A Gelatin Hydrogel Nonwoven Fabric Combined With Adipose Tissue-Derived Stem Cells Enhances Subcutaneous Islet Engraftment.

Subcutaneous islet transplantation is a promising treatment for severe diabetes; however, poor engraftment hinders its prevalence. We previously revealed that a gelatin hydrogel nonwoven fabric (GHNF) markedly improved subcutaneous islet engraftment. We herein investigated whether the addition of adipose tissue-derived stem cells (ADSCs) to GHNF affected the outcome. A silicone spacer sandwiched between two GHNFs with (AG group) or without (GHNF group) ADSCs, or a silicone spacer alone (Silicone group) was implanted into the subcutaneous space of healthy mice at 6 weeks before transplantation, then diabetes was induced 7 days before transplantation. Syngeneic islets were transplanted into the pretreated space. Intraportal transplantation (IPO group) was also performed to compare the transplant efficiency. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, and inflammatory mediators were evaluated. The results in the subcutaneous transplantation were compared using the Silicone group as a control. The results of the IPO group were also compared with those of the AG group. The AG group showed significantly better blood glucose changes than the Silicone and the IPO groups. The cure rate of AG group (72.7%) was the highest among the groups (GHNF; 40.0%, IPO; 40.0%, Silicone; 0%). The number of vWF-positive vessels in the subcutaneous space of the AG group was significantly higher than that in other groups before transplantation (P < 0.01). Lectin angiography also showed that the same results (P < 0.05). According to the results of the ADSCs tracing, ADSCs did not exist at the transplant site (6 weeks after implantation). The positive rates for laminin and collagen III constructed around the transplanted islets did not differ among groups. Inflammatory mediators were higher in the Silicone group, followed by the AG and GHNF groups. Pretreatment using bioabsorbable scaffolds combined with ADSCs enhanced neovascularization in subcutaneous space, and subcutaneous islet transplantation using GHNF with ADSCs was superior to intraportal islet transplantation.

A Recombinant Peptide Device Combined with Adipose Tissue-Derived Stem Cells Enhances Subcutaneous Islet Engraftment.

Subcutaneous space has been considered an attractive site for islet graft transplantation; however, the oxygen tension and vascularization are insufficient for islet graft survival. We investigated whether subcutaneous pre-implantation of a recombinant peptide (RCP) device with adipose tissue-derived stem cells (ADSCs) enhanced subcutaneous islet engraftment. RCP devices with/without syngeneic ADSCs were pre-implanted into the subcutaneous space of C57BL/6 mice. Syngeneic islets (300 or 120 islet equivalents (IEQs)) were transplanted into the pre-treated space after diabetes induction using streptozotocin. The cure rates of groups in which RCP devices were implanted four weeks before transplantation were significantly better than the intraportal transplantation group when 300 IEQs of islets were transplanted (p < 0.01). The blood glucose changes in the RCP+ADSCs-4w group was significantly ameliorated in comparison to the RCP-4w group when 120 IEQs of islets were transplanted (p < 0.01). Immunohistochemical analyses showed the collagen III expression in the islet capsule of the RCP+ADSCs-4w group was significantly enhanced in comparison to the RCP-4w and RCP+ADSCs-d10 groups (p < 0.01, p < 0.01). In addition, the number of von Willebrand factor-positive vessels within islets in the RCP+ADSCs-4w group was significantly higher than the RCP-4w group. These results suggest that using ADSCs in combination with an RCP device could enhance the restoration of the extracellular matrices, induce more efficient prevascularization within islets, and improve the graft function.

Ideal Duration of Pretreatment Using a Gelatin Hydrogel Nonwoven Fabric Prior to Subcutaneous Islet Transplantation.

Subcutaneous islet transplantation is a promising treatment for severe diabetes; however, poor engraftment hinders its prevalence. We previously revealed that a gelatin hydrogel nonwoven fabric (GHNF) markedly improved subcutaneous islet engraftment in comparison with intraportal islet transplantation. We herein investigated whether the duration of pretreatment using GHNF affected the outcome of subcutaneous islet transplantation. A silicone spacer with GHNF was implanted into the subcutaneous space of healthy mice at 2, 4, 6, or 8 weeks before transplantation, and then diabetes was induced 7 days before transplantation. Syngeneic islets were transplanted into the pretreated space. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, inflammatory mediators, and gene expression were evaluated. The 6-week group showed significantly better blood glucose changes than the other groups (P < 0.05). The cure rate of the 6-week group (60.0%) was the highest among the groups (2-week = 0%, 4-week = 50.0%, 8-week = 15.4%). The number of von Willebrand factor (vWF)-positive vessels in the 6-week group was significantly higher than in the other groups at pre-islet and post-islet transplantation (P < 0.01 [vs 2-and 4-week groups] and P < 0.05 [vs all other groups], respectively). Notably, this beneficial effect was also observed when GHNF was implanted into diabetic mice injected with streptozotocin 7 days before GHNF implantation. The positive rates for laminin, collagen III, and collagen IV increased as the duration of pretreatment became longer and were significantly higher in the 8-week group (P < 0.01). Inflammatory mediators, including interleukin (IL)-1b, granulocyte colony-stimulating factor (G-CSF), and interferon (IFN)-γ, were gradually downregulated according to the duration of GHNF pretreatment and re-elevated in the 8-week group. Taken together, the duration of GHNF pretreatment apparently had an impact on the outcomes of subcutaneous islet transplantation, and 6 weeks appeared to be the ideal duration. Islet graft revascularization, extracellular matrix compensation of the islet capsule, and the inflammatory status at the subcutaneous space would be crucial factors for successful subcutaneous islet transplantation.

A gelatin hydrogel nonwoven fabric improves outcomes of subcutaneous islet transplantation.

Subcutaneous islet transplantation is a promising treatment for severe diabetes; however, poor engraftment hinders its prevalence. We previously reported that a recombinant peptide (RCP) enhances subcutaneous islet engraftment. However, it is impractical for clinical use because RCP must be removed when transplanting islets. We herein investigated whether a novel bioabsorbable gelatin hydrogel nonwoven fabric (GHNF) could improve subcutaneous islet engraftment. A silicon spacer with or without GHNF was implanted into the subcutaneous space of diabetic mice. Syngeneic islets were transplanted into the pretreated space or intraportally (Ipo group). Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, CT angiography and gene expression were evaluated. The cure rate and glucose tolerance of the GHNF group were significantly better than in the control and Ipo groups (p < 0.01, p < 0.05, respectively). In the GHNF group, a limited increase of vWF-positive vessels was detected in the islet capsule, whereas laminin (p < 0.05), collagen III and IV were considerably enhanced. TaqMan arrays revealed a significant upregulation of 19 target genes (including insulin-like growth factor-2) in the pretreated space. GHNF markedly improved the subcutaneous islet transplantation outcomes, likely due to ECM compensation and protection of islet function by various growth factors, rather than enhanced neovascularization.

A Gelatin Hydrogel Nonwoven Fabric Enhances Subcutaneous Islet Engraftment in Rats.

Although subcutaneous islet transplantation has many advantages, the subcutaneous space is poor in vessels and transplant efficiency is still low in animal models, except in mice. Subcutaneous islet transplantation using a two-step approach has been proposed, in which a favorable cavity is first prepared using various materials, followed by islet transplantation into the preformed cavity. We previously reported the efficacy of pretreatment using gelatin hydrogel nonwoven fabric (GHNF), and the length of the pretreatment period influenced the results in a mouse model. We investigated whether the preimplantation of GHNF could improve the subcutaneous islet transplantation outcomes in a rat model. GHNF sheets sandwiching a silicone spacer (GHNF group) and silicone spacers without GHNF sheets (control group) were implanted into the subcutaneous space of recipients three weeks before islet transplantation, and diabetes was induced seven days before islet transplantation. Syngeneic islets were transplanted into the space where the silicone spacer was removed. Blood glucose levels, glucose tolerance, immunohistochemistry, and neovascularization were evaluated. The GHNF group showed significantly better blood glucose changes than the control group (p < 0.01). The cure rate was significantly higher in the GHNF group (p < 0.05). The number of vWF-positive vessels was significantly higher in the GHNF group (p < 0.01), and lectin angiography showed the same tendency (p < 0.05). The expression of laminin and collagen III around the transplanted islets was also higher in the GHNF group (p < 0.01). GHNF pretreatment was effective in a rat model, and the main mechanisms might be neovascularization and compensation of the extracellular matrices.

Development of a novel method for measuring tissue oxygen pressure to improve the hypoxic condition in subcutaneous islet transplantation.

Subcutaneous tissue is a promising site for islet transplantation, but poor engraftment, due to hypoxia and low vascularity, hinders its prevalence. However, oxygen partial pressure (pO2) of the subcutaneous space (SC) and other sites were reported to be equivalent in several previous reports. This contradiction may be based on accidental puncture to the indwelling micro-vessels in target tissues. We therefore developed a novel optical sensor system, instead of a conventional Clark-type needle probe, for measuring tissue pO2 and found that pO2 of the SC was extremely low in comparison to other sites. To verify the utility of this method, we transplanted syngeneic rat islets subcutaneously into diabetic recipients under several oxygenation conditions using an oxygen delivery device, then performed pO2 measurement, glucose tolerance, and immunohistochemistry. The optical sensor system was validated by correlating the pO2 values with the transplanted islet function. Interestingly, this novel technique revealed that islet viability estimated by ATP/DNA assay reduced to less than 75% by hypoxic condition at the SC, indicating that islet engraftment may substantially improve if the pO2 levels reach those of the renal subcapsular space. Further refinements for a hypoxic condition using the present technique may contribute to improving the efficiency of subcutaneous islet transplantation.

Strategy towards tailored donor tissue-specific pancreatic islet isolation.

BACKGROUND: Optimizing the collagenase G (ColG):collagenase H (ColH) ratio is a key strategy for achieving tailored donor-tissue specific islet isolation. Collagen V (Col V) and collagen III (Col III) are crucial target matrices of ColG and ColH, respectively. We herein investigated the relevance between the expression of target matrices in pancreatic tissues and influence of ColG:ColH ratio on islet isolation outcome. METHODS: Islet isolation was performed in Lewis and SD rats using different ColG:ColH ratios (5:1, 1:1 and 1:5; n = 7/group). The composition of Col III and Col V was examined using immunohistochemical staining, real-time polymerase chain reaction (PCR), Western blotting and mass spectrometry. Chain types in collagen I (Col I) were also assessed using mass spectrometry. RESULTS: No beneficial effects were observed by increasing the ColG amount, irrespective of the rat strain. In contrast, the islet yield in Lewis rats was considerably increased by high amounts of ColH but decreased in SD rats, suggesting that Lewis pancreas contains more Col III than SD pancreas. Neither immunohistochemical nor real-time PCR showed correlation with isolation outcome. However, Western blotting revealed that Lewis contained considerably higher amount of Col III than SD (p = 0.10). Likewise, Col-I(α1)/Col-III(α1) and Col-I(α2)/Col-III(α1) were significantly lower in Lewis than in SD rats (p = 0.007, respectively). Furthermore, the isolation outcome was considerably correlated with the composition of homotrimeric Col I. CONCLUSIONS: The Col III expression and the composition of homotrimeric Col I in pancreatic tissues determined using mass analyses appeared useful for optimizing the ColG:ColH ratio in islet isolation.

Collagen V Is a Potential Substrate for Clostridial Collagenase G in Pancreatic Islet Isolation.

The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans.

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate.

Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation.

BACKGROUND: Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. METHODS: The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. RESULTS: The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. CONCLUSIONS: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.

Collagenase H is crucial for isolation of rat pancreatic islets.

The role(s) of collagenase G (ColG) and collagenase H (ColH) during pancreatic islet isolation remains controversial, possibly due to the enzyme blends used in the previous studies. We herein examined the role of ColG and ColH using highly pure enzyme blends of recombinant collagenase of each subtype. Rat pancreases were digested using thermolysin, together with ColG, ColH, or ColG/ColH (n = 9, respectively). No tryptic-like activity was detected in any components of the enzyme blends. The efficiency of the collagenase subtypes was evaluated by islet yield and function. Immunohistochemical analysis, in vitro collagen digestion assay, and mass spectrometry were also performed to examine the target matrix components of the crucial collagenase subtype. The islet yield was highest in the ColG/ColH group (4,101 ± 460 islet equivalents). A substantial number of functional islets (2,811 ± 581 islet equivalents) was obtained in the ColH group, whereas no islets were retrieved in the ColG group. Mass spectrometry demonstrated that ColH reacts with collagen I and III. In the immunohistochemical analysis, both collagen I and III were located in exocrine tissues, although collagen III expression was more pronounced. The collagen digestion assay showed that collagen III was more effectively digested by ColH than by ColG. The present study reveals that ColH is crucial, while ColG plays only a supporting role, in rat islet isolation. In addition, collagen III appears to be one of the key targets of ColH.

Solution structure of clostridial collagenase H and its calcium-dependent global conformation change.

Collagenase H (ColH) from Clostridium histolyticum is a multimodular protein composed of a collagenase module (activator and peptidase domains), two polycystic kidney disease-like domains, and a collagen-binding domain. The interdomain conformation and its changes are very important for understanding the functions of ColH. In this study, small angle x-ray scattering and limited proteolysis were employed to reveal the interdomain arrangement of ColH in solution. The ab initio beads model indicated that ColH adopted a tapered shape with a swollen head. Under calcium-chelated conditions (with EGTA), the overall structure was further elongated. The rigid body model indicated that the closed form of the collagenase module was preferred in solution. The limited proteolysis demonstrated that the protease sensitivity of ColH was significantly increased under the calcium-chelated conditions, and that the digestion mainly occurred in the domain linker regions. Fluorescence measurements with a fluorescent dye were performed with the limited proteolysis products after separation. The results indicated that the limited proteolysis products exhibited fluorescence similar to that of the full-length ColH. These findings suggested that the conformation of full-length ColH in solution is the elongated form, and this form is calcium-dependently maintained at the domain linker regions.

Enhancement of the structural stability of full-length clostridial collagenase by calcium ions.

The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca(2+) in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca(2+) chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca(2+) was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca(2+) chelation, in comparison with the thermal melting point (T(m)). The melting point changed from 54 to 49°C by the Ca(2+) chelation, and it was restored to 54°C by the addition of excess Ca(2+). These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca(2+).

Uptake of AMP, ADP, and ATP in Escherichia coli W.

The uptake activity ratio for AMP, ADP, and ATP in mutant (T-1) cells of Escherichia coli W, deficient in de novo purine biosynthesis at a point between IMP and 5-aminoimidazole-4-carboxiamide-1-ß-D-ribofuranoside (AICAR), was 1:0.43:0.19. This ratio was approximately equal to the 5'-nucleotidase activity ratio in E. coli W cells. The order of inhibitory effect on [2-³H]ADP uptake by T-1 cells was adenine > adenosine > AMP > ATP. About 2-fold more radioactive purine bases than purine nucleosides were detected in the cytoplasm after 5 min in an experiment with [8-¹4C]AMP and T-1 cells. Uptake of [2-³H]adenosine in T-1 cells was inhibited by inosine, but not in mutant (Ad-3) cells of E. coli W, which lacked adenosine deaminase and adenylosuccinate lyase. These experiments suggest that AMP, ADP, and ATP are converted mainly to adenine and hypoxanthine via adenosine and inosine before uptake into the cytoplasm by E. coli W cells.

Roles of heme axial ligands in the regulation of CO binding to CooA.

CooA is a CO-dependent transcription factor of the bacterium Rhodospirillum rubrum that contains a six-coordinate heme. It has as its heme axial ligands Pro(2) and Cys(75) in the ferric state and Pro(2) and His(77) in the ferrous state. To probe the regulation of CO binding and the ligand switching mechanism in CooA, we have prepared site-directed mutants in which the residues contributing the axial ligands are substituted. The properties of these mutants were investigated by resonance Raman and CO titration methods. Wild-type CooA binds CO with a modest dissociation constant (K(d)) of 11 microm, this value being typical for gas-sensing heme proteins. The K(d) value was greatly decreased in the P2H mutant, indicating that Pro(2) coordination fine tunes CO sensing in CooA. The bound CO in P2H gives rise to a nu(Fe-CO) stretching Raman line at 490 cm(-1), which is similar to that in wild-type CooA. Thus, Pro(2) is the ligand that is replaced by exogenous CO. In the H77A mutant, equilibrium CO binding is biphasic, and at high CO pressures two CO molecules occupy both axial sites. The nu(Fe-CO) stretching Raman line for the first CO molecule was observed at 497 cm(-1). Some of the His(77) mutants showed an additional nu(Fe-CO) line at 525 cm(-1). The binding affinity of the second CO molecule correlates with the five-coordinate component in the ferrous His(77) mutants and also with the acidity of the side chain at position 77. Thus, we propose the Cys(75)-His(77) ligand switch is controlled by His(77) acting as a proton reservoir.

Desulfurization of 2,4,6,8-tetraethyl dibenzothiophene by recombinant Mycobacterium sp. strain MR65.

Recombinant Mycobacterium sp. strain MR65 harboring dszABCD genes was used to desulfurize alkyl dibenzothiophenes (Cx-DBTs) in n-hexadecane. The specific desulfurization activity for 2,4,6,8-tetraethyl DBT (C8-DBT) by DszC enzyme was about twice that for 4,6-dipropyl DBT (C6-DBT). However, the degradation rate of 2,4,6,8-tetraethyl DBT in n-hexadecane by resting cells of strain MR65 was only about 40% of that of 4,6-dipropyl DBT. These results indicated that the desulfurization ability for Cx-DBTs by resting cells depends on carbon number substituted at positions 4 and 6 and that the rate-limiting step in the desulfurization reaction of highly alkylated Cx-DBTs is the transfer process from the oil phase into the cell.

Enhanced desulfurization in a transposon-mutant strain of Rhodococcus erythropolis.

The dsz desulfurization gene cluster from Rhodococcus erythropolis strain KA2-5-1 was transferred into R. erythropolis strain MC1109, unable to desulfurize light gas oil (LGO), using a transposon-transposase complex. As a result, two recombinant strains, named MC0203 and MC0122, were isolated. Resting cells of strain MC0203 decreased the sulfur concentration of LGO from 120 mg l(-1) to 70 mg l(-1) in 2 h. The LGO-desulfurization activity of strain MC0203 was about twice that of strain MC0122 and KA2-5-1. The 10-methyl fatty acids of strain MC0203 were about 28%-41% that of strain MC1109. It is likely that strain MC0203 had a mutation involving alkylenation or methylation of delta9-unsaturated fatty acids caused by the transposon inserted in the chromosome, which increased the fluidity of cell membranes and enhanced the desulfurization activity.

Recombinant Pseudomonas putida carrying both the dsz and hcu genes can desulfurize dibenzothiophene in n-tetradecane.

Pseudomonas putida IFO13696, a recombinant strain with dsz desulfurization genes, desulfurized dibenzothiophene (DBT) in water but not in n-tetradecane. By introducing into this recombinant strain the hcuABC genes that take part in the uptake of DBT in the oil phase into the cell, 82% of 1 mM DBT in n-tetradecane was degraded in 24 h by resting cells. The products of hcuABC genes thus acted in the uptake of DBT in n-tetradecane into the cells and were effective in desulfurization of DBT in the hydrocarbon phase.

Selective cleavage of 10-methyl benzo[b]naphtho[2,1-d]thiophene by recombinant Mycobacterium sp. strain.

Recombinant Mycobacterium sp. strain MR65 carrying dszABCD genes was used for desulfurization of 10-methylbenzo[b]naphtho[2,1-d]thiophene (10-methyl BNT) in the hexadecane phase. The specific activity was 25% of that of dibenzothiophene (DBT). One of two major metabolites of 10-methyl BNT produced by strain MR65 was identified as 1-methoxy-2-(3-methylphenyl)naphthalene by 1H and 13C NMR. The other major metabolite and two minor metabolites were determined as 1-hydroxy-2-(3-methylphenyl)naphthalene, 2-(2-methoxy-3-methylphenyl)naphthalene and 2-(2-hydroxy-3-methylphenyl)naphthalene, respectively, by HPLC and GC-MS. The production ratio of the two desulfurization metabolite isomers was 0.99:0.01, calculated on the basis of peak GC areas. These results indicated that the C-S bond adjacent to the naphthalene skeleton was selectively cleaved to form the two major compounds.

Cloning of a rhodococcal promoter using a transposon for dibenzothiophene biodesulfurization.

The expression of biodesulfurization genes (dsz) in Rhodococcus erythropolis strain KA2-5-1 is repressed by sulfate which is the product of biodesulfurization. The application of a sulfate non-repressible promoter could be effective in enhancing biodesulfurization. A promoter-probe transposon was constructed using the promoterless, red-shifted green fluorescence protein gene (rsgfp). A 340 bp putative promoter element, designated kap1, was isolated from a strain KA2-5-1 recombinant that had shown high fluorescence intensity. The activity of kap1 was not affected by 1 mM sulfate. It gave about a 2-fold greater activity than the 16S ribosomal RNA promoter in R. erythropolis strain KA2-5-1 and is therefore useful for expressing desulfurization genes in rhodococcal strains.