共查询到20条相似文献,搜索用时 31 毫秒
1.
Hurle Mark R.; Anderson Stephen; Kuntz Irwin D. 《Protein engineering, design & selection : PEDS》1991,4(4):451-455
A major question in protein structural analysis concerns theapplicability of results from model systems to other proteins.Theoretical approaches seem the best manner of transferringinformation from one system to another, but their accuracy inthe model systems must first be tested with results from experiment.Since bovine pancreatic trypsin inhibitor (BPTI) is a modelsystem for the evaluation of energy minimization and moleculardynamics routines, we can use folding and stability measurementsto examine the reliability of these methods. All two-disulfidemutants of BPTI investigated thus far have two very slow foldingreactions which have characteristics of proline isomerization.These reactions may occur because the non-native cis form oftwo of the four prolines in BPTI significantly destabilizesthe folded state of the protein. Previous energy minimizationstudies of wild-type BPTI suggested that the cis form of Pro8was the most destabilizing of the four prolines [Levitt,M. (1981)J. Mol. Biol., 145, 251263]. In this paper, we show thatmutation of Pro8 - Gln in the two-disulfide bond mutant Val30Ala51results in a loss of the slowest folding reaction, consistentwith Levitt's prediction. 相似文献
2.
Sriprapundh Dinlaka; Vieille Claire; Zeikus J.Gregory 《Protein engineering, design & selection : PEDS》2003,16(9):683-690
The Thermotoga neapolitana xylose isomerase (TNXI) is extremelythermostable and optimally active at 95°C. Its derivative,TNXI Val185Thr (V185T), is the most active type II xylose isomerasereported, with a catalytic efficiency of 25.1 s1 mM1toward glucose at 80°C (pH 7.0). To further optimize TNXIspotential industrial utility, two rounds of random mutagenesisand low temperature/low pH activity screening were performedusing the TNXI V185T-encoding gene as the template. Two highlyactive mutants were obtained, 3A2 (V185T/L282P) and 1F1 (V185T/L282P/F186S).1F1 was more active than 3A2, which in turn was more activethan TNXI V185T at all temperatures and pH values tested. 3A2and 1F1s high activities at low temperatures were dueto significantly lower activation energies (57 and 44 kJ/mol,respectively) than that of TNXI and V185T (87 kJ/mol). MutationL282P introduced a kink in helix 相似文献
3.
Structural study of mutants of Escherichia coli ribonuclease HI with enhanced thermostability 总被引:2,自引:0,他引:2
Ishikawa Kohki; Kimura Shigenobu; Kanaya Shigenori; Morikawa Kosuke; Nakamura Haruki 《Protein engineering, design & selection : PEDS》1993,6(1):85-91
Systematic replacement of the amino acid residues in Escherichiacoli ribonuclease HI with those in the thermophilic counterparthas revealed that two mutations, His62Pro (H62P) andLys95Gly (K95G), increased the thermostability of the protein.These single-site mutant proteins, together with the mutantproteins His62Ala (H62A), Lys95Asn (K95N) and Lys95Ala (K95A),were crystallized and their structures were determined at 1.8Å resolution. The crystal structures of these mutant proteinsreveal that only the local structure around each mutation siteis essential for the increase in thermostability. For each mutantprotein, the stabilization mechanism is considered to be asfollows: (i) H62P is stabilized because of a decrease in theentropy of the unfolded state, without a change in the nativebackbone structure; (ii) K95G is stabilized since the straincaused by the left-handed backbone structure in the typical3:5 type loop is eliminated; and (iii) K95N is slightly stabilizedby a hydrogen bond formed between the side-chain N-atom of themutated aspargine residue and the main-chain carbonyl oxygenwithin the same residue. 相似文献
4.
Nishiyama Makoto; Suzuki Junko; Ohnuki Tatsuya; Chang Hae Choon; Horinouchi Sueharu; Turley Stewart; Adman Elinor T.; Beppu Teruhiko 《Protein engineering, design & selection : PEDS》1992,5(2):177-184
Pseudoazurin (a blue copper protein or cupredoxin) of a denitrifyingbacterium Alcaligenes faecalis S-6 is a direct electron carrierfor a Cu-containing nitrite reductase (NIR) of the same organism.Site-directed mutagenesis of the pseudoazurin was carried outusing an Escherichia coli expression system. Replacement ofTyr74 by Phe to remove an internal hydrogen bond in the ß-barrelcaused a slight decrease in heat stability as well as a requirementfor a higher concentration of Cu2+ for production in the E.colihost. Exchange of Ala for Pro80 adjacent to His81, one of thefour ligands binding a type I Cu atom, caused a marked increasein reduction potential by 139 mV without change in the opticalabsorption spectrum. The ability of the pseudoazurin to transferelectrons to NIR was markedly diminished but the apparent Kmof NIR for pseudoazurin was not affected by the mutation. X-raydiffraction data collected on the oxidized and reduced formsof the Pro80Ala mutant show that a water molecule occupies thepocket created by the absent side chain. This observation suggeststhat the increase in reduction potential may be caused due tothe increased solvent accessibility to the Cu atom. The electrondensity difference maps on these structures (at 2.0 Å)show that this water moves during the change in oxidation state,and that there are small, but localized, conformational changes>6.5 Å from the copper site, as well as movement ofboth the Cu2+ and the cysteinate sulfur. 相似文献
5.
Yasumasa Fukushima 《Polymer Bulletin》2000,45(3):237-244
Summary
Four sequential copolypeptides poly(X-Gly-Gly) with X being Ala, Tyr, Gln, or Leu were prepared as a model of glycine-rich
sequence of dragline spider silk produced by Nephila clavipes and their secondary structures in the solid state were characterized by FT-IR spectroscopy. Poly(Tyr-Gly-Gly), poly(Gln-Gly-Gly),
and poly(Ala-Gly-Gly) form the β-sheet structures, whereas poly(Leu-Gly-Gly) existed as a disordered conformation as a cast
film from formic acid. These results indicated that X-Gly-Gly sequences with Tyr, Gln, and Ala could contribute to the formation
of the β-sheet structure in glycine-rich sequence.
Received: 5 August 2000/Accepted: 24 August 2000 相似文献
6.
Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD
Agren Lena; Norin Martin; Lycke Nils; Lowenadler Bjorn 《Protein engineering, design & selection : PEDS》1999,12(2):173-178
Protein engineering of the cholera toxin A1 subunit (CTA1) fusedto a dimer of the Ig-binding D-region of Staphylococcus aureusprotein A (DD) was employed to investigate the effect of specificamino acid changes on solubility, stability, enzymatic activityand capacity to act as an adjuvant in vivo. A series of CTA1-DDanalogues were selected by a rational modeling approach, inwhich surface-exposed hydrophobic amino acids of CTA1 were exchangedfor hydrophilic counterparts modeled for best structural fit.Of six different mutants initially produced, two analogues,CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated tohave 50 and 70% increased solubility, respectively, at neutralpH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at leastthreefold more soluble, demonstrating an additive effect ofthe two mutations. Only the Phe132Ser analogue retained fullbiological activity and stability compared with the native CTA1-DDfusion protein. Two mutants, Pro185Gln and Phe31His mutations,exhibited unaltered ADP-ribosyltransferase activity in vitro,but demonstrated markedly reduced adjuvant function. Since thePro185 and Phe31 amino acids are located in close vicinity onthe distal side of the molecule relative to the enzymaticallyactive cleft, it is conceivable that this region is involvedin mediating a biological function, separate from the enzymaticactivity but intrinsic to the adjuvant activity of CTA1. 相似文献
7.
Dr. Miroslav Malešević Michael Schumann Dr. Günther Jahreis Prof. Gunter Fischer Dr. Christian Lücke 《Chembiochem : a European journal of chemical biology》2012,13(14):2122-2127
Turns are secondary‐structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four‐residue β‐turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI‐type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c‐(‐D Xaa‐Ser‐Pro‐D Xaa‐Lys‐Pro‐) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D ‐amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis‐Pro/cis‐Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space. 相似文献
8.
Saab-Rincon Gloria; Juarez Victor Rivelino; Osuna Joel; Sanchez Filiberto; Soberon Xavier 《Protein engineering, design & selection : PEDS》2001,14(3):149-155
A monomeric version of triosephosphate isomerase from Trypanosomabrucei, MonoTIM, has very low activity, and the same is truefor all of the additional monomeric variants so far constructed.Here, we subjected MonoTIM to directed evolution schemes toachieve an activity improvement. The construction of a suitablestrain for genetic selection provided an effective way to obtainactive catalysts from a diverse population of protein variants.We used this tool to identify active mutants from two differentstrategies of mutagenesis: random mutagenesis of the whole geneand randomization of loop 2. Both strategies converged in theisolation of mutations Ala43 to Pro and Thr44 to either Alaor Ser, when randomizing the entire gene or to Arg in the caseof randomization of loop 2. The kinetic characterization ofthe two more active mutants showed an increase of 11-fold inkcat and a reduction of 4-fold in Km for both of them, demonstratingthe sensitivity of the selection method. A small differencein growth rate is observed when both mutant genes are compared,which seems to be attributable to a difference in solubilityof the expressed proteins. 相似文献
9.
Mercer Kelly E.; Ahn Christina E.; Coke Amanda; Compadre Cesar M.; Drake Richard R. 《Protein engineering, design & selection : PEDS》2002,15(11):903-911
Understanding the functional and mechanistic properties of themulti-substrate herpes simplex virus type-1 thymidine kinase(HSV-1 TK) remains critical to defining its role as a majorpharmacological target in herpesvirus and gene therapies forcancer. An inherent limitation of the activity of HSV-TK isthe >70-fold difference in the Kms for phosphorylation ofthymidine over the pro-drug ganciclovir (GCV). To engineer anHSV-1 TK isoform that is specific for GCV as the preferred substrate,16 site-specific mutants were generated. The mutations wereconcentrated at conserved residues involved in nucleoside basebinding, Gln125 and near sites 3 and 4 involved in catalysisand substrate binding. The substrate preferences of each mutantenzyme were compared with wild-type HSV-1 TK. One mutant, termedQ7530 TK, had a lower Km for GCV than thymidine. Expressionof the Q7530 TK in tumor cells indicated comparable metabolismto and improved sensitivity to GCV over wild-type HSV-1 TK,with minimal thymidine phosphorylation activity. A molecularmodeling simulation of the different HSV-1 TK active-sites wasdone for GCV and thymidine binding. It was concluded that mutationsat Gln125 and near site 4, especially at Ala168, were responsiblefor loss of deoxypyrimidine substrate binding. 相似文献
10.
Sierks Michael R.; Ford Clark; Reilly Peter J.; Svensson Birte 《Protein engineering, design & selection : PEDS》1990,3(3):193-198
Asp176, Glu179 and Glu180 of Aspergillus awamori glucoamylaseappeared by differential labeling to be in the active site.To test their functions, they were replaced by mutagenesis withAsn, Gln and Gln respectively, and kinetic parameters and pHdependencies of all enzyme forms were determined. Glu179 Gln glucoamylase was not active on maltose or isomaltose, whilethe kcat for maltoheptaose hydrolysis decreased almost 2000-foldand the KM was essentially unchanged from wild-type glucoamylase.The Glu180 Gln mutation drastically increased the KMand moderately decreased the kcat with maltose and maltoheptaose,but affected isomaltose hydrolysis less. Differences in substrateactivation energies between Glu180 Gln and wild-typeglucoamylases indicate that Glu180 binds D-glucosyl residuesin subsite 2. The Asp176 Asn substitution gave moderateincreases and decreases in KM and kcat respectively, and thereforesimilar increases in activation energies for the three substrates.This and the differences in subsite binding energies betweenAsp176 Asn and wild-type glucoamylases suggest thatAsp176 is near subsite 1, where it stabilizes the transitionstate and interacts with Trp120 at subsite 4. Glu179 and Asp176are thus proposed as the general catalytic acid and base ofpKa 5.9 and 2.7 respectively. The charged Glu180 contributesto the high pKa value of Glul79. Received May 25, 1989; accepted October 19, 1989. 相似文献
11.
Hewson Roger; Newbold Richard J.; Whitford David 《Protein engineering, design & selection : PEDS》1993,6(8):953-964
The DNA sequence of bovine microsomal cytochrome b5 has beenamplified from a liver cDNA library using a polymerase chainreaction. The amplified cDNA when cloned into plasmids thatsupport the high-level production of cytochrome bs in E.colileads to protein overexpression and results in cell coloniesbearing a strong red colouration. Using cassette mutagenesis,truncated versions of the cytochrome b5 cDNA have been madethat encode the first 90 amino acid residues (Ala1-Lys90), thefirst 104 amino acids (Ala1-Ser104) and the complete protein(Ala1-Asnl33). The location of the overexpressed cytochromebs within prokaryotic cells is dependent on the overall lengthof the protein. Expression of the Ala-Lys90 and Alal-SerlO4variants leads to a location in the cytoplasmic phase of thebacteria whereas the whole protein, Alal-Asnl33, is found withinthe bacterial membrane fraction. The last 30 residues of cytochromebs therefore contain all of the necessary information to insertthe protein into E.coli membranes. The solubility of the Alal-SerlO4variant permits the solution structure and stability of thisprotein to be measured using 1- and 2-D 1HNMR methods and electronicspectroscopy. 1-D NMR studies show that the chemical shiftsof the haem and haem ligand resonances of the Alal - Ser 104variant exhibit only very slight perturbations to their magneticmicroenvlronments when compared with the tryptic fragment offerricytochrome b5. These results indicate an arrangement ofresidues in the haem pocket that is very similar in both theAlal-Ser 104 variant and the tryptic fragment and by 2-D NMRit is shown that this similarity extends to the conformationsof the poly peptide backbone and side chains. Electronic spectroscopyof this variant shows absorbance maxima for the Soret peaksat 423 run (reduced) and 413 nm (oxidized). From absorbancespectra the relative thermal stabilities of the Alal-Ser 104variant and the tryptic fragment were measured. In the oxidizedstate the Ala1 - Ser104 variant denatures in a single cooperativetransition with a midpoint temperature (Tm of 73°C thatis significantly higher than that of tryptic ferricytochromebs. The reduced form of the protein shows increased transitiontemperatures (Tm 78°C) reflected in the values of Hm, Smand (G) of 420 kj/mol, 1096 J/mol/K and 12.38 kj/mol respectively,estimated for this variant. The increased stability of the Alal-SerlO4variant and other recombinant forms of cytochrome bs is correlatedwith the presence of additional residues at the N- and C-termini.The subtle differences in reactivity, stability and targetingbetween variant forms of cytochrome bs and the tryptic fragmentare discussed in terms of the overall structure of the protein. 相似文献
12.
Hugo N.; Weidenhaupt M.; Beukes M.; Xu B.; Janson J.-C.; Vernet T.; Altschuh D. 《Protein engineering, design & selection : PEDS》2003,16(5):381-386
Predictive engineering of antibodies exhibiting fast kineticproperties could provide reagents for biotechnological applicationssuch as continuous monitoring of compounds or affinity chromatography.Based on covariance analysis of murine germline antibody variabledomains, we selected position L34 (Kabat numbering) for mutationalstudies. This position is located at the VL/VH interface, atthe base of the paratope but with limited antigen contacts,thus making it an attractive position for mild alterations ofantigen binding properties. We introduced a serine at positionL34 in two different antibodies: Fab (fragment antigen binding)57P (Asn34Ser) and scFv (single chain fragment variable) 1F4(Gln34Ser), that recognize peptides derived from the coat proteinof tobacco mosaic virus and the oncoprotein E6, respectively.Both mutated antibodies exhibited similar properties: (i) expressionlevels of active fragments in Escherichia coli were markedlyimproved; (ii) thermostability was enhanced; and (iii) dissociationrate parameters (koff) were increased by 2- and at least 57-foldfor scFv1F4 and Fab57P, respectively, while their associationrate parameters (kon) remained unchanged. The L34 Ala and Thrmutants of both antibody fragments did not possess these properties.This first demontration of similar effects observed in two antibodieswith different specificities may open the way to the predictivedesign of molecules with enhanced stability and fast dissociationrates. Received October 3, 2002; revised March 7, 2003; accepted April 4, 2003. 相似文献
13.
We have performed molecular dynamics simulation of Rhizomucormiehei lipase (Rml) with explicit water molecules present. Thesimulation was carried out in periodic boundary conditions andconducted for 1.2 ns in order to determine the concerted proteindynamics and to examine how well the essential motions are preservedalong the trajectory. Protein motions are extracted by meansof the essential dynamics analysis method for different lengthsof the trajectory. Motions described by eigenvector 1 convergeafter approximately 200 ps and only small changes are observedwith increasing simulation time. Protein dynamics along eigenvectorswith larger indices, however, change with simulation time andgenerally, with increasing eigenvector index, longer simulationtimes are required for observing similar protein motions (alonga particular eigenvector). Several regions in the protein showrelatively large fluctuations and in particular motions in theactive site lid and the segments Thr57Asn63 and the activesite hinge region Pro101Gly104 are seen along severaleigenvectors. These motions are generally associated with glycineresidues, while no direct correlations are observed betweenthese fluctuations and the positioning of prolines in the proteinstructure. The partial opening/closing of the lid is an exampleof induced fit mechanisms seen in other enzymes and could bea general mechanism for the activation of Rml. 相似文献
14.
Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase 总被引:2,自引:0,他引:2
Chen Hsiu-mei; Ford Clark; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1995,8(6):575-582
Both Dative Aspergillus niger glucoamylase and wild-type Aspergillusawamori glucoamylase expressed in Saccharo-myces cerevisiae,which have identical primary structures, undergo hydrolysisat aspartyl bonds at low pH values and elevated temperatures.In native A.niger enzyme the Aspl26Glyl27 bond was preferentiallycleaved at pH 3.5,while at pH 4.5 cleavage of the Asp257Pro258and Asp293Gly294 bonds was dominant. In wild-type A.awamoriglucoamylase, cleavage of the latter was dominant at both pH3.5 and 4.5. Site-directed mutations Aspl26Glu and Glyl27Alain wild-type enzyme decreased specific activities by 60 and30%, respectively, and increased irreversible thermoinactivationrates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Gluand Asp293 with Glu or Gin decreased specific activities by20%, but greatly reduced cleavage of the Asp257Pro258and Asp293Gly294 bonds. The Asp257Glu mutant was producedvery slowly and was more thermostable than wildtype glucoamylaseat pH 4.5up to 70°C. Replacement of Asp293 with either Gluor Gln significantly raised protein production and slightlyincreased thermostability at pH 3.5 and 4.5, but not at pH5. 相似文献
15.
Gaseidnes Sigrid; Synstad Bjornar; Jia Xiaohong; Kjellesvik Hege; Vriend Gert; Eijsink Vincent G.H. 《Protein engineering, design & selection : PEDS》2003,16(11):841-846
This paper describes attempts to increase the kinetic stabilityof chitinase B from Serratia marcescens (ChiB) by the introductionof semi-automatically designed rigidifying mutations of theGlyAla and XxxPro type. Of 15 single mutants, several displayedsignificant increases in thermal stability, whereas most mutantsshowed minor effects. All mutations with non-marginal effectson stability clustered in a limited, surface-exposed regionof the enzyme, indicating that this region is involved in apartial unfolding process that triggers irreversible thermalinactivation (aggregation). A double mutant containing two stabilizingmutations in this region (G188A, A234P) displayed a 10-foldincrease in half-life at 57°C and a 4.2°C increase inapparent Tm. These results show that entropic stabilizationworks well for ChiB and they pinpoint a region whose unfoldingmay be crucial for the kinetic stability of this enzyme. Received June 18, 2003; revised September 3, 2003; accepted September 12, 2003. 相似文献
16.
Analysis of structural determinants of the stability of thermolysinlike proteases by molecular modelling and site-directed mutagenesis 总被引:1,自引:0,他引:1
Veltman Oene R.; Vriend Gert; Middelhoven Pieter J.; van den Burg Bertus; Venema Gerard; Eijsink Vincent G.H. 《Protein engineering, design & selection : PEDS》1996,9(12):1181-1189
The thermolysin-like protease (TLP) produced by Bacillus stearothermophilusCU21 (TLP-ste) differs at 43 positions from the more thermallystable thermolysin (containing 316 residues in total). Of thesedifferences, 26 were analysed by studying the effect of replacingresidues in TLP-ste by the corresponding residues in thermolysin.Several stabilizing mutations were identified but, remarkably,considerable destabilizing mutational effects were also found.A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertionaldifference between the two enzymes) appeared to make an importantcontribution to the stability of the enzyme. Mutations withlarge effects on stability were all localized in the ßpleatedN-terminal domain of TLP-ste, confirming observations that thisdomain has a lower intrinsic stability than the largely -helicalC-terminal domain. Rigidifying mutations such as Gly58 Alaand Ala69 Pro were among the most stabilizing ones. Apart fromthis observation, the analyses did not reveal general rulesfor stabilizing proteins. Instead, the results highlight theimportance of context in evaluating the stability effects ofmutations. 相似文献
17.
A sequence motif in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization 总被引:2,自引:0,他引:2
Sternberg Michael J.E.; Gullick William J. 《Protein engineering, design & selection : PEDS》1990,3(4):245-248
A mechanism by which ligand binding to the extracellular domainof a growth factor receptor causes activation of its cytoplasmictyrosine kinase domain is that binding promotes receptor dimerization.Recently we proposed a model in which dimerization of the transmembrane-helices in one member of this family, rat neu, is mediatedby the presence of three specific residues. This paper showsthat a similar sequence motif is observed in 18 of the 20 transmembrane-helices of the tyrosine kinase family of growth factor receptors.The motif encompasses a five residue segment in which position0 (P0) requires a small side chain (Gly, Ala, Ser, Thr or Pro),P3 an aliphatic side chain (Ala, Val, Leu or Ile) and P4 onlythe smallest side chains (Gly or Ala). In addition other featuresof the transmembrane sequences are reported. It is concludedthat the dimerization of transmembrane -helices may be a generalmechanism of tyrosine kinase activation in this family of growthfactor receptors. 相似文献
18.
The influence of Glu44 and Glu56 of cytochrome b5 on the protein structure and interaction with cytochrome c 总被引:1,自引:0,他引:1
Sun Yu-Long; Xie Yi; Wang Yun-Hua; Xiao Gu-Tian; Huang Zhong-Xian 《Protein engineering, design & selection : PEDS》1996,9(7):555-558
The gene encoding trypsin-solubilized bovine liver microsomalcytochrome b5 (82 residues in length) has been mutated, in whichthe codons of Glu44 and Glu56 were changed to those of Ala.The mutated genes were expressed in Escherichia coli successfullyand three mutant proteins (E44A, E56A and E44/56A) were obtained.The UV-visible, CD and 1H NMR spectra of proteins have beenstudied. The results show that the mutagenesis at surface residuesdoes not alter the secondary and tertiary structures of cytochromeb5 significantly. The interactions between recombinant cytochromeb5 and its mutants with cytochrome c were studied by using opticaldifference spectra. The results demonstrated that both Glu44and Glu56 of cytochrome b5 participate in the formation of acomplex between cytochrome b5 and cytochrome c. 相似文献
19.
Liu Hsuan-Liang; Ford Clark; Reilly Peter J. 《Protein engineering, design & selection : PEDS》1999,12(2):163-172
Six previously constructed and nine newly constructed Aspergillusawamori glucoamylases with multiple mutations made by combiningexisting single mutations were tested for their ability to produceglucose from maltodextrins. Multiple mutations have cumulativeeffects on glucose yield, specific activity and thermostability.No general correlation between glucose yield and thermostabilitywas observed, although mutations that presumably impede unfoldingat high temperatures uniformly increase thermostability andgenerally increase glucose yield. Peak glucose yields decreasewith increasing temperature. The best combination of high glucoseyield, high specific activity and high thermostability occursin Asn20Cys/Ala27Cys/Ser30Pro/Gly137Ala glucoamylase. 相似文献
20.
In Aspergillus awamori glucoamylase, Ala27, Ala393, Ala435, Ser436 and
Ser460 were replaced with proline residues, in order to stabilize the
enzyme by forming more rigid peptide backbones. Specific activities were
unaffected except for a decrease in Ser460-->Pro glucoamylase.
Thermostability was increased in Ser436-->Pro glucoamylase, unchanged in
Ala435-->Pro glucoamylase and decreased in Ala27-->Pro,
Ala393-->Pro glucoamylases. As measured by circular dichroism, mutant
glucoamylases Ala435-->Pro and Ser436-->Pro resisted unfolding caused
by guanidine hydrochloride at pH 4.5 and 25 degrees C better than wild-type
glucoamylase, whereas mutant glucoamylases Ala27-->Pro, Ala393-->Pro
and Ser460-->Pro were more susceptible to unfolding than wild-type
glucoamylase, reaching a level of 50% unfolded enzyme at guanidine
hydrochloride concentrations 0.50-0.75 M lower than that of the wild- type
enzyme. Mutations Ala435-->Pro and Ser436-->Pro are located in a
non-regular structure, which is assumed to be stabilized by these
mutations. The Ala27-->Pro residue is partially buried, which may result
in unfavorable steric contact and/or regional strains; mutation
Ala393-->Pro results in loss of a hydrogen bond, since the N of the
proline residue does not have an extra hydrogen to act as donor; and
mutation Ser480-->Pro eliminates an O-glycosylation site, which could
explain how these mutations destabilized glucoamylase.
相似文献