Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region.

Modification of substrate specificity of an autoprocessing enzyme is accompanied by a risk of significant failure of self-cleavage of the pro-region essential for activation. Therefore, to enhance processing, we engineered the pro-region of mutant subtilisins E of Bacillus subtilis with altered substrate specificity. A high-activity mutant subtilisin E with Ile31Leu replacement (I31L) as well as the wild-type enzyme show poor recognition of acid residues as the P1 substrate. To increase the P1 substrate preference for acid residues, Glu156Gln and Gly166Lys/Arg substitutions were introduced into the I31L gene based upon a report on subtilisin BPN' [Wells et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1219-1223]. The apparent P1 specificity of four mutants (E156Q/G166K, E156Q/G166R, G166K, and G166R) was extended to acid residues, but the halo-forming activity of Escherichia coli expressing the mutant genes on skim milk-containing plates was significantly decreased due to the lower autoprocessing efficiency. A marked increase in active enzyme production occurred when Tyr(-1) in the pro-region of these mutants was then replaced by Asp or Glu. Five mutants with Glu(-2)Ala/Val/Gly or Tyr(-1)Cys/Ser substitution showing enhanced halo-forming activity were further isolated by PCR random mutagenesis in the pro-region of the E156Q/G166K mutant. These results indicated that introduction of an optimum arrangement at the cleavage site in the pro-region is an effective method for obtaining a higher yield of active enzymes.     

Molecular cloning of the gene encoding Vibrio metalloproteinase vimelysin and isolation of a mutant with high stability in organic solvents

Vimelysin is a unique metalloproteinase from Vibrio sp. T1800 exhibiting high activity at low temperature and high stability in organic solvents such as ethanol. A 1,821 bp open reading frame of the vimelysin gene encoded 607 amino acid residues consisting of an N-terminal pro-region, a mature enzyme, and a C-terminal pro-region. The mature enzyme region showed 80%, 57% and 35% sequence identity with the mature forms of vibriolysin from V. vulnificus, pseudolysin from Pseudomonas aeruginosa, and thermolysin from Bacillus thermoproteolyticus, respectively. The catalytic residues and zinc-binding motifs of metalloproteinases are well conserved in vimelysin. The vimelysin gene was expressed in E. coli JM109 cells and the recombinant enzyme was purified as a 38-kDa mature form from cell-free extracts. The purified recombinant enzyme is indistinguishable from the enzyme purified directly from Vibrio. To obtain mutants exhibiting higher stability in organic solvents, random mutations were introduced by error-prone PCR and 600 transformants were screened. The N123D mutant exhibits two times higher stability in organic solvents than the wild-type enzyme. A plausible mechanism for the stability of the N123D mutant in organic solvents was discussed based on homology models of vimelysin and the N123D mutant.     

Growth of bacteria on chitin, fungal cell walls and fungal biomass, and the effect of extracellular enzymes produced by these cultures on the antifungal activity of amphotericin B

Vibrio alginolyticus, Streptomyces griseus, Arthrobacter G12, Bacillus sp. and Cytophaga sp. were grown on solid and liquid media containing soluble and insoluble carbon sources. Arthrobacter G12, Bacillus sp. and Cytophaga sp. grew well on media which contained fungal cell walls or fungal biomass as the main carbon source. All bacteria produced extracellular proteases and all bacteria except Arthrobacter G12 produced extracellular chitinases. Growth of Cytophaga sp. on colloidal chitin was paralleled by the accumulated chitinase activity in the culture filtrate, and growth of Cytophaga sp. and Arthrobacter G12 on cell walls of Geotrichum candidum and cell walls of Candida pseudotropicalis was paralleled by the accumulation of laminarinase activity in the culture filtrate, but little or no extracellular chitinase activity was observed in these cultures. Mycolases purified from the culture filtrates of Cytophaga sp. grown on colloidal chitin on cell walls of C. pseudotropicalis potentiated the antifungal activity of amphotericin B.     

碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。     

Evaluation of in vitro Cr(VI) reduction potential in cytosolic extracts of three indigenous Bacillus sp. isolated from Cr(VI) polluted industrial landfill

Three efficient Cr(VI) reducing bacterial strains were isolated from Cr(VI) polluted landfill and characterized for in vitro Cr(VI) reduction. Phylogenetic analysis using 16S rRNA gene sequencing revealed that the newly isolated strains G1DM20, G1DM22 and G1DM64 were closely related to Bacillus cereus, Bacillus fusiformis and Bacillus sphaericus, respectively. The suspended cultures of all Bacillus sp. exhibited more than 85% reduction of 1000 microM Cr(VI) within 30 h. The suspended culture of Bacillus sp. G1DM22 exhibited an ability for continuous reduction of 100 microM Cr(VI) up to seven consecutive inputs. Assays with the permeabilized cells and cell-free extracts from each of Bacillus sp. demonstrated that the hexavalent chromate reductase activity was mainly associated with the soluble fraction of cells and expressed constitutively. The Cr(VI) reduction by the cell-free extracts of Bacillus sp. G1DM20 and G1DM22 was maximum at 30 degrees C and pH 7 whereas, Bacillus sp. G1DM64 exhibited maximum Cr(VI) reduction at pH 6. Addition of 1mM NADH enhanced the Cr(VI) reductase activity in the cell-free extracts of all three isolates. Amongst all three isolates tested, crude cell-free extracts of Bacillus sp. G1DM22 exhibited the fastest Cr(VI) reduction rate with complete reduction of 100 microM Cr(VI) within 100 min. The apparent K(m) and V(max) of the chromate reductase activity in Bacillus sp. G1DM22 were determined to be 200 microM Cr(VI) and 5.5 micromol/min/mg protein, respectively. The Cr(VI) reductase activity in cell-free extracts of all the isolates was stable in presence of different metal ions tested except Hg(2+) and Ag(+).     

Pectin methylesterase, a regulator of pollen tube growth

The apical wall of growing pollen tubes must be strong enough to withstand the internal turgor pressure, but plastic enough to allow the incorporation of new membrane and cell wall material to support polarized tip growth. These essential rheological properties appear to be controlled by pectins, which constitute the principal component of the apical cell wall. Pectins are secreted as methylesters and subsequently deesterified by the enzyme pectin methylesterase (PME) in a process that exposes acidic residues. These carboxyls can be cross-linked by calcium, which structurally rigidifies the cell wall. Here, we examine the role of PME in cell elongation and the regulation of its secretion and enzymatic activity. Application of an exogenous PME induces thickening of the apical cell wall and inhibits pollen tube growth. Screening a Nicotiana tabacum pollen cDNA library yielded a pollen-specific PME, NtPPME1, containing a pre-region and a pro-region. Expression studies with green fluorescent protein fusion proteins show that the pro-region participates in the correct targeting of the mature PME. Results from in vitro growth analysis and immunolocalization studies using antipectin antibodies (JIM5 and JIM7) provide support for the idea that the pro-region acts as an intracellular inhibitor of PME activity, thereby preventing premature deesterification of pectins. In addition to providing experimental data that help resolve the significance and function of the pro-region, our results give insight into the mechanism by which PME and its pro-region regulate the cell wall dynamics of growing pollen tubes.     

Cover Image,Volume 116, Number 3, March 2019

Transglutaminase (TGase) induces the cross-linking of proteins by catalyzing an acyl transfer reaction. TGase is a zymogen, activated by the removal of its pro-region. Because the pro-region is crucial for folding and inhibition of the TGase activity, the recombinant expression of the mature TGase (mTGase) without the pro-region, usually results in inactive inclusion bodies or low protein yield. Here, Streptomyces netropsis TGase was fused with Escherichia coli lysyl-tRNA synthetase (LysRS), as a module with chaperoning activity in an RNA dependent manner (chaperna). The TGase activity from purified fusion protein induced via the removal of LysRS by tev protease in vitro. Moreover, active mTGase was produced in E. coli via an intracellular cleavage system, wherein LysRS-mTGase was cleaved by the coexpressed tev protease in vivo. The results suggest that LysRS essentially mimics pro-region, which exerts a dual function—folding of TGase into active conformation and keeping it as dormant state—in an RNA-dependent manner. Thus, trans-acting RNAs, prompt the cis-acting chaperone function of LysRS, while being mechanistically similar to the intramolecular chaperone function of the pro-region. These results could be implemented and extended for the folding of “difficult-to-express” recombinant proteins, by harnessing the chaperna function.     

Role of the pre-pro-region of neutral protease in secretion in Bacillus subtilis

A thermostable neutral protease (NprT) from Bacillus stearothermophilus CU21 is translated as a precursor containing pre-pro-structure. Deletion and insertion mutations were introduced in the pro-region and the effects of the mutations on production of active NprT were studied. The pro-region was important for the production of active NprT and was effective only when it was combined with the signal sequence (pre-region) of NprT. To examine the role of the pre-region, a penicillinase gene from Bacillus licheniformis (penP) was fused with various nprT secretion vectors. It was also found that the pre-region of NprT can potentially function as a signal sequence but is more functional for the production of NprT when it was combined with a normal pro-region. We discuss how the processing between the pro-structure and the mature portion of the protease might be done autoproteolytically.     

Analysis of the flagellin (hag) gene of alkalophilic Bacillus sp. C-125.

Motility of the alkalophilic Bacillus sp. C-125, a flagellate bacterium, was demonstrated to be Na(+)- and pH-dependent. Flagellin protein from this strain was purified to homogeneity and the N-terminal sequence determined. Using the hag gene of Bacillus subtilis as a probe, the hag gene of Bacillus sp. C-125 was identified and cloned into Escherichia coli. Sequencing of this hag gene revealed that it encodes a protein of 272 amino acids (M(r) 29,995). The predicted N terminal sequence of this protein was identical to that determined by N-terminal sequencing of the flagellin protein from strain C-125. The alkalophilic Bacillus sp. C-125 flagellin shares homology with other known flagellins in both the N- and C-terminal regions. The middle portion, however, shows considerable differences, even from that of flagellin from the related species, B. subtilis.     

N capture by Plantago lanceolata and Brassica napus from organic material: the influence of spatial dispersion, plant competition and an arbuscular mycorrhizal fungus

This study investigated N capture by Plantago lanceolata L. and Brassica napus L. from complex organic material (dual-labelled with 15N/13C) added either as a thin concentrated layer (discrete patch treatment) or dispersed uniformly with the background sand:soil mix in a 10 cm band (dispersed treatment) when grown in monoculture or in interspecific competition and in the presence or absence of a mycorrhizal inoculum (Glomus mosseae). No 13C enrichments from the organic material were detected in the plant tissues, but 15N enrichments were present. Total plant uptake of N from the organic material on a microcosm basis was not affected by the spatial placement of the organic material, but Plantago monocultures captured less N than the species in interspecific competition (i.e. 23% versus 38% of the N originally added). N capture from Brassica monocultures was no different to either Plantago monocultures or both species in mixture. However, N capture from the organic material by both individual Plantago and Brassica plants was reduced when grown with Brassica plants (by 10-fold and by more than half, respectively). N capture from the organic material was directly related to the estimated root length produced in the sections containing the organic material: the individual that produced the greatest root length captured most N. Strikingly, when the organic material was added as a discrete patch the N captured by Brassica, a non-mycorrhizal species, actually increased when the G. mosseae inoculum was present compared to when G. mosseae was absent (i.e. 35% versus 19% of the N originally added).     

Multiple septation in variants of Bacillus cereus

Remsen, C. C. (Syracuse University, Syracuse, N.Y.), and D. G. Lundgren. Multiple septation in variants of Bacillus cereus. J. Bacteriol. 90:1426-1431. 1965.-Abnormal nonsporulating cells observed in cultures containing predominantly endospores were examined in an electron microscope. One or more septa were seen which consisted of cell-wall material between two unit membranes. Numerous membrane swirls were often seen associated with the septa. Some of the reported structural features of the variant cells were similar to those published for Bacillus cereus var. alesti and B. subtilis; however, the majority of the cells observed in this work were multiseptated.     

鲟源病原性嗜水气单胞菌拮抗芽孢杆菌的鉴定及其生物学特性

从养殖池污泥中分离筛选了1株优良的鲟源嗜水气单胞菌拮抗芽孢杆菌G1,其对鲟源嗜水气单胞菌S1产生的抑菌圈直径为18.50 mm。通过API50CH细菌鉴定系统以及16S rRNA序列分析法,菌株G1被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),GenBank登录号HM245965.1,其16S rRNA序列与基因库中芽孢杆菌属菌株的16S rRNA序列有99%100%的同源性,而且与解淀粉芽孢杆菌Ba-74501(GenBank登录号:DQ422953.1)的亲缘关系最近。菌株G1的最适生长pH值为7,最适生长温度为30°C,其在30°C、200 r/min条件下的生长曲线为:0 6 h为生长延迟期,6 54 h为对数生长期,54 90 h为稳定期,90 h以后为衰亡期。此外,菌株G1对其他实验选用的病原性嗜水气单胞菌也表现出良好的拮抗活性。本实验结果有利于填补嗜水气单胞菌拮抗菌在分类地位、生物学特性等方面的不足,为鲟鱼嗜水气单胞菌病的生物防控提供科学资料。     

Facultative alkalophilic bacteria from mangrove soil with varying buffering capacity and H+ conductance

Facultative alkalophilic bacteria Planococcus sp. (EMGA-26), Bacillus sp. (EMGA-29) and Corynebacterium spp. (EMGA-33 and 130) were isolated from mangrove soil samples. Neutrophiles were predominant than alkalophiles. Buffering capacity and membrane H+ conductance were investigated for the strains grown in PPYG medium at pH 10.5 using acid pulse technique. Bacillus sp. showed higher buffering capacity than Planococcus sp. and Corynebacterium spp. Buffering capacity was two-fold higher in Corynebacterium sp. EMGA-33 than in EMGA-130. The membrane H+ conductance was high in Bacillus sp. and was directly proportional to the buffering capacity values. The Bacillus sp. (EMGA-29) had higher cell membrane adaptability in high pH environment than the Planococcus sp. and Corynebacterium spp.     

Thermostable chitosanase from Bacillus sp. Strain CK4: cloning and expression of the gene and characterization of the enzyme

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80 degrees C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.     

InhA-like protease secreted by Bacillus sp. S17110 inhabited in turban shell

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around 50 degrees . Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of Ca2+, Zn2+, Mg2+, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.     

The autocatalytic processing of the subtilisin Carlsberg pro-region is independent of the primary structure of the cleavage site

Subtilisins are extracellular seryl-proteases produced by bacilli (Markland and Emil, 1971). In addition to signal sequences, these proteases have N-terminal extensions (pro-regions) which have also been identified in several other proteases (Silen et al., 1988; Vasantha et al., 1984; Polhner et al., 1987; Henderson et al., 1987; Yanagida et al., 1986; Takagi et al., 1985). The pro-region holds the pro-protease associated with the membrane and release of the protease takes place as a result of pro-region removal by autocatalytic processing (Egnell and Flock, 1991). In this report we describe the construction of four deletion-mutations in the gene encoding subtilisin Carlsberg at the junction between the pro-region and mature subtilisin Carlsberg. We found that the introduction of different deletions abolished the ability of subtilisin to undergo autocatalytic cleavage of the pro-region in cis, whereas cleavage by exogenous subtilisin could still occur in trans. Point mutations were also introduced in positions -5 to +4 around the pro-region and native subtilisin cleavage site. Processing of pro-subtilisin with the point mutations showed that the autocatalytic cleavage and recognition of this junction of the subtilisin Carlsberg pro-region is independent of the amino acid sequence around the cleavage site.     

Synthesis of Malto-Oligosaccharides Via the Acceptor Reaction Catalyzed by Cyclodextrin Glycosyltransferases

The disproportionation activity (intermolecular transglycosylation) of cyclomaltodextrin glycosyltransferases (CGTases) from Thermoanaerobacter sp. and Bacillus circulans strain 251 was studied. Using soluble starch as donor, the CGTase from Thermoanaerobacter sp. showed the highest transglycosylation activity with all the malto-oligosaccharides tested as acceptors. At ratios of starch: D-glucose from 2:1 to 1:2 (w/w), the formation of cyclodextrins was completely inhibited, and a homologous series of malto-oligosaccharides (G_n) was produced. The conversion of starch into acceptor products was in the range of 63-79% in 48 h. The degree of polymerisation of malto-oligosaccharides formed could be modulated by the ratio of starch: D-glucose provided; at a ratio of 1:2 (w/w), the reaction was quite selective for the formation of G2-G3.     

Lysosomal acid lipase as a preproprotein

Lysosomal acid lipase (LAL; EC 3.1.1.13) hydrolyzes intracellular triglycerides and cholesterol esters taken up by various cell-types. Previously, LAL purified from human liver tissue was described as a preproprotein with a 27 amino acid signal peptide and a 49 amino acid propeptide. Three mutants of the putative proregion of LAL were produced and expressed in Spodoptera frugiperda insect cells. Pulse-chase experiments demonstrated that LAL undergoes proteolytical processing. The deletion of the 49 amino acids led to a complete loss of the LAL activity. The two other mutants were produced at the C-terminus of the pro-region, at positions 49 and 50, by site-directed mutagenesis. Mutant K49R showed wild-type LAL activity, but mutant G50A showed significantly reduced enzyme activity compared to wild-type LAL and a greater reduction in culture medium than in detergent cell extracts. Kinetic data suggest that mutant G50A is less stable than wild-type LAL and mutant K49R. In contrast to K49, the highly conserved amino acid residue G50 seems to be in a very important position and its mutation influences both secretion and enzyme activity of LAL. A three-dimensional model of LAL shows that K49 and G50 are localized in the loop-region between two beta-sheets, highly accessible for proteolytic enzymes. These data together indicate that LAL is indeed a preproprotein, in which the pro-region is essential for its folding and stability, secretion, and enzyme activity.     

Endophytic bacillus sp. isolated from the interior of balloon flower root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G+C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.