Tatsuji Sakamoto

⌈Pectin, Pectinase, and Protopectinase: Production,⌈ Properties, and Applications

Publisher Summary This chapter discusses the applications, properties, and production of pectin, pectinase, and protopectinase. Pectic substances are acid polysaccharides of high molecular weight that are widespread in the plant kingdom. The size, charge density, charge distribution, and degree of substitution of pectin molecules may be changed biologically or chemically. The pectins in these beverages are considered to be responsible for the “veloute” or “moeilleux” (softness or silkiness), which give the fruit product its particular character. The pectin production process consists mainly of an acid extraction, followed by a (partial) purification of the extract and, eventually, precipitation and drying. However, this process has several disadvantages that are addressed in the chapter. Research has been initiated with the aim of developing an improved alternative microbial pectin production process by means of a specific enzyme. The chapter also deals with a specific enzyme, called “protopectinase,” which solubilizes protopectin forming highly polymerized soluble pectin.

Purification and characterisation of two exo-polygalacturonases from Aspergillus niger able to degrade xylogalacturonan and acetylated homogalacturonan.

Two exo-polygalacturonases (EC 3.2.1.67) were purified from a commercial Aspergillus niger enzyme preparation by ammonium sulfate precipitation, preparative electrofocusing, anion-exchange and size-exclusion chromatographies. The enzymes had molar masses of 82 kDa (exo-PG1) and 56 kDa (exo-PG2). Exo-PG1 was stable over wider pH and temperature ranges than exo-PG2. Addition of 0.01 mM HgCl(2) increased the exo-PG2 activity 3.4 times but did not affect exo-PG1. Analysis of the reaction products of (reduced) pentagalacturonate by high-performance anion-exchange chromatography revealed that both enzymes split the substrate from the non-reducing end in a multi-chain attack mode. Exo-PG1 had a broad specificity towards oligogalacturonates with different degrees of polymerisation, while digalacturonate was the most favorable substrate for exo-PG2. Both enzymes degraded xylogalacturonan from pea hull in an exo manner to produce galacturonic acid and Xyl-GalA disaccharide, as identified by electrospray ionization-ion trap mass spectrometry (ESI-ITMS). Moreover, exo-PGs split acetylated homogalacturonan in an exo manner, producing galacturonic acid and acetylated galacturonic acid, as shown by ESI-ITMS.

Purification and properties of two type-B α-l-arabinofuranosidases produced by Penicillium chrysogenum

Two distinct extracellular alpha-L-arabinofuranosidases (AFases; EC 3.2.1.55) were purified from the culture filtrate of Penicillium chrysogenum 31B. The molecular masses of the enzymes were estimated to be 79 kDa (AFQ1) and 52 kDa (AFS1) by SDS-PAGE. Both enzymes had their highest activities at 50 degrees C and were stable up to 50 degrees C. Enzyme activities of AFQ1 and AFS1 were highest at pH 4.0 to 6.5 and pH 3.3 to 5.0, respectively. Addition of 10 mg/ml arabinose to the reaction mixture decreased the AFS1 activity but hardly affected AFQ1. Both enzymes displayed broad substrate specificities; they released arabinose from branched arabinan, debranched arabinan, arabinoxylan, arabinogalactan, and arabino-oligosaccharides. AFS1 also showed low activity towards p-nitrophenyl-beta-D-xylopyranoside. An exo-arabinanase, which catalyzes the release of arabinobiose from linear arabinan at the nonreducing terminus, acted synergistically with both enzymes to produce L-arabinose from branched arabinan.

Identification of a GH62 α-L-arabinofuranosidase specific for arabinoxylan produced by Penicillium chrysogenum.

An arabinoxylan arabinofuranohydrolase (AXS5) was purified from the culture filtrate of Penicillium chrysogenum 31B. A cDNA encoding AXS5 (axs5) was isolated by in vitro cloning using the N-terminal amino acid sequence of the native enzyme as a starting point. The deduced amino acid sequence of the axs5 gene has high similarities with those of arabinoxylan arabinofuranohydrolases of Aspergillus niger, Aspergillus tubingensis, and Aspergillus sojae. Module sequence analysis revealed that a “Glyco_hydro_62” was present at position 28–299 of AXS5. This is a family of α-l-arabinofuranosidases which are all members of glycoside hydrolase family 62. Recombinant AXS5 (rAXS5) expressed in Escherichia coli was highly active on arabinoxylan but not on branched sugar beet arabinan. 1H-NMR analysis revealed that the rAXS5 cleaved arabinosyl side-chains linked to C-2 and C-3 of single-substituted xylose residues in arabinoxylan. Semi-quantitative RT-PCR analysis indicated that expression of the axs5 gene in P. chrysogenum 31B was strongly induced by adding d-xylose and arabinoxylan to the culture medium. Moreover, two binding sites of XlnR, a transcriptional activator that regulates the expression of the genes encoding xylanolytic enzymes, are present in the upstream region of the axs5 gene. These results suggest that AXS5 is involved in xylan degradation.

Analysis of structure of sugar-beet pectin by enzymatic methods

The structure of alkali-soluble pectin (ASP) prepared from sugar-beet pulp was determined. ASP was sequentially degraded by pure galactanase, rhamnogalacturonase and arabinosidase and sugar compositions and NMR analysis of the products used to assign the following tentative structure to ASP: a backbone based on units of -->4)-alpha-D-GalpA-(1--> 2)-alpha-L-Rhap-(1-->, and side chains both of arabinan and of arabinogalactan.

Molecular characterization of a Penicillium chrysogenum exo‐1,5‐α‐L‐arabinanase that is structurally distinct from other arabinan‐degrading enzymes

The nucleotide sequence of the abnx cDNA gene, which encodes an exo‐arabinanase (Abnx) of Penicillium chrysogenum 31B, was determined. Abnx was found to be structurally distinct from known arabinan‐degrading enzymes based on its amino acid sequence and a hydrophobic cluster analysis. The protein in the protein database with the highest similarity to Abnx was the Neurospora crassa conserved hypothetical protein. The abnx cDNA gene product expressed in Escherichia coli catalyzed the release of arabinobiose from α‐1,5‐L‐arabinan. The activity of the recombinant Abnx towards a series of arabino‐oligosaccharides, as expressed by k cat/K m value, was greatest with arabinohexaose.

Immunostimulatory activity of polysaccharides isolated from Caulerpa lentillifera on macrophage cells.

Polysaccharides were extracted from Caulerpa lentillifera by treating with water and then purified by size-exclusion chromatography. The purified polysaccharides, termed SP1, were found to be sulfated xylogalactans with a molecular mass of more than 100 kDa. Adding SP1 to murine macrophage RAW 264.7 cells increased the production of nitric oxide (NO) in a dose-dependent manner. NO was found by immunoblotting and RT-PCR analyses to be synthesized by an inducible NO synthase. SP1 caused the degradation of IκB-α and the nuclear translocation of nuclear factor (NF)-κB subunit p65 in macrophage cells. SP1 also increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results demonstrate that SP1 activated macrophage cells via both the NF-κB and p38 MAPK signaling pathways. Moreover, SP1 increased the expression of various genes encoding cytokines, and the phagocytic activity of macrophage cells. These combined results show that SP1 immunostimulated the activity of macrophage cells.

Protopectinase-T: a rhamnogalacturonase able to solubilize protopectin from sugar beet

Protopectinase-T (PPase-T), isolated from the culture broth of Trametes sanguinea, was able to release pectic substances from sugar beet by degrading rhamnogalacturonan. The smallest polysaccharide (SPS) that can serve as a substrate for PPase-T was prepared from sugar beet pulp by extraction with NaOH and digestion with alpha-L-arabinofuranosidase, alpha-L-arabinase, and beta-(1-->4)-D-galactanase. The reaction products of SPS with PPase-T were isolated by chromatography on DEAE-Toyopearl 650M and Toyopearl HW40-S columns, and analyzed by labeling the reducing ends with [3H]NaBH4 and by 13C NMR spectroscopy. The results indicated that PPase-T cleaved galactopyranosyluronic-rhamnopyranosyl linkages in SPS. Moreover, it was confirmed that the chemical structure of SPS contains a backbone based on units of [-->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rha p-(1-->].

The preventive effect of β-carotene on denervation-induced soleus muscle atrophy in mice.

Muscle atrophy increases the production of reactive oxygen species and the expression of atrophy-related genes, which are involved in the ubiquitin-proteasome system. In the present study, we investigated the effects of β-carotene on oxidative stress (100 μM-H2O2)-induced muscle atrophy in murine C2C12 myotubes. β-Carotene (10 μM) restored the H2O2-induced decreased levels of myosin heavy chain and tropomyosin (P< 0·05, n 3) and decreased the H2O2-induced increased levels of ubiquitin conjugates. β-Carotene reduced the H2O2-induced increased expression levels of E3 ubiquitin ligases (Atrogin-1 and MuRF1) and deubiquitinating enzymes (USP14 and USP19) (P< 0·05, n 3) and attenuated the H2O2-induced nuclear localisation of FOXO3a. Furthermore, we determined the effects of β-carotene on denervation-induced muscle atrophy. Male ddY mice (8 weeks old, n 30) were divided into two groups and orally pre-administered micelle with or without β-carotene (0·5 mg once daily) for 2 weeks, followed by denervation in the right hindlimb. β-Carotene was further administered once daily until the end of the experiment. At day 3 after denervation, the ratio of soleus muscle mass in the denervated leg to that in the sham leg was significantly higher in β-carotene-administered mice than in control vehicle-administered ones (P< 0·05, n 5). In the denervated soleus muscle, β-carotene administration significantly decreased the expression levels of Atrogin-1, MuRF1, USP14 and USP19 (P< 0·05, n 5) and the levels of ubiquitin conjugates. These results indicate that β-carotene attenuates soleus muscle loss, perhaps by repressing the expressions of Atrogin-1, MuRF1, USP14 and USP19, at the early stage of soleus muscle atrophy.

Purification, Characterization, and Overexpression of Thermophilic Pectate Lyase of Bacillus sp. RN1 Isolated from a Hot Spring in Thailand

A thermophilic pectate lyase, Pel SWU, was isolated from a culture filtrate of Bacillus sp. RN1 isolated from a hot spring in Ranong Province, Thailand. The enzyme was purified to homogeneity using cation-exchange and hydrophobic column chromatographies. The molecular mass of Pel SWU was estimated to be 33 kDa. The specific substrate was demethylated galacturonic acid. The enzyme was stable at pH 4.0–10.0 and at temperatures up to 70 °C in the presence of calcium and polygalacturonic acid (PGA). The optimum pH and temperature were 10.0 and 90 °C. The pel gene encoding Pel SWU was 1,023 bp, which corresponds to 341 amino acids. The properties of the recombinant enzyme was similar to those of Bacillus Pel SWU. Unsaturated di- and trigalacturonic acids were formed mainly as the final products of degradation by Pel SWU, as revealed by high-performance anion-exchange chromatography (HPAEC) and electrospray ionization mass spectrometry (ESI-MS) analyses. This thermophilic pectate lyase should be useful in the degradation of pectin networks at high temperature.