Applied Biochemistry and Biotechnology (v.170, #8)

Efficient Production of Ethanol from Empty Palm Fruit Bunch Fibers by Fed-Batch Simultaneous Saccharification and Fermentation Using Saccharomyces cerevisiae by Jang Min Park; Baek-Rock Oh; Jeong-Woo Seo; Won-Kyung Hong; Anna Yu; Jung-Hoon Sohn; Chul Ho Kim (1807-1814).
The concentration of ethanol produced from lignocellulosic biomass should be at least 40 g l−1 [about 5 % (v/v)] to minimize the cost of distillation process. In this study, the conditions for the simultaneous saccharification and fermentation (SSF) at fed-batch mode for the production of ethanol from alkali-pretreated empty palm fruit bunch fibers (EFB) were investigated. Optimal conditions for the production of ethanol were identified as temperature, 30 °C; enzyme loading, 15 filter paper unit g−1 biomass; and yeast (Saccharomyces cerevisiae) loading, 5 g l−1 of dry cell weight. Under these conditions, an economical ethanol concentration was achieved within 17 h, which further increased up to 62.5 g l−1 after 95 h with 70.6 % of the theoretical yield. To our knowledge, this is the first report to evaluate the economic ethanol production from alkali-pretreated EFB in fed-batch SSF using S. cerevisiae.
Keywords: Lignocellulosic Biomass; Empty Palm Fruit Bunch Fiber (EFB); SSF; Ethanol; Pretreatment

A New Metal-Chelated Cryogel for Reversible Immobilization of Urease by Murat Uygun; Begüm Akduman; Sinan Akgöl; Adil Denizli (1815-1826).
Poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [poly(HEMA-GMA)] cryogel was synthesized by cryopolymerization technique at frozen temperature. Iminodiacetic acid (IDA) was then attached covalently to the cryogel as a chelating agent. Then, poly(HEMA-GMA)-IDA cryogel was chelated with Ni(II) ions and this novel metal affinity support was used for adsorption of urease from its aqueous solution. Urease adsorption experiments were carried out in a continuous system by using a peristaltic pump. Maximum urease adsorption onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was found to be 11.30 mg/g cryogel at pH 5.0 acetate buffer and in 25 °C medium temperature. Urease adsorption capacity decreased with increasing ionic strength and increasing chromatographic flow rate. Adsorption kinetics of urease onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was also investigated and it was found that Langmuir adsorption model is applicable for this adsorption study. This novel immobilized metal affinity chromatography support was used 10 times without any decrease at their adsorption capacity. It was also observed that urease enzyme was repeatedly adsorbed and desorbed without significant lost in enzymatic activity.
Keywords: IMAC; Cryogel; Urease; Iminodiacetic acid

Cross-linked enzyme aggregates (CLEAs) have been recently proposed as an alternative to conventional immobilization methods on solid carriers. However, the low cross-linking efficiency causes the major activity loss and instability in the conventional protocol for CLEA preparation. Herein, the effects of bovine serum albumin and starch addition on the cross-linking efficiency of CLEAs of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis were evaluated. A co-aggregation strategy was developed to improve cross-linking efficiency by adding starch and bovine serum albumin (BSA). CLEAs of PAL prepared in the presence of BSA and starch (PSB-CLEAs) retained 36 % activity, whereas CLEAs prepared without BSA and starch (PAL-CLEAs) retained only 8 % activity of the starting enzyme preparation. Compared with PAL-CLEAs, the thermal stability of PSB-CLEAs has improved considerably, maintaining 30 % residual activity after 4 h of incubation at 70 °C, whereas the PAL-CLEAs have only 13 % residual activity. PSB-CLEAs also exhibited the expected increased stability of PAL against hydrophilic organic solvents, superior operability, and higher storage stability. The proposed technique of preparing CLEAs using co-aggregation with starch and BSA would rank among the potential strategies for efficiently preparing robust and highly stable enzyme aggregates.
Keywords: Phenylalanine ammonia lyase; Rhodotorula glutinis ; Cross-linked enzyme aggregates; Co-aggregation; Biocatalysts

The pretreatment of lignocellulosic biomass with white-rot fungi to produce bioethanol is an environmentally friendly alternative to the commonly used physico-chemical processes. After biological pretreatment, a solid substrate composed of cellulose, hemicellulose and lignin, the two latter with a composition lower than that of the initial substrate, is obtained. In this study, six microorganisms and four process configurations were utilised to ferment a hydrolysate obtained from wheat straw pretreated with the white-rot fungus Irpex lacteus. To enhance total sugars utilisation, five of these microorganisms are able to metabolise, in addition to glucose, most of the pentoses obtained after the hydrolysis of wheat straw by the application of a mixture of hemicellulolytic and cellulolytic enzymes. The highest overall ethanol yield was obtained with the yeast Pachysolen tannophilus. Its application in combination with the best process configuration yielded 163 mg ethanol per gram of raw wheat straw, which was between 23 and 35 % greater than the yields typically obtained with a conventional bioethanol process, in which wheat straw is pretreated using steam explosion and fermented with the yeast Saccharomyces cerevisiae.
Keywords: Bioethanol; Lignocellulosic biomass pretreatment; Alcoholic fermentation; Pachysolen tannophilus ; Wheat straw

Fibroblast growth factor receptor 2 (FGFR2) controls a wide range of biological functions by regulating the cellular proliferation, survival, migration and differentiation. A growing body of preclinical data demonstrated that deregulation of the FGFR signalling through genetic modification was observed in various types of cancers. However, the extent to which genetic modifications interfere with gene regulation and their involvement in cancer susceptibility remains largely unknown. In this work, we performed in silico profiling of harmful non-synonymous single nucleotide polymorphisms (SNPs) in the protein kinase domain of FGFR2. Tolerance index, position-specific independent count score, change in free energy score (ΔΔG), Eris and FoldX indicated that seven mutations were found to be deleterious and may alter the protein function and structure. Furthermore, based on physico-chemical properties, two mutations K659N and R747H were found to be most deleterious in protein kinase domain and taken for further structural analysis. Docking study showed a complete loss of binding affinity followed by interference in hydrogen bonding and surrounding residues due to K659N and R747H mutations. In order to elucidate the mechanism behind the impact of mutation that can generate a ripple effect throughout the protein structure and ultimately affect the function, in-depth molecular dynamics simulation and principal component analysis were performed. The obtained results indicate that K659N and R747H mutations have a distinct effect on the dynamic behaviour of FGFR2 protein. Our strategy may be helpful for understanding SNP effects on proteins with function and their role in human genetic diseases and for the development of novel pharmacological strategies.
Keywords: FGFR2; nsSNPs; Ponatinib; Docking study; Molecular dynamics simulations

The article describes preparation of new calixarene biopolymers consisting of the immobilization of convenience calixarene derivative onto cellulose and chitosan biopolymers, and the encapsulation of these calixarene biopolymers with Candida rugosa lipase within a chemical inert sol–gel supported by polycondensation with tetraethoxysilane and octyltriethoxysilane. The catalytic properties of immobilized lipase were evaluated into model reactions employing the hydrolysis of p-nitrophenylpalmitate and the enantioselective hydrolysis of naproxen methyl esters from racemic prodrugs in aqueous buffer solution/isooctane reaction system. The resolution studies using sol–gel support have observed more improvement in the enantioselectivity of naproxen E = 300 with Cel-Calix-E than with encapsulated lipase without calixarene-based materials. Furthermore, the encapsulated lipase (Cel-Calix-E) was still retained about 39 % of their conversion ratios after the fifth reuse in the enantioselective reaction.
Keywords: Lipase; Calixarene; Biopolymers; Enantioselectivity; Naproxen

Discarded Oranges and Brewer’s Spent Grains as Promoting Ingredients for Microbial Growth by Submerged and Solid State Fermentation of Agro-industrial Waste Mixtures by Theodoros Aggelopoulos; Argyro Bekatorou; Ashok Pandey; Maria Kanellaki; Athanasios A. Koutinas (1885-1895).
The exploitation of various agro-industrial wastes for microbial cell mass production of Kluyveromyces marxianus, kefir, and Saccharomyces cerevisiae is reported in the present investigation. Specifically, the promotional effect of whole orange pulp on cell growth in mixtures consisting of cheese whey, molasses, and potato pulp in submerged fermentation processes was examined. A 2- to 3-fold increase of cell mass was observed in the presence of orange pulp. Likewise, the promotional effect of brewer’s spent grains on cell growth in solid state fermentation of mixtures of whey, molasses, potato pulp, malt spent rootlets, and orange pulp was examined. The cell mass was increased by 3-fold for K. marxianus and 2-fold for S. cerevisiae in the presence of these substrates, proving their suitability for single-cell protein production without the need for extra nutrients. Cell growth kinetics were also studied by measurements of cell counts at various time intervals at different concentrations of added orange pulp. The protein content of the fermented substrates was increased substantially, indicating potential use of mixed agro-industrial wastes of negligible cost, as protein-enriched livestock feed, achieving at the same time creation of added value and waste minimization.
Keywords: Agro-industrial wastes; Discarded oranges; Brewer’s spent grains; Cell growth; Single-cell protein

The present study is an attempt to demonstrate the feasibility of sal (Shorea robusta) deoiled cake—a forest-based industrial by-product—as a cheaper media supplement for augmented protease production from Aeromonas sp. S1 and application of protease in the treatment of kitchen wastewater. Under optimized conditions, protease production could successfully be enhanced to 5.13-fold (527.5 U mL−1) on using sal deoiled seed cake extract (SDOCE), as medium additive, compared to an initial production of 102.7 U mL−1 in its absence. The culture parameters for optimum production of protease were determined to be incubation time (48 h), pH (7.0), SDOCE concentration (3 % (v/v)), inoculum size (0.3–0.6 % (v/v)), and agitation rate (100 rpm). The enzyme was found to have an optimum pH and temperature of 8.0 and 60 °C, respectively. The protease preparation was tested for treatment of organic-laden kitchen wastewater. After 96 h of wastewater treatment under static condition, enzyme preparation was able to reduce 74 % biological oxygen demand, 37 % total suspended solids, and 41 % oil and grease. The higher and improved level of protease obtained using sal deoiled seed cake-based media hence offers a new approach for value addition to this underutilized biomass through industrial enzyme production. The protease produced using this biomass could also be used as pretreatment tool for remediation of organic-rich food wastewater.
Keywords: Sal seed cake; Protease; Aeromonas sp. S1; Submerged fermentation; Media optimization; Wastewater treatment

Immobilization of Inulinase on Concanavalin A-Attached Super Macroporous Cryogel for Production of High-Fructose Syrup by Canan Altunbaş; Murat Uygun; Deniz Aktaş Uygun; Sinan Akgöl; Adil Denizli (1909-1921).
In this study, concanavalin A (Con A)-attached poly(ethylene glycol dimethacrylate) [poly(EGDMA)] cryogels were used for immobilization of Aspergillus niger inulinase. For this purposes, the monolithic cryogel column was prepared by radical cryocopolymerization of EGDMA as a monomer and N,N′-methylene bisacrylamide as a crosslinker. Then, Con A was attached by covalent binding onto amino-activated poly(EGDMA) cryogel via glutaraldehyde activation. Characterization of cryogels was performed by FTIR, EDX, and SEM studies. Poly(EGDMA) cryogels were highly porous and pore size was found to be approximately 50–100 μm. Con A-attached poly(EGDMA) cryogels was used in the adsorption of inulinase from aqueous solutions. Adsorption of inulinase on the Con A-attached poly(EGDMA) cryogel was performed in continuous system and the effects of pH, inulinase concentration, and flow rate on adsorption were investigated. The maximum amount of inulinase adsorption was calculated to be 27.85 mg/g cryogel at 1.0 mg/mL inulinase concentration and in acetate buffer at pH 4.0. Immobilized inulinase was effectively used in continuous preparation of high-fructose syrup. Inulin was converted to fructose in a continuous system and released fructose concentration was found to be 0.23 mg/mL at the end of 5 min of hydrolysis. High-fructose content of the syrup was demonstrated by thin layer chromatography.
Keywords: Inulinase; Cryogel; Concanavalin A; Fructose; Lectin affinity chromatography

Selective Production of 2,3-Butanediol and Acetoin by a Newly Isolated Bacterium Klebsiella oxytoca M1 by Sukhyeong Cho; Kyung Duk Kim; Jae-Hyung Ahn; Jinwon Lee; Seon-Won Kim; Youngsoon Um (1922-1933).
A newly isolated bacterium, designated as Klebsiella oxytoca M1, produced 2,3-butanediol (2,3-BDO) or acetoin selectively as a major product depending on temperature in a defined medium. K. oxytoca M1 produced 2,3-BDO mainly (0.32~0.34 g/g glucose) at 30 °C while acetoin was a major product (0.32~0.38 g/g glucose) at 37 °C. To investigate factors affecting product profiles according to temperature, the expression level of acetoin reductase (AR) that catalyzes the conversion of acetoin to 2,3-BDO was analyzed using crude protein extracted from K. oxytoca M1 grown at 30 and 37 °C. The AR expression at 37 °C was 12.8-fold lower than that at 30 °C at the stationary phase and reverse transcription PCR (RT-PCR) analysis of the budC (encoding AR) was also in agreement with the AR expression results. When AR was overexpressed using K. oxytoca M1 harboring pUC18CM-budC, 2,3-BDO became a major product at 37 °C, indicating that the AR expression level was a key factor determining the major product of K. oxytoca M1 at 37 °C. The results in this study demonstrate the feasibility of using K. oxytoca M1 for the production of not only 2,3-BDO but also acetoin as a major product.
Keywords: Klebsiella oxytoca ; 2,3-Butanediol; Acetoin

Synergistic Antibacterial and Antibiofilm Effect Between (+)-Medioresinol and Antibiotics In Vitro by Ji Hong Hwang; Hyemin Choi; In-sok Hwang; A. Ryun Kim; Eun-Rhan Woo; Dong Gun Lee (1934-1941).
In this study, antibacterial effects of (+)-Medioresinol isolated from stem bark of Sambucus williamsii and its synergistic activities in combination with antibiotics such as ampicillin, cefotaxime, and chloramphenicol were tested by antibacterial susceptibility testing and checkerboard assay. (+)-Medioresinol possessed antibacterial effects against antibiotics-susceptible- or antibiotics-resistant strains. Most of combinations between (+)-Medioresinol and each antibiotic showed synergistic interaction (fractional inhibitory concentration index ≤0.5) against bacterial strains including antibiotics-resistant Pseudomonas aeruginosa. Furthermore, the antibiofilm effect of (+)-Medioresinol alone or in combination with each antibiotic was investigated. The results indicated that not only (+)-Medioresinol but also its combination with each antibiotic had antibiofilm activities. It concludes that (+)-Medioresinol has potential as a therapeutic agent and adjuvant for treatment of bacterial infection.
Keywords: (+)-Medioresinol; Antibacterial activity; Synergistic effect; Antibiofilm effect; Antibiotic resistance

Steam-exploded corn stalk biomass was used as the substrate for succinic acid production via lignocellulose enzymatic hydrolysis and fermentation. Succinic acid fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). For the washed steam-exploded corn stalk at 30 % substrate concentration, i.e., 30 % water-insoluble solids (WIS), enzymatic hydrolysis yielded 97.5 g/l glucose solution and a cellulose conversion of 73.6 %, thus a high succinic acid level up to 38.6 g/l. With the unwashed steam-exploded corn stalk, though a cellulose conversion of 71.2 % was obtained in hydrolysis at 30 % solid concentration (27.9 % WIS), its hydrolysate did not ferment at all, and the hydrolysate of 25 % solid loading containing 3.8 g/l acetic acid and 168.2 mg/l furfural exerted a strong inhibition on succinic acid production.
Keywords: Succinic acid; Escherichia coli ; High solid concentration; Enzymatic hydrolysis; Fermentation

β-d-Xylosidase from Geobacillus thermoleovorans IT-08: Biochemical Characterization and Bioinformatics of the Enzyme by Anak Agung Istri Ratnadewi; Muchzainal Fanani; Sari Dewi Kurniasih; Makiko Sakka; Eddy Bagus Wasito; Kazuo Sakka; Zeily Nurachman; Ni Nyoman Tri Puspaningsih (1950-1964).
The gene encoding a thermostable β-d-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6–8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s−1 mM−1 on p-nitrophenyl-β-d-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.
Keywords: CBM36; Geobacillus thermoleovorans ; IT-08; GH 43; Xylanase; β-d-xylosidase

Enantioconvergent Biohydrolysis of Racemic Styrene Oxide to R-phenyl-1, 2-ethanediol by a Newly Isolated Filamentous Fungus Aspergillus tubingensis TF1 by Aparajita Duarah; Amrit Goswami; Tarun C. Bora; Madhumita Talukdar; Binod K. Gogoi (1965-1973).
An effort was made to isolate biocatalysts hydrolyzing epoxides from various ecological niches of northeast India, a biodiversity hot spot zone of the world and screened for epoxide hydrolase activity to convert different racemic epoxides to the corresponding 1, 2-vicinal diols. Screening of a total of 450 microorganisms isolated was carried out using NBP colorimetric assay. One of the strains TF1, after internal transcribed spacer sequence analysis, identified as Aspergillus tubingensis, showed promising enantioconvergent epoxide hydrolase activity. The hydrolysis of unsubstituted styrene oxide (1) occurred to give 97 % ee of R-(−)-1-phenylethane-1, 2-diol (6) with more than 99 % conversion within 45 min incubation. It is shown to be a cheap and practical biocatalyst for one step asymmetric synthesis of chiral R-diol. The other representative substrates (2–5), although underwent hydrolysis with more than 99 % conversion beyond 15 h, exhibited poor enantioselectivity.
Keywords: Epoxides; Epoxide hydrolase; NBP colorimetric assay; R-(−)-1-phenyl-ethane 1, 2-diol; Aspergillus ; Enantioconvergent

Enantioselective Hydrolysis of dl-Menthyl Benzoate by Cell-Free Extract of Newly Isolated Acinetobacter sp. ECU2040 by Minh-Thu Ngo-Thi; Jin-Gang Yin; Jiang Pan; Gao-Wei Zheng; Jian-He Xu (1974-1981).
Production of l-menthol by bioprocesses attracts increasing attention nowadays. Herein, we attempted to develop a bioresolution process for production of l-menthol through enantioselective hydrolysis of dl-menthyl benzoate using a newly isolated bacterium from soil samples. Among 129 active soil isolates screened rapidly by thin-layer chromatography, an outstanding bacterial strain numbered ECU2040, which was subsequently identified as Acinetobacter species, was finally selected as our target enzyme producer due to its highest activity and the best enantioselectivity toward l-substrate as confirmed by chiral gas chromatography. The catalytic performance of the cell-free extract from Acinetobacter sp. ECU2040 was preliminarily examined, indicating that its optimal pH and temperature for the reaction were 7.5 and 37 °C, respectively. Under the optimal conditions, the enzymatic reaction was performed on a 1-L scale, affording l-menthol in 48 % yield and 71 % ee.
Keywords: Acinetobacter ; Biocatalysis; Enzymatic hydrolysis; l-Menthol; dl-Menthyl benzoate

Autohydrolysis of Tropical Agricultural Residues by Compressed Liquid Hot Water Pretreatment by Saksit Imman; Jantima Arnthong; Vorakan Burapatana; Navadol Laosiripojana; Verawat Champreda (1982-1995).
Pretreatment is an essential step in biorefineries for improving digestibility of recalcitrant agricultural feedstocks prior to enzymatic hydrolysis to composite sugars, which can be further converted to fuels and chemicals. In this study, autohydrolysis by compressed liquid hot water (LHW) pretreatment of various tropical agricultural residues including sugarcane bagasse (BG), rice straw (RS), corn stover (CS), and empty palm fruit bunch (EPFB) was investigated. It was found that LHW pretreatment at 200 °C for 5–20 min resulted in high levels of hemicellulose solubilization into the liquid phase and marked improvement on enzymatic digestibility of the solid cellulose-enriched residues. The maximal yields of glucose and pentose were 409.8–482.7 mg/g and 81.1–174.0 mg/g of pretreated substrates, respectively. Comparative analysis based on severity factor showed varying susceptibility of biomass to LHW in the order of BG> RS> CS> EPFB. Structural analysis revealed surface modification of the pretreated biomass along with an increase in crystallinity index. Overall, 75.7–82.3 % yield of glucose and 27.4–42.4 % yield of pentose from the dried native biomass was recovered in the pretreated solid residues, while 18.3–29.7 % of pentoses were recovered in the liquid phase with dehydration by-product concentration under the threshold for ethanologens. The results suggest the potential of LHW as an efficient pretreatment strategy for implementation in biorefineries operated using various seasonal agricultural feedstocks.
Keywords: Biorefinery; Enzymatic hydrolysis; Lignocelluloses; Liquid hot water; Pretreatment

Molecular Dynamic and Docking Interaction Study of Heterodera glycines Serine Proteinase with Vigna mungo Proteinase Inhibitor by C. V. S. Siva Prasad; Saurabh Gupta; Alex Gaponenko; Murlidhar Tiwari (1996-2008).
Many plants do produce various defense proteins like proteinase inhibitors (PIs) to protect them against various pests. PIs function as pseudosubstrates of digestive proteinase, which inhibits proteolysis in pests and leads to amino acid deficiency-based mortality. This work reports the structural interaction studies of serine proteinase of Heterodera glycines (SPHG) with Vigna mungo proteinase inhibitor (VMPI). 3D protein structure modeling, validation of SPHG and VMPI, and their putative protein–protein binding sites were predicted. Protein–protein docking followed by molecular dynamic simulation was performed to find the reliable confirmation of SPHG–VMPI complex. Trajectory analysis of each successive conformation concludes better interaction of first loop in comparison with second loop. Lysine residues of first loop were actively participating in complex formation. Overall, this study discloses the structural aspects and interaction mechanisms of VMPI with SPHG, and it would be helpful in the development of pest-resistant genetically modified crops.
Keywords: Serine proteinase; Proteinase inhibitor; Heterodera glycines ; Vigna mungo ; Protein–protein interaction; Molecular dynamic simulation

The kinetic characteristics of two Rhizopus oryzae exo-polygalacturonases acting on galacturonic acid oligomers (GalpA) were determined using isothermal titration calorimetry (ITC). RPG15 hydrolyzing (GalpA)2 demonstrated a K m of 55 μM and k cat of 10.3 s−1 while RPG16 was shown to have greater affinity for (GalpA)2 with a K m of 16 μM, but lesser catalytic activity with a k cat of 3.9 s−1. Both enzymes were inhibited by the product, galacturonic acid, with app K i values of 886 and 501 μM for RPG15 and RPG16, respectively. RPG15 exhibited greater affinity for (GalpA)3 with a K m of 9.2 μM and a similar k cat at 10.7 s−1 relative to (GalpA)2. Catalytic constants for RPG16 hydrolyzing (GalpA)3 could not be determined; however, single-injection ITC assays suggest a distinct preference and catalytic rate for (GalpA)3 relative to (GalpA)2. Thermodynamic parameters of a series of galacturonic acid oligomers binding to RPG15 were determined and exhibited some distinct differences from RPG16 binding thermodynamics, providing potential clues to the differing kinetic characteristics of the two exo-polygalacturonase enzymes.
Keywords: Exo-polygalacturonase; Isothermal titration calorimetry; Oligogalacturonates; Kinetics; Substrate binding

The increased additive amount of adjuncts in the raw materials of Chinese beer requires the usage of protease to release more water-soluble proteins. Here, a metallo-neutral protease suited for brewing industry was purified from Bacillus amyloliquefaciens SYB-001. A 5.6-fold purification of the neutral protease was achieved with a 4-step procedure including ammonium sulfate precipitation, ion-exchange, hydrophobic interaction, and gel-filtration chromatography. The molecular mass of the enzyme was estimated to be 36.8 kDa. The protease was active and stable at a wide range of pH from 6.0–10.0 with an optimum at pH 7.0. The highest activity of the purified enzyme was found at 50 °C. The existence of manganese ion would specifically enhance the protease activity. Comparing with other commercial neutral proteases in China, adding the new neutral protease during mashing process would release more amino acids from wort such as aspartic acid, arginine, methione, and histidine, resulting in a better amino acid profile in wort. Moreover, the wort processed with the new neutral protease had a higher α-amino nitrogen concentration, which would ensure a vigorous yeast growth and better flavor. The study of the enzyme could lay a foundation for its industrial application and further research.
Keywords: Beer brewing; Adjuncts; Neutral protease; Mashing; Bacillus amyloliquefaciens

A Protein from Aloe vera that Inhibits the Cleavage of Human Fibrin(ogen) by Plasmin by Jaruwan Siritapetawee; Punchapat Sojikul; Siriwat Soontaranon; Wanwisa Limphirat; Sompong Thammasirirak (2034-2045).
A protease inhibitor protein with the molecular mass of 11,804.931 Da (analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) was isolated from Aloe vera leaf gel and designated as AVPI-12. The isoelectric point of the protein is about 7.43. The first ten amino acid sequence from the N-terminal was found to be R–D–W–A–E–P–N–D–G–Y, which did not match other protease inhibitors in database searches and other publications, indicating AVPI-12 is a novel protease inhibitor. The band protein of AVPI-12 migrated further on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than reducing SDS-PAGE. This result indicated that the molecule of AVPI-12 did not contain interchain disulfide bonds, but appeared to have intrachain disulfide bonds instead. AVPI-12 strongly resisted digestion by the serine proteases human plasmin and bovine trypsin. The protein could protect the γ-subunit of human fibrinogen from plasmin and trypsin digestion, similar to the natural plasma serine protease inhibitor α2-macroglobulin. The protein also could protect the γ-subunit of fibrinogen from the cysteine protease papain. AVPI-12 also exhibited dose-dependent inhibition of the fibrinogenolytic activity of plasmin, similar to α2-macroglobulin. The fibrinolytic inhibitory activity of AVPI-12 and the small-angle X-ray scattering showed that the protein could protect human fibrin clot from complete degradation by plasmin. The inhibition of the fibrinogenolytic and fibrinolytic activities of plasmin by AVPI-12 suggests that the inhibitor has potential for use in antifibrinolytic treatment.
Keywords: Aloe vera ; Fibrinogen; Fibrin; Protease inhibitor; Fibrinolytic; Fibrinogenolytic