Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101

A chitinase encoding gene from Bacillus sp. DAU101 was cloned in Escherichia coli. The nucleotide sequencing revealed a single open reading frame containing 1781 bp and encoding 597 amino acids with 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram. The chitinase was composed of three domains: a catalytic domain, a fibronectin III domain, and a chitin binding domain. The chitinase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 7.5 and 60 degrees C, respectively. The metal ions, Zn(2+), Cu(2+), and Hg(2+), were strongly inhibited chitinase activity. However, chitinase activity was increased 1.4-fold by Co(2+). Chisb could hydrolyze GlcNAc(2) to N-acetylglucosamine and was produced GlcNAc(2), when chitin derivatives were used as the substrate. This indicated that Chisb was a bifunctional enzyme, N-acetylglucosaminase and chitobiosidase. The enzyme could not hydrolyze glycol chitin, glycol chitosan, or CMC, but hydrolyzed colloidal chitin and soluble chitosan.     

Cloning and expression of an antifungal chitinase gene of a novel Bacillus subtilis isolate from Taiwan potato field

A chitinase producing Bacillus subtilis CHU26 was isolated from Taiwan potato field. This strain exhibited a strong extra-cellular chitinase activity on the colloidal chitin containing agar plate, and showed a potential inhibit activity against phytopathogen, Rhizoctonia solani. The gene encoding chitinase (chi18) was cloned from the constructed B. subtilis CHU26 genomic DNA library. The chi18 consisted of an open reading frame of 1791 nucleotides and encodes 595 amino acids with a deduced molecular weight of 64kDa, next to a promoter region containing a 9 base pair direct repeat sequence (ATTGATGAA). The deduced amino acid sequence of the chitinase from Bacillus subtilis CHU26 exhibits 62% and 81% similarity to those from B. circulans WL-12 and B. licheniformis, respectively. Subcloned chi18 into vector pGEM3Z and pYEP352 to construct recombinant plasmid pGCHI18 and pYCHI18, respectively, chitinase activity could be observed on the colloidal chitin agar plate from recombinant plasmid containing Escherichia coli transformant. Cell-free culture broth of pYCHI18 containing E. coli transformant decreased R. solani pathogenic activity more than 90% in the antagonistic test on the radish seedlings (Raphanus sativus Linn.).     

青海柴达木极端干旱沙地分离芽孢杆菌的分子鉴定及拮抗活性分析

【目的】研究高原极地环境微生物资源。【方法】采用rep-PCR指纹图谱分析、gyrB基因及16S rDNA基因序列分析等多项分子鉴定技术对分离自青海柴达木极端干旱沙地的8株芽孢杆菌菌株进行分类鉴定;通过平板对峙及接种离体叶片试验检测分离菌株的拮抗活性及对病原菌侵染的防效;采用MALDI-TOF-MS质谱分析生防菌株的活性成分。【结果】8株分离菌株鉴定为Bacillus amyloliquefaciens(6株)、Bacillus axarquiensis(1株)和Bacillus atrophaeus(1株);各菌株对油菜菌核病原真菌(Sclerotinia sclerotiorum)均具有显著的拮抗活性;接种离体叶片试验表明菌株对油菜菌核病菌的侵染具有较好防效;MALDI-TOF-MS质谱分析结果显示菌株DGL1(B.amyloliquefaciens)产生脂肽化合物Fengycin,菌株DGL6(B.axarquiensis)产生脂肽化合物Surfactin、BacillomycinsD和Fengycin,菌株DCD1(B.atrophaeus)产生脂肽化合物Surfactin、Fengycin。【结论】为高原干旱沙地极端环境微生物资源研究及生防菌资源开发和应用提供了研究材料。     

Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase

Bacillus subtilis W-118, a strain that produces antifungal materials, excreted a chitinase when cultured in a medium containing shrimp- and crab-shell powder as the major carbon source. This chitinase, purified by sequential chromatography, had a molecular mass of 20,600 Da and a pI of 6. The optimum pH, optimum temperature, and pH stability of the chitinase were pH 6, 37 degrees C, and pH 5-7, respectively. The unique characteristics of the purified chitinase include low molecular mass and acidic pI. In the investigation of the inhibitory activity, it was found that the growth of Fusarium oxysporum was 100% inhibited after incubation for 1 day with sterilized W-118 chitinase solution (5.6 units/mL). The chitinase hydrolyzates of chitin with low degrees of polymerization (DP 1-6) were analyzed by HPLC. Longer reaction times led to the generation of chitin oligosaccharides with lower DP. The chitin oligosaccharides were examined for their inhibitory effects on F. oxysporum and human leukemia cell lines.     

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.     

Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene

A chitosanase produced constitutively by Bacillus sp. MET 1299 was purified by SP-Sephadex column chromatography. The molecular weight was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzyme activity was observed at a pH of 5.5 and temperature of 60 degrees C. The purified chitosanase showed high activity on 90% deacetylated colloidal chitosan and beta-glucan, but not on hydrolyzed colloidal chitin, CMC, or their derivatives. The N-terminal amino acid sequence of the enzyme was determined. The cloned full length gene, 1362 bp in size, encoded a single peptide of 453 amino acids and had a conserved amino acid sequence of glycosyl hydrolase family 8. A search of the cDNA sequence with NCBI BLAST showed homology with chitosanase of Bacillus sp. KTCC 0377BP and Bacillus sp. No. 7-M. The recombinant protein was expressed in Escherichia coli, purified using affinity chromatography and characterized.     

一种来源于兼性嗜碱菌Bacillus pseudofirmus 703的β-N-乙酰氨基葡萄糖苷酶

N-乙酰氨基葡萄糖苷酶作用于肽聚糖或几丁质,从其非还原末端水解产生β-D-N-乙酰氨基葡萄糖单体,该酶在细胞壁代谢过程中起重要作用,在医药和生物技术领域也有广泛的应用。【目的】克隆表达来源于兼性嗜碱菌Bacillus pseudofirmus 703的β-N-乙酰葡糖胺糖苷酶NagZ703,为获得乙酰氨基葡萄糖单体奠定基础。【方法】以B.pseudofirmus703基因组DNA为模板,克隆得到了β-N-乙酰氨基葡萄糖苷酶基因NagZ703,通过构建pET28a-nagZ703表达载体,在大肠杆菌BL21(DE3)中诱导表达NagZ703,利用镍柱纯化得到NagZ703纯蛋白,并对其酶学和生化性质进行分析。【结果】NagZ703与其同源蛋白多序列比对分析结果表明,NagZ703属于糖苷水解酶3家族(GH3),由2个结构域构成,催化活性中心由位于N端结构域的Arg232-His234-Arg318组成,和研究最多的Bacillussubtilis168来源的BsNagZ氨基酸的序列相似性为37%。酶学性质分析表明,以对硝基酚-β-乙酰氨基葡萄糖苷(pNP-β-GlcNAc)为底物,NagZ703的最适反应温度和pH分别为60°C和pH 6.5,比酶活为10.79 U/mg,其Km和Vmax分别为0.276 mmol/L和0.612 mmol/(mg·min)。该酶具有较好的稳定性,在50°C处理30 min,或在pH 6.0–10.5条件下,4°C保存12 h后,仍保留80%以上的酶活力。EDTA不影响该酶的活性,推测其为非金属依赖酶,且Hg2+可完全抑制酶活性。【结论】本研究将兼性嗜碱菌Bacillus pseudofirmus 703来源的β-N-乙酰葡糖胺糖苷酶NagZ703在大肠杆菌中成功表达和纯化,并分析了其酶学性质;NagZ703的最适pH为6.5,没有表现出耐盐嗜碱的特征;NagZ703能水解胶体几丁质产生GlcNAc,为酶解生产GlcNAc提供了一条可行的思路。     

First report of a bifunctional chitinase/lysozyme produced by Bacillus pumilus SG2

Bacillus pumilus SG2 isolated from high salinity ecosystem in Iran produces two chitinases (ChiS and ChiL) and secretes them into the medium. In this study, chiS and chiL genes were cloned in pQE-30 expression vector and were expressed in the cytoplasm of Escherichia coli strain M15. The recombinant proteins were purified using Ni-NTA column. The optimum pH and optimum temperature for enzyme activity of ChiS were pH 6, 50°C; those of ChiL were pH 6.5, 40°C. The purified chitinases showed antifungal activity against Fusarium graminearum, Rhizoctonia solani, Magnaporthe grisea, Sclerotinia sclerotiorum, Trichoderma reesei, Botrytis cinerea and Bipolaris sp. Moreover, purified ChiS was identified as chitinase/lysozyme, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of cell walls with many kinds of bacteria (Xanthomonas translucens pv. hordei, Xanthomonas axonopodis pv. citri, Bacillus licheniformis, E. coli C600, E. coli TOP10, Pseudomonas aeruginosa and Pseudomonas putida). Strong homology was found between the three-dimensional structures of ChiS and a chitinase/lysozyme from Bacillus circulans WL-12. This is the first report of a bifunctional chitinase/lysozyme from B. pumilus.     

Determination of the efficacy of two building decontamination strategies by surface sampling with culture and quantitative PCR analysis

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus ("Bacillus subtilis subsp. niger," also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 10(5) to 10(6) CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.     

Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation

Fermentation of shrimp shell in jaggery broth using Bacillus subtilis for the production of chitin and chitosan was investigated. It was found that B. subtilis produced sufficient quantities of acid to remove the minerals from the shell and to prevent spoilage organisms. The protease enzyme in Bacillus species was responsible for the deprotenisation of the shell. The pH, proteolytic activity, extent of demineralization and deprotenisation were studied during fermentation. About 84% of the protein and 72% of the minerals were removed from the shrimp shell after fermentation. Mild acid and alkali treatments were given to produce characteristic chitin and their concentrations were standardized. Chitin was converted to chitosan by N-deacetylation and the properties of chitin and chitosan were studied. FTIR spectral analysis of chitin and chitosan prepared by the process was carried out and compared with spectra of commercially available samples.     

N-terminal region of chitinase I of Bacillus circulans KA-304 contained new chitin-biding domain

Chitinase I (CHI1) of Bacillus circulans KA-304 forms protoplasts from Schizophyllum commune mycelia when the enzyme is combined with α-1,3-glucanase of B. circulans KA-304. CHI1 consists of an N-terminal unknown region and a C-terminal catalytic region classified into the glycoside hydrolase family-19 type. An N-terminal region-truncated mutant of CHI 1 (CatCHI1), which was expressed in Escherichia coli Rosetta-gami B (DE3), lost colloidal chitin- and powder chitin-binding activities. The colloidal chitin- and the powder chitin-hydrolyzing activities of CatCHI1 were lower than those of CHI1, and CatCHI1 was not effective in forming the protoplast. A fusion protein of the N-terminal region of CHI1 and green fluorescent protein (Nterm-GFP) was expressed in E. coli, and the fusion protein was adsorbed to colloidal chitin, powder chitin, and chitosan. Fluorescence microscopy analysis showed that Nterm-GFP bound to the S. commune cell-wall.     

A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85 degrees C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.     

Following the biochemical and morphological changes of Bacillus atrophaeus cells during the sporulation process using Bioaerosol Mass Spectrometry

Bioaerosol Mass Spectrometry (BAMS), a real-time single cell analytical technique, was used to follow the biochemical and morphological changes within a group of Bacillus atrophaeus cells by measuring individual cells during the process of sporulation. A mutant of B. atrophaeus that lacks the ability to produce dipicolinic acid (DPA) was also analyzed. Single cell aerodynamic sizing was used to follow gross morphological changes, and chemical analysis of single cells by mass spectrometry was used to follow some biochemical changes of B. atrophaeus cells during endospore formation.     

Characterization of chitinases excreted by Bacillus cereus CH

Bacillus cereus CH was shown to excrete chitinases into the culture supernatant when cultivated in a medium containing 0.2% colloidal chitin, whereas the removal of colloidal chitin resulted in a low activity. After concentration of the culture supernatant by precipitation with ammonium sulfate, the induced chitinases were purified by sequential chromatography. Four different chitinases, A, B1, B2, and B3 with molecular masses of 35, 47, 58, and 64 kDa, respectively, were separated. All chitinases showed similarities in their kinetic parameters when observed with colloidal chitin, including an optimal pH of 5.0-7.5, and an optimal temperature between 50-60 degrees C. Chitinase A hydrolyzed glycol chitin and p-nitrophenyl-di-N-acetyl-beta-chitobioside at similar rates to that of colloidal chitin, whereas group B chitinases hydrolyzed both substrates in much lower rates. From analyses of the reaction products, it is most likely that chitinase A and all group B chitinases hydrolyze the substrates tested in an endo-fashion. However, group B chitinases were distinct from chitinase A in possessing high transglycosylation activity. From amino terminal sequencing, chitinases B1, B2, and B3 were shown to have almost identical sequences, which differed from that of chitinase A. The similarities in the reaction modes and amino terminal sequences among chitinases B1, B2, and B3 suggest that these chitinases may be derived from a presumptive precursor protein through C-terminal processing.     

Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake

Alkaline cellulase-producing actinomycete strains were isolated from mud samples collected from East African soda lakes. The strains were identified as novel Streptomyces spp. by 16S rDNA sequence analysis. A cellulase gene (cel12A) from Streptomyces sp. strain 11AG8 was cloned by expression screening of a genomic DNA library in Escherichia coli. From the nucleotide sequence of a 1.5-kb DNA fragment, an open reading frame of 1,113 nucleotides was identified encoding a protein of 371 amino acids. From computer analysis of the sequence, it was deduced that the Cel12A mature enzyme is a protein of 340 amino acids. The protein contained a catalytic domain, a glycine-rich linker region, and a cellulose-binding domain of 221, 12, and 107 amino acids, respectively. FASTA analysis of the catalytic domain of Cel12A classified the enzyme as a family 12 endoglucanase and the cellulose-binding domain as a family IIa CBD. Streptomyces rochei EglS was determined as nearest neighbor with a similarity of 75.2% and 61.0% to the catalytic domain and the cellulose-binding domain, respectively. The cell2A gene was subcloned in a Bacillus high-expression vector carrying the Bacillus amyloliquefaciens amylase regulatory sequences, and the construct was transformed to a Bacillus subtilis host strain. Crude enzyme preparations were obtained by ultrafiltration of cultures of the Bacillus subtilis recombinant strain containing the 11AG8 cell2A gene. The enzyme showed carboxymethylcellulase (CMCase) activities over a broad pH range (5-10) with an optimum activity at pH 8 and 50 degrees C. The enzyme retained more than 95% of its activity after incubation for 30 min under these conditions.     

Detection of molecular diversity in Bacillus atrophaeus by amplified fragment length polymorphism analysis

Phenotypically, Bacillus atrophaeus is indistinguishable from the type strain of Bacillus subtilis except by virtue of pigment production on certain media. Several pigmented variants of B. subtilis have been reclassified as B. atrophaeus, but several remain ambiguous in regard to their taxonomic placement. In this study, we examined strains within the American Type Culture Collection originally deposited as Bacillus globigii, B. subtilis var. niger, or Bacillus niger using 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis to determine the level of molecular diversity among these strains and their relationship with closely related taxa. The 16S rRNA gene sequences revealed little variation with one base substitution between the B. atrophaeus type strain ATCC 49337 and the other pigmented bacilli. AFLP analysis produced high-quality DNA fingerprints with sufficient polymorphism to reveal strain-level variation. Cluster analysis of Dice similarity coefficients revealed that three strains, ATCC 31028, ATCC 49760, and ATCC 49822, are much more closely related to B. atrophaeus than to B. subtilis and should be reclassified as B. atrophaeus. A very closely related cluster of B. atrophaeus strains was also observed; this cluster was genetically distinct from the type strain. The level of variation between the two groups was approximately the same as the level of variation observed between members of the two B. subtilis subspecies, subtilis and spizizenii. It is proposed that the cluster of strains typified by ATCC 9372 be designated a new subspecies, B. atrophaeus subsp. globigii.     

Cloning and DNA sequencing of xyaA, a gene encoding an endo-beta-1,4-xylanase from an alkalophilic Bacillus strain (N137).

The gene xyaA encoding an alkaline endo-beta 1,4-xylanase from an alkalophilic Bacillus sp. strain (N137) isolated in our laboratory was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1,656-bp DNA fragment containing xyaA was determined, revealing one open reading frame of 993 bp that encodes a xylanase (XyaA) of 39 kDa. This xylanase lacks a typical putative signal peptide, yet the protein is found in the Bacillus culture supernatant. In Escherichia coli, the active protein is located mainly in the periplasmic space. The xylanase activity of the cloned XyaA is an endo-acting enzyme that shows optimal activity at pH 8 and 40 degrees C. This activity is stable at a pH between 6 and 11. Incubations of XyaA at 40 degrees C for 1 h destroyed 45% of the activity.     

A novel beta-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose

A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine. The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.     

产几丁质酶的苏云金杆菌菌株筛选及酶合成条件研究

从本室保存的 64株苏云金芽孢杆菌中 ,筛选出一株几丁质酶活力较高的菌株WB 50。产酶条件研究表明 :在pH 7.0的基础培养基中添加 2 .0 %的细粉几丁质 ,1.0 %的酵母膏 ,2 2 0rpm 30℃下培养 72小时 ,几丁质酶的产出最大。     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.