Liquid chromatography-mass spectrometry to study chondroitin lyase action pattern

Liquid chromatography-mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A.     

Microbial pectic transeliminases

Pectic transeliminases, also known as pectic lyases or pectinases, are involved in the degradation of pectic substances. They have a wide range of applications in food and textile processing. Although Aspergillus and Penicillium spp. produce pectin lyases, bacteria are the major producers of polygalacturonate lyase. The yields of pectic transeliminases are less than other pectinases. Since new applications for pectic transeliminases are emerging, an improved process for the production of these enzymes is necessary.     

Hydroxynitrile lyases: Functions and properties

Plant hydroxynitrile lyases (Hnl) have attracted the attention of bioorganic scientists for more than 90 years. However, the most important increase in knowledge of this class of enzymes has only arisen in the recent decade. The industrial application of these enzymes as biocatalysts for the synthesis of enantiomerically pure α-cyanohydrins may be responsible for the growing interest in this area.
The Hnls are involved in the catabolic degradation of cyanogenic glycosides, releasing HCN which serves as defense agent against herbivores and microbial attack, or as a nitrogen source. Hydroxynitrile lyases from various plant families appear to represent a new example of enzymes that originated from the convergent evolution of different precursor proteins. The enzymes have been classified into non-FAD- and FAD-containing proteins. FAD-containing enzymes have been isolated exclusively from the Rosaceae, whereas the FAD-independent Hnls, which are more heterogenous in structure, have been characterized from various plant families (Poaceae, Euphorbiaceae, Linaceae, Olacaceae. Filitaceae). The aim of this review is to present a general survey of the natural function and localization of this class of enzymes and a comprehensive summary of the biochemical and genetic data of the isolated proteins.     

Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.

A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70°C with a growth rate of 0.23 h⁻¹ with pectin and 0.12 h⁻¹ with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70°–75°C), amylolytic as well as pullulolytic enzymes (highest activity at 80°–85°C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135 000 Da whereas lyase b consisted of two subunits with molecular masses of 93 000 Da and 158 000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80°C. The enzymes were very stable at 70°C and at 80°C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The K _m values of both lyases for pectate and citrus pectin were 0.5 g·l⁻¹ and 5.0 g·l⁻¹, respectively. After incubation with polygalacturonic acid, mono-, di-, and tri-galacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca²⁺. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases isolated and characterized from a thermophilic anaerobic bacterium. On the basis of the results of the 16S rRNA sequence comparisons and the observed phenotypic differences, we propose strain Ab9 as a new species of Thermoanaerobacter, namely Thermoanaerobacter italicus sp. nov. Received: May 25, 1997 / Accepted: June 5, 1997     

海藻酸盐裂解酶研究进展

海藻酸盐裂解酶是一类降解褐藻中海藻酸盐的酶。此酶已经在多种有机体中得到分离。对海藻酸盐裂解酶的生物特性、研究方法及其生物学功能进行了介绍。在酶学特性研究的基础上 ,通过酶解构建新型海藻酸盐多聚物 ,可增强和扩展海藻酸盐裂解酶在工业、农业、医药领域中的应用 ,使其在海藻多糖的高值化应用中发挥重要的作用。概述了海藻酸盐和海藻酸盐裂解酶过去和现在的研究状况 ,展望了海藻酸盐和海藻酸盐裂解酶将来的应用前景。     

Characterization of galactose-induced extracellular and intracellular pectolytic activities from the exo-1 mutant strain of Neurospora crassa

Pectolytic enzymes from the hyperproducer exo-1 mutant of Neurospora crassa are induced either by pectin or galactose. Galactose-induced pectinases, in contrast with pectin-induced enzymes, are not affected by glucose repression. Here, the pectolytic enzymes induced by galactose were purified and characterized. Extracellular pectolytic activities were separated into two main fractions. Pool I contained lyases, and a polygalacturonase (PG) copurifying as a complex of about 80 kDa (gel filtration). Pool II contained PG only. Under urea-SDS-PAGE the lyases and polygalacturonase from pool I migrated with an apparent MW of 56.2 kDa, and 34.3 kDa, respectively. PG from pool II exhibited an apparent MW of 44.7 kDa. Cell extracts contained PG free of lyase activities. Purified intracellular PG migrated (SDS-PAGE) as a single band of apparent MW of 31.5 kDa. All pectinases were glycoproteins (18.5–39% carbohydrate), with stability and optimum pH at 5–6 and 9–10 for PG and lyases, respectively. Temperature optima were 40–50°C, respectively. All enzymes were inactivated at 60°C, with a half-life from 1.5 to 5 min. Activation energy (Ea) values for extracellular and intracellular PG varied between 0.45 and 2.0 Kcal mol⁻¹. Pool II and intracellular PG and lyases, exhibited a random mechanism of hydrolysis. Pool I PG exhibited an exo character. Received 20 October 1997/ Accepted in revised form 28 February 1998     

Polysaccharide lyases: recent developments as biotechnological tools

Polysaccharide lyases, which are polysaccharide cleavage enzymes, act mainly on anionic polysaccharides. Produced by prokaryote and eukaryote organisms, these enzymes degrade (1,4) glycosidic bond by a beta elimination mechanism and have unsaturated oligosaccharides as major products. New polysaccharides are cleaved only by their specific polysaccharide lyases. From anionic polysaccharides controlled degradations, various biotechnological applications were investigated. This review catalogues the degradation of bacterial, plant and animal polysaccharides (neutral and anionic) by this family of carbohydrate acting enzymes.     

Biofilm inhibitory effect of alginate lyases on mucoid P. aeruginosa from a cystic fibrosis patient

Chronic mucoid Pseudomonas aeruginosa infections are a major scourge in cystic fibrosis patients. Mucoid P. aeruginosa displays structured alginate-rich biofilms that are resistant to antibiotics. Here, we have assessed the efficacy of a panel of alginate lyases in combating mucoid P. aeruginosa biofilms in cystic fibrosis. Albeit we could not demonstrate alginate degradation by alginate lyases in sputum, we demonstrate that the endotypic alginate lyases, CaAly (from Cellulophaga algicola) and VspAlyVI (from Vibrio sp. QY101) and the exotypic alginate lyases, FspAlyFRB (from Falsirhodobacterium sp. alg1), and SA1-IV (from Sphingomonas sp. A1), indeed inhibit biofilm formation by a mucoid P. aeruginosa strain isolated from the sputum of a cystic fibrosis patient with comparative effect to that of the glycoside hydrolase PslG, a promising candidate for biofilm treatment. We believe that these enzymes should be explored for in vivo efficacy in future studies.     

海藻工具酶——褐藻胶裂解酶研究进展

从海洋生物中筛选提取有价值的酶类,开发海洋多糖降解产物,已成为海洋生物资源开发的一个重要方面。因此,近年来对于海藻工具酶之一的褐藻胶裂解酶及其降解产物——褐藻寡糖的研究日益受到人们的普遍关注。从褐藻胶裂解酶的来源、分类、底物专一性、作用方式及结构与机理研究、酶活力测定和酶学性质等方面,结合本课题组的研究工作综述近十年来有关褐藻胶裂解酶的研究进展。     

Analysis of bacterial carbapenem antibiotic production genes reveals a novel β-lactam biosynthesis pathway

Carbapenems are β-lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug-resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora , makes the simple β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the β-lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so-called sulphur-containing β-lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC , encode proteins which show significant homology with proteins encoded by the Streptomyces clavuligerus gene cluster responsible for the production of the β-lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the β-lactamase inhibitor. Our observations are consistent with the evolution of a second major biosynthetic route to the production of β-lactam-ring-containing antibiotics.     

The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein

Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66’s structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.     

cDNA cloning of an alginate lyase from a marine gastropod Aplysia kurodai and assessment of catalytically important residues of this enzyme

Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5′- and 3′-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed ∼40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg; however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1.     

Cloning and Sequence Analysis of the Gene (alyII) Coding for an Alginate Lyase of Pseudomonas sp. OS-ALG-9

Pseudomonas sp. OS-ALG-9 produces several kinds of alginate-degrading enzymes both intra- and extracellularly. As a second alginate lyase of this bacterium, the gene encoding alyII has been cloned in Escherichia coli JM109 by shotgun techniques and then sequenced. The alyII gene has an open reading frame of 2141 bp encoding 713 amino acid residues with a calculated molecular mass of 79,803 Da. The deduced amino acid sequence did not show any extensive similarity with those of other known alginate lyases, however, hydrophobic cluster analysis showed that alyII belonged to class 3 of alginate lyases. The alginate lyase from E. coli harboring the alyII gene showed a single active band, which coincided with one of four major alginate lyases from the crude cell extracts of Pseudomonas sp. OS-ALG-9 on a zymogram.     

Structural and Biochemical Studies Elucidate the Mechanism of Rhamnogalacturonan Lyase from Aspergillus aculeatus

We present here the first experimental evidence for bound substrate in the active site of a rhamnogalacturonan lyase belonging to family 4 of polysaccharide lyases, Aspergillus aculeatus rhamnogalacturonan lyase (RGL4). RGL4 is involved in the degradation of rhamnogalacturonan-I, an important pectic plant cell wall polysaccharide. Based on the previously determined wild-type structure, enzyme variants RGL4_H210A and RGL4_K150A have been produced and characterized both kinetically and structurally, showing that His210 and Lys150 are key active-site residues. Crystals of the RGL4_K150A variant soaked with a rhamnogalacturonan digest gave a clear picture of substrate bound in the − 3/+ 3 subsites. The crystallographic and kinetic studies on RGL4, and structural and sequence comparison to other enzymes in the same and other PL families, enable us to propose a detailed reaction mechanism for the β-elimination on [-,2)-α-l-rhamno-(1,4)-α-d-galacturonic acid-(1,-]. The mechanism differs significantly from the one established for pectate lyases, in which most often calcium ions are engaged in catalysis.     

海洋微生物裂解二甲基巯基丙酸内盐(DMSP)的酶促催化机制

二甲基巯基丙酸内盐(dimethylsulfoniopropionate,DMSP)是全球硫循环和碳循环的重要载体物质。海洋浮游植物、大型藻类和临海被子植物是DMSP的主要生产者。每年DMSP的产量可以达到1×10~9吨。在北大西洋表面的某些区域,DMSP的产量可以达到碳固定总量的10%。微生物介导的DMSP的裂解是全球硫循环和碳循环的重要步骤。目前,8种参与裂解DMSP的DMSP裂解酶已被报道。在已发现的8种DMSP裂解酶中,3种DMSP裂解酶的催化机制得到了研究和阐明。本文根据国内外研究成果,主要对DMSP裂解过程的酶促催化机制的研究进展进行综述,认为在今后工作中需要继续发现新的DMSP裂解酶,并进一步揭示海洋微生物裂解DMSP的分子机制。     

GLYCOSIDASES IN NORMAL AND SCRAPIE MOUSE BRAIN

Abstract— The pH optima of ten glycosidases have been determined in normal and scrapie-affected mouse brain. The enzymes α-mannosidase, α-glucosidase and β-glucosidase displayed two peaks of enzyme activity over the pH range examined.
There is a significant increase in the activity of the enzymes α-mannosidase, β-glucuronidase, N -acetyl β-D-glucosaminidase, N -acetyl β-galactosaminidase, β-glucosidase (pH 4.1), α-fucosidase and β-xylosidase in the brains of mice clinically affected with scrapie, whilst only α-mannosidase (pH 4.1), β-glucuronidase, N -acetyl-β-D-glucosinidase and N- acetyl-β-D-gaiactosaminidase are elevated before mice exhibit signs of the disease.     

Polysaccharases for microbial exopolysaccharides

Microbial exopolysaccharides (EPS) are the substrates for a wide range of enzymes most of which are highly specific. The enzymes are either endoglycanases or polysaccharide lyases and their specificity is determined by carbohydrate structure with uronic acids often playing a major role. The presence of various acyl substituents frequently has little effect on the action of many of the polysaccharases but markedly inhibits some of the polysaccharide lyases including alginate and gellan lyases. The commonest sources of such enzymes can be either microorganisms or bacteriophages. These specific polysaccharide-degrading enzymes can yield oligosaccharide fragments, which are amenable to NMR and other analytical techniques. They have thus proved to be extremely useful in providing information about microbial polysaccharide structures and were routinely used in many such studies. Complex systems containing various mixtures of enzymes may also be effective in the absence of single enzymes but may be difficult to obtain with reproducible activities. Such preparations may also cause extensive degradation of the polysaccharide structure and thus prove less useful in providing information. Commercially available enzyme preparations have seldom proved capable of degrading microbial heteropolysaccharides, although some are active against bacterial alginates and homopolysaccharides including bacterial cellulose and curdlan.     

Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34 805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50°C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl. Received: June 10, 2000 / Accepted: October 3, 2000     

Alginate lyase: Structure,property, and application

Alginate is a linear polysaccharide in which β-D-mannuronate (M) and its epimer, α-L-guluronate (G), are covalently (1–4)-linked in different sequences. Alginate is mainly used as a food additive to modify food texture due to its high viscosity and gelling property. Alginate lyase can degrade alginate by cleaving the glycosidic bond through a β-elimination reaction, generating oligomer with 4-deoxy-L-erythro-hex-4-enepyranosyluronate at the nonreducing end. Alginate oligosaccharides have been shown to stimulate the growth of human endothelial cells and the secretion of cytotoxic cytokines from human macrophage. Alginate can be converted into unsaturated monosaccharide by saccharification process using endolytic and exolytic alginate lyases, thus alginate lyases have potential as key biocatalyst for application of alginate as a renewable source for biochemicals and biofuels in near future. In this paper, structures and functions of various alginate lyases are reviewed. Prospects on future applications of alginate lyases are also discussed.     

Structural insights into a novel Ca2+-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution

Alginate lyases have a wide range of industrial applications, such as oligosaccharide preparation, medical treatment, and bioconversion. Therefore, the discovery and characterization of novel alginate lyases are extremely important. PL-6 alginate lyases are classified into two groups: those with a single domain or two domains. However, only one structure of a two-domain alginate lyase has been determined to date. In this study, we characterized a novel single-domain PL-6 alginate lyase (named AlyF). According to the biochemical analysis, AlyF possesses unique features compared with other PL-6 enzymes, including (1) a Ca²⁺-independent catalytic mechanism and (2) a PolyG-specific cleavage specificity that predominantly produces trisaccharides. The structures of AlyF and its complexes described here reveal the structural basis for these unique features and substrate binding mechanisms, which were further confirmed using mutagenesis. More importantly, we determined the possible subsites specifying the predominantly trisaccharide products of AlyF, which may facilitate the rational design of AlyF for potential applications in preparing a single alginate oligomer.