Sequencing,cloning, and heterologous expression of cyclomaltodextrin glucanotransferase of Bacillus firmus strain 37 in Bacillus subtilis WB800

Bacillusfirmus strain 37 produces the cyclomaltodextrin glucanotransferase (CGTase) enzyme and CGTase produces cyclodextrins (CDs) through a starch cyclization reaction. The strategy for the cloning and expression of recombinant CGTase is a potentially viable alternative for the economically viable production of CGTase for use in industrial processes. The present study used Bacillus subtilis WB800 as a bacterial expression host for the production of recombinant CGTase cloned from the CGTase gene of B. firmus strain 37. The CGTase gene was cloned in TOPO-TA^® plasmid, which was transformed in Escherichia coli DH5α. The subcloning was carried out with pWB980 plasmid and transformation in B. subtilis WB800. The 2xYT medium was the most suitable for the production of recombinant CGTase. The enzymatic activity of the crude extract of the recombinant CGTase of B. subtilis WB800 was 1.33 µmol β-CD/min/mL, or 7.4 times greater than the enzymatic activity of the crude extract of CGTase obtained from the wild strain. Following purification, the recombinant CGTase exhibited an enzymatic activity of 157.78 µmol β-CD/min/mL, while the activity of the CGTase from the wild strain was 9.54 µmol β-CD/min/mL. When optimal CDs production conditions for the CGTase from B. firmus strain 37 were used, it was observed that the catalytic properties of the CGTase enzymes were equivalent. The strategy for the cloning and expression of CGTase in B. subtilis WB800 was efficient, with the production of greater quantities of CGTase than with the wild strain, offering essential data for the large-scale production of the recombinant enzyme.

     

Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis

Summary The cyclomaltodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) gene from an alkalophilic Bacillus circulans var. alkalophilus ATCC21783 was cloned into Escherichia coli and B. subtilis. When cloned from E. coli to B. subtilis, the entire insert containing the CGTase gene was, depending on the plasmid construction, either unstable or the recombinant B. subtilis did not secrete the enzyme in significant amounts. To achieve efficient enzyme production in B. subtilis, the gene was placed under the control of the B. amyloliquefaciens -amylase promoter. In one of the constructions, both the promoter and the signal sequence of the gene were replaced with those of B. amyloliquefaciens, whereas in another construction only the promoter area was exchanged. The recombinant B. subtilis clones transformed with these plasmid constructions secreted CGTase into the culture medium 14 times as much as did the parental strain in shake flask cultures. In fermentor cultures in an industrially feasible medium the enzyme production was substantially higher, yielding 1.2 g/l of CGTase, which is about 33 times the amount of the enzyme produced by the parental strain in corresponding fermentations. Both of the plasmid constructions were stable when grown over 50 generations without antibiotic selection.     

Cyclodextrin production and genetic characterization of cyclodextrin glucanotranferase of <Emphasis Type="Italic">Paenibacillus graminis</Emphasis>

Paenibacillus graminis strains were described recently as cyclodextrin (CD) producers. Cyclodextrins are produced by cyclodextrin glucanotransferase (CGTase) which has not been characterized in P. graminis. Similar amounts of α- and β-CDs were produced by P. graminis (MC22.13) and P. macerans (LMD24.10^T). Primers were designed to sequence the gene encoding CGTase from P. graminis. A phylogenetic tree was constructed and P. graminis CGTase protein showed to be closer (79.4% protein identity) to P. macerans |P31835|. Hybridization studies suggested that the gene encoding CGTase is located in different positions in the genomes of P. macerans and P. graminis.     

Thermostable enzymes for industrial applications

Summary The variety of thermostable (TS) enzymes has been steadily increasing for use in industrial applications, mainly as replacements for thermolabile (TL) enzymes. For example, TS amylases fromBacillus licheniformis andBacillus stearothermophilus have replaced TL amylases fromBacillus subtilis. TS enzymes also have advantages in new areas such as cyclodextrin production. The TS cyclodextrin glycosyl transferase (CGTase) fromThermoanaerobacter sp. (95°C optimum) gives a higher productivity than the CGTase fromBacillus macerans (55°C optimum). In the area of enzymatic bleach boosting of wood pulps, a TS xylanase (Myceliophera thermophila) would be advantageous over a TL xylanase (Trichoderma reesei), due to the high temperature of the incoming pulp. Not all TS enzymes are from thermophiles; the mesophileCandida antarctica produces a TS lipase which has a temperature optimum of 90°C when immobilized. The characterization of these enzymes will be described along with comparisons to some newly described TS enzymes.     

Application of LFH-PCR for the disruption ofSpoIIIE andSpoIIIG ofB. subtilis

The application of LFH-PCR (long flanking homology region-PCR) forBacillus subtilis gene disruption is presented. Without plasmid- or phage-vector construction, only by PCR, based on a DNA sequence retrieved fromB. subtilis genome data base, kanamycin resistance gene was inserted into two genes ofB. subtilis involved in sporulation,spoIIIE andspoIIIG. The effect of gene disruption on subtilisin expression was examined and the sporulation frequency of two mutants was compared to that of the host strain. For this purpose, only 2 or 3 rounds of PCR were required with 4 primers. We first demonstrated the possibility of LFH-PCR for rapid gene disruption to characterize an unknown functional gene ofB. subtilis or other prokaryote in the genomic era.     

A zymogram method to detect endoglucanases fromBacillus subtilis,Myrothecium verrucaria andTrichoderma reesei

Summary A purified endoglucanase (Eg) fromBacillus subtilis and crude Eg preparations fromMyrothecium verrucaria andTrichoderma reesei were denatured by heat in the presence of SDS and separated by using SDS polyacrylamide gel electrophoresis. Eg zymogram activity bands were detected directly in carboxymethylcellulose-laden separating gels after enzyme renaturation. Denaturing zymogram analysis was a simple method for the separation and detection of Egs.     

Production of cyclodextrin glycosyltransferase by alkaliphilic <Emphasis Type="Italic">Bacillus circulans</Emphasis> in submerged and solid-state cultivation

Cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19) is an industrially important enzyme, which is used to produce cyclodextrins (CDs). In this research, we report the use of experimental factorial design to find the best conditions of pH and temperature for CGTase production by Bacillus circulans var. alkalophilus. The optimized calculated values for the tested variables were, respectively, pH 9.7 and temperature 36^oC, with a CGTase activity of 615 U mL⁻¹. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC) and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on SC was 1,155 U mL⁻¹ under aerobic conditions. Cell growth and CGTase synthesis in SSC using SIFR as substrate was excellent, with CGTase activity of 32,776 U g_(SIFR) ⁻¹. These results strongly support the use of SIFR for CGTase production since it is a non-expensive residue.     

Selective degradation of β- and γ-cyclodextrin by co-digestion using a CGTase fromBacillus ohbensis and α-glucosidase for efficient α-cyclodextrin recovery

A simple and specific recovery method for α-cyclodextrin (α-CD) was developed by employing co-digestion of CD reaction mixtures with CGTase fromBacillus ohbensis and α-glucosidase. The combination of CGTase fromB. ohbensis and α-glucosidase, such as α-amylase, β-amylase, or glucoamylase was examined for the selective degradation of β-and γ-CD in the CD reaction mixture formed by CGTase fromB. macerans. The co-digestion of the CD mixture with Taka-amylase and the CGTase resulted in α-CD and maltodextrins, the combination with β-amylase resulted in α-CD and maltose, and that with glucoamylase resulted in α-CD and glucose. The conditions of selective degradation of β- and γ-CD by co-digestion with the CGTase and glucoamylase were optimized as follows: the incubation pH, 5.5; incubation temperature, 50°C; CGTase concentration, 15 u/g of substrate; glucoamylase, 10 u/g of substrate; substrate concentration, 10% (w/v); the incubation time was fixed for 18 hr from the stand point of operation convenience. Most part of the content was presented in poster session at the 7th International Cyclodextrin Symposium, Tokyo, April 1994.     

Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.     

Preprosubtilisin Carlsberg processing and secretion is blocked after deletion of amino acids 97-101 in the mature part of the enzyme

Summary During an investigation into the substrate specificity and processing of subtilisin Carlsberg fromBacillus licheniformis, two major independent findings were made: (i) as has been shown previously, a stretch of five amino acids (residues 97–101 of the mature enzyme) that loops out into the binding cleft is involved in substrate binding by subtilisin Carlsberg. In order to see whether this loop element also determines substrate specificity, the coding region for these five amino acids was deleted from the cloned gene for subtilisin Carlsberg by site-directed mutagenesis. Unexpectedly the resulting mutant preproenzyme (P42c, M_r=42 kDa) was not processed to the mature form (M_r=30 kDa) and was not released into the medium by a proteasedeficientB. subtilis host strain; rather, it accumulated in the cell membrane. This result demonstrates that the integrity of this loop element, which is very distant from the processing cleavage sites in the preproenzyme, is required for secretion of subtilisin Carlsberg. (ii) In culture supernatants fromB. subtilis harbouring the cloned wild-type subtilisin Carlsberg gene the transient appearance (at 0–3 h after onset of stationary phase) of a processing intermediate (P38c, M_r=38 kDa) of this protease could be demonstrated. P38c very probably represents a genuine proform of subtilisin Carlsberg.     

Pyrimidine ribonucleoside monophosphokinase and the mode of RNA turnover in Bacillus subtilis

A protein catalyzing the phosphorylation of CMP to CDP was purified and characterized. Kinase activity for UMP copurified during ammonium sulfate fractionation, DEAE-cellulose and hydroxylapatite chromatography, and gel filtration on Sephadex G-75, the ratios of activities for the two substrates remaining constant. The purified product, possessing both activities was homogeneous as judged by the single band following polyacrylamide gel electrophoresis. The protein showed no kinase activity against purine nucleoside monophosphates or the other pyrimidine nucleoside monophosphates: dCMP, dUMP, and dTMP. Thus unlike the enteric bacteria, Escherichia coli and Salmonella typhimurium which have distinct enzymes which phosphorylate UMP and CMP, Bacillus subtilis produces a single pyrimidine ribonucleoside monophosphokinase. The K _mvalues of this enzyme from B. subtilis are 0.04 and 0.25 mM for CMP and UMP, respectively, and 0.04 and 0.4 mM for ATP at saturating concentrations of CMP and UMP, respectively. The properties of this enzyme and the differences between enteric bacteria and B. subtilis with respect to the enzymes which phosphorylate CMP are consistent with the measurements which indicate that turnover of messenger RNA is largely hydrolytic in E. coli but largely phosphorolytic in B. subtilis.Non-Standard Abbreviations PRMK Pyridine ribonucleoside monophosphokinase This paper is affectionately dedicated to Professor R. Y. Stanier     

Regulated expression of heterologous genes inBacillus subtilis using the Tn10 encodedtet regulatory elements

Summary TheEscherichia coli-derivedtet regulatory elements from Tn10 have been used to construct vectors allowing the regulated, inducible, high-level expression of foreign genes inBacillus subtilis. While the wild-typetet promoters are inactive inB. subtilis, a synthetic mutanttet sequence with improved promoter consensus sequences and upstream poly A blocks shows activity inB. subtilis. The expression of an indicatorcat gene is inducible by sublethal amounts of tetracycline, indicating that the Tet repressor protein and thetet operator sequences are functional. However, the inducibility and maximal expression are not sufficient in this construct. To improve these properties atet operator sequence was placed between the —35 and —10 boxes of theB. subtilis-derived very strongxyl promoter. In the presence of atetR gene this construct is about 100-fold inducible and has high promoter strength, but some basal expression. This is avoided by placing a secondtet operator downstream resulting in no detectable basal expression at the expense of reduced inducibility. Using the system with a singletet operator inducible expression of glucose dehydrogenase fromB. megaterium was obtained at a very high level, and inducible expression of human single-chain urokinase-like plasminogen activator was achieved at the same level as inE. coli. Unlike inE. coli, the product was not degraded up to 4 h after induction inB. subtilis. These results demonstrate that the regulated expression vector described here should be very useful for production of foreign gene products fromB. subtilis cultures.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Secretory expression in Escherichia coli and Bacillus subtilis of human interferon alpha genes directed by staphylokinase signals

Summary A DNA segment covering the signal sequence coding region, the ribosome binding site, and the promoter of the staphylokinase (sak) 42D gene (Behnke and Gerlach 1987) was cloned into pUC19 to form a portable expression-secretion unit (ESU). Fusion of human interferon α₁ (hIFNα₁) and hybrid hIFNα_1/2 genes to thissak ESU resulted in secretory expression of the two gene products in bothEscherichia coli andBacillus subtilis. While most of the IFNα was exported to the periplasmic space ofE. coli, about 99% was secreted to the culture medium by recombinantB. subtilis strains. The total yield inE. coli was 1.2×10⁵ IU/ml. This level of expression and export led to instability of the recombinant strains that was spontaneously relieved in vivo by inactivation of thesak ESU through insertion of an IS1 element. No such instability was observed withB. subtilis although expression and secretion levels reached even 3×10⁶ IU/ml. Proteolytic degradation of IFNα by extracellular proteases was avoided by a combination of constitutive expression and secretion during the logarithmic growth phase and the use of exoprotease-reduced host strains. The IFNα₁ protein purified fromB. subtilis culture supernatant was correctly processed, carried the expected 11 amino acid N-terminal elongation that resulted from DNA manipulations and proved to be homogenous in Western blotting experiments. The same recombinant plasmid that directed efficient secretion of hIFNα₁ inB. subtilis gave poor yields when introduced intoStreptococcus sanguis.     

Application of fibrin zymography for determining the optimum culture time for protease activity

The culture time for maximal enzyme production by bacteria was determined by SDS-fibrin zymography. Bacillus subtilis and B. amyloliquefaciens secreted several extracellular proteases during the cellular lysis after 40 h and 30 h, respectively. Using this technique, we determined which protein possessed activity and would be isolated before the process of purification began. This proposed method offers an effective harvesting time in which to obtain the proteins having maximal activity.     

Microbial transformation of immunosuppressive compounds III. Glucosylation of immunomycin (FR 900520) and FK 506 byBacillus subtilis ATCC 55060

Summary The regiospecific glucosylation of FK 506 and immunomycin (FR 900520) at the 24-hydroxy position was performed using resting cells ofBacillus subtilis ATCC 55060. 24-Glucopyranosyl FK 506 and 24-glucopyranosyl immunomycin were isolated by methylene chloride extraction and purification using reverse phase HPLC. The metabolite structures were established using spectroscopic techniques including MS and NMR. The glucose conjugate was further confirmed by chemical degradation. Enzymatic glucosylation was demonstrated using cell-free extracts derived fromBacillus subtilis ATCC 55060. The 24-glucosyltransferase, which appears UDP-glucose dependent, was solubilized from cell membranes by treatment with 0.1% Nonidet P-40 detergent. The optimal conditions for assay of the enzyme have been determined.     

Overexpression,purification and characterization of a recombinant secretary catalase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

A recombinant Bacillus subtilis strain (KN25) was generated for the large-scale preparation of catalase. The B. subtilis katA gene encoding for catalase was cloned into the shuttle vector PRB374, downstream of the constitutively active vegII promoter, followed by transformation of the B. subtilis strain WB600 with the plasmid. The transformant strain, KN25 secretes high levels (3,500 U/ml) of catalase, which facilitates its purification. Three simple purification steps yielded nearly homogeneous catalase, with ∼70% recovery. The purified recombinant catalase has a specific activity of 34,600 U/mg under optimal conditions, and is more resistant to acidic conditions than bovine liver catalase.     

Changes in the antibiotic production by co-culture of Rhizopus peka P8 and Bacillus subtilis IFO3335

The co-culture of Bacillus subtilis IFO 3335 with Rhizopus peka P8 or Rhizopus oligosporus P12 in liquid medium was found to increase production of antibiotic activity and to alter the spectrum of activity relative to the pure cultures. However, a mixed culture of Rhizopus arrhizus P7 and Rhizopus oryzae P17 did not produce antibiotic activity. The concentration, ratio, and time of addition of B. subtilis to the R. peka culture was found to influence antibiotic yields. Solid-state fermentations using mixed cultures of R. peka and B. subtilis were investigated. The growth of Escherichia coli IFO 3792 as a target bacterium was inhibited by the mixed culture. These results suggest the possibility of biopreservation of fermented foods by novel co-culture systems.     

The expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase gene in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strains

The gene encoding for B. intermedius glutamyl endopeptidase (gseBi) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius glutamyl endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on endopeptidase expression. Activity of glutamyl endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius glutamyl endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

枯草芽孢杆菌中高效响应N-乙酰神经氨酸生物传感器的构建

枯草芽孢杆菌(Bacillus subtilis)是公认的食品安全菌株,目前已被用于多种高附加值产品的生物合成,包括被广泛用作营养化学品和药物中间体的N-乙酰神经氨酸(N-acetylneuraminic acid, NeuAc)。响应目标产物的生物传感器被广泛用于代谢工程中的动态调控和高通量筛选等方面,以提高生物合成效率。但是,枯草芽孢杆菌中缺乏可高效响应NeuAc的生物传感器。因此,本文首先测试和优化了能将胞外NeuAc转运进胞内的转运蛋白,获得了一系列具有不同转运能力的菌株,以用于后续响应NeuAc的生物传感器的验证;随后将响应NeuAc的转录因子Bbr_NanR的结合位点插入枯草芽孢杆菌组成型启动子的不同位置,筛选具有活性的杂合启动子;接下来,通过在具有NeuAc转运能力的枯草芽孢杆菌中表达Bbr_NanR,选择能响应NeuAc的杂合启动子,并进一步通过优化Bbr_NanR表达量获得了一系列动态范围广、激活倍数高的生物传感器,其中生物传感器P535-N2能灵敏地响应胞内NeuAc浓度的变化,具有最大的动态范围,为(180–20 245) AU/OD;P566-N2则具有最高的激活倍数,为122倍,是已报道的枯草芽孢杆菌中响应N-乙酰神经氨酸的生物传感器的2倍。本文构建的响应NeuAc的生物传感器可用于高产NeuAc的酶突变体和枯草芽孢杆菌菌株的筛选,为枯草芽孢杆菌生物合成NeuAc提供了高效、灵敏的分析和调控工具。