Detection of Novel Proline 3-Hydroxylase Activities in Streptomyces and Bacillus spp. by Regio- and Stereospecific Hydroxylation of l-Proline

Abstract

During the screening of microbial proline hydroxylases, novel proline 3-hydroxylase activities, which hydroxylate free L-proline to free cis-3-hydroxy-L-proline, were detected in whole cells of Streptomyces sp. strain TH1 and Bacillus sp. strains TH2 and TH3 from 3,000 strains isolated from soil. The reaction product was purified from a reaction mixture of Streptomyces sp. strain TH1, and its chemical structure was identified as cis-3-hydroxy-L-proline by instrumental analyses. Proline 3-hydroxylase activity was also detected in Streptomyces canus ATCC 12647 which produces the 3-hydroxyproline-containing peptide antibiotic telomycin. Bacillus sp. strains TH2 and TH3 were found to accumulate cis-3-hydroxy-L-proline in culture media at 426 and 352 M, respectively. It was suggested that hydroxylation occurred in a highly regio-and stereospecific manner at position 3 of L-proline because no hydroxylation product other than cis-3-hydroxy-L-proline was observed. Proline 3-hydroxylases of these strains were first characterized on crude enzyme preparations. Since 2-oxoglutarate and ferrous ion were required for hydroxylation of L-proline, these 3-hydroxylases were thought to belong to a family of 2-oxoglutarate-related dioxygenases. The reaction was inhibited by Co 2؉ , Zn 2؉ , and Cu 2؉. L-Ascorbic acid accelerated the reaction. The optimum pH and temperature were 7.5 and 35؇C, respectively.

Microbial Proline 4-Hydroxylase Screening and Gene Cloning

“…These five actinomycete strains produced no etamycin as far as we could determine (Table 1). During the screening study we also found that Streptomyces and Bacillus strains produced proline 3-hydroxylases as well as telomycin-producing Streptomyces strains produce these enzymes (20). Our preliminary results suggested that the strains identified in this screening study produced peptidelike compounds containing hydroxyprolines other than etamycin and telomycin (20).…”

“…Screening for microbial proline 4-hydroxylases. Screening for proline 4-hydroxylases was done by the whole-cell reaction method as described previously (20). The screening procedure was focused mainly on actinomycetes grown on HT medium to which hydroxyproline-containing actinomycin I or proline analogs were sometimes added.…”

“…Culture medium and cultural conditions. HT medium and SR3 medium have been described previously (20). Df1 medium consisted of 50 g of soluble starch per liter, 15 g of soy bean meal per liter, 0.5 g of KH 2 PO 4 per liter, 0.5 g of MgSO 4 ⅐ 7H 2 O per liter, and 5 g of calcium carbonate per liter, and the pH was adjusted to 7.0.…”

“…Cellular proline 4-hydroxylase activities were measured by the whole-cell reaction procedure. Whole-cell reactions were performed as described previously (20). Cell-free enzyme activities were assayed as follows.…”

Construction and optimization of trans‐4‐hydroxy‐L‐proline production recombinant E. coli strain taking the glycerol as carbon source

“…The Hyp content was measured by a method utilizing chloramine‐T method . The P4H activity of whole cells was tested by the whole‐cell reaction procedure, which was implemented as described above . All of the results are the mean values of at least three measurements (the accuracy is considered to be better than ± 5%) for every experimental point.…”