Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune

KA-prep, 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 alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.     

Cloning and expression of chitinase A gene from Streptomyces cyaneus SP-27: the enzyme participates in protoplast formation of Schizophyllum commune

Chitinase A of Streptomyces cyaneus SP-27 or chitinase I of Bacillus circulans KA-304 showed the protoplast-forming activity when combined with alpha-1,3-glucanase of B. circulans KA-304. The gene of chitinase A was cloned. It consisted of 903 nucleotides encoding 301 amino acid residues, including a putative signal peptide (35 amino acid residues). The deduced N-terminal moiety of chitinase A showed sequence homology with the chitin-binding domain of chitinase F from Streptomyces coelicolor and chitinase 30 from Streptomyces olivaceoviridisis. The C-terminal moiety also showed high sequence similarity to the catalytic domain of several Streptomyces family 19 chitinases as well as that of chitinase I of B. circulans KA-304. Recombinant chitinase A was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the recombinant enzyme were almost the same as those of chitinase A purified from a culture filtrate of S. cyaneus SP-27. The recombinant enzyme was superior to B. circulans KA-304 chitinase I not only in respect to protoplast forming activity in a mixture containing alpha-1,3-glucanase, but also to antifungal activity and powder chitin-hydrolyzing activity.     

Purification and characterization of chitinase A of Streptomyces cyaneus SP-27: an enzyme participates in protoplast formation from Schizophyllum commune mycelia

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. alpha-1,3-Glucanase and chitinase I, which were isolated from the filtrate, did not form the protoplast by itself while a mixture of them showed protoplast-forming activity. Streptomyces cyaneus SP-27 was isolated based on the productivity of chitinase. The culture filtrate of S. cyaneus SP-27 did not form S. commune protoplasts, but addition of it to alpha-1,3-glucanase of B. circulans KA-304 brought about protoplast-forming activity. Chitinase A isolated from the S. cyaneus SP-27 culture filtrate was more effective than chitinase I of B. circulans KA-304 for the protoplast formation in combination with alpha-1,3-glucanase. The N-terminal amino acid sequence of chitinase A (MW 29,000) has a sequential similarity to those of several Streptomycete family 19 chitinases. Chitinase A adsorbed to chitinous substrate and inhibited the growth of Trichoderma reesei mycelia. Anomer analysis of the reaction products also suggested that the enzyme is a family 19 chitinase.     

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.     

A chitinase indispensable for formation of protoplast of Schizophyllum commune in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304

KA-prep, 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. alpha-1,3-Glucanase, which was isolated from an ammonium sulfate fraction of 0-30% saturation of KA-prep, gave the protoplast-forming activity to an ammonium sulfate fraction of 30-50% saturation of KA-prep, which contained chitinase(s) and beta-glucanase(s) but was inactive in the protoplast formation. Chitinase(s) and beta-glucanase(s) in the ammonium sulfate fraction of 30-50% saturation were separated by DEAE-cellulofine A-500 column chromatography, and the protoplast-forming activity appeared when the chitinase preparation was mixed with the alpha-1,3-glucanase. The beta-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of alpha-1,3-glucanase and the chitinase preparation. The chitinase preparation contained two chitinases (chitinase I and II). Chitinase I showed the protoplast-forming activity with alpha-1,3-glucanase, but chitinase II did not. Chitinase I, a monomeric protein with a molecular weight of 41,000, was active toward colloidal chitin and ethylene glycol chitin. Chitinase I produced predominantly N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose from colloidal chitin, and the enzyme was inactive to p-NP-beta-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme. The N-terminal amino acid sequence of chitinase I (A L A T P T L N V S A S S G M) had no sequential identity to those of known chitinases.     

Occurrence of a specific protein in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304 inductively with a cell-wall preparation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation (CWP) of Schizophyllum commune, has been reported to have an activity to form protoplasts from S. commune mycelia. The SDS-polyacrylamide gel electrophoreses described here demonstrated that a specific proteinous component (molecular weight: 150,000) occurred in KA-prep. The protein (P150T) was also formed in culture filtrates with CWP of several basidiomycetes, which could release the protoplasts, suggesting that the component was an indispensable factor for protoplast formation. P150T, isolated from an ammonium sulfate fraction of KA-prep (0-30% saturation), did not have any protoplast-forming activity. Results were obtained indicating that P150T participates in protoplast formation together with chitinase(s) and beta-glucanase(s) in KA-prep. The N-terminal amino acid sequence indicated an analogy of P150T to mutanase (alpha-1,3-glucanase) from Bacillus sp. RM1, and actually P150T hydrolyzed mutan as well as S-(alpha-1,3) glucan from S. commune.     

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.     

Mutanase from a Paenibacillus isolate: nucleotide sequence of the gene and properties of recombinant enzymes

A mutanase (alpha-1,3-glucanase)-producing microorganism was isolated from a soil sample and was identified as a relative of Paenibacillus sp. The mutanase was purified to homogeneity from culture, and its molecular mass was around 57 kDa. The gene for the mutanase was cloned by PCR using primers based on the N-terminal amino acid sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of 3651-bp open reading frame that encoded a predicted 1217-amino acid polypeptide including a 43-amino acid signal peptide. The mature enzyme showed similarity to mutanases RM1 of Bacillus sp. strain RM1 and KA-304 of Bacillus circulans with 65.6% and 62.7% identity, respectively. The predicted molecular mass of the mutanase was 123 kDa. Thus, the enzyme purified from the isolate appears to be truncated by proteolysis. The genes for the full-length and truncated mutanases were expressed in Bacillus subtilis cells, and the corresponding recombinant enzymes were purified to homogeneity. The molecular masses of the two enzymes were 116 and 57 kDa, respectively. The specific activity was 10-fold higher for the full-length enzyme than for the truncated enzyme. The optimal pH and temperature for both recombinant enzymes was pH 6.4 in citrate buffer and 45 degrees C to 50 degrees C. Amongst several tested polysaccharides, the recombinant full-length enzyme specifically hydrolyzed mutan.     

Nucleotide and deduced amino acid sequences of mutanase-like genes from Paenibacillus isolates: proposal of a new family of glycoside hydrolases

Three mutanase (alpha-1,3-glucanase)-producing microorganisms isolated from soil samples were identified as a relatives of Paenibacillus. A mutanase was purified to homogeneity from cultures of each, and the molecular masses of the purified enzymes were approximately 132, 141, and 141kDa, respectively. The corresponding three genes for mutanases were cloned by PCR using primers designed from each N-terminal amino acid sequence. Another mutanase-like gene from one strain was also cloned by PCR using primers designed from conserved amino acid sequences among known mutanases. Consequently, four mutanase-like genes were sequenced. The genes contained long open reading frames of 3411 to 3915bp encoding 1136 to 1304 amino acids. The deduced amino acid sequences of the mutanases showed relatively high similarity to those of a mutanase (E16590) from Bacillus sp. RM1 with 46.9% to 73.2% identity and an alpha-1,3-glucanase (AB248056) from Bacillus circulans KA-304 with 46.7% to 70.4% identity. Phylogenetic analysis based on the amino acid sequences of the enzymes showed bacterial mutanases form a new family between fungal mutanases (GH family 71) and Streptomycetes mycodextranases (GH family 87).     

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both beta-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 beta-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 beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Mutanase from a Paenibacillus isolate: Nucleotide sequence of the gene and properties of recombinant enzymes

A mutanase (α-1,3-glucanase)-producing microorganism was isolated from a soil sample and was identified as a relative of Paenibacillus sp. The mutanase was purified to homogeneity from culture, and its molecular mass was around 57 kDa. The gene for the mutanase was cloned by PCR using primers based on the N-terminal amino acid sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of 3651-bp open reading frame that encoded a predicted 1217-amino acid polypeptide including a 43-amino acid signal peptide. The mature enzyme showed similarity to mutanases RM1 of Bacillus sp. strain RM1 and KA-304 of Bacillus circulans with 65.6% and 62.7% identity, respectively. The predicted molecular mass of the mutanase was 123 kDa. Thus, the enzyme purified from the isolate appears to be truncated by proteolysis. The genes for the full-length and truncated mutanases were expressed in Bacillus subtilis cells, and the corresponding recombinant enzymes were purified to homogeneity. The molecular masses of the two enzymes were 116 and 57 kDa, respectively. The specific activity was 10-fold higher for the full-length enzyme than for the truncated enzyme. The optimal pH and temperature for both recombinant enzymes was pH 6.4 in citrate buffer and 45 °C to 50 °C. Amongst several tested polysaccharides, the recombinant full-length enzyme specifically hydrolyzed mutan.     

Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin

A chitinase gene of Bacillus circulans WL-12 was cloned into Escherichia coli by transforming HB101 cells with a recombinant plasmid composed of chromosomal DNA fragments prepared from B. circulans WL-12 and the plasmid vector pKK223-3. DNA sequencing analysis revealed that the region necessary for the normal expression of chitinase activity contained one open reading frame of 2097 base pairs which codes for the precursor of chitinase A1. The precursor of chitinase A1 contained a long signal sequence of 41 amino acids with an extremely long N-terminal hydrophilic segment of 15 amino acids. Cloned chitinase produced in E. coli had at its N terminus an additional 8 amino acids that were not found in B. circulans mature chitinase A1. The N-terminal two-thirds of the deduced amino acid sequence of chitinase A1 showed a 33% amino acid match to chitinase A of Serratia marcescens. This region of chitinase A1 is immediately followed by tandemly repeating 95-amino acid segments that are 70% homologous to each other. Statistical analysis revealed that these repeating segments are homologous to the type III homology units of fibronectin, a multifunctional extracellular matrix and plasma protein of higher eukaryotes. This observation indicates that type III homology units originated prior to the emergence of eukaryotes and may be distributed in a wide range of organisms.     

Purification and characterization of a Bacillus cereus exochitinase

Five extracellular chitinases of Bacillus cereus 6E1 were detected by a novel in-gel chitinase assay using carboxymethyl-chitin-remazol brilliant violet 5R (CM-chitin-RBV) as a substrate. The major chitinase activity was associated with a 36-kDa (Chi36) gel band. Chi36 was purified by a one-step, native gel purification procedure derived from the new in-gel chitinase assay. The purified Chi36 has optimal activity at pH 5.8 and retains some enzymatic activity between pH 2.5-8. The temperature optimum for Chi36 was 35 degrees C, but the enzyme was active between 4-70 degrees C. Based on its ability to hydrolyze mainly p-nitrophenyl-(N-acetyl-beta-D-glucosaminide)(2), Chi36 is characterized as a chitobiosidase, a type of exochitinase. The N-terminal amino acid sequence of mature Chi36 was determined (25 amino acids). Alanine is the first N-terminal amino acid residue indicating the cleavage of a signal peptide from a Chi36 precursor to form the mature extracellular Chi36. The N-terminal sequence of Chi36 demonstrated highest similarity with Bacillus circulans WL-12 chitinase D and significant similarity with several other bacterial chitinases.     

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.).     

The aman6 gene encoding a yeast mannan backbone degrading 1,6-alpha-D-mannanase in Bacillus circulans: cloning, sequence analysis, and expression

A gene (aman6) encoding endo-1,6-alpha-D-mannanase, a yeast mannan backbone degrading enzyme from Bacillus circulans was cloned. The putative aman6 was 1,767 base pairs long and encoded a mature 1,6-alpha-D-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids. The purified mature 1,6-alpha-D-mannanase from the Escherichia coli transformant showed 61-kDa protein, and N-terminal amino acid sequence and other general properties of the recombinant enzyme were identical to those of 1,6-alpha-D-mannanase from Bacillus circulans TN-31.     

Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma.

A method is described for the quantitative and, possibly, large-scale extraction of astaxanthin from the yeast Phaffia rhodozyma. The method utilizes extracellular enzymes produced by the bacterium Bacillus circulans WL-12, which partially digests the yeast cell wall and renders the carotenoid pigments extractable by acetone or ethanol. Complete recovery of astaxanthin from heat-killed P. rhodozyma cells was obtained after growing B. circulans WL-12 on these yeast cells for 26 h and then extracting the yeast-bacterium mixture with acetone. A bacteria-free lytic system, which gave quantitative extraction of astaxanthin from P. rhodozyma, was obtained by concentrating the culture broth from the growth of B. circulans WL-12 on P. rhodozyma cells. Hydrolytic enzyme activities detected in this concentrate included beta-(1 leads to 3)-glucanase, beta-(1 leads to 6)-glucanase, alpha-(1 leads to 3)-glucanase, xylanase, and chitinase. The lytic system was found to work most efficiently at pH 6.5 and with low concentrations of yeast.     

Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma.

A method is described for the quantitative and, possibly, large-scale extraction of astaxanthin from the yeast Phaffia rhodozyma. The method utilizes extracellular enzymes produced by the bacterium Bacillus circulans WL-12, which partially digests the yeast cell wall and renders the carotenoid pigments extractable by acetone or ethanol. Complete recovery of astaxanthin from heat-killed P. rhodozyma cells was obtained after growing B. circulans WL-12 on these yeast cells for 26 h and then extracting the yeast-bacterium mixture with acetone. A bacteria-free lytic system, which gave quantitative extraction of astaxanthin from P. rhodozyma, was obtained by concentrating the culture broth from the growth of B. circulans WL-12 on P. rhodozyma cells. Hydrolytic enzyme activities detected in this concentrate included beta-(1 leads to 3)-glucanase, beta-(1 leads to 6)-glucanase, alpha-(1 leads to 3)-glucanase, xylanase, and chitinase. The lytic system was found to work most efficiently at pH 6.5 and with low concentrations of yeast.     

环状芽孢杆菌几丁质酶基因序列分析、表达和生物活性测定

真菌病害一直是影响作物的主要病害之一 ,每年造成巨大经济损失。几丁质酶可水解许多病原真菌细胞壁所含有的主要成分—几丁质 ,是研究得最多的抗真菌蛋白质。许多几丁质酶基因已从微生物中克隆到 ,芽孢杆菌是一类重要的几丁质酶产生菌。环状芽孢杆菌可产生并分泌多种多糖降解酶类 ,包括几丁质酶、β 1 ,3 葡聚糖酶、β 1 ,6葡聚糖酶和半纤维素酶[1] 。Ｗａｔａｎａｂｅ克隆了环状芽孢杆菌ＷＬ 1 2菌株的几丁质酶基因ｃｈｉＡ和ｃｈｉＤ ,对该几丁质酶基因的结构和功能进行了深入研究[2～ 4 ] 。我国的陈三凤克隆了黄杆菌的几丁质酶基因 ,…     

Molecular cloning, expression, and characterization of endo-beta-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans.

Endo-beta-1,4-glucanase genes from Bacillus circulans and from B. polymyxa were cloned by direct expression by using bacteriophage M13mp9 as the vector. The enzymatic activity of the gene products was detected by using either the Congo red assay or hydroxyethyl cellulose dyed with Ostazin Brilliant Red H-3B. The B. circulans and B. subtilis PAP115 endo-beta-1,4-glucanase genes were shown to be homologous by the use of restriction endonuclease site mapping, DNA-DNA hybridization, S1 nuclease digestion after heteroduplex formation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein products. Analysis of the nucleotide sequence of 3.1 kilobase pairs of cloned B. polymyxa DNA revealed two convergently transcribed open reading frames (ORFs) consisting of 398 codons (endoglucanase) and 187 codons (ORF2) and separated by 374 nucleotides. The coding region of the B. polymyxa endoglucanase gene would theoretically produce a 44-kilodalton preprotein. Expression of the B. polymyxa endoglucanase in Escherichia coli was due to a fusion of the endoglucanase gene at codon 30 with codon 9 of the lacZ alpha-peptide gene. The B. polymyxa endoglucanase has 34% amino acid similarity to the Clostridium thermocellum celB endoglucanase sequence but very little similarity to endoglucanases from other Bacillus species. ORF2 has 28% amino acid similarity to the NH2-terminal half of the E. coli lac repressor protein, which is responsible for DNA binding.