Protein engineering of subtilisin

Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology

Volumn 1543, Issue 2, 2000, Pages 203-222

  Bryan, P N ^a  

^a UNIVERSITY OF MARYLAND BIOTECHNOLOGY INSTITUTE   (United States)

Author keywords

Design; Directed evolution; Folding; Site directed mutagenesis; Stability

Indexed keywords

AMINO ACID; INDUSTRIAL ENZYME; PROTEIN; SUBTILISIN;

CATALYSIS; ENZYME MODIFICATION; GENE EXPRESSION; GENE MUTATION; GENETIC ENGINEERING; MOLECULAR CLONING; PRIORITY JOURNAL; PROTEIN FOLDING; PROTEIN STABILITY; PURIFICATION; REVIEW; SITE DIRECTED MUTAGENESIS;

EID: 0034731382     PISSN: 01674838     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0167-4838(00)00235-1     Document Type: Review

References (194)

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3. Selection for improved subtiligases by phage display
4. Improvement of thermal stability of subtilisin J by changing the primary autolysis site
5. Designing subtilisin BPN′ to cleave substrates containing dibasic residues
6. Furilisin: A variant of subtilisin BPN′ engineered for cleaving tribasic substrates
7. Introduction of a free cysteinyl residue at position 68 in the subtilisin savinase, based on homology with proteinase K
8. Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference
9. Significance of hydrophobic S4-P4 interactions in subtilisin 309 from Bacillus lentus
10. Kinetic studies on the peroxidase activity of selenosubtilisin
11. Peroxide dependence of the semisynthetic enzyme selenosubtilisin
12. Mapping the active site of papain with the aid of peptide substrates and inhibitors
13. Crystal structure of the alkaline proteinase savinase from Bacillus lentus at 1.4Å resolution
14. The high-resolution x-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin C, an elastase inhibitor from the leech Hirudo medicinalis
15. The importance of a distal hydrogen bonding group in stabilizing the transition state in subtilisin BPN′
16. Incorporation of a stabilizing Ca-binding loop into subtilisin BPN′
17. Subtilisin BPN′ variants: Increased hydrolytic activity on surface-bound substrates via decreased surface activity
18. Enzyme behavior at surfaces. Site-specific variants of subtilisin BPN′ with enhanced surface stability
19. Energetics of folding subtilisin BPN′
20. Site-directed mutagenesis and the role of the oxyanion hole in subtilisin
21. Catalysis of a protein folding reaction: Mechanistic implications of the 2.0Å structure of the subtilisin-prodomain complex
22. Proteases of enhanced stability: Characterization of a thermostable variant of subtilisin
23. Probing the mechanism and improving the rate of substrate-assisted catalysis in subtilisin BPN′
24. Engineering subtilisin BPN′ for site-specific proteolysis
25. Engineering enzyme specificity by 'substrate-assisted catalysis'
26. Dissecting the catalytic triad of a serine protease
27. Functional interaction among catalytic residues in subtilisin BPN′
28. Tuning the activity of an enzyme for unusual environments: Sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide
29. The 2 Å crystal structure of subtilisin E with PMSF inhibitor
30. Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure
31. The controlled introduction of multiple negative charge at single amino acid sites in subtilisin Bacillus lentus
32. A strategy for the isolation of catalytic activities from repertoires of enzymes displayed on phage
33. Site-directed mutagenesis combined with chemical modification as a strategy for altering the specificity of the S1 and S1′ pockets of subtilisin Bacillus lentus
34. Probing the altered specificity and catalytic properties of mutant subtilisin chemically modified at position S156C and S166C in the S1 pocket
35. Toward tailoring the specificity of the S1 pocket of subtilisin B. lentus: Chemical modification of mutant enzymes as a strategy for removing specificity limitations
36. Electric fields in active sites: Substrate switching from null to strong fields in thiol- and selenol-subtilisins
37. Folding of subtilisin BPN′: Characterization of a folding intermediate
38. Folding of subtilisin BPN′: Role of the pro-sequence
39. Engineering surface charges in a subtilisin: The effects on electrophoretic and ion-exchange behaviour
40. Effects of engineered salt bridges on the stability of subtilisin BPN′
41. Probing steric and hydrophobic effects on enzyme-substrate interactions by protein engineering
42. Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation
43. Understanding and increasing protein stability
44. Calcium-free subtilisin by design
45. The prosegment-subtilisin BPN′ complex: Crystal structure of a specific foldase
46. Stability of subtilisins and related proteinases (subtilases)
47. Strategy and implementation of a system for protein engineering
48. Engineered Bacillus lentus subtilisins having altered flexibility
49. A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification
50. Studies of binding sites in the subtilisin from Bacillus lentus by means of site directed mutagenesis and kinetic investigations
51. Interdependency of the binding subsites in subtilisin
52. Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching
53. Calcium binding to thermitase
54. Reasoning enantioselectivity and kinetics of seleno-subtilisin from the subtilisin template
55. From detergent additive to semisynthetic peroxidase-simplified and up-scaled synthesis of seleno-subtilisin
56. Chemical engineering of enzymes: Altered catalytic activity, predictable selectivity and exceptional stability of the semisynthetic peroxidase seleno-subtilisin
57. Semisynthetic enzymes in asymmetric synthesis: Enantioselective reduction of racemic hydroperoxides catalyzed by seleno-subtilisin
58. Renaturation of the mature subtilisin BPN′ immobilized on agarose beads
59. Increasing thermal stability of subtilisin from mutations suggested by strongly interacting side-chain clusters
60. Binding rates, O-S substitution effects, and the pH dependence of chymotrypsin reactions
61. Further evidence for the structure of the subtilisin propeptide and for its interactions with mature subtilisin
62. A covalently trapped folding intermediate of subtilisin E: Spontaneous dimerization of a prosubtilisin E Ser49Cys mutant in vivo and its autoprocessing in vitro
63. Improving the activity of immobilized subtilisin by site-specific attachment to surfaces
64. Denaturation of subtilisin BPN′ and its derivatives in aqueous guanidine hydrochloride solutions
65. Requirement of pro sequence for the production of active subtilisin in Escherichia coli
66. Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis
67. The crystal structure of an autoprocessed Ser221Cys-subtilisin E-propeptide complex at 2.0 Å resolution
68. Effect of the weak Ca(2+)-binding site of subtilisin J by site-directed mutagenesis on heat stability
69. Identification of autoproteolytic cleavage site in the Asp-49 mutant subtilisin J by site-directed mutagenesis
70. Scoring functions for computational algorithms applicable to the design of spiked oligonucleotides
71. Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis
72. Rapid analysis of single-cysteine variants of recombinant proteins
73. Breaking the low barrier hydrogen bond in a serine protease
74. A weak calcium binding site in subtilisin BPN′ has a dramatic effect on protein stability
75. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides
76. Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second site suppressor mutation within the subtilisin molecule
77. Display of active subtilisin 309 on phage: Analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors
78. Engineering proteases with altered specificity
79. Functional analysis of the propeptide of subtilisin E as an intramolecular chaperone for protein folding. Refolding and inhibitory abilities of propeptide mutants
80. Autoprocessing of prothiolsubtilisin E in which active-site serine 221 is altered to cysteine
81. The mechanism of autoprocessing of the propeptide of prosubtilisin E: Intramolecular or intermolecular event?
82. Binding of amino acid side-chains to S1 cavities of serine proteinases
83. Interaction of subtilisin BPN′ and recombinant Streptomyces subtilisin inhibitors with substituted P1 site residues
84. Identification of amino acid residues responsible for the changes of absorption and fluorescence spectra on the binding of subtilisin BPN′ and Streptomyces subtilisin inhibitor
85. Achievement of renaturation of subtilisin BPN′ by a novel procedure using organic salts and a digestible mutant of Streptomyces subtilisin inhibitor
86. Structural comparison of two serine proteinase-protein inhibitor complexes: Eglin-C-subtilisin Carlsberg and CI-2-subtilisin novo
87. Engineering subtilisin YaB: Restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala
88. Protein engineering of disulfide bonds in subtilisin BPN′
89. Exploring nonnatural evolutionary pathways by saturation mutagenesis: Rapid improvement of protein function
90. Free energy perturbation calculations on binding and catalysis after mutating threonine 220 in subtilisin
91. Peptide segment coupling catalyzed by the semisynthetic enzyme thiolsubtilisin
92. Enhanced stability of subtilisin by three point mutations
93. Subtilisin from psychrophilic antarctic bacteria: Characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold
94. The conversion of serine at the active site of subtilisin to cysteine: A 'chemical mutation'
95. Properties of thiol-subtilisin. The consequences of converting the active serine residue to cysteine in a serine protease
96. DNA shuffling of subgenomic sequences of subtilisin
97. 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct
98. UCLA Symposium on Protein Structure, Folding and Design
99. Conformational stabilities and activity of ribonuclease T1 with zero, one and two intact disulfide bonds
100. Unusual ligand binding at the active site domain of an engineered mutant of subtilisin BL
101. Proteins designed for challenging environments and catalysis in organic solvents
102. Protein engineering of subtilisin BPN′: Stabilization through the introduction of two cysteines to from a disulfide bond
103. Large increases in general stability for subtilisin BPN′ through incremental changes in the free energy of unfolding
104. The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: Subtilisin as a test case
105. Structural basis of substrate specificity in the serine proteases
106. Nonessential active site residues modulate selenosubtilisin's kinetic mechanism
107. Kinetics of subtilisin and thiolsubtilisin
108. Comparison of the kinetic specificity of subtilisin and thiolsubtilisin toward n-alkyl p-nitrophenyl esters
109. Modulation of esterase and amidase activity of subtilisin Bacillus lentus by chemical modification of cysteine mutants
110. Statistical mechanics of noncovalent bonds in polyamino acids. VIII. Covalent loops in proteins
111. The reactivity of thiol-subtilisin, an enzyme containing a synthetic functional group
112. Chromatography and activity of thiol-subtilisin
113. Free energy perturbation calculations on binding and catalysis after mutating Asn 155 in subtilisin
114. Engineering a novel specificity in subtilisin BPN′
115. Variants of subtilisin BPN′ with altered specificity profiles
116. Engineering thermostability in subtilisin BPN′ by in vitro mutagenesis
117. Rapid folding of calcium-free subtilisin by a stabilized pro-domain mutant
118. Stabilizing the subtilisin BPN′ pro-domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?
119. Rational modification of enzyme catalysis by engineering surface charge
120. Electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering
121. Engineering the independent folding of the subtilisin BPN′ prodomain: Analysis of two-state folding vs. protein stability
122. Thermostable variants of subtilisin selected by temperature-gradient gel electrophoresis
123. Preprosubtilisin Carlsberg processing and secretion is blocked after deletion of amino acids 97-101 in the mature part of the enzyme
124. Engineering subtilisin for peptide coupling: Studies on the effects of counterions and site-specific modifications on the stability and specificity of the enzyme
125. Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization
126. Engineering new functions and altering existing functions
127. Random-priming in vitro recombination: An effective tool for directed evolution
128. A pathway for conformational diversity in proteins mediated by intramolecular chaperones
129. Folding mediated by an intramolecular chaperone: Autoprocessing pathway of the precursor resolved via a substrate assisted catalysis mechanism
130. Folding pathway mediated by an intramolecular chaperone: Characterization of the structural changes in pro-subtilisin E coincident with autoprocessing
131. Propeptide-mediated folding in subtilisin: The intramolecular chaperone concept
132. Protein memory through altered folding mediated by intramolecular chaperones
133. Mutational replacements of the amino acid residues forming the hydrophobic S4 binding pocket of subtilisin 309 from Bacillus lentus
134. Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis
135. The effect on subtilisin activity of oxidizing a methionine residue
136. Prediction of electrostatic effects of engineering of protein charges
137. Prediction of electrostatic effects of engineering of protein charges
138. Directed evolution of a subtilisin with calcium-independent stability
139. An engineered disulfide crosslink accelerates the refolding rate of calcium-free subtilisin by 850-fold
140. Catalysis of a protein folding reaction: Thermodynamic and kinetic analysis of subtilisin BPN′ interactions with its propeptide fragment
141. Crystal structure of selenosubtilisin at 2.0-Å resolution
142. Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system
143. A cold-adapted protease engineered by experimental evolution system
144. The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis
145. Restriction of substrate specificity of subtilisin E by introduction of a side chain into a conserved glycine residue
146. Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis
147. The role of Pro-239 in the catalysis and heat stability of subtilisin E
148. Construction of novel subtilisin E with high specificity, activity and productivity through multiple amino acid substitutions
149. Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease
150. Random mutagenesis into the conserved Gly154 of subtilisin E: Isolation and characterization of the revertant enzymes
151. Thermal denaturation of streptomyces subtilisin inhibitor, subtilisin BPN′, and the inhibitor-subtilisin complex
152. P1 specificity of aqualysin I (a subtilisin-type serine protease) from Thermus aquaticus YT-1, using P1-substituted derivatives of Streptomyces subtilisin inhibitor
153. Engineering of S2 site of aqualysin I; alteration of P2 specificity by excluding P2 side chain
154. Identification and designing of the S3 site of aqualysin I, a thermophilic subtilisin-related serine protease
155. Improvement of a useful enzyme (subtilisin BPN′) by an experimental evolution system
156. Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: Functional and structural consequences of mutation at the S4 substrate binding pocket
157. Tailoring the pH dependence of enzyme catalysis using protein engineering
158. Length of the acyl carbonyl bond in acyl-serine proteases correlates with reactivity
159. Protein engineering
160. Genes for alkaline and neutral protease from Bacillus amyloliquefaciens contain a large open-reading frame between the regions coding for signal sequence and mature protein
161. Evidence for intramolecular processing of prosubtilisin sequestered on a solid support
162. Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair
163. Protein engineering of subtilisins to improve stability in detergent formulations
164. Role of bound calcium in thermostable, proteolytic enzymes. Separation of intrinsic and calcium ion contributions to the kinetic thermal stability
165. Engineering the independent folding of the subtilisin BPN′ pro-domain: Correlation of pro-domain stability with the rate of subtilisin folding
166. Prodomain mutations at the subtilisin interface: Correlation of binding energy and the rate of catalyzed folding
167. Probing enzymic transition state hydrophobicities
168. Additivity of mutational effects in proteins
169. Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin
170. Recruitment of substrate-specificity properties from one enzyme into a related one by protein engineering
171. Cloning, sequencing and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis
172. In vivo formation and stability of engineered disulfide bonds in subtilisin
173. Designing substrate specificity by protein engineering of electrostatic interactions
174. Details of the acyl-enzyme intermediate and the oxyanion hole in serine protease catalysis
175. Enzymes in organic synthesis: Use of subtilisin and a highly stable mutant derived from multiple site-specific mutations
176. Enzymatic catalysts in organic synthesis
177. Enzymatic catalysis in organic synthesis
178. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide
179. Functional and nonfunctional mutations distinguished by random recombination of homologous genes
180. Directed evolution converts subtilisin E into a functional equivalent of thermitase
181. Molecular evolution by staggered extension process (StEP) in vitro recombination
182. Strategy for the directed evolution of a peptide ligase
183. Protein engineering on subtilisin E