氨基酰化酶在LDS溶液中的失活与去折叠的比较研究

氨基酰化酶在阴离子去圬剂十二烷基硫酸锂（ＬＤＳ）溶液中的失活与去折叠的研究结果表明,在低浓度的ＬＤＳ溶液（０．６ｍｍｏｌ／Ｌ）中变性时,以荧光和紫外差吸收方法监测的酶分子构象尚未发生明显变化。而酶的活力已经大部分或几乎全部丧失。当ＬＤＳ浓度达１．６ｍｍｏｌ／Ｌ时,此时酶分子的构象变化才达到最大程度,在实验使用的ＬＤＳ的浓度范围内,用远紫外ＣＤ光谱监测的二级结构没有发生明显的变化。从上述研究结果,可以认为含锌氨基酰化酶的活性部位也具有相对的柔性。     

Alteration of metal ions improves the activity and thermostability of aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

Recombinant l-aminoacylase (PhoACY) from a hyperthermophilic archeon, Pyrococcus horikoshii, is a zinc-containing metalloenzyme. When the zinc was substituted by Mn²⁺ or Ni²⁺, its specific activity was significantly increased with acetyl-l-methionine as a substrate. The thermostability of PhoACY was improved when it was incubated with 1 mM Zn²⁺, Mn²⁺ or Ni²⁺. The enzyme with external Zn²⁺ addition had no significant loss of the activity when held at 90°C for up to 12 h and moreover had more than a 10-fold longer half-life even at 100°C, compared to the enzyme without Zn²⁺ addition. A thermostable structure of the enzyme associated with zinc binding is described based on differential scanning calorimetry.     

Identification and characterisation of a gene encoding aminoacylase activity from Lactococcus lactis MG1363

Analysis of the sequence of a randomly cloned chromosomal DNA fragment (3.2 kb) from Lactococcus lactis revealed the presence of part of an open reading frame, designated amd1, which specifies a protein displaying significant similarity to aminoacylases from various bacteria. The presence of an immobilised copy of an IS982 element immediately upstream of the coding region of amd1 has probably resulted in the displacement of amd1's native promoter. This genetic organisation was shown to be retained in seven other dairy strains, one of which was only slightly different. The amd1 gene was overexpressed in L. lactis NZ9800 under the control of the inducible nisA promoter and the deacetylating capacity of its gene product was measured on a number of substrates.     

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α‐position and peptides on N‐terminal position

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N‐α or ε‐acetyl‐L‐lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε‐position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N‐acylation on the α‐position of the lysine or of the amino‐acid in N‐terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N‐α‐acetyl‐L‐lysine could be attributed to the product of SAM23877_1734 gene.     

产氨基酰化酶米曲霉高产菌株的选育

采用亚硝基瓜抑菌圈法对米曲霉菌株3042,AF92011,AF93018,AF93020,AF93022,AF93332进行诱变处理,从诱变菌株中筛选到几个高产氨基酰化酶菌株(3042-5,AF93020-2,AF93020-6和AF93022-5),对DL-蛋氨酸的拆分活力分别比出发菌株提高34.1%、157.6%、152.4%和145.4%,其中米曲霉菌株3042的诱变菌株3042-5最高酶活力达203.1U/ml     

Aminoacylase I from porcine kidney: Identification and characterization of two major protein domains

The domain structure of hog-kidney aminoacylase I was studied by limited proteolytic digestion with trypsin and characterization of the resulting fragments. In the native enzyme, the sequences from residue 6 to 196 and 307 to 406 are resistant to trypsin and remain tightly bound in nondenaturing solvents, while the intervening sequence (197–306) is efficiently degraded by trypsin. We conclude that the N-terminal half of the molecule and its C-terminal fourth form two independently folded domains. Both contain a peculiar PWW(A,L) sequence motif preceded by several strongly polar residues. We propose that these sequences form surface loops that mediate the membrane association of aminoacy clase I. We further show that the three free cysteine residues and the essential Zn²⁺ ion reside in the trypsin-resistant domains, while the intervening sequence contains the only disulfide H bond of the protein.     

Nitrile- and Amide-converting Microbial Enzymes: Stereo-, Regio- and Chemoselectivity

Biotransformations using microbial nitrile- and amide-converting enzymes have developed considerably in the recent years. Most processes profited from the stereo-, regio- and chemoselectivity of nitrile hydratases, nitrilases and amidases specific for primary or secondary amides. The aim of this review is to discuss the developments in this branch of biotransformations in the last ca. 5 years by taking highlights from research journals, patents and industrial applications.     

Inactivation and unfolding of aminoacylase during denaturation in sodium dodecyl sulfate solutions

During denaturation by sodium dodecyl sulfate (SDS), aminoacylase shows a rapid decrease in activity with increasing concentration of the detergent to reach complete inactivation at 1.0 mM SDS. The denatured minus native-enzyme difference spectrum showed two negative peaks at 287 and 295 nm. With the increase of concentration of SDS, both negative peaks increased in magnitude to reach maximal values at 5.0 mM SDS. The fluorescence emission intensity of the enzyme decreased, whereas there was no red shift of emission maximum in SDS solutions of increasing concentration. In the SDS concentration regions employed in the present study, no marked changes of secondary structure of the enzyme have been observed by following the changes in far-ultraviolet CD spectra. The inactivation of this enzyme has been followed and compared with the unfolding observed during denaturation in SDS solutions. A marked inactivation is already evident at low SDS concentration before significant conformational changes can be detected by ultraviolet absorbance and fluorescence changes. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by the kinetics method of the substrate reaction in the presence of inactivator previously described by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. It was found that substrate protects against inactivation and at the same SDS concentrations, the inactivation rate of the free enzyme is much higher than the unfolding rate. The above results show that the active sites of metal enzyme containing Zn²⁺ are also situated in a limited and flexible region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Nitrile- and Amide-converting Microbial Enzymes: Stereo-, Regio- and Chemoselectivity

Biotransformations using microbial nitrile- and amide-converting enzymes have developed considerably in the recent years. Most processes profited from the stereo-, regio- and chemoselectivity of nitrile hydratases, nitrilases and amidases specific for primary or secondary amides. The aim of this review is to discuss the developments in this branch of biotransformations in the last ca. 5 years by taking highlights from research journals, patents and industrial applications.     

Kinetics of the course of reactivation of aminoacylase reconstituted using Mn2+ ions

The kinetic theory of the substrate reaction during irreversible change of enzyme activity previously described by Tsou (Tsou (1988),Adv. Enzymol. Relat. Areas Mol. Biol.61, 381–436] has been applied to a study of the kinetics of the course of reactivation during reconstitution of apo-aminoacylase using Mn²⁺ or Zn²⁺. The kinetic parameters for Mn²⁺-and Zn²⁺-reconstituted enzymes and the microscopic rate constants for reactivation during reconstitution were determined. The kinetic analysis suggests the presence of a second Mn²⁺ binding site in Mn²⁺-reconstituted aminoacylase.