Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.     

Significance of bile salt hydrolytic activities of lactobacilli

Bile salt hydrolase (BSH) activity was shown to be constitutive and substrate-specific: the BSH isogenic Lactobacillus plantarum wild type (LP80 WT) and BSH overproducing LP80 (pCBH1) strains preferentially hydrolysed glycodeoxycholic acid (GDCA), whereas the hamster Lact. animalis isolates H362 and H364 showed a higher affinity for taurodeoxycholic acid (TDCA). In viability studies in the presence of nutrients, it was demonstrated that GDCA exerted a higher toxicity than TDCA in a pH-dependent manner. This toxicity was inversely proportionate to the BSH activity level of the strains tested, indicating that BSH activity contributed towards bile salt resistance when appropriate nutrients were available. The high toxicity of GDCA relative to TDCA was suggested to be caused by their weak and strong acid properties respectively. It was therefore hypothesized that the protonated form of bile salts exhibited toxicity as it imported protons in the cell. This puts an energy-burden on BSH⁻ lactobacilli which undergo intracellular acidification. BSH⁺ cells primarily protect themselves through the formation of the weaker DCA compound, which can help negate the pH-drop by recapturing and exporting the co-transported proton. However, since DCA is more toxic than its conjugated counterparts, an additional energy-dependent detoxification of DCA is suggested.     

Relationship between bile salt hydrolase activity, changes in the internal pH and tolerance to bile acids in lactic acid bacteria

The effect of the conjugated bile acid (BA) on the microbial internal pH (pHin) values in lactic acid bacteria with and without ability to hydrolyze bile salts (BSH[+] and BSH[-] strains, respectively) was evaluated. BSH(+) strains showed a gradual increase in the pHin following the addition of conjugated BA; this behavior was more pronounced with GDCA than with TDCA may be due to the higher affinity of BSH for the glyco-conjugates acids. Conversely, the BSH(-) strains showed a decrease in internal pH probably as a consequence of weak acid accumulation. As expected, a decrease in the cytoplasmatic pH affected the cell survival in this last group of strains, while the BSH(+) strains were more resistant to the toxic effect of BA. PURPOSE OF WORK: To evaluate bile salt hydrolase activities, changes in the internal pH and cell survival to bile acids in lactic acid bacteria to establish the relationship between these parameters.     

Identification of Genes Encoding Conjugated Bile Salt Hydrolase and Transport in Lactobacillus johnsonii 100-100

Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, α and β, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in Escherichia coli. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3′ partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5′ and 3′ deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3′ of the cloned segment. The predicted amino acid sequence of the 5′ partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5α cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [¹⁴C]taurocholic acid that were increased as much as threefold over control levels. [¹⁴C]Cholic acid was taken up in similar amounts by strain DH5α pSportI (control) and DH5α p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.     

A New Insight into the Physiological Role of Bile Salt Hydrolase among Intestinal Bacteria from the Genus Bifidobacterium

This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.     

Purification and Characterization of Conjugated Bile Salt Hydrolase from Bifidobacterium longum BB536

Bifidobacterium species deconjugate taurocholic, taurodeoxycholic, taurochenodeoxycholic, glycocholic, glycodeoxycholic, and glycochenodeoxycholic acids. The enzyme level increases in the growth phase. No increase in activity is observed for the cytoplasmic enzyme after addition of conjugated bile acids to a stationary-phase culture. Conjugated bile salt hydrolase (BSH) was purified from Bifidobacterium longum BB536. Its apparent molecular mass in denaturing polyacrylamide gel electrophoresis was ca. 40,000 Da. The intact enzyme had a relative molecular weight of ca. 250,000 as determined by gel filtration chromatography, suggesting that the native BSH of B. longum is probably a hexamer. The purified enzyme is active towards both glycine and taurine conjugates of cholate, deoxycholate, and chenodeoxycholate. The pH optimum is in the range of 5.5 to 6.5. A loss of BSH activity is observed after incubation at temperatures higher than 42(deg)C; at 60(deg)C, 50% of the BSH activity is lost. The importance of free sulfhydryl groups at the enzyme active center is suggested. For B. longum BB536, no significant difference in the initial rate of deconjugation and enzymatic efficiency appears between bile salts. The enzymatic efficiency is higher for B. longum BB536 than for other genera. In this paper, a new method which permits a display of BSH activity directly on polyacrylamide gels is described; this method confirms the molecular weight obtained for B. longum BB536 BSH.     

Localization and primary characterization of bile salt hydrolase from Lactobacillus reuteri

The bile salt hydrolase (BSH) of Lactobacillus reuteri CRL 1098 is a single, constitutive, intracellular enzyme which is only detectable in stationary phase cells. It has optimal activity at pH 4.5–5.5 and 37–45 °C. The enzyme (80 kDa apparent mass) has sulphydryl groups in the catalytic active site and hydrolyzes both glycine and taurine conjugated bile acids with higher affinity for glyco-conjugates.     

Cholesterol-lowering probiotics: in vitro selection and in vivo testing of bifidobacteria

Thirty-four strains of bifidobacteria belonging to Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium pseu-docatenulatum were assayed in vitro for the ability to assimilate cholesterol and for bile salt hydrolase (BSH) against glycocholic and taurodeoxycholic acids (GCA and TDCA). Cholesterol assimilation was peculiar characteristic of two strains belonging to the species B. bifidum (B. bifidum MB 107 and B. bifidum MB 109), which removed 81 and 50 mg of cholesterol per gram of biomass, being the median of specific cholesterol absorption by bifidobacteria 19 mg/g. Significant differences in BSH activities were not established among bifidobacterial species. However, the screening resulted in the selection of promising strains able to efficiently deconjugate GCA and TDCA. No relationship was recognized between BSH phenotype and the extent of cholesterol assimilation. On the basis of cholesterol assimilation or BSH_GCA and BSH_TDCA activities, B. bifidum MB 109 (DSMZ 23731), B. breve MB 113 (DSMZ 23732), and B. animalis subsp. lactis MB 2409 (DSMZ 23733) were combined in a probiotic mixture to be fed to hypercholesterolemic rats. The administration of this probiotic formulation resulted in a significant reduction of total cholesterol and low-density cholesterol (LDL-C), whereas it did not affect high-density cholesterol (HDL-C) and HDL-C/LDL-C ratio.     

Bile salt deconjugation by Lactobacillus plantarum 80 and its implication for bacterial toxicity

The effects of bile salts on the survival of lactobacilli were investigated using glycocholic acid, cholic acid and deoxycholic acid as model compounds and the bile salt hydrolase active Lactobacillus plantarum 80 (BSH⁺) and its BSH negative mutant. The detrimental effects of cholic acid, i.e. growth inhibition and cytotoxicity at a concentration of 1 and 5 mmol l⁻¹, respectively, were considered to be due to the hydrophobic protonated form of the molecule, which brings about membrane damage. The conversion of glycocholic acid to cholic acid by the BSH active L. plantarum 80 caused a growth inhibition which was comparable with the inhibition observed in the broth supplemented with 1 mmol l⁻¹ cholic acid. Deoxycholic acid caused toxicity through membrane damage when the compound was in solution. Its toxicity disappeared in the culture broth as the molecule precipitated. In case of cholic acid, the toxicity could be removed by buffering the solution at pH 7·0. It was calculated that at this pH most of the cholic acid molecules were ionized. The results led to the formulation of an extended hypothesis about the ecological significance of bile salt transformations. Primary deconjugation is carried out to counteract intracellular acidification. Yet, the deconjugated molecule can be harmful at moderately acidic pH-values. In this case, the BSH⁺ strains could effectively profit from their activity in case they are associated with 7α-dehydroxylating bacteria which dehydroxylate the deconjugated bile salts. The dehydroxylated molecule has a low solubility and precipitates at moderately acidic pH.     

CbsT2 from Lactobacillus johnsonii 100-100 is a transport protein of the major facilitator superfamily that facilitates bile acid antiport

We previously identified two conjugated bile acid transporters, CbsT1 and CbsT2, in Lactobacillus johnsonii 100-100 and Lactobacillus acidophilus KS-13 that are gene duplicates encoded in tandem with a conjugated bile salt hydrolase (BSH) [Elkins and Savage, J. Bacteriol. 180:4344-4349, 1998; Elkins et al., Microbiology 147: 3403-3412, 2001]. CbsT2 from 100-100 was shown to increase taurocholic acid (TCA) uptake in Escherichia coli; however, higher levels were achieved when an extracellular factor (EF) from 100-100 was present in the assay medium (spent medium from 100-100, pH 4.2). We continued this study here to determine the role of EF in this transport system. Kinetic studies revealed that the previously observed CbsT2- and EF-mediated TCA accumulation is rapid (< 15 s) but not saturable, suggesting that EF is limiting. In addition, uptake of TCA by E. coli expressing CbsT2 was insensitive to ionophores, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, and thus, is independent of the proton motive force. Since BSH converts [24-(14)C]TCA to [24-(14)C]cholic acid (CA), we measured net radiolabel uptake in E. coli cells expressing transporter(s) and BSH. Interestingly, such cells accumulated less 14C radiolabel (by approximately half) than cells expressing CbsT2 alone. These data can be explained if CA diffuses out of E. coli through the transporter(s). We, therefore, added exogenous, unlabeled CA to EF-spent media, which under our assay conditions, performed similarly to EF+ culture supernatant in TCA and CA uptake assays. Thus, unlabeled CA (a protonated, neutral lipophile) can partition directly into E. coli cells especially at low pH. These findings were validated in uptake assays with [3H]TCA, which yields [3H]taurine (a hydrophilic moiety) upon hydrolysis by the BSH. Amounts of cell-associated 3H radiolabel remained similar in cells expressing CbsT2 and BSH versus cells expressing only CbsT2, both of which were higher than in cells expressing BSH alone. Our data support a hypothesis that these transporters, which comprise a new subfamily of the major facilitator superfamily, facilitate antiport of TCA and CA.     

Identification and Characterization of a Bile Salt Hydrolase from Lactobacillus salivarius for Development of Novel Alternatives to Antibiotic Growth Promoters

Antibiotic growth promoters (AGPs) have been used as feed additives to improve average body weight gain and feed efficiency in food animals for more than 5 decades. However, there is a worldwide trend to limit AGP use to protect food safety and public health, which raises an urgent need to discover effective alternatives to AGPs. The growth-promoting effect of AGPs has been shown to be highly correlated with the decreased activity of intestinal bile salt hydrolase (BSH), an enzyme that is produced by various gut microflora and involved in host lipid metabolism. Thus, BSH inhibitors are likely promising feed additives to AGPs to improve animal growth performance. In this study, the genome of Lactobacillus salivarius NRRL B-30514, a BSH-producing strain isolated from chicken, was sequenced by a 454 GS FLX sequencer. A BSH gene identified by genome analysis was cloned and expressed in an Escherichia coli expression system for enzymatic analyses. The BSH displayed efficient hydrolysis activity for both glycoconjugated and tauroconjugated bile salts, with slightly higher catalytic efficiencies (k_cat/K_m) on glycoconjugated bile salts. The optimal pH and temperature for the BSH activity were 5.5 and 41°C, respectively. Examination of a panel of dietary compounds using the purified BSH identified some potent BSH inhibitors, in which copper and zinc have been recently demonstrated to promote feed digestion and body weight gain in different food animals. In sum, this study identified and characterized a BSH with broad substrate specificity from a chicken L. salivarius strain and established a solid platform for us to discover novel BSH inhibitors, the promising feed additives to replace AGPs for enhancing the productivity and sustainability of food animals.     

Hydrolysis of Conjugated Bile Acids by Cell-Free Extracts from Aerobic Bacteria

By means of an aerobic enrichment culture technique, several bacteria that hydrolyze conjugated bile acids and modify the formed deconjugates were isolated from feces of man, rat, and chicken and from soil. Hydrolase activity was intracellular and extractable, and the yield of the enzymes was increased by adding the conjugated bile acids to the culture media. The hydrolase from bacterium of human origin was stable, having a pH optimum at about 7.0. All bile acid conjugates were hydrolyzed linearly as a function of time.     

降胆固醇乳酸菌的筛选和分析比较

目的比较7株乳酸菌各项生理特性,筛选高效、安全降解胆固醇的益生菌株,为后期开发应用奠定基础。方法对选定菌株依次进行体外降胆固醇、胆盐水解酶(BSH)活力、人工胃肠液及胆盐耐受性、细胞粘附性能的测定和比较,并分析抗生素耐药性和抑制病原菌活性,最后利用16S rRNA基因测序确定细菌种属。结果不同菌株各项生理指标存在差异。其中菌株ZL-2、JQI-7和PC-26的BSH酶活分别为0.010、0.015和0.030 U,可高效清除体外胆固醇,3株菌体外降解胆固醇率在58%以上;并且耐酸、耐胆盐,具备较好的HT--29细胞粘附性,不存在抗生素耐药性,可有效抑制大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌和阴沟肠杆菌生长。结论菌株ZL-2、JQI-7和PC-26满足降胆固醇益生菌株的生物学特性,可用于进一步研究和开发。     

Measurements of Mutagenic and Antimutagenic Activities of Bile Acids by Rec-assay

To study the carcinogenic activity of bile acids, we examined the mutagenic activity of bile acids by Rec-assay using B. subtilis H17 and M45 strains. Cholic, chenodeoxycholic, lithocholic, and glycolithocholic acids exerted much weaker mutagenicity than mitomicin C (MMC), and deoxycholic and glycodeoxycholic acids showed toxicity toward the bacteria. Most of the conjugated bile acids (glycocholic, taurocholic, and taurodexycholic acids) and their amino acid components (glycine and taurine) were neither toxic nor mutagenic. No bile acids enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), but glycine enhanced both toxicity and mutagenicity of MNNG in a dose-dependent manner. On the other hand, taurine decreased the mutagenicity of MNNG, and most of the bile acids decreased the mutagenicity of MMC. Furthermore, taurocholic acids decreased toxicity and/or mutagenicity of other bile acids. These results suggested that the mutagenic and comutagenic activities of bile acids can be disregarded, but they are antimutagenic in some situations.     

Screening lactic acid bacteria from swine origins for multistrain probiotics based on in vitro functional properties

Lactic acid bacteria originated from swine feces and intestines were selected for potential probiotics based on their bile-salt resistance, low pH tolerance, potential adhesion to epithelial cells and especially functional properties, including production of antimicrobial substances, bile-salt hydrolase (BSH) and amylolytic activity. Results showed 7 isolates with antimicrobial activity, 5 with BSH activity and 3 with amylolytic activity were preliminarily selected from 485 lactic acid bacteria based on their highest potential with functional properties in vitro. The 15 isolates were further assayed on the essential characteristics as potential probiotics. All isolates were fully tolerant to 0.3% bile salts and 11 of them were able to resist pH 3 for 3 h without loss of viable cells. The eleven isolates were then evaluated on their adhesion capability. Wide variation in the hydrophobic character and specific adhesion efficiency was observed and three isolates G1-1, G22-2 and G8-5, with respective antimicrobial, BSH and amylolytic activities were finally selected. In addition, the three isolates were compatible in the coexistence assay. Isolate G1-1 was identified as Lactobacillus salivarius by API system and a 16S rRNA gene sequence analysis. Both G8-5 and G22-2 showed the closest homology to Lactobacillus reuteri according to their 16S rRNA gene sequences (99%). From the study, the three Lactobacilli strains were shown to share the functional properties necessary for probiotics use in animal additives. Their compatibility with respective in vitro activities was expected to show enhanced in vivo efficacy after combination for multistrain probiotics use.     

Structure and function of a highly active Bile Salt Hydrolase (BSH) from Enterococcus faecalis and post-translational processing of BSH enzymes

Bile Salt Hydrolase (BSH), a member of Cholylglycine hydrolase family, catalyzes the de-conjugation of bile acids and is evolutionarily related to penicillin V acylase (PVA) that hydrolyses a different substrate such as penicillin V. We report the three-dimensional structure of a BSH enzyme from the Gram-positive bacteria Enterococcus faecalis (EfBSH) which has manifold higher hydrolase activity compared to other known BSHs and displays unique allosteric catalytic property. The structural analysis revealed reduced secondary structure content compared to other known BSH structures, particularly devoid of an anti-parallel β-sheet in the assembly loop and part of a β-strand is converted to increase the length of a substrate binding loop 2. The analysis of the substrate binding pocket showed reduced volume owing to altered loop conformations and increased hydrophobicity contributed by a higher ratio of hydrophobic to hydrophilic groups present. The aromatic residues F18, Y20 and F65 participate in substrate binding. Thus, their mutation affects enzyme activity. Docking and Molecular Dynamics simulation studies showed effective polar complementarity present for the three hydroxyl (–OH) groups of GCA substrate in the binding site contributing to higher substrate specificity and efficient catalysis. These are unique features characteristics of this BSH enzyme and thought to contribute to its higher activity and specificity towards bile salts as well as allosteric effects. Further, mechanism of autocatalytic processing of Cholylglycine Hydrolases by the excision of an N-terminal Pre-peptide was examined by inserting different N-terminal pre-peptides in EfBSH sequence. The results suggest that two serine residues next to nucleophile cysteine are essential for autocalytic processing to remove precursor peptide. Since pre-peptide is absent in EfBSH the mutation of these serines is tolerated. This suggests that an evolution-mediated subordination of the pre-peptide excision site resulted in loss of pre-peptide in EfBSH and other related Cholylglycine hydrolases.     

Bile salt hydrolase and cholesterol removal effect by Bifidobacterium bifidum NRRL 1976

Summary Growing cells of Bifidobacterum bifidum NRRL 1976 exhibited an ability to remove cholesterol in the presence of bile salts. The cholesterol removal by Bifidobacterium bifidum was due to a co-precipitation together with unconjugated bile acids, which was linked to the bile salt hydrolase (BSH) activity of the cells at pH values lower than 5.0 and the cholesterol removed was partially recovered when the cells were washed with phosphate buffer at pH 7, while the remaining cholesterol was extracted from the cells. It is concluded that the removal of cholesterol from the growth medium by Bifidobacterium bifidum strain is due to both bacterial assimilation and precipitation of cholesterol.     

唾液乳杆菌BSH1及其突变体的底物特异性

为了解析胆盐水解酶催化中心中关键氨基酸位点与其底物特异性的关系,以大肠杆菌pET-20b(+)表达系统为分子改造平台,采用理性设计,结合氨基酸定点突变的方法,成功构建了唾液乳杆菌Lactobacillus salivarius胆盐水解酶BSH1的7种突变体。通过对比L.salivarius BSH1及其突变体对6种结合胆盐的底物特异性表明,7种突变体对不同的结合胆盐的水解活性有所改变。结果说明,Cys2和Thr264分别是BSH1催化TCA和GCA的关键残基,且对酶的催化活性的保持具有关键作用。其中,高保守性的氨基酸位点Cys2不是BSH1唯一的活性位点,而其他突变的氨基酸位点可能作为BSH1的结合位点参与了底物的结合,也可能影响了底物进入BSH1活性中心的通道或底物结合口袋的体积与形状,进而影响了BSH1对不同结合胆盐的水解活性。