Streptomyces Caespitosus Research Articles

Augmented trabeculectomy - The surgical journey and road trip forward.

Augmented trabeculectomy - The surgical journey and road trip forward.

Tracing of Acyl Carrier Protein-channeled Mitomycin Intermediates in Streptomyces caespitosus Facilitates Characterization of the Biosynthetic Steps for AHBA-GlcN Formation and Processing.

Mitomycins are a family of naturally occurring, potent alkylating agents in which the C member has been clinically used for cancer chemotherapy for over 5 decades. In Streptomyces caespitosus, mitomycins are derived from an N-glycoside composed of a 3-amino-5-hydroxybenzoic acid (AHBA) unit and a d-glucosamine (GlcN) unit; however, how this N-glycoside is formed and rearranged to a mitosane, for example, the compact polycyclic ring system of mitomycin C, remains elusive. Benefiting from the development of a method used to trace the mitomycin intermediates that accumulate on an acyl carrier protein (ACP), we here dissect the enzymatic steps for AHBA-GlcN formation and processing to underlie the mitosane structure. Following the N-glycosylation of AHBA with activated N-acetyl-GlcN, deacetylation occurs on ACP to provide AHBA-GlcN. Then, the sugar portion of this N-glycoside is transformed into a linear aminodiol that terminates with an epoxyethane, yielding an ACP-channeled intermediate that is ready for mitosane formation through crosslinking between the AHBA and linearized sugar units. This transformation is unusual and relies on the functional association of a dihydronicotinamide adenine dinucleotide (phosphate)-dependent protein with a radical S-adenosyl-l-methionine protein. Characterization of these ACP-based enzymatic steps for AHBA-GlcN formation and processing sheds light on the poorly understood biosynthetic pathway of mitomycins.

Improving the Results of Endonasal Dacryocystorhinostomy with Mitomycin C Application: A Prospective Case-Control Study.

Many surgical advancement paved to surgical success in endonasal endoscopic dacryocystorhinostomy. Mitomycin C is a systemic chemotherapeutic agent derived from Streptomyces caespitosus that inhibits the synthesis of DNA, cellular RNA, and protein by inhibiting the synthesis of collagen by fibroblasts. The objective of the study was to evaluate the advantage of mitomycin C in primary endoscopic endonasal dacryocystorhinostomy without stenting. Randomised case control design. Tertiary academic centre in central India. 112 patients who presented with epiphora and diagnosed as chronic dacryocystitis after syringing by ophthalmologist, were selected and randomised into two groups with or without mitomycin C intraoperative application. 112 eyes underwent endoscopic-dacryocystorhinostomy. Additional surgeries were done according to necessity. Mitomycin C was applied in concentration of 0.4mg/dl for 5min. Patients were followed up on OPD-basis at the end of 1st week, 1st month, 3rd month and over phone call at end of 1year. Symptomatic improvement assessed by verbal enquiry and clinically by syringing by ophthalmologist. 8 patients underwent septal surgery in case group and 4 in control group along with 1 concha bullosa correction in the case group (p < 0.05). Nil intraoperative complications (p < 0.05) and nil postoperative complication noted (p < 0.05). Functional and anatomical patency found to be 100% at the end of 1st week in both groups, 96.5% at 3rd month in case group and 96.4% and 92.9% at 1month and 3month respectively in control group. Nasal endoscopy of all surgical failures showed restenosis in both case group and control group except 1 patient with granulation in control group. Eventhough intraoperative mitomycin C application is effective in increasing the success rate of endonasal DCR surgery in standard nasolacrimal duct obstruction, and with no significant complications from its use, the study did not show added benefit in the primary endoscopic endoscopic dacryocystorhinostomy without stenting.

Mitomycin-C in dacryocystorhinostomy: From experimentation to implementation and the road ahead: A review.

Dacryocystorhinostomy (DCR) is the procedure of choice in patients with epiphora due to primary acquired nasolacrimal duct obstruction. The evolution of surgical tools, fiber-optic endoscopes, effective anesthesia techniques, and the adjunct use of antimetabolites intraoperatively; namely mitomycin-C (MMC) have significantly contributed to the advancement of DCR surgery. MMC is a systemic chemotherapeutic agent derived from Streptomyces caespitosus that inhibits the synthesis of DNA, cellular RNA, and protein by inhibiting the synthesis of collagen by fibroblasts. Even the cellular changes in the human nasal mucosal fibroblasts induced by MMC at an ultrastructural level have been documented. There, however, seems to be a lack of consensus regarding MMC: The dosage, the route of delivery/application, the time of exposure and subsequently what role each of these variables plays in the final outcome of the surgery. In this review, an attempt is made to objectively examine all the evidence regarding the role of MMC in DCR. MMC appears to improve the success rate of DCR.

English

Endonasal dacrocystohinostomy (DCR) is the surgery used for chronic dacrocystitis, where the patient has epiphora caused by blocking of nasolacrimal duct, leading to collection of lacrimal fluid in the sac which ends in inflammation of the lacrimal sac. As the success rate of endonasal DCR varies from 50---97%, various different methods are used to have high success rate. It is very important to make the ostium at the level of the common canaliculus. If the exposure of lacrimal sac is not sufficient then even though Mitomycin is applied the rate of failure remains high. INTRODUCTION: Epiphora is the most bothersome complication of lacrimal system obstruction. Other than it relapsing dacrocystitis, dacryocele and dacryolithiasis, latter being quite rare, are the other indications of dacrocystorhinostomy. DCR is a surgical procedure in management of obstructive epiphora. Obstructive epiphora finds today his best solution with a surgical procedure Dacrocystorhinostomy by nasal endoscope. The lacrimal flow is diverted into nasal cavity through an artificial opening made at the level of lacrimal sac. DCR can also be performed by an external approach. It is called external DCR. In comparison of both, endonasal DCR has certainly many advantages; minimally invasive procedure, better aesthetic results with no cutaneous scar, preserves lacrimal pump system, faster resumption to daily activities, any intranasal pathology that might have caused obstruction can be addressed at the same time, active infection of the lacrimal system is not a contraindication to the surgery, it is much less bloody than the external approach, the per-operative time is shorter. If the obstruction is in puncta or canaliculus, endonasal; DCR will not help. Mitomycin—C is an alkaline agent used as anticancer, isolated from streptomyces Caespitosus. It inhibits the synthesis of DNA, cellular RNA and proteins in the rapidly growing cells. It has the ability to suppress fibrosis and vascular growth hence can reduce possibility of stenosis of the newly created ostium. We used Mitomycin –C (0.5 mg/ml) for 5 minutes over osteotomy site. MATERIAL AND METHODS: Three hundred patients (300), who presented with epiphora, were selected and operated for Endonasal DCR. The patients ranged from 5 years to 85 years of age. One hundred and thirty nine (139) were males and one hundred and sixty one (161) were females. All of them had epiphora which was not responding to medical line of treatment. Some of them had relapsing dacrocystitis, some had persistent purulent, sticky discharge from eye, and some had lacrimal abscess or fistula. All the patients were examined by ophthalmologist prior to surgery. The clinical evaluation included probing of nasolacrimal system and lacrimal irrigation. The nasal cavity had an adequate examination using 0 degree Endoscope and intranasal pathology that might have caused the obstruction was determined preoperatively. The indication for endonasal approach included distal nasolacrimal duct obstruction. All the patients were examined by an ophthalmologist prior to the surgery. DOI: 10.14260/jemds/2014/2641

English

The soil environment is a vast reservoir that harbours microorganisms that represent virtually all the microbial groups including actinomycetes. An investigation was carried out to determine the species of antibiotic-producing actinomycetes found in the soil environment of Keffi Metropolis, Nasarawa State, Nigeria. Soil samples were obtained from ten different locations of Keffi town for the isolation of actinomycetes. Eight species belonging to two genera were isolated from the soil samples and these include Actinomyces israelii, Actinomyces viscosus, Streptomyces antibioticus, Streptomyces aureofiens, Streptomyces caespitosus, Streptomyces exfoliatus, Streptomyces griseus and Streptomyces hygroscopicus. All except S. aureofiens were found to have antimicrobial activity against at least one of the four pathogenic organisms tested for sensitivity. The pathogenic organisms tested for sensitivity were Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The results of this investigation revealed that species of antibiotic-producing actinomycetes are found in the soil environment of Keffi Metropolis, and these could be harnessed for the industrial production of novel antibiotics. Key words: Antibiotic-producing actinomycetes, soil, Keffi, Nigeria

Bronchoscopic Application of Mitomycin-C as Adjuvant Treatment for Benign Airway Stenosis

The management of benign stenosis of the central airways continues to be challenging. Acquired benign airway stenosis can result from a variety of injuries to the airway wall: ischemia related to endotracheal intubation, surgical procedures such as tracheotomy or airway resection, chemical or thermal injury, direct mechanical trauma after bacterial or mycobacterial infections, from inflammatory diseases affecting the airways such as Wegener granulomatosis or sarcoidosis, after radiotherapy, stent-stimulated granulation tissue, and idiopathic when the cause can not be identified. After any of the afore-mentioned damages to the mucosa, the following inflammatory process activates fibroblasts to participate in wound healing. Fibroblasts synthesize several factors, such as transforming growth factor b1 and basic fibroblast growth factor, that stimulate the production of extracellular matrix components, leading to scar formation and contraction at the stenosis site. The same mechanism takes place in restenosis. Current treatment includes surgical resection as the first option. However, when surgery is unsuitable because of the patient’s clinical or respiratory conditions or airway issues, endoscopic approaches need to be considered. Airway patency can be reestablished by means of mechanical dilation with the bevel of the rigid bronchoscope, balloon inflation, laser ablation, electrocautery or argon plasma coagulator, stent insertion, or usually, a combination of any of the above. However, a high rate of restenosis ranging from 40% to 70% has been reported after the endoscopic treatment of tracheal stenosis, urging a need for therapies aimed to obtain better results. Treatments studied to reduce relapses include steroids, 5-fluorouracil, halofuginone, tamoxifen, and mitomycin-C (MMC). Mitomycin is an antibiotic that was isolated from the bacteria Streptomyces caespitosus in 1956. Its C-form is an alkylating agent that inhibits deoxyribonucleic acid synthesis. It was first used as an anticancer drug. It has also been used in ophthalmologic procedures to reduce corneal scarring and recently in the treatment of benign airway stenosis.

On the Relevance of the Met-turn Methionine in Metzincins

The metzincins are a clan of metallopeptidases consisting of families that share a series of structural elements. Among them is the Met-turn, a tight 1,4-turn found directly below the zinc-binding site, which is structurally and spatially conserved and invariantly shows a methionine at position 3 in all metzincins identified. The reason for this conservation has been a matter of debate since its discovery. We have studied this structural element in Methanosarcina acetivorans ulilysin, the structural prototype of the pappalysin family, by generating 10 mutants that replaced methionine with proteogenic amino acids. We compared recombinant overexpression yields, autolytic and tryptic activation, proteolytic activity, thermal stability, and three-dimensional structure with those of the wild type. All forms were soluble and could be purified, although with varying yields, and three variants underwent autolysis, could be activated by trypsin, and displayed significant proteolytic activity. All variants were analyzed for the thermal stability of their zymogens. None of the mutants analyzed proved more stable or active than the wild type. Both bulky and small side chains, as well as hydrophilic ones, showed diminished thermal stability. Two mutants, leucine and cysteine, crystallized and showed three-dimensional structures that were indistinguishable from the wild type. These studies reveal that the Met-turn acts as a plug that snugly inserts laterally into a core structure created by the protein segment engaged in zinc binding and thus contributes to its structural integrity, which is indispensable for function. Replacement of the methionine with residues that deviate in size, side-chain conformation, and chemical properties impairs the plug-core interaction and prejudices molecular stability and activity.

Use of Mitomycin C in the treatment of conjunctival and corneal squamous cell carcinoma. a case report

Abstract Purpose: Corneal conjunctival squamous carcinoma is a rare form of slowly progressive carcinoma of low malignant potential. Mitomycin C (MMC) is a chemiotherapic antibiotic isolated from Streptomyces Caespitosus and medical literature reports efficacy of MMC in the treatment of squamous cell carcinoma. We present a case of conjunctival and corneal squamous cell carcinoma which was treated with both surgical excision and topical use of MMC 0.02%. Methods: A 78‐year old man with a red‐grey vascularized elevated epithelial lesion that started at the conjunctiva and extended past the corneoscleral limbus on to the cornea for about 1.5 mm.The patient was given surgical exeresis of the conjunctival neoformation and the histological exam highlighted a corneal conjunctival squamous cell carcinoma. after three monthes a recurrence of the neoformation was displayed and we therefore decided to start topical MMC 0.02% eye drops treatment four times daily for 7 days. Four cycles of treatment were done with a one‐week interval during which only sodium hyaluronate 0.4% lubricant eye drops were administered. Results: During the period of treatment with the MMC 0.02% eye drops, the patient did not display any adverse systemic or ocular reactions. At the end of the treatment we found a complete regression of the disease. Twelve months after treatment he was without conjunctival and corneal epithelial dysplasia. Conclusions: This case highlights the efficacy of MMC 0.02% eye drops in the treatment of conjunctival and corneal squamous cell carcinoma that can avoid the topical use with sponge of MMC at higher concentration that it would present a not predictable long term effects on the conjunctival fornix and on lachrymal secretion.

Purification to Homogeneity of a Neutral Metalloproteinase fromStreptomyces caespitosus

We established an improved purification procedure for Streptomyces caespitosus neutral protease (ScNP) from culture supernatants of S. caespitosus. The procedure comprises sequential ammonium sulfate fractionation and column chromatography procedures with anion exchange chromatography, followed by hydrophobic-interaction chromatography and gel filtration. Purified ScNP revealed a single band with a molecular mass of 14 kDa by SDS-PAGE under reduced conditions and did not contain any detectable pigment, which has not been completely removed by other methods. We also purified another protease with a molecular mass of 40 kDa from the culture supernatants. The pure preparation of ScNP obtained by this procedure is suitable for spectrophotometric measurement of its catalytic activity.

Effects of Neutral Salts and Alcohols on the Activity of Streptomyces caespitosus Neutral Protease

Streptomyces caespitosus neutral protease (ScNP) is one of the smallest metalloproteinase with a molecular mass of 14 kDa. Effects of solvent composition on ScNP activity were examined using a peptide substrate. The k(cat)/K(m) values of ScNP exhibited bell-shaped pH-dependence with the optimal pH of 6.4-7.0 and the pK(a) values of 5.0 +/- 0.1 and 8.3 +/- 0.1. ScNP activity increased in an exponential fashion with increasing [NaCl]. The relative k(cat)/K(m) value at 3.6 M NaCl to that at 0 M NaCl was 3.7, and the degree of the activation at x M NaCl was expressed as 1.2 (x) (x < 2.0) and 1.4(x) (x > 2.0). On the other hand, ScNP activity decreased with increasing concentrations of LiCl, KCl, NaBr, LiBr, KBr and NaClO(4). Alcohols inhibited ScNP activity with the IC(50) values, the concentration required for decreasing the activity at 50% of the maximum, of 0.77-6.54 M. The order of the inhibitory potency was 1-butanol, 2-methyl-1-propanol, 2-methyl-2-butanol > 2-methyl-2-propanol, 2-butanol, 1-propanol > 2-propanol >> ethanol >> methanol. The activities recovered completely by the dilution of alcohols, suggesting that the ScNP inhibition by alcohols is reversible. These characteristics of ScNP are compared with those of human matrix metalloproteinase 7 and thermolysin.

A flow cytometry-optimized assay using an SOS–green fluorescent protein (SOS–GFP) whole-cell biosensor for the detection of genotoxins in complex environments

Whole-cell biosensors have become popular tools for detection of ecotoxic compounds in environmental samples. We have developed an assay optimized for flow cytometry with detection of genotoxic compounds in mind. The assay features extended pre-incubation and a cell density of only 10 6–10 7 cells/mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS–green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS-induction in whole soil samples. Soil microcosms were spiked with a dilution-series of crude broth extract from the mitomycin C-producing streptomycete Streptomyces caespitosus. Biosensors extracted from these microcosms after 1 day of incubation at 30 °C were easily distinguished from extracts of non-contaminated soil particles when using flow cytometry, and induction of the biosensor by mitomycin C was detectable at concentrations as low as 2.5 ng/g of soil.

Topical application of mitomycin-C in corrosive esophageal strictures

<b>Introduction</b>: Corrosive ingestion is the major cause of esophageal stricture in our country. Mitomycin-C, an anthracycline derived from <i>Streptomyces caespitosus, </i> has both antineoplastic and antiproliferative properties. Use of mitomycin-C have been well documented in various ophthalmologic procedures and laryngo-tracheal conditions where it has been shown that mitomycin-C inhibited fibroblast proliferation during the postoperative phase without damaging the mucosal and epithelial growth. This preliminary report documents the use of mitomycin-C in caustic stricture esophagus. <b><br>Materials and Methods:</b> A prospective study was carried out in ten known patients of caustic esophageal strictures. After taking informed consent patients were subjected to antegrade or retrograde esophageal dilatation under general anesthesia and mitomycin-C was applied topically by a 10 mm cottonoid sponge soaked in mitomycin-C solution (0.4 mg/ml) for 5 minutes at the stricture site. The procedure was repeated at 4 weekly intervals thereafter and need for further dilatation was assessed. Outcomes in terms of symptomatic relief and radiological evaluation were assessed after therapy and results were analyzed for effectiveness of mitomycin-C and compared with retrospective results of previous cases of corrosive ingestion managed at the same institution. <b><br>Results:</b> Total number of dilatations required were 34 (mean - 3.4) Two patients required single dilatation while two required six sessions. Symptomatic as well as radiological improvement was present in nine patients. <b><br>Conclusions:</b> Use of mitomycin-C seems to improve the outcome in patients of corrosive esophageal strictures. Patients who have undergone esophageal replacement also seem to benefit from mitomycin-C.

A Novel Double-headed Proteinaceous Inhibitor for Metalloproteinase and Serine Proteinase

A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (Streptomyces caespitosus neutral proteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K(i) value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN' (K(i) = 1.4 nm) (EC ). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M(r) = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN' was identified to be Met(71)-Tyr(72) bond by specific cleavage. To identify the reactive site for ScNP, Tyr(33) and Tyr(72), which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN' but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN' was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN'.

The Detoxin Complex. — A Naturally Occurring Safener

The detoxin complex is a group of depsipeptide metabolites produced by the organism Streptomyces caespitosus var. detoxicus 7072 GC 1 as well as other strains of streptomyces. The complex is the first isolated natural product which displays a unique detoxification effect in plant and animal cells as its biological activity. The structural investigations carried out to elucidate the structures of the congeners and the stereochemistry of the stereogenic centers are described. Structure-activity relationships are discussed. The different synthetic approaches to the parent amino acid, (-)detoxinine, are compared. The syntheses of detoxins (-)-B 1 , (-)-B 3 , (-)-D 1 , and (+)-valyldetoxinine are described

Protease inhibitors from jackfruit seed (Artocarpus integrifolia)

Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7.6. A minor fraction (I) that did not bind to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1.9:1.0. Fraction II bound least tightly to the ion exchanger eluting with 0.05 M NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and IV eluted successively with 0.10 M NaCl and 0.15 M NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin in the ratio 1.0: 0.53:0.55 and 1.0:8.9:9.8 respectively. Fraction V, most strongly bound to the matrix eluting with 0.3 M NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose, xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had very poor action on subtilisin BPN′, porcine elastase, pronase,Streptomyces caespitosus protease andAspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate oxidation also decreased the inhibitory activity.

Activation of Hageman Factor and Prekallikrein and Generation of Kinin by Various Microbial Proteinases

Activation of the Hageman factor-kallikrein-kinin system by serratial 56-kDa proteinase was previously demonstrated (Matsumoto, K., Yamamoto, T., Kamata, T., and Maeda, H. (1984) J. Biochem. (Tokyo) 96, 739-749; Kamata, R., Yamamoto, T., Matsumoto, K., and Maeda, H. (1985) Infect. Immun. 48, 747-753). To investigate whether the activation of the system is specific for 56-kDa proteinase or is found similarly with other microbial proteinases, 11 proteinases of microbial origins were studied; the 56-kDa proteinase was the control. For in vitro studies, activation of guinea pig Hageman factor and prekallikrein was examined in purified systems as well as in plasma as a zymogen source. Specific antibodies and inhibitors confirmed the activation steps of the cascade. In the in vivo study the enhancement of vascular permeability in guinea pig skin and its sensitivity to inhibitors of activated Hageman factor, plasma kallikrein, or a kininase were examined. The results from the in vivo experiments were consistent with those in vitro. Taking all the data together, we classified the 11 microbial proteinases into three groups as follows: 1) Serratia marcescens 56-, 60-, and 73-kDa proteinases, Pseudomonas aeruginosa alkaline proteinase and elastase, and Aspergillus melleus proteinase (this group activated Hageman factor but not prekallikrein); 2) Vibrio vulnificus proteinase, subtilisin from Bacillus subtilis, and thermolysin from Bacillus stearothermophilus (this group activated both Hageman factor and prekallikrein); 3) Streptomyces caespitosus proteinase and V8 proteinase from Staphylococcus aureus (this group activated neither Hageman factor nor prekallikrein, but generated kinin from high molecular weight kininogen directly).

Studies on the coagulation of soymilk-protein by commercial proteinases.

A coagulation test of soymilk-protein was carried out with various commercial proteinases originating from microorganisms, plants and animals. The soymilk-protein was coagulated by the following proteinases: Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus polymyxa, Streptomyces griseus, Streptomyces caespitosus, Aspergillus oryzae, Aspergillus sojae, Endothia parasitica, Rhizopus sp., Mucor miehei, bromelain, papain and trypsin. However, such proteinases as Aspergillus saitoi, rennin and pepsin did not coagulate the protein. The coagulability of enzymes decreased as the pH of the soymilk was increased from 5.9 to 6.7. The optimum temperature for soymilk-clotting activity varied widely with the enzyme origin; for example, 55°C in the case of proteinases of Bacillus polymyxa and Rhizopus sp., 65°C in that of Aspergillus oryzae, Aspergillus sojae and trypsin, 75°C in that of Bacillus amyloliquefaciens, Streptomyces griseus, Streptomyces caespitosus and Endothia paracitica, and 85°C in that of Bacillus subtilis and Bacillus thermoproteolyticus. The coagulum made with the enzyme was separated into soymilk-curd and whey in a volume ratio of 1 to 1 by centrifugation at about 1600 × g. The moisture content of the curd was about 87%.

A new site-specific endonuclease, ScaI, from Streptomyces caespitosus.

A new site-specific endonuclease has been isolated from Streptomyces caespitosus and named ScaI. Based on analysis of sequences around the restriction sites in pBR322 and pBR325, the recognition sequence of ScaI endonuclease was deduced to be a new hexanucleotide 5'-AGTACT-3'. The cleavage site was determined by comparing the ScaI-cleaved product of a primer-extended M13mp18-SCA DNA, which contains an AGTACT sequence, with dideoxy chain terminator ladders of the same DNA. ScaI was found to cleave the recognition sequence between the internal T and A, leaving flush ends to the cleaved fragments.

New mitomycin, 10-decarbamoyloxy-9-dehydromitomycin B from Streptomyces caespitosus.

New mitomycin, 10-decarbamoyloxy-9-dehydromitomycin B from Streptomyces caespitosus.