Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

Download Full-text

A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

Applied Sciences ◽

10.3390/app11125521 ◽

2021 ◽

Vol 11 (12) ◽

pp. 5521

Author(s):

Li Liu ◽

Shuhuai Yu ◽

Wei Zhao

Keyword(s):

Ph Value ◽

Wild Type ◽

Michaelis Constant ◽

Wild Type Enzyme ◽

Maximum Reaction Rate ◽

E Coli ◽

Maximum Reaction ◽

Conversion Rates ◽

Raoultella Terrigena ◽

Sucrose Isomerase

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (Km) and maximum reaction rate (Vmax) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.

Download Full-text

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

Biochemical Journal ◽

10.1042/bj1790099 ◽

1979 ◽

Vol 179 (1) ◽

pp. 99-107 ◽

Cited By ~ 5

Author(s):

Jeffrey D. Hillman

Keyword(s):

Escherichia Coli ◽

Simple Procedure ◽

Rabbit Antiserum ◽

Double Diffusion ◽

Wild Type ◽

Wild Type Enzyme ◽

Glycolytic Intermediate ◽

E Coli ◽

Catalytic Function ◽

Mutant Strains

NAD+-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD+ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD+ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.

Download Full-text

New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation

Biochemical Journal ◽

10.1042/bj2600491 ◽

1989 ◽

Vol 260 (2) ◽

pp. 491-497 ◽

Cited By ~ 21

Author(s):

L Hederstedt ◽

L O Hedén

Keyword(s):

Bacillus Subtilis ◽

Succinate Dehydrogenase ◽

Active Site ◽

Amino Acid Position ◽

Biochemical Properties ◽

Cysteine Residue ◽

Wild Type ◽

Wild Type Enzyme ◽

Succinate Oxidation ◽

E Coli

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.

Download Full-text

Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.3.1037-1046.2003 ◽

2003 ◽

Vol 47 (3) ◽

pp. 1037-1046 ◽

Cited By ~ 47

Author(s):

Christian H. Gross ◽

Jonathan D. Parsons ◽

Trudy H. Grossman ◽

Paul S. Charifson ◽

Steven Bellon ◽

...

Keyword(s):

Amino Acid ◽

Atp Hydrolysis ◽

Dna Gyrase ◽

Dna Supercoiling ◽

Wild Type ◽

Wild Type Enzyme ◽

Mutant Proteins ◽

E Coli ◽

Novobiocin Resistance

ABSTRACT DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

Download Full-text

Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis

Biochemical Journal ◽

10.1042/bj3580423 ◽

2001 ◽

Vol 358 (2) ◽

pp. 423-430 ◽

Cited By ~ 11

Author(s):

Alisdair B. BORASTON ◽

R. Antony J. WARREN ◽

Douglas G. KILBURN

Keyword(s):

Pichia Pastoris ◽

Association Constant ◽

Mutational Analysis ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Wild Type ◽

Cellulomonas Fimi ◽

E Coli ◽

Glycosylation Sites ◽

High Mannose

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc2Man8 to GlcNAc2Man14. The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (Ka) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The Ka of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.

Download Full-text

Improvements of the productivity and saccharification efficiency of the cellulolytic β-glucosidase D2-BGL in Pichia pastoris via directed evolution

Biotechnology for Biofuels ◽

10.1186/s13068-021-01973-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Mu-Rong Kao ◽

Su-May Yu ◽

Tuan-H ua David Ho

Keyword(s):

Pichia Pastoris ◽

Directed Evolution ◽

Specific Activity ◽

Substrate Inhibition ◽

Catalytic Efficiency ◽

Single Amino Acid ◽

Cellulolytic Enzyme ◽

Glycoside Hydrolase Family ◽

Wild Type ◽

Wild Type Enzyme

Abstract Background β-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) β-glucosidase isolated from Chaetomella raphigera with high substrate affinity, and is an efficient β-glucosidase supplement to Trichoderma reesei cellulase mixtures for the saccharification of lignocellulosic biomass. Results We have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in Saccharomyces cerevisiae. Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in Pichia pastoris. Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in Pichia pastoris resulted in 2.7-fold higher production of recombinant protein, higher Vmax and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in P. pastoris. When combined with the T. reesei cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2. Conclusions D2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 β-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher Vmax than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in P. pastoris. Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.

Download Full-text

Biochemical and structural studies of mutants indicate concerted movement of the dimer interface and ligand-binding region ofMycobacterium tuberculosispantothenate kinase

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17015667 ◽

2017 ◽

Vol 73 (11) ◽

pp. 635-643 ◽

Cited By ~ 1

Author(s):

A. Paul ◽

P. Kumar ◽

A. Surolia ◽

M. Vijayan

Keyword(s):

Ligand Binding ◽

Wild Type ◽

Binding Region ◽

Wild Type Enzyme ◽

Dimer Interface ◽

E Coli ◽

Structural Differences ◽

Feedback Inhibitor ◽

Reduced Activity ◽

Independent Mutant

Two point mutants and the corresponding double mutant ofMycobacterium tuberculosispantothenate kinase have been prepared and biochemically and structurally characterized. The mutants were designed to weaken the affinity of the enzyme for the feedback inhibitor CoA. The mutants exhibit reduced activity, which can be explained in terms of their structures. The crystals of the mutants are not isomorphous to any of the previously analysed crystals of the wild-type enzyme or its complexes. The mycobacterial enzyme and its homologousEscherichia colienzyme exhibit structural differences in their nucleotide complexes in the dimer interface and the ligand-binding region. In three of the four crystallographically independent mutant molecules the structure is similar to that in theE. colienzyme. Although the mutants involve changes in the CoA-binding region, the dimer interface and the ligand-binding region move in a concerted manner, an observation which might be important in enzyme action. This work demonstrates that the structure of the mycobacterial enzyme can be transformed into a structure similar to that of theE. colienzyme through minor perturbations without external influences such as those involving ligand binding.

Download Full-text

F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues

Journal of Bacteriology ◽

10.1128/jb.187.24.8267-8277.2005 ◽

2005 ◽

Vol 187 (24) ◽

pp. 8267-8277 ◽

Cited By ~ 15

Author(s):

Trevor C. Elton ◽

Samantha J. Holland ◽

Laura S. Frost ◽

Bart Hazes

Keyword(s):

Disulfide Bond ◽

Enteric Bacteria ◽

Type Iv Secretion ◽

Site Directed Mutagenesis ◽

Secretion Systems ◽

E Coli ◽

Type Iv ◽

Transfer Region ◽

Periplasmic Proteins ◽

Disulfide Bond Isomerase

ABSTRACT F and R27 are conjugative plasmids of enteric bacteria belonging to the IncF and IncHI1 plasmid incompatibility groups, respectively. Based on sequence analysis, two genes of the F transfer region, traF and trbB, and three genes of the R27 transfer region, trhF, dsbC, and htdT, are predicted to encode periplasmic proteins containing a C-terminal thioredoxin fold. The C-X-X-C active-site motif of thioredoxins is present in all of these proteins except TraFF. Escherichia coli carrying a dsbA mutation, which is deficient in disulfide bond formation, cannot synthesize pili and exhibits hypersensitivity to dithiothreitol (DTT) as monitored by mating ability. Overproduction of the E. coli disulfide bond isomerase DsbC, TrbBF, DsbCR27, or HtdTR27, but not TraFF or TrhFR27, reverses this hypersensitivity to DTT. Site-directed mutagenesis established that the C-X-X-C motif was necessary for this activity. Secretion into the periplasm of the C-terminal regions of TrbBF and DsbCR27, containing putative thioredoxin folds, but not TrhFR27, partially complemented the host dsbA mutation. A trbBF deletion mutant showed a 10-fold-lower mating efficiency in an E. coli dsbC null strain but had no phenotype in wild-type E. coli, suggesting redundancy in function between TrbBF and E. coli DsbC. Our results indicate that TrbBF, DsbCR27, and HtdTR27 are putative disulfide bond isomerases for their respective transfer systems. TraFF is essential for conjugation but appears to have a function other than disulfide bond chemistry.

Download Full-text

Random mutagenesis suggests that sequence errors are not a major cause of variation in the activity of individual molecules of β-galactosidase

Biochemistry and Cell Biology ◽

10.1139/o2012-006 ◽

2012 ◽

Vol 90 (4) ◽

pp. 540-547 ◽

Cited By ~ 10

Author(s):

Douglas B. Craig ◽

Thomas Schwab ◽

Reinhard Sterner

Keyword(s):

Single Molecule ◽

Random Sequence ◽

Random Mutagenesis ◽

Error Rates ◽

Wild Type ◽

Wild Type Enzyme ◽

E Coli ◽

Significant Difference ◽

Gene Libraries ◽

Sequence Errors

Wild-type Escherichia coli lacZ was subjected to error-prone PCR to generate two plasmid-encoded gene libraries containing approximately 2.6 (SD 1.9) nucleotide exchanges resulting in 1.8 (SD 1.4) amino-acid substitutions. The libraries were used, along with a plasmid containing wild-type lacZ, to transform E. coli lacking genomic lacZ. Cells expressing functional β-galactosidase were identified by blue/white screening. Cell lysates containing the populations of heterogeneously mutagenized β-galactosidase were subjected to single molecule assays using a capillary electrophoresis laser-induced fluorescence-based protocol. There was no significant difference in the average catalytic rate between the random mutagenized and wild-type enzyme populations. Furthermore, there was no clear pattern between error rates and the variances in the population catalytic rates. This suggests that random sequence errors are not a substantial source of the catalytic heterogeneity of this enzyme.

Download Full-text

Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3500537 ◽

2000 ◽

Vol 350 (2) ◽

pp. 537-545 ◽

Cited By ~ 19

Author(s):

Motoyoshi NAKAMURA ◽

Tasuku NAKAJIMA ◽

Yasunori OHBA ◽

Seigo YAMAUCHI ◽

Byung Rho LEE ◽

...

Keyword(s):

Atomic Absorption ◽

Atomic Absorption Spectrophotometry ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

E Coli ◽

Absorption Spectrophotometry ◽

Acid Shock ◽

Copper Ligands

Copper ligands of the recombinant tyrosinase from the fungus Aspergillus oryzae expressed in Saccharomyces cerevisiae or Escherichia coli were identified by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase and L-dopa oxidase at pH 5.5 after acid shock at pH 3.0. Replacements of His-63, His-84, His-93, His-290, His-294, His-332 or His-333 with asparagine resulted in mutant enzymes exhibiting no activities. The site-directed mutant Cys82Ala showed that Cys-82 was also an essential residue for the activity. We obtained homogeneous preparations of activated tyrosinases from mutated thioredoxin fusion gene products expressed in E. coli by acid shock. The copper contents of engineered mutants and wild-type enzyme expressed in E. coli were determined by atomic absorption spectrophotometry. The wild-type enzyme contained 2 g-atoms of copper/mol of the subunit. The His63Asn, His84Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala substitution decreased copper binding by approx. 50%, indicating that the mutants contain only approx. 1 g-atom of copper/mol of the subunit. The five mutants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g‖ and CuA‖, we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedral geometric environment. In further atomic absorption spectrophotometry experiments, no copper atom was observed in the seven double mutants His63Asn/His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, Cys82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a new structure of active sites of tyrosinase from A. oryzae: the most likely binding sites of tyrosinase for Cu(A) are His-63, His-84 and His-93, with the remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded by four histidine residues, His-290, His-294, His-332 and His-333, is identified as new binding motif of Cu(B).

Download Full-text