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.     

Microbial alkaline pectinases and their industrial applications: a review

The biotechnological potential of pectinolytic enzymes from microorganisms has drawn a great deal of attention from various researchers worldwide as likely biological catalysts in a variety of industrial processes. Alkaline pectinases are among the most important industrial enzymes and are of great significance in the current biotechnological arena with wide-ranging applications in textile processing, degumming of plant bast fibers, treatment of pectic wastewaters, paper making, and coffee and tea fermentations. The present review features the potential applications and uses of microbial alkaline pectinases, the nature of pectin, and the vast range of pectinolytic enzymes that function to mineralize pectic substances present in the environment. It also emphasizes the environmentally friendly applications of microbial alkaline pectinases thereby revealing their underestimated potential. The review intends to explore the potential of these enzymes and to encourage new alkaline pectinase-based industrial technology. Electronic Publication     

Biotechnological potential of pectinolytic complexes of fungi

Plant cell wall-degrading enzymes, such as cellulases, hemicellulases and pectinases, have been extensively studied because of their well documented biotechnological potential, mainly in the food industry. In particular, lytic enzymes from filamentous fungi have been the subject of a vast number of studies due both to their advantages as models for enzyme production and their characteristics. The demand for such enzymes is rapidly increasing, as are the efforts to improve their production and to implement their use in several industrial processes, with the goal of making them more efficient and environment-friendly. The present review focuses mainly on pectinolytic enzymes of filamentous fungi, which are responsible for degradation of pectin, one of the major components of the plant cell wall. Also discussed are the past and current strategies for the production of cell wall-degrading enzymes and their present applications in a number of biotechnological areas.     

Wax removal for accelerated cotton scouring with alkaline pectinase

A rational approach has been applied to design a new environmentally acceptable and industrially viable enzymatic scouring process. Owing to the substrate specificity, the selection of enzymes depends on the structure and composition of the substrate, i.e. cotton fibre. The structure and composition of the outer layers of cotton fibre has been established on the basis of thorough literature study, which identifies wax and pectin removal to be the key steps for successful scouring process. Three main issues are discussed here, i.e. benchmarking of the existing alkaline scouring process, an evaluation of several selected acidic and alkaline pectinases for scouring, and the effect of wax removal treatment on pectinase performance. It has been found that the pectinolytic capability of alkaline pectinases on cotton pectin is nearly 75% higher than that of acidic pectinases. It is concluded that an efficient wax removal prior to pectinase treatment indeed results in improved performance in terms of hydrophilicity and pectin removal. To evaluate the hydrophilicity, the structural contact angle (theta) was measured using an auto-porosimeter.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of pectin lyase and pectinase sequences

A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.     

Pectin: cell biology and prospects for functional analysis

Pectin is a major component of primary cell walls of all land plants and encompasses a range of galacturonic acid-rich polysaccharides. Three major pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II) are thought to occur in all primary cell walls. This review surveys what is known about the structure and function of these pectin domains. The high degree of structural complexity and heterogeneity of the pectic matrix is produced both during biosynthesis in the endomembrane system and as a result of the action of an array of wall-based pectin-modifying enzymes. Recent developments in analytical techniques and in the generation of anti-pectin probes have begun to place the structural complexity of pectin in cell biological and developmental contexts. The in muro de-methyl-esterification of homogalacturonan by pectin methyl esterases is emerging as a key process for the local modulation of matrix properties. Rhamnogalacturonan-I comprises a highly diverse population of spatially and developmentally regulated polymers, whereas rhamnogalacturonan-II appears to be a highly conserved and stable pectic domain. Current knowledge of biosynthetic enzymes, plant and microbial pectinases and the interactions of pectin with other cell wall components and the impact of molecular genetic approaches are reviewed in terms of the functional analysis of pectic polysaccharides in plant growth and development.     

Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization

Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a ‘smooth’ region and a ‘hairy’ region. The ‘smooth’ region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The ‘hairy’ region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of β-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40 % the viscosity of pectin with a degree of esterification ≥85 %. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8 % esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.     

Viscometric method for assaying of total endodepolymerase activity of pectinases

An improved method for assaying of the total endodepolymerase activity of pectinases has been developed. The method is based on the determination of the viscosity of a citrus pectin solution in the presence of the enzyme using an Ostwald viscometer. The depolymerizing activity of different pectinases can be detected including polygalacturonase, polymethylgalacturonase, pectin lyase, and pectate lyase. One unit of the endodepolymerase activity corresponds to the activity resulting in 50% decrease in the relative viscosity of 0.5% citrus pectin solution for 5 min at 40°C and the appropriate pH. Depending on the pH-optima of the enzymes, two modifications of the method are described: 1) for acid pectinases at pH 5.0, and 2) for neutral (mildly alkaline) pectinases at pH 8.0. The modifications differed in the control and in the calculation of the activity. Six enzyme preparations were used to demonstrate the applicability of the method. The parameter used for the calculation of the enzymatic activity was directly proportional to the enzyme concentration (the dependence was linear in the range of at least 10-fold change in the enzyme concentration). The relative error of the method did not exceed 10%.     

Formation of an extracellular system of enzymes during growth of Geotrichum candidum 3C on cell walls isolated from cereal grain capsules

The activities of extracellular systems of hemicellulases, pectinases, and cellulases was studied during a 72-h cultivation of Geotrichum candidum 3C. The culture was grown on a medium containing 3% cell walls isolated from wheat grain capsules, which served as the sole carbon source. Enzymes catalyzing the degradation of pectin substances (beet pectin, alpha-L-arabinan, and 1,4-beta-D-galactan), as well as beta-D-galactosidase and alpha-L-arabinofuranosidase involved in their hydrolysis, were formed first (4 h after the beginning of cultivation). Enzymes hydrolyzing 4-O-methyl-alpha-D-glucurono-beta-D-xylan and sodium carboxymethyl xylan were also found in the culture liquid after 4 h of fungal growth. The contents of pectin-degrading and xylanolytic enzymes reached their maximum levels after 52-56 and 72 h of growth, respectively. Cellulolytic enzymes were detected after 8-28 h of cultivation. Enzymes degrading alpha-D-galacto-beta-D-mannan were found 24 h after the beginning of growth; their content was maximum after 72 h of cultivation.     

Partial characterization ofAspergillus fumigatus polygalacturonases for the degumming of natural fibers

Summary Aspergillus fumigatus strain 4, cultured on citrus pectin as the sole carbon source, produced polygalacturonases whose activity was optimum at 65°C and pH 3.5–4.5. The enzymes presented a bimodal thermostability for 10 min, but not 60 min, of incubation. Polygalacturonases showed pH stability between 3.0 to 9.0. The enzymes were stable when stored at 4–6°C for 90 days, but their activity was reduced by 24% when they were stored at 26–30°C. Orange pulp was the best pectic carbon source tested for the production of pectinases capable of retting ramie fibers. The reutilization of these enzymes was possible, suggesting the viability of industrial use of pectinases for degumming ramie fibers.     

果胶酶在麻类脱胶中的应用及其作用机理

脱胶是麻类产业链中的难题。生物脱胶则是解决麻类加工技术难题的发展方向。果胶酶在生物脱胶中的应用一直是研究的重点。本文通过分析国内外有关果胶酶和产酶微生物在选育、发酵、酶学性质、基因工程与脱胶工艺等方面的研究进展,阐明了果胶酶在麻类脱胶中的作用机理。果胶酶是麻类生物脱胶不能缺少的关键酶之一,但不能独立完成麻类脱胶;根据麻类纤维原料特性,采用基因操作等技术构建复合酶高产菌株是未来的重点研究方向。     

微生物果胶酶研究进展

果胶酶是一类分解果胶质的酶的总称,它能将复杂的果胶分解为半乳糖醛酸等小分子。目前果胶酶在食品、纺织、医药、造纸、环境、生物技术、饲料等领域得到广泛应用。果胶酶主要来自微生物。综述了微生物果胶酶生产菌的菌种、选育、鉴定、发酵方法和发酵条件优化,酶的分离纯化、酶学性质和分子生物学方面的研究进展,并介绍了果胶酶的应用进展,最后展望了微生物果胶酶研究的广阔前景。     

Sequential production of pectinases byPenicillium frequentans

The pectinases produced byPenicillium frequentans were subjected to pectin- or sodium polypectate-PAGE. The fungus secreted one endo- and one exo-polygalacturonase during the first 10 h of incubation, either in media supplemented with pectin or in the absence of carbohydrate source. After 17 h of cultivation, another two exo-and three endo-polygalacturonases were detected in cultures supplemented with pectin. The results indicate that two constitutive polygalacturonases are secreted initially, followed by the synthesis of other inducible pectinases, in a sequential production of the pectinolytic complex. Monogalacturonic acid and sodium polypectate were better inducers for exo- than for endo-polygalacturonases, indicating that these enzymes are independently controlled.The authors are with the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Avenida do Café s/n^o, 14040-903 Ribeirão Preto, São Paulo, Brazil     

Applications of pectinases in the commercial sector: a review

Pectinases are one of the upcoming enzymes of fruit and textile industries. These enzymes break down complex polysaccharides of plant tissues into simpler molecules like galacturonic acids. The role of acidic pectinases in bringing down the cloudiness and bitterness of fruit juices is well established. Recently, there has been a good number of reports on the application of alkaline pectinases in the textile industry for the retting and degumming of fiber crops, production of good quality paper, fermentation of coffee and tea, oil extractions and treatment of pectic waste water. This review discusses various types of pectinases and their applications in the commercial sector.     

Pectin-modifying enzymes and pectin-derived materials: applications and impacts

Pectins are complex branched polysaccharides present in primary cell walls. As a distinctive feature, they contain high amount of partly methyl-esterified galacturonic acid and low amount of rhamnose and carry arabinose and galactose as major neutral sugars. Due to their structural complexity, they are modifiable by many different enzymes, including hydrolases, lyases, and esterases. Their peculiar structure is the origin of their physicochemical properties. Among others, their remarkable gelling properties make them a key additive for food industries. Pectin-degrading enzymes and -modifying enzymes may be used in a wide variety of applications to modulate pectin properties or produce pectin derivatives and oligosaccharides with functional as well as nutritional interests. This paper reviews the scientific information available on pectin structure, pectin-modifying enzymes, and the use of enzymes to produce pectin with controlled structure or pectin-derived oligosaccharides, with functional or nutritional interesting properties.     

Conidial and mycelial-bound exo-pectinase of Aspergillus sp.

Abstract Intact conidia of Aspergillus sp. were able to degrade pectin 'in vitro' even when protein synthesis was inhibited, thus indicating the presence of cell bound pectinases. At least an exo-pectinase was found and this enzyme was also present in the mycelium of Aspergillus sp. Its presence was not dependent on the carbon source used for growth, suggesting its constitutive nature. This exo-pectinase could be released from conidia or mycelium by incubation at different pH values and the amount of enzyme released could be increased by treatments with chemical agents and hydrolytic enzymes.     

Pectinolytic Systems of Two Aerobic Sporogenous Bacterial Strains with High Activity on Pectin

Strains Paenibacillus sp. BP-23 and Bacillus sp. BP-7, previously isolated from soil from a rice field, secreted high levels of pectinase activity in media supplemented with pectin. Production of pectinases in strain Paenibacillus sp. BP-23 showed catabolite repression, while in Bacillus sp. BP-7 production of pectin degrading enzymes was not negatively affected by glucose. The two strains showed lyase activities as the predominant pectinases, while hydrolase activity was very low. Analysis of Paenibacillus sp. BP-23 in SDS–polyacrylamide gels and zymograms showed five pectinase activity bands. The strict requirement of Ca²⁺ for lyase activity of the strain indicates that correspond to pectate lyases. For Bacillus sp. BP-7, zymograms showed four bands of different size. The strain showed a Ca²⁺ requirement for lyase activity on pectate but not on pectin, indicating that the pectinolytic system of Bacillus sp. BP-7 is comprised of pectate lyases and pectin lyases. The results show differences in pectin degrading systems between the two aerobic sporogenous bacterial strains studied.