaris. VI. Mitt. Zur Herstellung eines Trockenpraparates im Labor‐ und kleintechnischen Mnßstab unter besonderer Berücksichtigung des Öleinflusses

Wirkung von Ölen und Fettsäuren auf Wachstum und Enzymbildung bei Thermoactinomyces vulgaris. IV. Einfluß von Art und Menge des Öles sowie des Zeitpunktes der Ölzugabe

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