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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #5)

Comparison of the Properties of Lipase Immobilized onto Mesoporous Resins by Different Methods by Wenqin Wang; Yanjun Jiang; Liya Zhou; Jing Gao (pp. 561-572).

Genipin, a natural cross-linking agent, was used for the immobilization of lipase from Candida sp. 99-125 by cross-linking to two kinds of mesoporous resins. Under optimum conditions, the activity recovery of immobilized lipase on resin NKA-9 could reach up to 96.99% when the genipin concentration was 0.5%, and it could reach up to 86.18% for S-8 with a genipin concentration of 0.25%. Compared with using glutaraldehyde as a cross-linking agent, the immobilized lipase using genipin showed better pH and thermal stability, storage stability, and reusability. The residual activity of immobilized lipase using genipin as cross-linker remained more than 60% of its initial activity after six hydrolytic cycles, whereas only about 35% activity remained by using glutaraldehyde as cross-linker.

Keywords: Immobilized lipase; Cross-linking; Genipin; Glutaraldehyde; Stability

Exploring Applications of Procerain B, a Novel Protease from Calotropis procera, and Characterization by N-Terminal Sequencing as well as Peptide Mass Fingerprinting by Abhay Narayan Singh; Vikash Kumar Dubey (pp. 573-580).

Procerain B is a novel cysteine protease isolated from Calotropis procera by our group and published recently. We have further characterized the enzyme by N-terminal sequencing and peptide mass fingerprinting. Procerain B showed maximum sequence similarity (80%) with Asclepain. Moreover, the characteristic VDWR motif of cysteine proteases is present in procerain B. The N-terminal and peptide mass fingerprinting analysis showed a distinct nature of the enzyme. Various applications of the enzyme were also evaluated. Procerain B is very effective in milk-clotting and may be a potential candidate for this process in the cheese industry. Additionally, the enzyme has potential application as dietary supplement to aid digestion. Effects of various metal ions on milk-clotting activity were also studied. The milk-clotting activity was increased in case of few metals while others have a negative effect. It is worth mentioning that the easy availability of plant material and simple purification method makes industrial production of the enzyme feasible. A protease with easy purification and suitable properties for application is always desired.

Keywords: Cysteine protease; Food industry; Calotropis procera

Gene Cloning, Heterologous Expression, and Characterization of a High Maltose-Producing α-Amylase of Rhizopus oryzae by Song Li; Zhirui Zuo; Dandan Niu; Suren Singh; Kugenthiren Permaul; Bernard A. Prior; Guiyang Shi; Zhengxiang Wang (pp. 581-592).

A putative α-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the α-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4–6 and 60 °C, respectively. The enzyme was stable at pH ranges of 4.5–6.5 and temperatures below 50 °C. Purified RoAmy had a K _m and V _max of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity was strongly inhibited by 5 mM Cu²⁺ and 5 mM Fe²⁺, whereas 5 mM Ca²⁺ showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K _m = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration of 40 mM.

Keywords: α-Amylase; Maltose; Rhizopus oryzae ; Heterologous expression; Enzyme characterization

Purification and Characterization of a Novel Thermostable Xylose Isomerase from Opuntia vulgaris Mill by Sambandam Ravikumar; Jeyaraman Vikramathithan; Kotteazeth Srikumar (pp. 593-603).

Thermophilic xylose isomerase from the xerophytic eukaryote Opuntia vulgaris can serve as a good alternate source of enzyme for use in the production of high fructose corn syrup. The existence of two temperature stable isoforms having optimal activity at temperatures 70 °C (T₇₀) and 90 °C (T₉₀), respectively, is reported here. These isoforms were purified to homogeneity using column chromatography and SDS-polyacrylamide gel electrophoretic techniques. Only the T₉₀ isoform was subjected to full biochemical characterization thereafter. The purified T₉₀ isoform was capable of converting glucose to fructose with high efficiency under the assay conditions. The enzyme at pH 7.5 exhibited a preference to yield the forward isomerization reaction. The melting temperature of the native enzyme was determined to be 90 °C employing differential scanning colorimetery. Thermostability of the enzyme protein was established through temperature-related denaturation kinetic studies. It is suggested that the thermostability and the wide pH activity of this eukaryotic enzyme will make it an advantageous and dependable alternate source of catalytic activity for protected use in the high fructose corn syrup sweetener industry.

Keywords: Xylose isomerase; Isoenzyme; Opuntia vulgaris ; Thermophilic; Purification

Cloning of Two Genes (LAT1,2) Encoding Specific l-Arabinose Transporters of the l-Arabinose Fermenting Yeast Ambrosiozyma monospora by Ritva Verho; Merja Penttilä; Peter Richard (pp. 604-611).

We identified and characterized two genes, LAT1 and LAT2, which encode specific l-arabinose transporters. The genes were identified in the l-arabinose fermenting yeast Ambrosiozyma monospora. The yeast Saccharomyces cerevisiae had only very low l-arabinose transport activity; however, when LAT1 or LAT2 was expressed, l-arabinose transport was facilitated. When the LAT1 or LAT2 were expressed in an S. cerevisiae mutant where the main hexose transporters were deleted, the l-arabinose transporters could not restore growth on d-glucose, d-fructose, d-mannose or d-galactose. This indicates that these sugars are not transported and suggests that the transporters are specific for l-arabinose.

Keywords: Sugar transport; l-Arabinose transport; l-Arabinose metabolism; Pentose fermentation; Metabolic engineering; Yeast

Propyl Gallate Synthesis Using Acidophilic Tannase and Simultaneous Production of Tannase and Gallic Acid by Marine Aspergillus awamori BTMFW032 by P. S. Beena; Soorej M. Basheer; Sarita G. Bhat; Ali H. Bahkali; M. Chandrasekaran (pp. 612-628).

Marine Aspergillus awamori BTMFW032, recently reported by us, produce acidophilic tannase as extracellular enzyme. Here, we report the application of this enzyme for synthesis of propyl gallate by direct transesterification of tannic acid and in tea cream solubilisation besides the simultaneous production of gallic acid along with tannase under submerged fermentation by this fungus. This acidophilic tannase enabled synthesis of propyl gallate by direct transesterification of tannic acid using propanol as organic reaction media under low water conditions. The identity of the product was confirmed with thin layer chromatography and Fourier transform infrared spectroscopy. It was noted that 699 U/ml of enzyme could give 60% solubilisation of tea cream within 1 h. Enzyme production medium was optimized adopting Box–Behnken design for simultaneous synthesis of tannase and gallic acid. Process variables including tannic acid, sodium chloride, ferrous sulphate, dipotassium hydrogen phosphate, incubation period and agitation were recognized as the critical factors that influenced tannase and gallic acid production. The model obtained predicted 4,824.61 U/ml of tannase and 136.206 μg/ml gallic acid after 48 h of incubation, whereas optimized medium supported 5,085 U/ml tannase and 372.6 μg/ml of gallic acid production after 36 and 84 h of incubation, respectively, with a 15-fold increase in both enzyme and gallic acid production. Results indicated scope for utilization of this acidophilic tannase for transesterification of tannic acid into propyl gallate, tea cream solubilisation and simultaneous production of gallic acid along with tannase.

Keywords: Aspergillus awamori ; Acidophilic tannase; Propyl gallate synthesis; Gallic acid

Development and Characterization of an Environmentally Friendly Process Sequence (Autohydrolysis and Organosolv) for Wheat Straw Delignification by Héctor A. Ruiz; Denise S. Ruzene; Daniel P. Silva; Fernando F. Macieira da Silva; António A. Vicente; José A. Teixeira (pp. 629-641).

The present work describes the delignification of wheat straw through an environmentally friendly process resulting from sequential application of autohydrolysis and organosolv processes. Wheat straw autohydrolysis was performed at 180°C during 30 min with a liquid–solid ratio of 10 (v/w); under these conditions, a solubilization of 44% of the original xylan, with 78% of sugars as xylooligosaccharides of the sum of sugars solubilized in the autohydrolysis liquors generated by the hemicellulose fraction hydrolysis. The corresponding solid fraction enrichment with 63.7% of glucan and 7.55% of residual xylan was treated with a 40% ethanol and 0.1% NaOH aqueous solution at a liquid–solid ratio of 10 (v/w), with the best results obtained at 180°C during 20 min. The highest lignin recovery, measured by acid precipitation of the extracted lignin, was 3.25 g/100 ml. The lignin obtained by precipitation was characterized by FTIR, and the crystallinity indexes from the native cellulose, the cellulose recovered after autohydrolysis, and the cellulose obtained after applying the organosolv process were obtained by X-ray diffraction, returning values of 21.32%, 55.17%, and 53.59%, respectively. Visualization of the fibers was done for all the processing steps using scanning electron microscopy.

Keywords: Organosolv process; Delignification; Wheat straw; Autohydrolysis; Extraction

Effect of the Conformational Stability of the CH2 Domain on the Aggregation and Peptide Cleavage of a Humanized IgG by Daisuke Kameoka; Tadashi Ueda; Taiji Imoto (pp. 642-654).

To examine the effect of the conformational stability of the CH2 domain on aggregation and peptide cleavage of a humanized IgG1, we carried out size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of incubated sample solutions. By comparing the residual percentage of monomer after incubation at 60 and 80°C at various pH levels, we found that aggregation and peptide cleavage of the humanized IgG1 occurred during long incubation at 60°C under acidic conditions. Next, we confirmed cleavage of the Asp272-Pro273 peptide bond in the CH2 domain. Comparison of the cleavage rates of the IgG1 monomer and a peptide containing the same Asp-Pro sequence revealed that the conformational stability of the CH2 domain retards cleavage of the Asp272-Pro273 peptide bond at 60°C and pH 4.0. The finding of aggregation and peptide cleavage of the humanized IgG1 after long incubation at 60°C under acidic conditions was supported by another finding: there were lower unfolding temperatures of the CH2 domain at pH 4.0 and 5.0. We conclude that the conformational stability of the CH2 domain is closely related to aggregation and peptide cleavage of the humanized IgG1 under acidic conditions. We also found that the 2-[N-morpholino] ethane sulfonate buffer inhibits aggregation of the IgG1 at pH 4.0–5.0 and 7.0–8.0.

Keywords: pH; Protein deterioration; Higher-order structure; Structural stability; Asp-Pro peptide bond; Chemical cleavage; Unfolding temperature

Production of Astaxanthin from Cellulosic Biomass Sugars by Mutants of the Yeast Phaffia rhodozyma by Justin Montanti; Nhuan P. Nghiem; David B. Johnston (pp. 655-665).

Astaxanthin is a potential high-value coproduct in an ethanol biorefinery. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were tested for their capability of utilizing the major sugars that can be generated from cellulosic biomass, including glucose, xylose, and arabinose, for astaxanthin production. While all three strains were capable of metabolizing these sugars, individually and in mixtures, JTM185 demonstrated the greatest sugar utilization and astaxanthin production. Astaxanthin yield by this strain (milligrams astaxanthin per gram of sugar consumed) was highest for xylose, followed by arabinose and then glucose. The kinetics of sugar utilization by strain JTM185 was studied in fermenters using mixtures of glucose, xylose, and arabinose at varied concentrations. It was found that glucose was utilized preferentially, followed by xylose, and lastly, arabinose. Astaxanthin yield was significantly affected by sugar concentrations. Highest yields were observed with sugar mixtures containing the highest concentrations of xylose and arabinose. Hydrolysates produced from sugarcane bagasse and barley straw pretreated by the soaking in aqueous ammonia method and hydrolyzed with the commercial cellulase preparation, Accellerase™ 1000, were used for astaxanthin production by the mutant strain JTM185. The organism was capable of metabolizing all of the sugars present in the hydrolysates from both biomass sources and produced similar amounts of astaxanthin from both hydrolysates, although these amounts were lower when compared to yields obtained with reagent grade sugars.

Keywords: Phaffia rhodozyma ; Astaxanthin; Barley straw; Sugarcane bagasse; Cellulosic biomass

Cellulase Production by Pink Pigmented Facultative Methylotrophic Strains (PPFMs) by Shanmugam Jayashree; Rajendran Lalitha; Ponnusamy Vadivukkarasi; Yuko Kato; Sundaram Seshadri (pp. 666-680).

Pink pigmented facultative methylotrophs (PPFM) isolated from water samples of Cooum and Adyar rivers in Chennai and soil samples of forests located in various districts of Tamil Nadu, India were screened for cellulase production using carboxymethylcellulose agar (CMC agar) medium. The strains showed wide variations in the production of clearing zones around the colonies on CMC agar medium flooded with Congo red. CMCase and filter paper assays were used to quantitatively measure the cellulase activity of 13 PPFM strains. Among the strains, Methylobacterium gregans, MNW 60, MHW 109, MSF 34, and MSF 40 showed cellulolytic activity ranging from 0.73 to 1.16 U mL⁻¹ with wide temperature (35–65°C) and pH (5 to 8) tolerance. SDS-PAGE analysis of the crude enzyme of PPFM strain MNW 60 exhibited several protein bands, and zymogram analysis revealed two dimeric cellulase bands with molecular mass of ~92 and 42 kDa. Scanning electron microscopic studies revealed significant morphological differences between the cells grown in normal and CMC amended medium. The strain MNW 60 was identified as Methylobacterium sp. based on biochemical, physiological, and morphological analyses, and the methylotrophic nature was authenticated by the presence of mxaF gene, encoding methanol dehydrogenase as a key indicator enzyme of methylotrophs, with 99% similarity to Methylobacterium lusitanum. With the 16S ribosomal RNA sequence showing 97% similarity to M. lusitanum strain MP2, this can be proposed as a novel taxon of the genus Methylobacterium. The study forms the first detailed report on the extracellular cellulase production by pink pigmented Methylobacterium sp., and it is expected that this might be the basis for further studies on cellulase production by PPFMs to explore the molecular mechanism, strain improvement, and large-scale cellulase production for its application.

Keywords: CMC; CMCase; FPase; mxaF ; Methylobacterium

Actinobacterial Peroxidases: an Unexplored Resource for Biocatalysis by Marilize le Roes-Hill; Nuraan Khan; Stephanie Gail Burton (pp. 681-713).

Peroxidases are redox enzymes that can be found in all forms of life where they play diverse roles. It is therefore not surprising that they can also be applied in a wide range of industrial applications. Peroxidases have been extensively studied with particular emphasis on those isolated from fungi and plants. In general, peroxidases can be grouped into haem-containing and non-haem-containing peroxidases, each containing protein families that share sequence similarity. The order Actinomycetales comprises a large group of bacteria that are often exploited for their diverse metabolic capabilities, and with recent increases in the number of sequenced genomes, it has become clear that this metabolically diverse group of organisms also represents a large resource for redox enzymes. It is therefore surprising that, to date, no review article has been written on the wide range of peroxidases found within the actinobacteria. In this review article, we focus on the different types of peroxidases found in actinobacteria, their natural role in these organisms and how they compare with the more well-described peroxidases. Finally, we also focus on work remaining to be done in this research field in order for peroxidases from actinobacteria to be applied in industrial processes.

Keywords: Actinobacteria; Biocatalysis; Peroxidases

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #5)