Purification and Some Properties of Urethane Hydrolase II from Lactobacillus casei ω ATCC 7469

The β-1,3-glucanase (1,3-β-d-glucan glucanohydrolase, EC 3.2.1.6) gene from Flavobacterium dormitator var. glucanolyticae was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKUβG1 (8.2 kb), showed a high β-1,3-glucanase activity and a lytic activity on viable yeast cells. These activities were found in the peripiasmic space of E. coli clone cells. Southern hybridization analysis showed that the cloned gene was derived from F. dormitator chromosomal DNA. The gene products were purified from the periplasmic fraction of E. coli by ammonium sulfate fractionation and ion-exchange chromatography. The purified enzymes were demonstrated to be identical with a lytic endo-β-1,3-glucanase II and a nonlytic endo-β-1,3-glucanase I from F. dormitator from their enzymological and immunological properties. In the E. coli cells, endo-β-1,3-glucanase I was also formed by a proteolytic digestion of endo-β-1,3-glucanase II during the cultivation as in F. dormitator. Thus, the only endo-β-1,3-glucanase II was coded for in the cloned gene.     

Purification,Characterization, and Gene Analysis of Cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both β-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the β-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to β-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Cloning and Expression of a Gene for an 87-kDa β-1,3-Glucanase of Bacillus circulans IAM1165 in Escherichia coli K-12

Partially Sau3AI-digested fragments of chromosomal DNA from Bacillus circulans IAM1165, a high producer of β-1,3-glucanases able to lyse fungal cell walls, were inserted into a BamHI site of the plasmid vector pHSG399. A gene for the glucanase was cloned in Escherichia coli K-12 by the shotgun method. An 8-kb inserted DNA directed synthesis of an 87-kDa endo-β-1,3-n-glucanase in E. coli. The β-glucanase gene was in a 2.6-kb EcoRI-SmaI segment within the insert DNA. The enzyme activity was found mainly in the periplasmic fraction of E. coli carrying the gene.     

Glucanases and chitinases ofBacillus circulans WL-12

Summary Lysis of cell walls of various yeast species by -1,3- and -1,6-glucanases ofBacillus circulans WL-12 was investigated. Selective enzymolysis of cell walls ofPyricularia oryzae by single and combined actions of -1,3-, -1,6-glucanases and chitinase was followed. Chemical structure of the cell wall glucan ofP. oryzae was determined by chemical and enzymatic methods. Multiple component nature of glucanases ofB. circulans WL-12, their induction and lytic actions on cell walls of various yeasts were studied. Genes specifying glucanases and chitinases ofB. circulans WL- 12 were cloned inE. coli, and their nucleotide sequences were determined. Fibronectin type III modules were found in the chitinases. Functions of the domains of the deduced structures of the glucanases and the chitinases were studied by various methods including molecular genetic techniques.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan.

Limited proteolysis of beta-1,3-glucanase A1 by three different proteases, trypsin, chymotrypsin, and papain, gave three major active fragments. The sizes of the three major fragments generated by each protease treatment were identical to those of beta-1,3-glucanase A2, A3, and A4 detected in both the culture supernatant of Bacillus circulans WL-12 and the periplasmic space of Escherichia coli carrying a cloned glcA gene. These results indicate a four-domain structure for the enzyme. At the N terminus of the glucanase, duplicated segments of approximately 100 amino acids were observed. N-terminal amino acid sequence analysis revealed that the active fragments with sizes corresponding to those of A2 and A3 lack the first segment (domain) and both duplicated segments (domains), respectively. The fragment corresponding to A4 lacks both duplicated segments and the following ca. 120-amino-acid region. By losing the first, second, and third (corresponding to the segment of 120 amino acids) domains, beta-1,3-glucanase progressively lost the ability to bind to pachyman, beta-1,3-glucan. An active fragment which did not have the three N-terminal domains did not show significant binding to pachyman. Thus, all three N-terminal domains contribute to binding to beta-1,3-glucan, and the presence of three domains confers the highest binding activity on the glucanase. The loss of these binding domains remarkably decreased pachyman-hydrolyzing activity, indicating that the binding activity is essential for the efficient hydrolysis of insoluble beta-1,3-glucan.     

Cloning and Expression of an α-1,3-Glucanase Gene from Bacillus circulans KA-304: The Enzyme Participates in Protoplast Formation of Schizophyllum commune

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of α-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity.

The gene of α-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of α-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of α-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (α-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.

The recombinant α-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant α-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-β-D-thiogalactopyranoside. The recombinant α-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.     

Overproduction and secretion of Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium

A gene coding for endo--1,3-1,4-glucanase (lichenase) containing a recombinant plasmid, pLL200K, was transferred from Bacillus circulans into a new shuttle plasmid, pLLS920, by ligating linearized DNAs of pLL200K and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLLS920 produced the endo--1,3-1,4-glucanase. The enzyme was produced during active growth with maximum activity. The B. subtilis (pLLS920) enzyme was 83 times (8522 mU ml^–1) more active than that of the gene donor cells (103 mU ml^–1). The B. megaterium (pLLS920) enzyme was 7 times (735 mU ml^–1) more active than that of the gene donor cells. While E. coli secreted only about 10% of the produced enzyme, B. subtilis excreted the enzyme completely into the medium and B. megaterium by about 98%. The plasmid pLLS920 was stable in B. megaterium (98%), and in B. subtilis (51%) but not in E. coli (29%).     

Cloning and expression of a β -1,3-glucanase gene from Bacillus circulans KCTC3004 in Escherichia coli

Summary A new gene encoding the -1,3-glucanase(laminarinase) of Bacillus circulans KCTC3004 was cloned into Escherichia coli using pUC19 as a vector. The gene localized in the 5.3 kb PstI DNA fragment was expressed independently of its orientation in the cloning vector showing enzyme activity about 33 times greater than that produced by the original B. circulans. The optimum pH and temperature of the cloned enzyme were pH 5.4 and 50°C, respectively. The molecular weight of the enzyme was about 38,000 and the processing of the enzyme molecule within the E. coli cell was not observed. The enzyme hydrolyzed laminarin to produce laminaritriose, laminaribiose, and glucose as main products, but it was inactive for lichenan, CMC, or xylan.     

Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

A Bacillus subtilis gene coding for an endo-β-1,3-1,4-glucanase has been transferred to Escherichia coli by molecular cloning using bacteriophage λ and plasmid vectors. The gene is contained within a 1.6-kb EcoRI-PvuI DNA fragment and directs the synthesis in E. coli of a β-glucanase which specifically degrades barley glucan and lichenan. A novel dye-staining method has been developed to detect β-glucanase activity in colonies on agar plates.     

Additional Carbohydrate-Binding Modules Enhance the Insoluble Substrate-Hydrolytic Activity of β-1,3-Glucanase from Alkaliphilic Nocardiopsis sp. F96

β-1,3-Glucanase (BglF) from Nocardiopsis sp. F96 is composed of only a catalytic domain. To improve the enzymatic properties of BglF, we attempted to construct chimeric enzymes consisting of BglF and some carbohydrate-binding modules, such as the C-terminal additional domain (CAD) and the N-terminal additional domain (NAD) of β-1,3-glucanase H from Bacillus circulans IAM1165 and the chitin-binding domain (ChBD) of chitinase from alkaliphilic Bacillus sp. J813. CAD-fused BglF (BglF-CAD), NAD-fused BglF (NAD-BglF), both NAD- and CAD-fused BglF (NAD-BglF-CAD) and ChBD-fused BglF (BglF-ChBD) were constructed and characterized. The addition of CAD caused increases in binding abilities and hydrolytic activities toward insoluble β-1,3-glucans. As well as BglF-CAD, the binding ability and hydrolytic activity of BglF-ChBD toward pachyman were also increased. The hydrolytic activity of BglF-CAD at pH 9–10 was higher than that of BglF. The relative activities of BglF-CAD and BglF-ChBD at around 50–70 °C were higher than that of BglF.     

β-1, 3-Glucanase Produced by Alkalophilic Bacteria Bacillus No. K-12-5

Bacillus No. K–12–5 isolated from soil produced a β-1,3-glucanase in alkaline media. The characteristic point of this bacteria was especially good growth in alkaline media, and no growth was observed in neutral media such as nutrient broth. The β-1,3-glucanase of Bacillus No. K–12–5 was purified by DEAE-cellulose, Sephadex G–100 and hydroxyl apatite columns. The enzyme was most active at pH 5.5 ~ 8.0 which was much broader and higher than those of Bacillus criculans enzyme. The sedimentation constant was about 3.6 and molecular weight was about 40,000. The isoelectric point was about pH 3.5 and the enzyme was most stable at pH 7. Calcium ion was not effective to stabilize the enzyme. The enzyme did not hydrolyse laminaritriose. Laminaritetraose was hydrolysed, and glucose and laminaritriose were detected in the hydrolysate. The enzyme split laminaran at random and yielded glucose, laminaribiose, laminaritriose and higher oligosaccharides. If the enzyme is a single entity, it is a type of endo-β-1,3-glucanase. However, activity of hydrolysis of fungal cell walls was lower than that of B. circulans enzyme.     

Subcloning and functional analysis of aBacillus subtilis β-1,3-1,4-glucanase gene (bglS) inEscherichia coli

This article describes a 7.1kb EcoRI DNA fragment carrying aBacillus subtilis -1,3-1,4-glucanase gene (bglS). By subcloning, a 1.5kb EcoRI-PstI DNA fragment was inserted into the polylinker cloning sites of the plasmid pUC19 and transferred toEscherichia coli JM101. The fragment directed the synthesis inE. coli of a-1,3-1,4-glucanase that specifically degrades barley glucan and lichenan. The largest proportion (>50%) of the total enzyme activity was cellular enzyme; 25% of the-1,3-1,4-glucanse activity was present in the extracellular fractions. Through enzyme analysis, the enzymes purified fromE. coli or fromBacillus subtilis 1.88 were proved to be identical.     

Cloning and Structural Analysis of bglM Gene Coding for the Fungal Cell Wall-lytic β-1,3-Glucan-hydrolase BglM of Bacillus circulans IAM1165

Bacillus circulans IAM1165 produces isoforms of β-1,3-glucan-hydrolases. Of these enzymes, the 42-kDa enzyme BglM degrades Aspergillus oryzae cell walls the most actively. A gene coding for a BglM precursor consisting of 411 amino acid residues was cloned. The 27 N-terminal amino acid sequence of the precursor is a signal peptide. The 141 C-terminal amino acid sequence showed a motif of carbohydrate-binding module family 13. This domain bound to pachyman, lichenan, and A. oryzae cell walls. The central domain showed a bacterial β-1,3-glucan-hydrolase motif belonging to glycosyl hydrolase family 16. By removal of the C-terminal domain in the IAM1165 culture, mature BglM was processed to several 27-kDa fragments that hydrolyze a soluble β-1,3-glucan.     

Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the recombinant enzyme

An endoglycanase gene of Paenibacillus cookii SS-24 was cloned and sequenced. This Pgl8A gene had an open reading frame of 1,230 bp that encoded a putative signal sequence (31 amino acids) and mature enzyme (378 amino acids: 41,835 Da). The enzyme was most homologous to a β-1,3-1,4-glucanase of Bacillus circulans WL-12 with 84% identity. The recombinant enzyme hydrolyzed carboxymethyl cellulose, swollen celluloses, chitosan and lichenan but not Avicel, chitin powder or xylan. With chitosan as the substrate, the optimum temperature and hydrolysis products of the recombinant enzyme varied at pH 4.0 and 8.0. This is the first report that characterizes chitosanase activity under different pH conditions.     

Microcalorimetric studies on the polyhydroxyalkanoates production of recombinant Escherichia Coli

The thermogenic curves of metabolism of two strains of Escherichia coli pUC19cab/XL-IBlue and XL-IBlue have been determined by using a LKB-2277 bioActivity Monitor and ampoule method at 37°C. pUC19cab/XL-IBlue was a recombinant E. coli strain bearing a foreign plasmid pUC19cab which brought the polyhydroxyalkanoates (PHAs) production. XL-IBlue was a host bacterium without any foreign DNA. Our studies reveal that the PHA production of recombinant E. coli has an apparent influence on their thermogenic curves of metabolism and therefore the initial time of PHAs production can be determined from these thermogenic curves. The text was submitted by the authors in English.     

Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli

Hen egg white lysozyme was expressed as a protein fusion with the OmpA signal sequence and an octapeptide linker in Escherichia coli. The expression yielded soluble and enzymatically active lysozyme. Lysozyme activity was detected in the periplasmic space, in the cytosol and in the insoluble cytosolic fraction of E. coli. The results indicate that the environmental conditions in both the cytosol and the periplasmic space of E. coli were sufficient for correct protein folding and disulphide bond formation of eukaryotic recombinant lysozyme. However, the expression of active enzyme in E. coli consequently led to bacterial cell lysis due to hydrolysis of the peptidoglucan. Correspondence to: B. Fischer     

Multiple β-1,3 Glucanases in the Lytic Enzyme Complex of Bacillus circulans WL 12

The lytic enzyme complex produced by Bacillus circulans WL 12 upon induction with the myceiia of Piricularia oryzae P₂ was analyzed by large scale polyacrylamide gel electrophoresis, Six β-1,3 glucanases were separated. Activity profiles were obtained of these multiple β-1,3 glucanases against P. oryzae P₂ cell walls, Saccharomyces cerevisiae walls, laminarin, oat glucan, Phytophthora glucan and pachyman.     

Enzymatic properties,crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans

A high-isoelectric-point (pI), alkaline endo-1,4-β-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 °C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-β-glucanase. However, this enzyme was not active on p-nitrophenyl β-d-cellotrioside and p-nitrophenyl β-d-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl₂ as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 Å resolution. It belongs to the space group P2₁2₁2₁ with unit cell parameters of a=62.5 Å, b=71.7 Å, and c=88.6 Å.     

Vectors for cloning in cyanobacteria: construction and characterization of two recombinant plasmids capable of transformation of Escherichia coli K12 and Anacystis nidulans R2

Summary Two plasmids were constructed consisting of the E. coli vector pACYC184 and the cyanobacterial plasmid pUC1. These recombinants, designated pUC104 and pUC105, can be transformed to E. coli K12 as well as to the cyanobacterium Anacystis nidulans R2 and in both hosts they express their antibiotic markers. pUC104 and pUC105 differ with respect to the location and the orientation of the pACYC184 segment in pUC1. pUC104 was found to be stable under all circumstances. Transformation of pUC105 to A. nidulans R2 gave intact plasmids when chloramphenicol was the selective agent, but upon ampicillin selection a deletion derivative was produced identical to pUC1. Further characteristics of pUC104 and pUC105 are described and their usefulness as cloning vectors is discussed.     

Expression of an 87-kD-β-1,3-Glucanase of Bacillus circulans IAM1165 in Saccharomyces cerevisiae by Low-temperature Incubation

A DNA segment encoding a signal peptide from yeast invertase was fused in frame to. hglH. gene encoding 87-kD- β-1,3-glucanase from Bacillus circulans IAM1165 and was expressed in the yeast Saccharomyces cerevisiae under the control of the GAL1 gene promoter. Yeast cells contain.ng this fused gene produced active β -1,3-glucanase in the medium after a long period of incu ation at low temperature. The enzyme produced by yeast was heterogeneous in size, and larger than the enzyme produced by Escherichia coli.