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1.
Bian Y  Liang X  Fang N  Tang XF  Tang B  Shen P  Peng Z 《FEBS letters》2006,580(25):6007-6014
Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.  相似文献   

2.
By directed evolution and subsequent site‐directed mutagenesis, cold‐adapted variants of WF146 protease, a thermophilic subtilase, have been successfully engineered. A four‐amino acid substitution variant RTN29 displayed a sixfold increase in caseinolytic activity in the temperature range of 15–25°C, a down‐shift of optimum temperature by ~15°C, as well as a decrease in thermostability, indicating it follows the general principle of trade‐off between activity and stability. Nevertheless, to some extent RTN29 remained its thermophilic nature, and no loss of activity was observed after heat‐treatment at 60°C for 2 h. Notably, RTN29 exhibited a lower hydrolytic activity toward suc‐AAPF‐pNA, due to an increase in Km and a decrease in kcat, in contrast to other artificially cold‐adapted subtilases with increased low‐temperature activity toward small synthetic substrates. All mutations (S100P, G108S, D114G, M137T, T153A, and S246N) identified in the cold‐adapted variants occurred within or near the substrate‐binding region. None of these mutations, however, match the corresponding sites in naturally psychrophilic and other artificially cold‐adapted subtilases, implying there are multiple routes to cold adaptation. Homology modeling and structural analysis demonstrated that these mutations led to an increase in mobility of substrate‐binding region and a modulation of substrate specificity, which seemed to account for the improvement of the enzyme's catalytic activity toward macromolecular substrates at lower temperatures. Our study may provide valuable information needed to develop enzymes coupling high stability and high low‐temperature activity, which are highly desired for industrial use. Biotechnol. Bioeng. 2009; 104: 862–870. © 2009 Wiley Periodicals, Inc.  相似文献   

3.
Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90°C) and Pyrococcus furiosus (optimal growth temperature, 98°C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca2+, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140°C. The addition of Ca2+ also affected substrate binding, as evidenced by a decrease in Km for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the α-1,6 linkages in pullulan, α-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller α-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.  相似文献   

4.
Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.  相似文献   

5.
High thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants—P35C-G426C, G116C-Q120C, and R436C-M480C—of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (kcat/Km) increased from 1.8 × 104 to 2.4 × 104 liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.  相似文献   

6.
WF146 protease, a thermophilic subtilase from thermophile Bacillus sp. WF146, suffers excessive autolysis in the presence of reducing agents. In this report, two autolytic sites of WF146 protease were modified by site-directed mutagenesis. The introduction of prolines into the autolytic sites increased the autolysis resistance of the enzyme under reducing conditions. The double mutant N63P/A66P displayed a 2.8-fold longer half-life at 80°C and higher hydrolytic activities than wild-type enzyme toward soluble (casein) and insoluble (keratin azure) substrates at high temperatures. In the presence of reducing agents, N63P/A66P was able to degrade feather at 80°C (∼3 h), with hydrolysis efficiency comparable to that of proteinase K at 50°C (∼24 h). Meanwhile, the mutant N63P/A66P had the ability to hydrolyze PrPSc-like prion protein at high temperatures. In virtue of these properties, N63P/A66P is of great interest to be used in recycling of keratinous wastes, such as feather, and disinfection of medical apparatus. In addition, our study may provide useful information needed to explore keratinolytic potential of thermophilic subtilases, even if they are produced by non-keratinolytic microorganisms.  相似文献   

7.
The proform of the WF146 protease, an extracellular subtilase produced by thermophilic Bacillus sp. WF146, matures efficiently at high temperatures. Here we report that the proform, which contains an N-terminal propeptide composed of a core domain (N*) and a linker peptide, is intrinsically able to mature via multiple pathways. One autocatalytic pathway is initiated by cis-processing of N* to generate an autoprocessed complex N*-IWT, and this step is followed by truncation of the linker peptide and degradation of N*. Another autocatalytic pathway is initiated by trans-processing of the linker peptide followed by degradation of N*. Unlike most reported subtilases, the maturation of the WF146 protease occurs not only autocatalytically but also hetero-catalytically whereby heterogeneous proteases accelerate the maturation of the WF146 protease via trans-processing of the proform and N*-IWT. Although N* acts as an intramolecular chaperone and an inhibitor of the mature enzyme, the linker peptide is susceptible to proteolysis, allowing the trans-processing reaction to occur auto- and hetero-catalytically. These studies also demonstrate that the WF146 protease undergoes subtle structural adjustments during the maturation process and that the binding of Ca2+ is required for routing the proform to mature properly at high temperatures. Interestingly, under Ca2+-free conditions, the proform is cis-processed into a unique propeptide-intermediate complex (N*-IE) capable of re-synthesis of the proform. Based on the basic catalytic principle of serine proteases and these experimental results, a mechanism for the cis-processing/re-synthesis equilibrium of the proform and the role of the linker peptide in regulation of this equilibrium has been proposed.  相似文献   

8.
Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp. WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region. The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins. However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions. Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution. The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form. After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide. The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 degrees C, and lost activity with a half-life of 30 min at 80 degrees C in the presence of 10 mM CaCl2.  相似文献   

9.
Photosynthetic and respiratory rates of two psychrophilic diatoms   总被引:1,自引:0,他引:1       下载免费PDF全文
The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3-10 and Nitzschia sp. K3-3 for cells grown at 0°C were 8 to 10 microliters O2 evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10°C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0°C, and 6 to 10 for cells grown at 10°C. When cells grown at 0°C were immediately shifted to 10°C or cells grown at 10°C were shifted to 0°C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O2 evolution showed much less immediate adaptation, especially on the up shift, 0° to 10°C. The chlorophyll a content of 0°C grown cells was about 0.5% of dry weight, in 10°C grown cells 1.3% (strain K3-10) and 2.2% (strain K3-3). In addition to a diminished chlorophyll a content in 0°C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10°C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0°C grown cells of Chaetoceros sp. strain K3-10 as compared to 10°C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0°C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins.  相似文献   

10.
Improving enzyme thermostability is of importance for widening the spectrum of application of enzymes. In this study, a structure-based rational design approach was used to improve the thermostability of a highly active, wide-pH-range-adaptable, and stable endopolygalacturonase (PG8fn) from Achaetomium sp. strain Xz8 via the optimization of charge-charge interactions. By using the enzyme thermal stability system (ETSS), two residues—D244 and D299—were inferred to be crucial contributors to thermostability. Single (D244A and D299R) and double (D244A/D299R) mutants were then generated and compared with the wild type. All mutants showed improved thermal properties, in the order D244A < D299R < D244A/D299R. In comparison with PG8fn, D244A/D299R showed the most pronounced shifts in temperature of maximum enzymatic activity (Tmax), temperature at which 50% of the maximal activity of an enzyme is retained (T50), and melting temperature (Tm), of about 10, 17, and 10.2°C upward, respectively, with the half-life (t1/2) extended by 8.4 h at 50°C and 45 min at 55°C. Another distinguishing characteristic of the D244A/D299R mutant was its catalytic activity, which was comparable to that of the wild type (23,000 ± 130 U/mg versus 28,000 ± 293 U/mg); on the other hand, it showed more residual activity (8,400 ± 83 U/mg versus 1,400 ± 57 U/mg) after the feed pelleting process (80°C and 30 min). Molecular dynamics (MD) simulation studies indicated that mutations at sites D244 and D299 lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability.  相似文献   

11.
The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k cat /K m) by 290% at 0°C. In the presence of 1 M TMAO, the thermostability (t 1/2) of MCP-01 increased by two- to fivefold at 60∼40°C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40°C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T m) of MCP-01 by 10.5°C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.  相似文献   

12.
Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60–64°C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.  相似文献   

13.
In environments with temperatures above 60°C, thermophilic prokaryotes are the only metabolically active life-forms. By using the 35SO42- tracer technique, we studied the activity of sulfate-reducing microorganisms (SRM) in hot sediment from a hydrothermal vent site in the northern part of freshwater Lake Tanganyika (East Africa). Incubation of slurry samples at 8 to 90°C demonstrated meso- and thermophilic sulfate reduction with optimum temperatures of 34 to 45°C and 56 to 65°C, respectively, and with an upper temperature limit of 80°C. Sulfate reduction was stimulated at all temperatures by the addition of short-chain fatty acids and benzoate or complex substrates (yeast extract and peptone). A time course experiment showed that linear thermophilic sulfate consumption occurred after a lag phase (12 h) and indicated the presence of a large population of SRM in the hydrothermal sediment. Thermophilic sulfate reduction had a pH optimum of about 7 and was completely inhibited at pH 8.8 to 9.2. SRM could be enriched from hydrothermal chimney and sediment samples at 60 and 75°C. In lactate-grown enrichments, sulfide production occurred at up to 70 and 75°C, with optima at 63 and 71°C, respectively. Several sporulating thermophilic enrichments were morphologically similar to Desulfotomaculum spp. Dissimilatory sulfate reduction in the studied hydrothermal area of Lake Tanganyika apparently has an upper temperature limit of 80°C.  相似文献   

14.
In the vast number of random mutagenesis experiments that have targeted protein thermostability, single amino acid substitutions that increase the apparent melting temperature (Tm) of the enzyme more than 1 to 2°C are rare and often require the creation of a large library of mutated genes. Here we present a case where a single beneficial mutation (R236F) of a hemp fiber-processing pectate lyase of Xanthomonas campestris origin (PLXc) produced a 6°C increase in Tm and a 23-fold increase in the half-life at 45°C without compromising the enzyme's catalytic efficiency. This success was based on a variation of sequence alignment strategy where a mesophilic amino acid sequence is matched with the sequences of its thermophilic counterparts that have established Tm values. Altogether, two-thirds of the nine targeted single amino acid substitutions were found to have effects either on the thermostability or on the catalytic activity of the enzyme, evidence of a high success rate of mutation without the creation of a large gene library and subsequent screening of clones. Combination of R236F with another beneficial mutation (A31G) resulted in at least a twofold increase in specific activity while preserving the improved Tm value. To understand the structural basis for the increased thermal stability or activity, the variant R236F and A31G R236F proteins and wild-type PLXc were purified and crystallized. By structure analysis and computational methods, hydrophobic desolvation was found to be the driving force for the increased stability with R236F.  相似文献   

15.
Dewatered sewage sludge was composted in a laboratory-scale autothermal reactor in which a constant temperature of 60°C was kept as long as possible by regulating the air feed rate. The change in CO2 evolution rate was measured continuously from the start up through the cessation of compositing. The succession of mesophilic bacteria, thermophilic bacteria, and thermophilic actinomycetes was also observed during the composting. Specific CO2 evolution rates of thermophilic bacteria and actinomycetes in the constant-temperature region of 60°C were assessed quantitatively. It was found that the CO2 evolution rate was attributed to thermophilic bacteria at the initial stage of 60°C and to thermophilic actinomycetes at the later stage of 60°C.  相似文献   

16.
The thermal dependencies of the apparent Km of the glutathione reductases from spinach (Spinacia oleracea L.) corn (Zea mays L.), and cucumber (Cucumis sativus L.) were determined. The apparent Km of the enzymes were found to vary up to 9-fold between 12.5 and 45°C. Values of the apparent Km in excess of 200% of the observed minimum are suggested to be detrimental to the normal function of the enzyme. We propose the term “thermal kinetic window” to describe to the range of temperatures over which the apparent Km of the glutathione reductase is within 200% of its minimum and suggest that it may be a useful indicator of the limits of thermal stress for a given species. The thermal kinetic windows determined in this study are: <16°C for spinach, 23 to 32°C for corn, and 35 to 41°C for cucumber.  相似文献   

17.

Background

Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°C.

Results

A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50–70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in Tm) and improved half-life at 60°C (t1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.

Conclusion

Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0118-z) contains supplementary material, which is available to authorized users.  相似文献   

18.
High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C) and thermophilic (55°C) conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2) were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production.  相似文献   

19.
Limited secretion capacity remains a drawback of using Escherichia coli as the host for the production of recombinant proteins. In this report, random mutagenesis was performed within the N-terminal propeptide of thermostable WF146 protease, a subtilase from thermophilic Bacillus sp. WF146, generating a variant named WBMMT with improved capacity for extracellular production when expressed in E. coli. Two mutations, L(-57)Q and E(-10)D, were identified within the N-terminal propeptide. The amount of WBMMT in the culture medium was found to be about three times higher than that of wild type. Besides, the introduction of mutations L(-57)Q/E(-10)D into the N-terminal propeptide also accelerated the maturation of the enzyme. Biochemical analysis indicated that the thermostability and the catalytic activity of mature WBMMT were similar to those of wild type. Far-UV CD spectra analysis and limited proteolysis experiments suggested that the mutations L(-57)Q/E(-10)D resulted in a structural change in the N-terminal propeptide of the proform, and the N-terminal propeptide became more flexible, which might be beneficial for the proform to keep in a translocation-competent state. Our result indicates that N-terminal propeptide engineering may be a valuable approach for improving extracellular production of recombinant subtilases expressed in E. coli.  相似文献   

20.
Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072–4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8- and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 × 108 and 449.3 × 108 U · mol−1, respectively, which are 0.94- and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7- and 1.3-fold that of the DM enzyme. Kinetic studies showed that the Km values of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and the Kcat/Km values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced d-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes.  相似文献   

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