Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.     

Purification and characterisation of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent: Allicin

Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of alliin with the enzyme alliinase (EC 4.4.1.4). A bacterium Cupriavidus necator with the ability of alliinase production was isolated from a soil sample and was identified by morphological, biochemical and 16S rRNA sequence. Alliinase production was optimised and it was further purified to apparent homogeneity with 103-fold purification and specific activity of 209 U/mg of protein by using DEAE Cellulose and Sephadex G-100 chromatography. The enzyme is a homodimer of molecular weight 110 kDa with two subunits of molecular weight 55 kDa each. The optimum activity of the purified enzyme was found at pH 7 and the optimum temperature was 35 °C. The enzyme exhibited maximum reaction rate (V_max) at 74.65 U/mg and Michaelis–Menten constant (K_m) was determined to be 0.83 mM when alliin was used as a substrate. The cytotoxic activity of in-situ generated allicin using purified alliinase and alliin was assessed on MIA PaCa-2 cell line using MTT assay and Acridine orange–ethidium bromide staining. This approach of in-situ allicin generation suggests a novel therapeutic strategy wherein alliin and alliinase work together synergistically to produce cytotoxic agent allicin.     

Lectin and alliinase are the predominant proteins in nectar from leek (Allium porrum L.) flowers

Analysis of nectar from leek (Allium porrum) flowers by SDS-PAGE revealed the presence of two major polypeptide bands of 50 kDa and 13 kDa, respectively. Using a combination of agglutination tests, enzyme assays and N-terminal sequencing, the polypeptides have been identified as subunits of alliin lyase (alliinase, EC 4.4.1.4) and mannose-binding lectin, respectively. The latter protein is particularly abundant since it represents about 75% of the total nectar protein. Honey produced by bees foraging on flowering leek plants still contains biologically active lectin and alliinase. However, the levels of both proteins are strongly reduced as compared to those in the original nectar. It is evident, therefore, that the lectin as well as the alliinase are inactivated/degraded during the conversion of nectar into honey. Received: 24 May 1996 / Accepted: 19 August 1996     

Immobilization of alliinase with a water soluble–insoluble reversible N-succinyl-chitosan for allicin production

N-Succinyl-chitosan (NSC), a pH-sensitive polymer of reversibly soluble–insoluble characteristics with pH change, was prepared by modification of the chitosan backbone with succinic anhydride and employed as carrier for alliinase immobilization. The obtained NSC is soluble at pH above 4.8 and insoluble at pH below 4.4. The characteristics of NSC were evaluated using Fourier transform IR spectrophotometer, the X-ray diffraction spectrometry and thermogravimetric analyzer. Under an optimized condition (glutaraldehyde 0.8% (v/v), 31.2 U alliinase), the enzyme immobilization yield was 75.6%. The maximum activity of NSCA was achieved at 40 °C, pH 7, while the free enzyme exhibited maximum activity at 30 °C, pH 6. The Michaelis–Menten constant of NSCA was lower than that of free alliinase, indicating higher affinity of immobilized enzyme toward its substrate. The NSCA retained 85% of its initial activity even after being recycled 5 times. The immobilized alliinase in reversibly soluble NSC is suitable to catalyze the conversion of alliin to allicin, as active ingredient of pharmaceutical compositions and food additive.     

A spectrophotometric assay for allicin,alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates

Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.     

新鲜大蒜中蒜氨酸酶的分离纯化及性质

用葡聚糖凝胶G-200层析柱分离纯化了新鲜大蒜(Allium sativum)中的蒜氨酸酶,SDS-PAGE结果为单一条带,分子量为53 kD在35℃下以蒜氨酸为底物,Km为0.693 mmol·L-1,Vmax为0.353 mmol·min-1,最适反应温度为30℃,热稳定的温度在50℃以下.Zn2 对酶有抑制作用,Mn2 使酶活力增加.     

蒜氨酸酶的固定化及其酶学性质研究

为了提高蒜氨酸酶的稳定性并实现酶的反复利用,研究了影响蒜氨酸酶固定化的因素及固定化蒜氨酸酶的酶学性质。蒜氨酸酶的固定化以壳聚糖微球为载体,戊二醛为交联剂,固定化的最适条件为：戊二醛浓度4%,给酶量20.2U,交联时间2h。固定化蒜氨酸酶的最适pH值7.0,最适温度35℃,米氏常数Km 7.9 mmol/L,操作稳定性比较好,连续使用10次后酶活力损失低于10%。     

3H]Allicin: preparation and applications

Allicin (diallylthiosulfinate), the active substance of garlic, has been shown to possess a variety of biological activities. Mechanistic and pharmacokinetic studies of allicin and its derivatives raise the need for a labeled compound. However, labeling of this volatile and unstable liquid requires delicate handling. Here, we describe a simple method for the preparation of (3)H-labeled allicin. This was achieved by applying synthetic [(3)H]alliin ([2,3-(3)H]allylcysteine sulfoxide) to a column containing immobilized alliinase [EC 4.1.1.4.] from garlic. Purification of [(3)H]allicin was done by differential adsorbtion of the reaction components on a neutral polystyrene resin, Porapak Q. Thiol-containing compounds are known to be the main target of allicin. In this work we demonstrated that [(3)H]allicin can be used for the synthesis of labeled [(3)H]allylmercapto derivatives of SH peptides and proteins. Thus, we prepared [(3)H]S-allylmercaptoglutathione which can be used in metabolic studies. Moreover, we showed that incubation of alliinase with [(3)H]allicin led to modification of 1.4 cysteine residues per subunit of the enzyme.