This document provides instructions for isolating and characterizing two alginate lyase isozymes (AkAly28 and AkAly33) from the common sea hare Aplysia kurodai. Both enzymes are endolytic polymannuronate lyases that degrade alginate and preferentially produce unsaturated tri- and disaccharides. While they have similar optimal pH and temperature, they differ in their NaCl requirement and activity toward oligosaccharide substrates. AkAly28 requires higher NaCl concentrations and hardly degrades oligosaccharides smaller than tetrasaccharides, whereas AkAly33 is more active without NaCl and can degrade disaccharides. Analysis indicates both enzymes belong to the polysaccharide lyase family
Biochemical components of three marine macroalgae (Padina pavonica, Ulva lact...Innspub Net
Green macroalgae Ulva lactuca, brown macroalgae Taonia atomaria and Padina pavonica are spread in the Turkish Levantine Sea. There is limited information about antioxidant activities and fatty acid composition of these species from Levantine Sea. In this study was to determine and compare antioxidant activities, vitamin and fatty acid (FA) composition of U. lactuca, T. atomaria and P. pavonica. The analysis was made with HPLC and GC device. g. Then, the results were analyzed using SPSS software. The results showed; palmitic acid (C16:0) as the most abundant saturate fatty acid (21-41%). The green algae was rich palmitic acid (C16:0) (41.68%). Monounsaturated fatty acids (MUFAs) were major components (39.81–42.89%). The total MUFA content for U. lactuca was 40.63%, P. pavonica 42.89% and for T. atomaria 38.81%. Oleic acid (C18:1 n-9) was the most abundant MUFA in all the species analyzed. Eicosapentaenoic acid ( C20:5 n-3) and arahidonic acid (C20:4 n-6) were found in significant levels in T. atomaria. P. pavonica and T. atomaria showed similar amounts of C18 and C20 PUFAs contents. In T. atomaria eicosopentaenoic acid (EPA, C20:5n3) accounted 4.78% of total fatty acids. PUFA/SFA ratio in T. atomaria was 1.10%, U. lactuca; 0.26% and for P. pavonica 0.68%.The total phenolic contents ranged from 0.96 to 2.22 mg gallic acid equivalents per 1 g of dry macroalgae material. Phenolic content of the water extract of T. atomaria (2.22 mg GAE /g) was higher than that of the water extract of P. pavonica and U. lactuca. It has been thought that the amount of α-tocoferol was higher than the other lipophilic vitamins in all the three species tested. In Conclusion; these species can be used as food and in food industry.
Biochemical components of three marine macroalgae (Padina pavonica, Ulva lact...Innspub Net
Green macroalgae Ulva lactuca, brown macroalgae Taonia atomaria and Padina pavonica are spread in the Turkish Levantine Sea. There is limited information about antioxidant activities and fatty acid composition of these species from Levantine Sea. In this study was to determine and compare antioxidant activities, vitamin and fatty acid (FA) composition of U. lactuca, T. atomaria and P. pavonica. The analysis was made with HPLC and GC device. g. Then, the results were analyzed using SPSS software. The results showed; palmitic acid (C16:0) as the most abundant saturate fatty acid (21-41%). The green algae was rich palmitic acid (C16:0) (41.68%). Monounsaturated fatty acids (MUFAs) were major components (39.81–42.89%). The total MUFA content for U. lactuca was 40.63%, P. pavonica 42.89% and for T. atomaria 38.81%. Oleic acid (C18:1 n-9) was the most abundant MUFA in all the species analyzed. Eicosapentaenoic acid ( C20:5 n-3) and arahidonic acid (C20:4 n-6) were found in significant levels in T. atomaria. P. pavonica and T. atomaria showed similar amounts of C18 and C20 PUFAs contents. In T. atomaria eicosopentaenoic acid (EPA, C20:5n3) accounted 4.78% of total fatty acids. PUFA/SFA ratio in T. atomaria was 1.10%, U. lactuca; 0.26% and for P. pavonica 0.68%.The total phenolic contents ranged from 0.96 to 2.22 mg gallic acid equivalents per 1 g of dry macroalgae material. Phenolic content of the water extract of T. atomaria (2.22 mg GAE /g) was higher than that of the water extract of P. pavonica and U. lactuca. It has been thought that the amount of α-tocoferol was higher than the other lipophilic vitamins in all the three species tested. In Conclusion; these species can be used as food and in food industry.
International Journal of Engineering and Science Invention (IJESI)inventionjournals
International Journal of Engineering and Science Invention (IJESI) is an international journal intended for professionals and researchers in all fields of computer science and electronics. IJESI publishes research articles and reviews within the whole field Engineering Science and Technology, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
In this ppt the students will be able to know about different inclusions of plant cells and correlate their constituents. There are different types of metabolites produce in the plant which are not useful for them but are having great medicinal property for human being.
Portion covered:
1. Cell Inclusions
2. Reserve food of plants
3. Inorganic Materials
4. Secretory Products
5. Excretory Products.
Phytochemical and Biological Evaluation of Cassia tora, L. Seedsiosrjce
In the present study the total and the defatted alcoholic extracts of seeds of Cassia tora Linn.
(Leguminosae) were screened for hepatoprotective activity using adult Wister albino rats (120-170 g) as the
experimental animals. Hepatic injury caused by carbon tetra chloride, was analyzed through estimation of AST,
ALT, ALB and platelets in blood samples taken from the veins of orbital plexus of each animal as well as the
histopathological examination of the liver.The effects of the extracts were comparable with standardhepatoprotective drug Silymarin. On the other hand GC-MS analysis was performed on the fatty acid
composition of the lipoidal fraction for the seeds. The separated fatty acids were converted to their methyl ester
and then subjected to the analysis.
1 functional characterization of chitin and chitosanDuy Thanh Tran
Chitin and its deacetylated derivative chitosan are natural polymers composed of randomly distributed -(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitin is insoluble in aqueous
media while chitosan is soluble in acidic conditions due to the free protonable amino groups present in the D-glucosamine
units. Due to their natural origin, both chitin and chitosan can not be defined as a unique chemical structure but as a family of polymers which present a high variability in their chemical and physical properties. This variability is related not
only to the origin of the samples but also to their method of preparation. Chitin and chitosan are used in fields as different
as food, biomedicine and agriculture, among others. The success of chitin and chitosan in each of these specific applications is directly related to deep research into their physicochemical properties. In recent years, several reviews covering
different aspects of the applications of chitin and chitosan have been published. However, these reviews have not taken
into account the key role of the physicochemical properties of chitin and chitosan in their possible applications. The aim
of this review is to highlight the relationship between the physicochemical properties of the polymers and their behaviour.
A functional characterization of chitin and chitosan regarding some biological properties and some specific applications
(drug delivery, tissue engineering, functional food, food preservative, biocatalyst immobilization, wastewater treatment,
molecular imprinting and metal nanocomposites) is presented. The molecular mechanism of the biological properties such
as biocompatibility, mucoadhesion, permeation enhancing effect, anticholesterolemic, and antimicrobial has been updated.
Assimilation of ammonium ions is the ultimate aim of nitrogen metabolism in plants. this is the source of nitrogen for various organic compounds of structural and functional importance for the living world
STUDIES ON EXTRACTION METHODS OF CHITIN FROM CRAB SHELL AND INVESTIGATION OF ...IAEME Publication
This paper describes the most common methods for recovery of chitin from crab shell. Deproteinization, demineralization and deacetylation are the main processes for the extraction of chitin and chitosan. The mechanical properties were investigated to recognize their mechanical applications. Chitin is the most widespread biopolymer in nature, after cellulose. It has great economic value because of their biological activities and their industrial and biomedical applications. Chitin can be extracted from three sources, namely crustaceans, insects and microorganisms. However, the main commercial sources are shells of shrimps, crabs, lobsters and krill that are supplied in large quantities by the shellfish processing industries. Extraction of chitin involves two steps, demineralization and deproteinisation, which can be processed by two methods, chemical or biological. Acids and bases are required for chemical method, while the biological method involves microorganisms. The mechanical properties of isolated crab chitin are highly susceptible to the effects of hydration. Philippine blue swimming crab were used for the extraction of chitin. The extracted chitin was used to form polymer films at different conditions. Polymer films were also formed from commercially acquired chitin. It was observed that the films prepared at different conditions have greater ultimate tensile strengths as compared to the commercially-available films..The Chitin discussed in the present study is analyzed mechanically. Thus ensuring the extracted Chitin and Chitosan could be considered for further applications. This study therefore, intends to extract and investigate the mechanical performance of chitin from crab shell.
Debunking the myths of organizational change managementaccenture
Over the past 15 years, we have studied 250 major change initiatives at more than 150 organizations, including dozens of Fortune Global 500 corporations. We have collected data from more than 850,000 employees, from front-line staffers through leadership at all levels. The resulting analysis of that dataset—representing the cumulative wisdom of experienced change travelers—has dispelled many long-held myths about organization change. It has brought new insights to help leaders and the workforce of the future embark on insight-driven change. Learn more at http://www.accenture.com/MythsofChange
International Journal of Engineering and Science Invention (IJESI)inventionjournals
International Journal of Engineering and Science Invention (IJESI) is an international journal intended for professionals and researchers in all fields of computer science and electronics. IJESI publishes research articles and reviews within the whole field Engineering Science and Technology, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
In this ppt the students will be able to know about different inclusions of plant cells and correlate their constituents. There are different types of metabolites produce in the plant which are not useful for them but are having great medicinal property for human being.
Portion covered:
1. Cell Inclusions
2. Reserve food of plants
3. Inorganic Materials
4. Secretory Products
5. Excretory Products.
Phytochemical and Biological Evaluation of Cassia tora, L. Seedsiosrjce
In the present study the total and the defatted alcoholic extracts of seeds of Cassia tora Linn.
(Leguminosae) were screened for hepatoprotective activity using adult Wister albino rats (120-170 g) as the
experimental animals. Hepatic injury caused by carbon tetra chloride, was analyzed through estimation of AST,
ALT, ALB and platelets in blood samples taken from the veins of orbital plexus of each animal as well as the
histopathological examination of the liver.The effects of the extracts were comparable with standardhepatoprotective drug Silymarin. On the other hand GC-MS analysis was performed on the fatty acid
composition of the lipoidal fraction for the seeds. The separated fatty acids were converted to their methyl ester
and then subjected to the analysis.
1 functional characterization of chitin and chitosanDuy Thanh Tran
Chitin and its deacetylated derivative chitosan are natural polymers composed of randomly distributed -(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitin is insoluble in aqueous
media while chitosan is soluble in acidic conditions due to the free protonable amino groups present in the D-glucosamine
units. Due to their natural origin, both chitin and chitosan can not be defined as a unique chemical structure but as a family of polymers which present a high variability in their chemical and physical properties. This variability is related not
only to the origin of the samples but also to their method of preparation. Chitin and chitosan are used in fields as different
as food, biomedicine and agriculture, among others. The success of chitin and chitosan in each of these specific applications is directly related to deep research into their physicochemical properties. In recent years, several reviews covering
different aspects of the applications of chitin and chitosan have been published. However, these reviews have not taken
into account the key role of the physicochemical properties of chitin and chitosan in their possible applications. The aim
of this review is to highlight the relationship between the physicochemical properties of the polymers and their behaviour.
A functional characterization of chitin and chitosan regarding some biological properties and some specific applications
(drug delivery, tissue engineering, functional food, food preservative, biocatalyst immobilization, wastewater treatment,
molecular imprinting and metal nanocomposites) is presented. The molecular mechanism of the biological properties such
as biocompatibility, mucoadhesion, permeation enhancing effect, anticholesterolemic, and antimicrobial has been updated.
Assimilation of ammonium ions is the ultimate aim of nitrogen metabolism in plants. this is the source of nitrogen for various organic compounds of structural and functional importance for the living world
STUDIES ON EXTRACTION METHODS OF CHITIN FROM CRAB SHELL AND INVESTIGATION OF ...IAEME Publication
This paper describes the most common methods for recovery of chitin from crab shell. Deproteinization, demineralization and deacetylation are the main processes for the extraction of chitin and chitosan. The mechanical properties were investigated to recognize their mechanical applications. Chitin is the most widespread biopolymer in nature, after cellulose. It has great economic value because of their biological activities and their industrial and biomedical applications. Chitin can be extracted from three sources, namely crustaceans, insects and microorganisms. However, the main commercial sources are shells of shrimps, crabs, lobsters and krill that are supplied in large quantities by the shellfish processing industries. Extraction of chitin involves two steps, demineralization and deproteinisation, which can be processed by two methods, chemical or biological. Acids and bases are required for chemical method, while the biological method involves microorganisms. The mechanical properties of isolated crab chitin are highly susceptible to the effects of hydration. Philippine blue swimming crab were used for the extraction of chitin. The extracted chitin was used to form polymer films at different conditions. Polymer films were also formed from commercially acquired chitin. It was observed that the films prepared at different conditions have greater ultimate tensile strengths as compared to the commercially-available films..The Chitin discussed in the present study is analyzed mechanically. Thus ensuring the extracted Chitin and Chitosan could be considered for further applications. This study therefore, intends to extract and investigate the mechanical performance of chitin from crab shell.
Debunking the myths of organizational change managementaccenture
Over the past 15 years, we have studied 250 major change initiatives at more than 150 organizations, including dozens of Fortune Global 500 corporations. We have collected data from more than 850,000 employees, from front-line staffers through leadership at all levels. The resulting analysis of that dataset—representing the cumulative wisdom of experienced change travelers—has dispelled many long-held myths about organization change. It has brought new insights to help leaders and the workforce of the future embark on insight-driven change. Learn more at http://www.accenture.com/MythsofChange
BigWeatherGear Group and Corporate Services Brochure 2013Kristin Matson
Thank you for your interest in Bigweathergear.com Group Sales. We have been in business for over 20 years selling high quality outdoor gear. We specialize in Government, Corporate, and Group volume orders. Our staff of experts can help you fill your gear needs whether they are basic or very specific. We have custom logo applications available on most of the products we carry.
Lightning Talk #9: How UX and Data Storytelling Can Shape Policy by Mika Aldabaux singapore
How can we take UX and Data Storytelling out of the tech context and use them to change the way government behaves?
Showcasing the truth is the highest goal of data storytelling. Because the design of a chart can affect the interpretation of data in a major way, one must wield visual tools with care and deliberation. Using quantitative facts to evoke an emotional response is best achieved with the combination of UX and data storytelling.
Content personalisation is becoming more prevalent. A site, it's content and/or it's products, change dynamically according to the specific needs of the user. SEO needs to ensure we do not fall behind of this trend.
Succession “Losers”: What Happens to Executives Passed Over for the CEO Job?
By David F. Larcker, Stephen A. Miles, and Brian Tayan
Stanford Closer Look Series
Overview:
Shareholders pay considerable attention to the choice of executive selected as the new CEO whenever a change in leadership takes place. However, without an inside look at the leading candidates to assume the CEO role, it is difficult for shareholders to tell whether the board has made the correct choice. In this Closer Look, we examine CEO succession events among the largest 100 companies over a ten-year period to determine what happens to the executives who were not selected (i.e., the “succession losers”) and how they perform relative to those who were selected (the “succession winners”).
We ask:
• Are the executives selected for the CEO role really better than those passed over?
• What are the implications for understanding the labor market for executive talent?
• Are differences in performance due to operating conditions or quality of available talent?
• Are boards better at identifying CEO talent than other research generally suggests?
The impact of innovation on travel and tourism industries (World Travel Marke...Brian Solis
From the impact of Pokemon Go on Silicon Valley to artificial intelligence, futurist Brian Solis talks to Mathew Parsons of World Travel Market about the future of travel, tourism and hospitality.
We’re all trying to find that idea or spark that will turn a good project into a great project. Creativity plays a huge role in the outcome of our work. Harnessing the power of collaboration and open source, we can make great strides towards excellence. Not just for designers, this talk can be applicable to many different roles – even development. In this talk, Seasoned Creative Director Sara Cannon is going to share some secrets about creative methodology, collaboration, and the strong role that open source can play in our work.
TDD is the elengant way of designing software. People scares from it so much, because software design is hard and it requires discipline. In this talk, I tried to describe what TDD is from software design perspective.
The Great State of Design with CSS Grid Layout and FriendsStacy Kvernmo
For far too long we've been forced to reuse layout patterns that have worked in the past, creating a web full of sites that all look the same. Narrow timelines, browser support restrictions and lack of a true grid system have led us to create work that is "good enough".
I've spent years exploring how we can make the web a more unique space. With some of the newer CSS techniques available, we can start to make more creative designs. CSS Grid Layout is on the horizon and will play a major role in the design of our sites. Finally having a true, 2 dimensional grid will give our layouts much more flexibility and it is on us to explore the possibilities.
This talk was presented at CSS Day 2016.
The Six Highest Performing B2B Blog Post FormatsBarry Feldman
If your B2B blogging goals include earning social media shares and backlinks to boost your search rankings, this infographic lists the size best approaches.
Protective Effects of Alpha Lipoic Acid (α -LA) Against Lead Neuro-Toxicity i...inventionjournals
Aim of the work: The present study was conducted to elucidate the possible protective effect of alpha lipoic acid (α-LA) against the deleterious effect perturbation induced in rat brain exposed to lead acetate. Methods: 32 Wistar male rats (weighing 130 ± 10 g) were divided into four groups (n=8): (1) normal control group (C); (2) Initiation group (Pb as lead acetate 20 mg/kg.b.wt, i.p. for 2 wks); (3) treatment group (α-LA 20 mg/kg.b.wt, i.p. for 3 wks); (4) post-initiation treatment group (Pb for 2 wks then followed by α-LA for 3 wks). Levels of monoamines (norepinephrine NE and dopamine DA), the level of Ache activity and finally adenosine triphosphate (ATP), were estimated in the hippocampus and cerebral cortex, in addition, a Morris water maze and the histological study were performed after completion of the experiments. Results: The results of the present work demonstrated that Pb inhibited neurotransmitters releases and decrease the level of Ache activity, as well as it inhibited energy production ATP. Pb impaired performance on Morris Water Maze of rats and histological degeneration. However, treatment with α-LA significantly attenuated the behavioral impairment and biochemical parameters in rat treated with Pb. And amelioration of histological changes. Conclusion: As a conclusion, treatment with α-LA can improve the Pb-induced toxicity via antioxidant activity.
Protective Effects of Alpha Lipoic Acid (Α-LA) Against Lead Neuro-Toxicity in...inventionjournals
Aim of the work: The present study was conducted to elucidate the possible protective effect of alpha lipoic acid (α-LA) against the deleterious effect perturbation induced in rat brain exposed to lead acetate. Methods: 32 Wistar male rats (weighing 130 ± 10 g) were divided into four groups (n=8): (1) normal control group (C); (2) Initiation group (Pb as lead acetate 20 mg/kg.b.wt, i.p. for 2 wks); (3) treatment group (α-LA 20 mg/kg.b.wt, i.p. for 3 wks); (4) post-initiation treatment group (Pb for 2 wks then followed by α-LA for 3 wks). Levels of monoamines (norepinephrine NE and dopamine DA), the level of Ache activity and finally adenosine triphosphate (ATP), were estimated in the hippocampus and cerebral cortex, in addition, a Morris water maze and the histological study were performed after completion of the experiments. Results: The results of the present work demonstrated that Pb inhibited neurotransmitters releases and decrease the level of Ache activity, as well as it inhibited energy production ATP. Pb impaired performance on Morris Water Maze of rats and histological degeneration. However, treatment with α-LA significantly attenuated the behavioral impairment and biochemical parameters in rat treated with Pb. And amelioration of histological changes. Conclusion: As a conclusion, treatment with α-LA can improve the Pb-induced toxicity via antioxidant activity.
Biochemical evaluation of antioxidant activity in extracts and polysaccharide...GJESM Publication
In the present study ethanol and water extracts of 15 seaweeds, Dictyota dichotoma var. velutricata,
Dictyota indica, Iyengaria stellata, Padina pavonia, Sargassum swartzii, Sargassum variegatum, Stoechospermum marginatum, Stokeyia indica, Jolyna laminarioides, Caulerpa taxifolia, Halimeda tuna, Ulva fasciata, Ulva lactuca, Solieria robusta, and elanothamnus afaqhusainii, were evaluated for their antioxidant potential by ABTS, superoxide and total antioxidant capacity (TAC) assays. The activity was concentration dependent and the variation in antioxidant potential was also observed by different assays in both extracts. Ethanol extract ofD. dichotoma var. velutricata,D. indica and S. marginatum demonstrated highest activity by TAC assay. The antioxidant potential in organic solvent fractions of
seaweeds namely P. pavonia, S. swartzii, S. marginatum andM. afaqhusainii was also determined and chloroform fraction of all the four seaweeds showed highest activity by superoxide assay. Antioxidant activity of extracted fractions of polysaccharides from S. indica, C. taxifolia and D. dichotoma var. velutricata was also evaluated by superoxide method. Polysaccharide fractions of S. indica obtained from HCl (at 70 0C and room temperature) and water extract demonstrated highest activity respectively. All the polysaccharide fractions of C. taxifolia showed excellent activity except CaClF70 °C. Polysaccharide fractions of D. dichotoma var. velutricata also exhibited very good activity.
Objectives: The present study aisms to determine the effect of salt stress on the total lipid composition for two varieties of banana (Musa acuminata) viz., great dwarf and small dwarf variety. The presence of different concentrations viz.,. triglycerides and diglycerides did not influenced the increasing salt concentration in the medium. However, monoglycerides and free fatty acids were more affected by the effect of salinity.
Regarding the large dwarf variety, the absence of triglycerides noted, especially in the stressed plants and also in the control plants. In the light of our results we saw that the membrane lipids in the vast dwarf were less affected by salinity compared to the small dwarf
Original articleGamma radiation effect on quality changes .docxgerardkortney
Original article
Gamma radiation effect on quality changes in vacuum-packed squid
(Illex argentinus) mantle rings during refrigerated (4–5 �C) storage
Alejandra Tomac* & Marı́a Isabel Yeannes
Grupo de Investigación Preservación y Calidad de Alimentos, Facultad de Ingenierı́a, Universidad Nacional de Mar del Plata, Consejo Nacional de
Investigaciones Cientı́ficas y Técnicas (CONICET), Juan B. Justo 4302, B7608FDQ, Mar del Plata, Argentina
(Received 14 October 2011; Accepted in revised form 21 February 2012)
Summary The effect of gamma radiation (0, 1.8, 3.3 and 5.8 kGy) on microbiological, chemical and colour
characteristics of vacuum-packed squid (Illex argentinus) mantle rings was studied. Total viable counts;
psychrotrophic bacteria counts, Escherichia Coli, Staphylococcus aureus and Clostridium perfringens; total
volatile basic nitrogen (TVBN) and colour differenceDE�ab were analysed during 29 days of storage at
4–5 �C. Higher doses of gamma radiation significantly reduced Total Viable, phychrotrophic counts and
TVBN production (P < 0.05) in a dose-dependent way, delaying squid spoilage. Colour difference of non-
irradiated samples with respect to first day significantly increased while it was constant in radiated samples
during 22 days (P < 0.05). Independently from the dose, radiation avoided colour changes of squid rings.
Gamma irradiation was effective in delaying deterioration reactions, improving microbiological, chemical
and colour quality of vacuum-packed squid rings stored at 4–5 �C.
Keywords Colour, Illex argentinus, ionising radiation, microbial activity, quality, refrigeration.
Introduction
Food irradiation has been widely studied as a food
preservation method for the last five decades. It has
certainly proved its toxicological safety as well as it
efficiency in shelf life extension by decreasing microbial
counts. At present, more than 60 countries have
approved irradiation of one or more foods (WHO,
1994, 1999, Diehl, 2002; Sommers & Fan, 2006).
Nutritional adequacy of irradiated food has also been
largely investigated. Irradiation can induce changes in
proteins, lipids, carbohydrates and vitamins due mainly
to free radicals produced by water radiolysis. However,
no significant losses of the nutritional quality of lipid,
carbohydrate and protein constituents have been re-
ported at irradiation doses intended for food preservation
(£10 kGy) (Josephson et al., 1978; Kilcast, 1995; Giroux
& Lacroix, 1998; ICGFI, 1999; ADA Report, 2000).
Among lipids, polyunsaturated fatty acids (PUFAs)
are more sensitive to oxidation by free radicals. The
absence of oxygen can minimise this effect, as observed
by Kim et al. (2002) in raw beef, turkey and pork meats.
Erkan & Özden (2007) concluded that irradiation had
only marginal effects on the lipids of fishery products,
including the essential alpha-linolenic acid. Abreu et al.
(2010) found that irradiation doses up to 6 kGy did not
compromise negatively the fatty acid.
The bioactive phytochemicals in Gouania longipetala was determined using GCMS analysis. The
extract was prepared using Soxhlet`s extraction method and concentrated at 35oC in hot air oven. GCMS
analyzes phytochemicals in plant by demonstrating the structures of the chemical compounds in it. The gas
chromatogram showed the presence of eight phytochemicals. The molecular mass of the phytochemicals were
established based on the molecular ion in the mass spectra. Identification of the phytochemicals was based on
comparison with National Institute of Standards and Technology (NIST) database. The identified
phytochemicals with their peak area percentages are 11,14-octadecadienoic acid (1.72%), Hexadecanoic acid
also known as Palmitic acid (19.86%), 9,11-octadecadienoic acid (1.33%), 9,12,15-Octadecatrien-1-ol (2.92%),
9-Octadecenoic acid (56.40%), Ethyl palmitate (9.42%), 17-carboxyheptadec-9-en-1-ylium (1.70%) and
Glutaric acid, isobutyl 2-nitrophenyl ester (6.65%). These identified compounds exhibited the following
bioactivities; inhibition of uric acid, urine acidifiers, amino acid decarboxylase activity, arachidonic acid
inhibitor, oligosaccharide provider, decrease endothelial leukocyte and platelet adhesion . Gouania longipetala
therefore contain active phytochemicals that may be beneficial in pharmacognosy. We recommend further work
to be done on its isolation and synthesis.
Dr. Abhijit Mitra, Associate Professor and former Head, Dept. of Marine Science, University of Calcutta (INDIA) has been active in the sphere of Oceanography since 1985. He obtained his Ph.D as NET qualified scholar in 1994. Since then he joined Calcutta Port Trust and WWF (World Wide Fund), in various capacities to carry out research programmes on environmental science, biodiversity conservation, climate change and carbon sequestration. Presently Dr. Mitra is serving as the advisor of Oceanography Division of Techno India University, Kolkata. He has to his credit about 388 scientific publications in various National and International journals, and 34 books of postgraduate standards. Dr. Mitra has successfully completed about 16 projects on biodiversity loss in fishery sector, coastal pollution, alternative livelihood, climate change and carbon sequestration. Dr. Mitra also visited as faculty member and invited speakers in several foreign Universities of Singapore, Kenya, Oman and USA. In 2008, Dr. Mitra was invited as visiting fellow at University of Massachusetts at Dartmouth, USA to deliver a series of lecture on Climate Change. Dr. Mitra also successfully guided 29 Ph.D students. Presently his domain of expertise includes environmental science, mangrove ecology, sustainable aquaculture, alternative livelihood, climate change and carbon sequestration.
At Taste Of Middle East, we believe that food is not just about satisfying hunger, it's about experiencing different cultures and traditions. Our restaurant concept is based on selecting famous dishes from Iran, Turkey, Afghanistan, and other Arabic countries to give our customers an authentic taste of the Middle East
Roti Bank Hyderabad: A Beacon of Hope and NourishmentRoti Bank
One of the top cities of India, Hyderabad is the capital of Telangana and home to some of the biggest companies. But the other aspect of the city is a huge chunk of population that is even deprived of the food and shelter. There are many people in Hyderabad that are not having access to
Ang Chong Yi Navigating Singaporean Flavors: A Journey from Cultural Heritage...Ang Chong Yi
In the heart of Singapore, where tradition meets modernity, He embarks on a culinary adventure that transcends borders. His mission? Ang Chong Yi Exploring the Cultural Heritage and Identity in Singaporean Cuisine. To explore the rich tapestry of flavours that define Singaporean cuisine while embracing innovative plant-based approaches. Join us as we follow his footsteps through bustling markets, hidden hawker stalls, and vibrant street corners.
1. Instructions for use
Title
Isolation and characterization of two alginate lyase isozymes,
AkAly28 and AkAly33, from the common sea hare Aplysia
kurodai
Author(s)
Rahman, Mohammad Matiur; Inoue, Akira; Tanaka, Hiroyuki;
Ojima, Takao
Citation
Comparative Biochemistry and Physiology Part B:
Biochemistry and Molecular Biology, 157(4): 317-325
Issue Date 2010-12
Doc URL http://hdl.handle.net/2115/44107
Right
Type article (author version)
Additional
Information
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
2. 1
Isolation and characterization of two alginate lyase isozymes, AkAly28 and AkAly33, from the
common sea hare Aplysia kurodai
Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, and Takao Ojima*
Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido
University, Hakodate, Hokkaido, 041-8611, Japan
*Corresponding author. Tel/Fax: +81 138 40 8800.
E-mail address: ojima@fish.hokudai.ac.jp
Keywords: Aplysia kurodai, gastropod, alginate lyase, PL-14, alginate, brown seaweed
3. 2
Abstract
Two alginate lyase isozymes, AkAly28 and AkAly33, with approximate molecular masses of 28
kDa and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare, Aplysia
kurodai. Both of AkAly28 and AkAly33 were regarded as the endolytic polymannuronate (poly(M)) lyase
(EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate producing unsaturated tri- and
disaccharides and rapidly decreased the viscosity of sodium alginate solution in the initial phase of
degradation. Optimal pH and temperature of the two enzymes were similarly observed at pH 6.7 and 40 o
C,
respectively. Temperature that caused a half inactivation of the two enzymes during 20-min incubation was
also similar to each other, i.e., 38 o
C. However, NaCl requirement and activity toward oligosaccharide
substrates of the two enzymes were significantly different from each other. Namely, AkAly28 showed
practically no activity in the absence of NaCl and the maximal activity at NaCl concentrations higher than
0.2 M. While AkAly33 showed ~20% of maximal activity despite the absence of NaCl and the maximal
activity at around 0.1 M NaCl. AkAly28 hardly degraded oligosaccharides smaller than tetrasaccharide,
while AkAly33 could degrade oligosaccharides larger than disaccharide producing disaccharide and 2-keto-
3-deoxy-gluconaldehyde (an open chain form of unsaturated monosaccharide). Analysis of the N-terminal
and internal amino-acid sequences of AkAly28 and AkAly33 indicated that both of the two enzymes
belong to polysaccharide lyase family 14.
4. 3
1. Introduction
Herbivorous marine invertebrates such as sea urchin, abalone, and sea hare feed on seaweeds (Mai
et al., 1995; Takami et al., 1998; Johnston et al., 2005). To obtain carbohydrate nutrients, these
invertebrates digest seaweeds’ structural and storage polysaccharides, e.g., cellulose, alginate, mannan,
starch and laminarin, with appropriate polysaccharide-degrading enzymes in their digestive fluid (Suzuki et
al., 2003; Shimizu et al., 2003; Suzuki et al., 2006; Ootsuka et al., 2006; Nishida et al., 2007; Hata et al.,
2009; Nikapitiya et al., 2009; Kumagai and Ojima, 2009 and 2010; Zahura et al., 2010). The thus produced
oligosaccharides and monosaccharides are assimilated directly by animals themselves or through the
fermentation by intestinal bacteria (Erasmus et al., 1997; Sawabe at al., 2003). Among the seaweeds’
polysaccharides, alginate in brown seaweeds appears to be the most abundant carbohydrate. For example,
the alginate content in the frond of Laminaria sp. is usually more than 20% (w/w) in dry weight while other
polysaccharides are less than 5%. Correspondingly, an alginate-degrading enzyme, i.e., alginate lyase (EC
4.2.2.3), is also abundant in the digestive fluid of abalone and turban shell which are fond of brown
seaweeds (Shimizu et al., 2003 and Muramatsu et al., 1977). Alginate is a heteropolyuronide comprising
1,4-linked β-D-mannuronate (M) and its C5 epimer α-L-guluronate (G). These uronide units are arranged
as homopolymeric G and M blocks, and heteropolymeric MG blocks (Haug et al., 1967; Gacesa P., 1988;
Gacesa P., 1992; Wong et al., 2000). Alginate lyase splits the glycosyl linkages of alginate by the β-
elimination mechanism producing oligosaccharides possessing an unsaturated uronic acid (4-deoxy-L-
erythro-hex-4-eno-pyranosyl-uronic acid) at the newly formed non-reducing terminus. To date general
properties of alginate lyases from several gastropods have been investigated; however, the physiological
significance of this enzyme in the assimilation of alginate in gastropods still remains obscure.
To enrich the general information about the physiological roles of alginate lyases in gastropods, it
seems necessary to study comparatively the enzymatic properties of various gastropod alginate lyases and
5. 4
the reaction products produced by the gastropod enzymes. To date gastropod alginate lyases have been
isolated from abalone Haliotis rufescens and H. corrugate (Nakada et al., 1967), H. tuberculata (Boyen et
al., 1990; Heyraud et al., 1996), H. discus hannai (Shimizu et al., 2003; Suzuki et al., 2006), H. iris (Hata et
al., 2009); turban shell Turbo cornutus (Muramatsu et al., 1977); small marine snail Littorina sp. (Elyakova
et al., 1974), Omphalius rusticus and L. brevicula (Hata et al., 2009); and sea hare Dolabella auricular
(Nisizawa et al., 1968). In addition, alginate lyase activities were detected in sea hare Aplysia depilans, A.
californica and A. juliana ((Boyen et al., 1990; Wakabayashi et al., 1999). Most of the above gastropod
enzymes have been identified as an endolytic polymannuronate lyase (poly(M) lyase (EC 4.2.2.3)) which
produces unsaturated tri- and disaccharide as major products. Exceptionally, one enzyme that exolytically
acts on polymer substrate, i.e., HdAlex, has been isolated from abalone H. discus hannai (Suzuki et al.,
2006). This enzyme degraded not only polymer alginate but also unsaturated trisaccharide, which had been
produced by the abalone endolytic enzyme HdAly, to unsaturated disaccharide and 2-keto-3-deoxy-
gluonaldehyde (an open chain form of unsaturated monosaccharide; term -keto acid in the present paper).
The primary structures of HdAly and HdAlex were analyzed by the cDNA method and the amino-acid
identity between the two deduced sequences was 67% (Shimizu et al., 2003; Suzuki et al., 2006). On the
other hand, the amino-acid sequence of an endolytic enzyme SP2 from turban shell was determined by the
protein method (Muramatsu et al., 1996). Amino-acid identity among the above three gastropod enzymes
was approximately 60%. According to the hydrophobic cluster analysis of the primary structure (Henrissat
and Davies, 1997), these gastropod enzymes were classified to polysaccharide-lyase family 14 (PL-14)
(http://www.cazy.org/). Except for the above abalone and turban-shell enzymes, other gastropod alginate
lyases have not so extensively investigated.
The common sea hare, A. kurodai, is a typical herbivorous marine gastropod possessing various
polysaccharide-degrading enzymes in its digestive fluid. Recently we have succeeded to isolate two kinds
6. 5
of polysaccharide-degrading enzyme, i.e., -1,3-glucanase and -1,4-mannanase, from the digestive fluid
of this animal (Kumagai and Ojima, 2009; Zahura et al., 2010). During the purification of these enzymes,
we detected considerably high alginate lyase activity in the crude enzyme preparation. Although alginate
lyase activities were detected in the digestive fluid of A. depilans and A. californica (Boyen et al., 1990)
and buccal juice of A. juliana (Wakabayashi et al., 1999), no alginate lyase from Aplysia sp. has been
purified. Therefore, in the present study, we isolated alginate lyases from A. kurodai and characterized their
basic properties.
2. Materials and methods
2.1. Materials
The animal, A. kurodai (body length and weight, ~12 cm and ~150 g, respectively), was collected
from the coast of Hakodate, Hokkaido Prefecture of Japan, in July 2008. Approximately 150 mL of
digestive fluid was obtained from the gastric lumen of 20 animals by squeezing the stomach after dissection.
The digestive fluid was dialyzed against 2 mM sodium phosphate buffer (pH 7.0) for 2 h and centrifuged at
10,000×g for 10 min to remove insoluble materials. The supernatant was used as a crude enzyme for the
purification of alginate lyase. TOYOPEARL DEAE-650M was purchased from Toyo Soda Mfg, Co.
(Tokyo, Japan), and Mono-S 5/50GL and Mono-Q 5/50GL were from GE Healthcare UK Ltd. (Little
Chalfont, Bucking Hamshire, England). Sodium alginate (Macrocystis pyrifera origin) was purchased from
Sigma-Aldrich (St. Louis, USA). The other chemicals used were reagent grade from Wako Pure Chemical
Industries Ltd. (Osaka, Japan).
2.2. Substrates
7. 6
Sodium alginate was dissolved in 10 mM sodium phosphate buffer (pH 7.0) to make 1% (w/v) and
heated at 90 o
C for 1 h before use. Poly(M)-rich, Poly(G)-rich, and poly(MG)-rich substrates were prepared
by the method of Gacesa and Wusteman (1990). Mannuronate and guluronate contents in the substrates
were estimated by the method of Morris and coworkers (1980). The mannuronate content in the original
alginate was estimated to be 60%, while those in the poly(M)-rich substrate and the poly(MG)-rich
substrate were 86% and 64%, respectively. The guluronate content in the poly(G)-rich substrate was 99%.
Unsaturated oligomannuronates (unsaturated disaccharide–hexasaccharide, ΔM–ΔM6) were prepared by
the digestion of poly(M)-rich substrate with the abalone endolytic enzyme HdAly as described previously
(Shimizu et al., 2003; Suzuki et al., 2006).
2.3. Alginate lyase activity
Alginate lyase activity was assayed in a 1-mL reaction mixture containing 0.15% (w/v) substrate,
0.15 M NaCl, 10 mM sodium phosphate (pH 7.0) and an appropriate amount of enzyme (usually 5–10 units)
at 30 o
C. The progress of the reaction was monitored by measuring the absorbance at 235 nm with a Model
3010 spectrophotometer (HITACHI, Tokyo, Japan) equipped by a temperature-control device SP-12R
(TAITEC, Tokyo, Japan). One unit (U) of alginate lyase was defined as the amount of enzyme that
increases Abs235nm to 0.01 for 1 min. pH dependence of the enzyme was determined at 30 o
C in reaction
mixtures adjusted to pH 4–11 with 50 mM sodium phosphate buffer. Temperature dependence was
measured at 10–60 o
C in 10 mM sodium phosphate (pH 7.0). Thermal stability was assessed by measuring
the activity remaining after the heat treatment of the enzyme at 15–45 o
C for 20 min. NaCl dependence of
the enzyme was measured in reaction mixtures containing 0–0.5 M NaCl.
2.4. SDS-PAGE
8. 7
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with 10%
(w/v) polyacrylamide slab gel containing 0.1% (w/v) SDS according to the method of Porzio and Pearson
(1977). After the electrophoresis, the gel was stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 in
50% (v/v) methanol–10% (v/v) acetic acid, and the background of the gel was destained with 5% (v/v)
methanol–7% (v/v) acetic acid. Protein Marker, Broad Range (New England BioLabs, Ipswich, MA, USA)
was used as a molecular mass marker.
2.5. Thin-layer chromatography
The reaction mixture (70 μL) containing 1.0% (w/v) poly(M)-rich substrate or alginate
oligosaccharides, 0.15 M NaCl, 10 mM sodium phosphate buffer (pH 7.0) and 0.5 U enzyme was incubated
at 30 °C. At appropriate time intervals aliquots (each 10 μL) of the reaction mixture were withdrawn and
heated in boiling water for 2 min to terminate the reaction. The reaction products were then subjected to
thin-layer chromatography (TLC) using TLC-60 plate. The degradation products were developed with 1-
butanol–acetic acid–water (2:1:1, v:v:v) and the sugars fractionated on the plate were stained by heating the
TLC plate at 110 °C for 5 min after spraying with 10% (v/v) sulfuric acid in ethanol. To detect the
unsaturated sugars and α-keto acid on the plate, thiobarbituric acid (TBA) staining was carried out
according to the method of Lanning and Cohen (1951). Unsaturated oligosaccharide markers were prepared
by the digestion of poly(M)-rich substrate with abalone crude enzyme (Suzuki et al., 2006).
2.6. Analysis of oligosaccharides by anion-exchange chromatography
Production of oligosaccharides by alginate lyase was analyzed by anion-exchange chromatography
with a Shimadzu LC-20AT HPLC (Shimadzu, Kyoto, Japan) equipped with a TSK-GEL DEAE-2SW (4.6
mm × 25 cm) column (Tosoh Corporation, Japan). The degradation products of poly(M)-rich substrate
9. 8
produced by alginate lyases was subjected to the HPLC and the oligosaccharides adsorbed were eluted with
a linear gradient of 0-0.15 M NaCl. Elution of the oligosaccharides was detected by monitoring absorbance
at 235 nm with a Shimadzu SPD-20A UV detector.
2.7. Determination of partial amino-acid sequences
The N-terminal amino-acid sequence of alginate lyase was determined with an ABI Procise 492
protein sequencer (Applied Biosystems, Foster City, CA, USA). Internal amino-acid sequences were
determined with peptide fragments prepared either by tryptic digestion or by digestion with
lysylendopeptidase at 37 o
C for 12 hours. The tryptic fragments were subjected to a matrix-assisted laser
desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using an ABI Proteomics
Analyzer 4700 (Applied Biosystems, Foster city, CA, USA) and the amino-acid sequences of the fragments
were determined by MS/MS mode with DeNovo Explorer software. While the lysylendopeptidyl fragments
were separated by reverse phase HPLC quipped with a Mightysil Rp-18 (4.6 mm × 150 mm) column and
fragments were subjected to both MALDI TOF-MS and ABI Procise 492 protein sequencer. Homology
searches for the amino-acid sequences on databases were performed with the FASTA and BLAST
programs (http://fasta.ddbj.nig.ac.jp/top-j.html, http://blast.ddbj.nig.ac.jp/top-j.html) provided by DNA
Data Bank of Japan.
2.8. Protein determination
Protein concentration was determined by the biuret method (Gornall et al., 1949) or the method of
Lowry et al. (1951) using bovine serum albumin fraction V as a standard protein.
3. Results
10. 9
3.1. Purification of alginate lyase from A. kurodai
The sea hare alginate lyase was isolated as follows. The crude enzyme from A. kurodai was
subjected to ammonium sulfate fractionation and the precipitates formed between 60-90% saturation of
ammonium sulfate were collected by centrifugation at 10,000×g for 15 min. The precipitates were
dissolved in and dialyzed against 10 mM sodium phosphate buffer (pH 8.0) and applied to a
TOYOPEARL-DEAE 650M column (2.5 cm × 42 cm) pre-equilibrated with the same buffer. The adsorbed
proteins were eluted with a linear gradient of 0-0.2 M NaCl (total 600 mL) in 10 mM sodium phosphate
buffer (pH 8.0) at a flow rate of 15 mL/h and the eluent was collected as 5-mL fractions. Alginate lyase
activity was detected in two peaks separately eluted at 0.05 M and 0.10 M NaCl. The first peak fractions
consisted of 3–4 major proteins with ~30 kDa according to SDS-PAGE while the second peak fractions 7-8
protein bands with 25–40 kDa. Thus in the present study we used the first peak fractions for further
purification because of their higher purity. The fractions were pooled, lyophilized and dialyzed against 2
mM sodium phosphate buffer (pH 8.0) and subjected to an AKTA-FPLC (GE Healthcare UK Ltd., Little
Chalfont, Bucking Hamshire, England) equipped with a Mono-Q 5/50GL column pre-equilibrated with the
same buffer. The proteins were eluted with a linear gradient of 0–0.2 M NaCl (total 40 mL) at a flow rate of
1.0 mL/min and the eluent was collected as 1-mL fractions. In this chromatography, alginate lyase was
eluted in both passed through fractions and fractions eluted by 0.08 M NaCl (Fig. 1A). The latter fractions
showed a single band with ~33 kDa on SDS-PAGE and thus we used these fractions as a purified Aplysia
alginate lyase, AkAly33 (Fig. 2). While the passed through fractions were dialyzed against 2 mM sodium
phosphate buffer (pH 6.0) and subjected to a Mono-S 5/50GL column pre-equilibrated with same buffer.
The adsorbed proteins were eluted with a linear gradient of 0–0.5 M NaCl (total 30 mL) at a flow rate of
1.0 mL/min, and the alginate lyase showing a single band with ~28 kDa on SDS-PAGE was eluted at 0.5 M
NaCl after the linear gradient (Figs. 1B and 2). Thus, we named this enzyme AkAly28 as another Aplysia
11. 10
alginate lyase. By the above purification procedure, AkAly33 was purified 26-fold at a yield of 3.8% and
the specific activity 2057 U/mg, while AkAly28 was purified 73-fold at a yield of 4.9% and the specific
activity 5741 U/mg (Table 1).
3.2. Basic properties of AkAly28 and AkAly33
Optimal pH and temperature of both AkAly28 and AkAly33 were similarly observed at 6.7 and 40
o
C, respectively (Fig. 3A & B). The temperature that caused a half inactivation of AkAly28 and AkAly33
during 20-min incubation was also similar to each other, i.e., 38 o
C (Fig. 3C). On the other hand, the effect
of NaCl on the activity was significantly different between two enzymes. Namely, AkAly28 showed no
activity in the absence of NaCl and required NaCl at concentration higher than 0.2 M for the maximal
activity (Fig. 3D). On the other hand, AkAly33 showed ~20% of maximal activity even in the absence of
NaCl and the maximal activity at around 0.25 M NaCl. These results indicated that NaCl acted as a strong
activator for both AkAly28 and AkAly33; however, the NaCl dependency was much heavier in AkAly28
than AkAly33.
3.3. Degradation of polymer substrates by AkAly28 and AkAly33
To examine substrate specificity of AkAly28 and AkAly33, sodium alginate, poly(M)-rich,
poly(G)-rich and poly(MG)-rich substrates were subjected to lyase reaction. As shown in Fig. 4A and B,
both AkAly28 and AkAly33 exhibited the highest activity toward poly(M)-rich substrate, moderate activity
toward sodium alginate and weak activity toward poly(MG)-rich substrate, but no activity toward poly(G)-
rich substrate. These results indicate that both enzymes are classified to poly(M) lyase (EC 4.2.2.3). When
degraded sodium alginate, AkAly28 and AkAly33 rapidly decreased its viscosity in the early phase of the
reaction (Fig. 5). Accordingly, actions of these enzymes were regarded as endolytic.
12. 11
The degradation products of poly(M)-rich substrate produced by AkAly28 and AkAly33 were
analyzed by TLC. As shown in Fig. 6A and B, both AkAly28 and AkAly33 produced tri- and disaccharide
as major degradation products along with various sizes of intermediary oligosaccharides. However, relative
amounts of tri- and disaccharide produced by the two enzymes were significantly different in the prolonged
reaction time. Namely, the amount of disaccharide produced by AkAly33 in reaction time 2–6 h was
obviously larger than that by AkAly28 (Fig. 6A and B). This difference may be ascribable to the difference
in the oligosaccharide-degrading activity between AkAly28 and AkAly33. Namely, AkAly33 appeared to
degrade poly(M)-rich substrate into various sizes of oligosaccharides in the reaction time up to 1h and
further degraded the thus formed oligosaccharides to disaccharide and monosaccharide (-keto acid) in the
reaction time 2–6 h (Fig. 6B). On the other hand, AkAly28 readily degraded poly(M)-rich substrate to
oligosaccharides; however, this enzyme was considered to be incapable of degrading trisaccharide even by
the prolongation of reaction time. To confirm the difference in the trisaccharide-degrading activity between
AkAly28 and AkAly33, we further investigated the degradation produces by anion-exchange
chromatography using a TSK-GEL DEAE-2SW column. As shown in Fig. 7A, trisaccharide was produced
by AkAly28 as a major product and disaccharide was not so much produced even by the prolongation of
reaction time to 6 h. On the other hand, both di- and trisaccharide were readily produced by AkAly33 in 1-h
reaction and much higher amount of disaccharide was produced in 6-h reaction (Fig. 7B). These results
strongly suggested that AkAly33 degraded trisaccharide in the prolonged stage of the reaction. It should be
noted that -keto acid was also efficiently produced along with the production of disaccharide according to
the TBA-stained TLC (see Fig. 6). However, the -keto acid was hardly detected in HPLC (Fig. 7) since
the open chain form of -keto acid, i.e., 2-keto-3-deoxy-gluconaldehyde, does not exhibit absorbance at
235 nm.
13. 12
Then, we further examined the activities of AkAly28 and AkAly33 toward various sizes of
oligosaccharides, i.e., unsaturated disaccharide–heptasaccharide (ΔM–ΔM6). As shown in Fig. 8B,
AkAly28 could degrade oligosaccharides larger than pentasaccharides producing trisaccharide as a major
product; however, it hardly degraded oligosaccharides smaller than pentasaccharides. On the other hand,
AkAly33 could degrade oligosaccharides larger than disaccharide producing disaccharide and -keto acid
(Fig. 8C). From these results, we may conclude that AkAly28 is the enzyme that preferably degrades
substrates larger than trisaccharide while AkAly33 is the enzyme that can degrade not only polymer
substrate but also oligosaccharides producing disaccharide and -keto acid. By the actions of these two
enzymes, Aplysia may efficiently degrade alginate substrate to disaccharide and -keto acid in the digestive
fluid.
3.4. N-terminal and internal amino-acid sequences of AkAly28 and AkAly33
The N-terminal amino-acid sequences of 40 residues for AkAly28 and AkAly33 were determined
by the protein sequencer (Table 2). These sequences showed 57.5% amino-acid identity to each other;
however, they showed no appreciable similarity with the sequences of known alginate lyases. This suggests
that AkAly28 and AkAly33 are structurally related isozymes but distinct from other gastropod alginate
lyases. On the other hand, proteolytic fragments of AkAly28 and AkAly33 showed considerable sequence
identity with those previously determined in abalone and turban shell alginate lyases. Namely, the
sequences of two tryptic fragments of AkAly28, KGSFSPLHDKR (T-1) and GRFKFK (T-2), showed 27%
and 50-67% identities to the residues 51-61 and 159-164, respectively, of both abalone HdAly (Shimizu et
al., 2003) and turban shell SP2 (Muramatsu et al., 1996). In addition, the amino-acid sequences of two
lysylendopeptidyl fragments, MPGLFGGEDGDGAYK (L-1) and WNSVSEEVHINTVGK (L-2), showed
47% identity to the residues 96-111 and 170-184, respectively, of both HdAly and SP2. In these sequences,
14. 13
residues 97-102 and 178-181 are known as the highly conserved regions among PL-14 enzymes (Suzuki et
al., 2006; Yamamoto et al., 2008).
In case of AkAly33, the sequence GMFFSTFFGGSKK of a tryptic fragment T-3 showed 69% and
77% identities to the residues 216-228 and 233-245 of HdAly and HdAlex, respectively. This region is also
highly conserved among PL-14 enyzmes. The sequence LPGLFGGEK of a lysylendopeptidyl fragments L-
3 showed 67% and 78% identity to the residues 96-104 in the catalytic domains of HdAly and HdAlex,
respectively. The sequence YDVYFENFGFGIGGK of a lysylendopeptidyl fragment L-4 showed 73%
identity to the residues 80-95 in the catalytic domains of HdAly and HdAlex. The K95 was predicted as a
key residue for the catalytic action of HdAly (Yamamoto et al. 2008).
These amino-acid identities between Aplysia enzymes and abalone and turban shell PL-14
enzymes indicate that both AkAly28 and AkAly33 also belong to PL-14.
4. Discussion
Alginate from brown seaweeds has been widely used in various industrial fields such as food and
pharmaceutical industries because of its ability to form highly viscous solution as sodium salt and elastic
gel upon chelating divalent metal ions (Onsøyen E., 1996). Recently, degradation products of alginate
produced by alginate lyase were shown to exhibits various biological activities, e.g., promotion of root
growth in higher plants (Tomoda et al., 1994; Sutherland IW., 1995; Xu et al., 2003), acceleration of
growth rate of Bifidobacterium sp. (Akiyama et al., 1992), induction of production of cytotoxic cytokines in
human mononuclear cells (Natsume et al., 1994; Iwamoto et al., 2003), suppression of IgE (Yoshida et al.,
2004), antitumour and antibacterial effects (Hu et al., 2004 and 2005). These facts led us to consider that
alginate lyase is applicable to extend practical uses of alginate and its oligosaccharides. Besides the
practical uses, alginate lyase is known as an important enzyme for the seaweed-feeding gastropods like
15. 14
abalone, turban shell and sea hare. This enzyme is considered to provide carbon and energy sources for the
gastropods through the degradation of seaweeds’ alginate. Compared with abalone and turban shell alginate
lyases, other gastropod enzymes have not been so well characterized. Thus, in the present study, we
isolated alginate lyases from the common sea hare A. kurodai and determined their general properties.
Two alginate lyase isozymes, AkAly28 and AkAly33, were successfully isolated from the
digestive fluid of A. kurodai. The specific activity of AkAly28 was 2-times higher than that of AkAly33
and the yield of AkAly28 was also higher than that of AkAly33 (Fig. 2 and Table 1). In the TOYOPEARL
CM-650M chromatography, another alginate lyase(s) showing endolytic poly(M) lyase activity was also
detected. Although we have not purified this enzyme yet, this suggests that at least three kinds of poly(M)
lyases are present in the digestive fluid of A. kurodai. The presence of multiple alginate lyases may relate to
the specific food habit of sea hare, i.e., this animal usually feeds various kinds of brown seaweeds
belonging to Laminariales and Fucales. Simultaneous actions of multiple alginate lyases may be
advantageous on the degradation of alginate in the cell-wall matrices with different structures from various
seaweeds.
The optimum pH of both AkAly28 and AkAly33 was at around 6.7 whereas those of other
gastropod alginate lyases were usually observed at weak alkaline pH, e.g., optimal pHs of alginate lyases
from H. discus hannai, H. iris, and O. rusticus were at 8.0-8.5, and that of a Littorina enzyme was pH 7.5
(Hata et al., 2009). It is noteworthy that AkAly33 retained relatively high activity in a wide pH range
compared with AkAly28, e.g., it showed more than 15% of maximal activity at pH 5.0 - 9.0 and about 40%
even at pH 5.5 where AkAly28 showed no activity. Similar acid-tolerance property was also reported in a
Littorina enzyme, which retained 90% or higher activity even after the incubation at 30 o
C for 15 min at pH
3-11 (Hata et al., 2009). The optimum temperature of both AkAly28 and AkAly33 were 40 o
C and the
temperature that caused a half inactivation of these enzymes during 20-min incubation was 38 o
C. These
16. 15
values are fairly consistent with those of other gastropod alginate lyases. Both AkAly28 and AkAly33
showed high NaCl dependency. In the absence of NaCl, AkAly33 showed ~20% maximal activity but
AkAly28 no activity, and both enzymes required 0.2-0.25 M NaCl to exhibit maximal activities. Similar
NaCl requirement was also shown in a Littorina enzyme, e.g., it showed 20% maximal activity in the
absence of NaCl and maximal activity at 0.05 M NaCl (data not shown). Whereas less NaCl requirement
was observed in abalone enzyme HdAly which exhibited 70% maximal activity in the absence of NaCl and
maximal activity above 0.05 M NaCl (data not shown). Thus, NaCl requirement seemed to be a common
property among the molluscan alginate lyases although its extent differs depending on the animal species.
Activation of alginate lyase by monovalent and divalent metal ions was previously reported in the
enzymes from marine bacteria (Hu et. al., 2006). Whereas, alginate lyases from soil and terrestrial
bacteria did not require metal ions to express their optimum activities (Preston et. al., 2000; Cao et.
al., 2007). These facts may reflect an aspect of physiological adaptation of the alginate lyases to sea
water environment which contains various divalent metal ions and ~0.6 M NaCl. Thus, it is reasonable
to consider that the NaCl requirement of molluscan alginate lyases is also ascribable to the adaptation
to sea water environment. In case of an enzyme from halophilic bacteria, mechanisms for the
activation by NaCl were explained by the changes in the enzyme structure into a suitable form for
approaching to, or binding with, the substrate (Lanyi, 1974). Activation mechanisms for molluscan
alginate lyase by NaCl are still remained obscure.
AkAly28 and AkAly33 preferably degraded poly(M)-rich substrates and rapidly decreased the
viscosity of alginate solution, thus both of the two enzymes were regarded as endolytic poly(M) lyase (EC
4.2.2.3) like other gastropod alginate lyases (Muramatsu et. al., 1977; Heyraud at al., 1996; Shimizu et al.,
2003; Hata et al., 2009). However, the activities toward oligosaccharides were considerably different
17. 16
between two enzymes. Namely, AkAly28 degraded tetra- and pentasaccharide producing tri- and
disaccharide but not degraded trisaccharide, while AkAly33 could degrade trisaccharide to disaccharide
and -keto acid (Fig. 8B and C). These differences between AkAly28 and AkAly33 may imply that the
roles of two enzymes are somewhat different in the digestive fluid. For example, AkAly28 acts on larger
alginate substrates producing mainly trisaccharide and AkAly33 degrades the trisaccharide producing
disaccharide and -keto acid. We previously isolated two alginate lyase isozymes from the digestive fluid
of H. discus hannai, i.e., endolytic HdAly and exolytic HdAlex (Shimizu et al., 2003; Suzuki et al., 2006).
HdAly produced unsaturated trisaccharide and HdAlex degraded the trisaccharide to disaccharide and -
keto acid. Thus, AkAly28 and AkAly33 of A. kurodai may correspond to HdAly and HdAlex of H. discus
hannai, respectively.
The partial amino-acid sequences of AkAly28 and AkAly33 indicated that these enzymes are the
members of PL-14 which includes abalone and turban-shell enzymes (Muramatsu et al., 1996; Shimizu et
al., 2003; Suzuki et al., 2006). The N-terminal amino-acid sequences of AkAly28 and AkAly33 showed
practically no identities with those of abalone and turban shell enzymes; however, the internal sequences of
AkAly28 and AkAly33 showed considerably high similarity with the corresponding sequences of abalone
and turban shell enzymes (Table 2). The K95, which was predicted as a key residue for the catalytic action
of HdAly (Yamamoto et al. 2008), was conserved in a lysylendopeptidyl fragment (L-4) of AkAly33. The
importance of this lysine residue was also reported in the Chlorella virus PL-14 enzyme (Ogura et al, 2009).
These amino-acid sequence analyses for AkAly28 and AkAly33 indicate that these enzymes are the
members of PL-14 like abalone and turban shell enzymes although the sequences AkAly28 and AkAly33
may be somewhat diverged from other gastropod enzymes. To determine the structural characteristics of
Aplysia alginate lyases and investigate the regions relating to their catalytic actions, we recently cloned
18. 17
cDNAs encoding the Aplysia alginate lyases. Complete primary structure of Aplysia alginate lyase will be
published elsewhere.
Acknowledgements
This study was supported in part by Regional Innovation Cluster Program (Global Type) and a Grant-in-
Aid for Scientific Research (No. 19380117) of the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
References
Akiyama, H., Endo, T., Nakakita, R., Murata, K., Yonemoto, Y., Okayama, K., 1992. Effect of
depolymerized alginates on the growth of Bifidobacteria. Biosci. Biotechnol. Biochem. 56, 355-356.
Boyen, C., Kloareg, B., Polne-Fuller, M., Gibor, A., 1990. Preparation of alginate lyases from marine
molluscs for protoplast isolation in brown algae. Phycol. 29, 173-181.
Cao, L., Xie, L., Xue, X., Tan, H., Liu, Y., Zhou, S., 2007. Purification and characterization of alginate
lyase from Streptomyces species strain A5 isolated from banana rhizosphere. J Agric. Food Chem. 55,
5113-5117.
Elyakova, L.A., Favarov, V.V., 1974. Isolation and certain properties of alginate lyase VI from the mollusk
Littorina sp. Biochim. Biophys. Acta. 358, 341-354.
Erasmus, J.H., Cook, P.A., Coyne, V.E., 1997. The role of bacteria in the digestion of seaweed by the
abalone Haliotis midae. Aquaculture. 155, 377-386.
Gacesa, P., 1988. Alginates. Carbohydr. Polym. 8, 161–182.
Gacesa, P., 1992. Enzymatic degradation of alginates. Int. J. Biochem. 24, 545–552.
19. 18
Gacesa, P., Wusteman, F.S., 1990. Plate assay for simultaneous detection of alginate lyases and
determination of substrate specificity. Appl. Environ. Microbiol. 56, 2265–2267.
Gornall, A.G., Bardawill, C.J., David, M.M., 1949. Determination of serum proteins by means of the biuret
reaction. J. Biol. Chem. 177, 751-766.
Hata, M., Kumagai, Y., Rahman, M.M., Chiba, S., Tanaka, H., Inoue, A., Ojima, T., 2009. Comparative
study on general properties of alginate lyases from some marine gastropod mollusks. Fish. Sci. 75,
755-763.
Haug, A., Larsen, B., Smidsrød, O., 1967. Studies on the sequence of uronic acid residues in alginic acid.
Acta. Chem. Scand. 21, 691–704.
Henrissat, B., Davies, G., 1997. Structural and sequence-based classification of glycoside hydrolases. Curr.
Opin. Struct. Biol. 7, 637-644.
Heyraud, A., Colin-Morel, P., Girond, S., Richard, C., Kloareg, B., 1996. HPLC analysis of saturated or
unsaturated oligoguluronates and oligomannuronates. Application to the determination of the action
pattern of Haliotis tuberculata alginate lyase. Carbohydr. Res. 291, 115-126.
Hu, X., Jiang, X., Gong, J., Hwang, H., Liu, Y., Guan, H., 2005. Antibacterial activity of lyase-
depolymerized products of alginate. J. Appl. Phyco. 17, 57-60.
Hu, X., Jiang, X., Hwang, H., 2006. Purification and characterization of an alginate lyase from marine
bacterium Vibrio sp. mutant strain 510-64. Curr. Microbiol. 53, 135–140.
Hu, X., Jiang, X., Hwang, H., Liu, S., Guan, H., 2004. Antitumour activities of alginate-derived
oligosaccharides and their sulphated substitution derivatives. Eur. J. Phycol. 39, 67-71.
Iwamoto, Y., Xu, X., Tamura, T., Oda, T., Muramatsu, T., 2003. Enzymatically depolymerized alginate
20. 19
oligomers that cause cytotoxic cytokine production in human mononuclear cells. Biosci. Biotechnol.
Biochem. 67, 258-263.
Johnston, D., Moltschaniwskyj, N., Wells, J., 2005. Development of the radula and digestive system of
juvenile blacklip abalone (Haliotis rubra): potential factors responsible for variable weaning success
on artificial diets. Aquaculture. 250, 341–355.
Kumagai, Y., Ojima, T., 2009. Enzymatic properties and the primary structure of a β-1,3-glucanases from
the digestive fluid of the Pacific abalone Haliotis discus hannai. Comp. Biochem. Physiol. B. 154,
113-120.
Kumagai, Y., Ojima, T., 2010. Isolation and characterization of two types of beta-1,3-glucanases from the
common sea hare Aplysia kurodai. Comp. Biochem. Physiol. B. 155, 138-144.
Lanning, M.C., Cohen, S.S., 1951. The detection and estimation of 2-ketohexonic acids. J. Biol. Chem. 189,
109–114.
Lanyi, J.K., 1974. Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriol. Rev.
38, 272-290.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol
reagent. J. Biol. Chem. 193, 265-275.
Mai, K., Mercer, J.P., Donlon, J., 1995. Comparative studies on the nutrition of two species of abalone,
Haliotis tuberculata L. and Haliotis discus hannai Ino. III. Response of abalone to various levels of
dietary lipid. Aquaculture. 134, 65–80.
Morris, E.R., Rees, D.A., Thom, D., 1980. Characterisation of alginate composition and block-structure by
circular dichroism. Carbohydr. Res. 81, 305-314.
21. 20
Muramatsu, T., Hirose, S., Katayose, M., 1977. Isolation and properties of alginate lyase from the mid-gut
gland of wreath shell Turbo cornutus. Agric. Biol. Chem. 41, 1939-1946.
Muramatsu, T., Komori, K., Sakurai, N., Yamada, K., Awasaki, Y., Fukuda, K., Oda, T., 1996. Primary
structure of mannuronate lyases SP1 and SP2 from Turbo cornutus and involvement of the
hydrophobic C-terminal residues in the protein stability. J. Protein Chem. 15, 709-719.
Nakada, H.I., Sweeny, P.C., 1967. Alginic Acid Degradation by Eliminases from Abalone Hepatopancreas.
J. Biol. Chem. 242, 845-851.
Natsume, M., Kamo, Y., Hirayama, M., Adachi, T., 1994. Isolation and characterization of alginate-derived
oligosaccharides with root growth-promoting activities. Carbohydr. Res. 258, 187-197.
Nikapitiya, C., Oh, C., Whang, I., Kim, C.G., Lee, Y.H., Kim, S.J., Lee, J., 2009. Molecular
characterization, gene expression analysis and biochemical properties of α-amylase from the disk
abalone, Haliotis discus discus. Comp. Biochem. Physiol. B. 152, 271–281.
Nishida, Y., Suzuki, K., Kumagai, Y., Tanaka, H., Inoue, A., Ojima, T., 2007. Isolation and primary
structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus. Biochimie. 89, 1002-
1011.
Nisizawa, K., Fujibayashi, S., Kashiwabara, Y., 1968. Alginate lyases in the hepatopancreas of a marine
mollusk, Dolabella auricula Solander. J. Biochem (Tokyo). 64, 25-37.
Ogura, K., Yamasaki, M., Yamada, T., Mikami, B., Hashimoto, W., Murata, K., 2009. Crystal structure of
family 14 polysaccharide lyase with pH-dependent modes of action. J. Biol. Chem. 284, 35572–35579.
Onsøyen, E., 1996. Commercial applications of alginates. Carbohydr. Eur. 14, 26–31.
Ootsuka, S., Saga, N., Suzuki, K.I., Inoue, A., Ojima, T., 2006. Isolation and cloning of an endo-β-1,4-
22. 21
mannanase from pacific abalone Haliotis discus hannai. J. Biotechnol. 125, 269–280.
Porzio, M.A., Pearson, A.M., 1977. Improved resolution of myofibrillar proteins with sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Biochim. Biophys. Acta. 490, 27–34.
Preston, L.A., Wong, T.Y., Bender C.L., Schiller, N. L., 2000. Characterization of Alginate Lyase from
Pseudomonas syringae pv. Syringae. J. Bacteriol. 182, 6268-6271.
Sawabe, T., Setoguchi, N., Inoue, S., Tanaka, R., Ootsubo, M., Yoshimizu, M., Ezura, Y., 2003. Acetic
acid production of Vibrio halioticoli from alginate: a possible role for establishment of abalone-Vibrio
halioticoli association. Aquaculture. 219, 671-679.
Shimizu, E., Ojima, T., Nishita, K., 2003. cDNA cloning of an alginate lyase from abalone, Haliotis discus
hannai. Carbohydr. Res. 338, 2841-2852.
Sutherland, I.W., 1995. Polysaccharide lyases. FEMS Microbiol. Rev. 16, 323-347.
Suzuki, H., Suzuki, K., Inoue, A., Ojima, T., 2006. A novel oligoalginate lyase from abalone, Haliotis
discus hannai, that releases disaccharide from alginate polymer in an exolytic manner. Carbohydr. Res.
341, 1809-1819.
Suzuki, K., Ojima, T., Nishita, K., 2003. Purification and cDNA cloning of a cellulase from abalone
Haliotis discus hannai. Eur. J. Biochem. 270, 771-778.
Takami, H., Kawamura, T., Yamashita, Y., 1998. Development of polysaccharide degradation activity in
postlarval abalone Haliotis discus hannai. J. Shellfish Res. 17, 723–727.
Tomoda, Y., Umemura, K., Adachi, T., 1994. Promotion of barley root elongation under hypoxic
conditions by alginate lyase-lysate. Biosci. Biotechnol. Biochem. 58, 202-203.
23. 22
Wakabayashi, T., Kuboi, T., Tuboi, T., Kaji, M., Hara, M., 1999. Preparation of high yields of algal
protoplasts using buccal juice of sea hare and commercial cellulase. Mar. Biotechnol. 1, 407-410.
Wong, T.Y., Preston, L.A., Schiller, N.L., 2000. Alginate lyase: Review of major sources and enzyme
characteristics, structure-function analysis, biological roles, and applications. Annu. Rev. Microbiol.
54, 289–340.
Xu, X., Iwamoto, Y., Kitamura, Y., Oda, T., Muramatsu, T., 2003. Root growth-promoting activity of
unsaturated oligomeric uronates from alginate on carrot and rice plants. Biosci. Biotechnol. Biochem.
67, 2022-2025.
Yamamoto, S., Sahara, T., Sato, D., Kawasaki, K., Ohgiya, S., Inoue, A., Ojima, T., 2008. Catalytically
important amino-acid residues of abalone alginate lyase HdAly assessed by site-directed mutagenesis.
Enzyme Microb. Tech. 43, 396-402.
Yoshida, T., Hirano, A., Wada, H., Takahashi, K., Hattori, M., 2004. Alginic acid oligosaccharide
suppresses Th2 development and IgE production by inducing IL-12 production. Int. Arch. Allergy
Immunol. 133, 239-247.
Zahura, U.A., Rahman, M.M., Inoue, A., Tanaka, H., Ojima, T., 2010. An endo-ß -1,4 mannanase, AkMan,
from the common sea hare Aplysia kurodai. Comp. Biochem. Physiol. B 157, 137-143.
24. 23
Legends to figures
Fig. 1. Purification of alginate lyases from A. kurodai by AKTA-FPLC. (A) Mono-Q column
chromatography of the active fractions obtained by TOYOPEARL-DEAE 650M chromatography. (B)
Mono-S column chromatography of the passed through fractions in Mono-Q column chromatography.
Conditions for the chromatographies are described under “Materials and Methods”. Activity levels for
fractions are indicated with shaded bars.
Fig. 2. SDS-PAGE for the Aplysia alginate lyase in various purification steps. Mk, molecular mass markers;
Lane 1, Proteins precipitated between 60 and 90% saturation of ammonium sulfate; Lane 2, The first peak
fractions obtained by DEAE TOYOPEARL-650 M chromatography; Lane 3, AkAly33 purified by Mono-Q
column chromatography; Lane 4, AkAly28 purified by Mono-S column chromatography.
Fig. 3. Effects of pH, temperature, and NaCl on AkAly28 and AkAly33. (A) pH dependence was measured
at 30 o
C in reaction mixtures adjusted to pH 4-11 with 50 mM sodium phosphate buffer. (B) Temperature
dependence was measured at 10-60 o
C in a reaction mixture containing 0.15% sodium alginate, 0.15 M
NaCl and 10 mM sodium phosphate (pH 7.0). (C) Thermal stability was assessed by measuring the activity
remaining after the heat-treatment at 15-45 o
C for 20 min. (D) NaCl dependence was measured in a
reaction mixture containing various concentrations of NaCl, 0.15% sodium alginate, 10 mM sodium
phosphate (pH 7.0) and 1 U/mL enzyme at 30 o
C. ○, AkAly28; ●, AkAly33.
Fig. 4. Substrate preference of AkAly28 and AkAly33. Activity was measured in a reaction mixture
containing either the sodium alginate (○), poly(M)-rich substrate (●), poly(MG)-rich substrate (Δ) or
25. 24
poly(G)-rich substrate (▲) in a concentration of 0.15% (w/v). Degradation of substrates was monitored by
measuring the increase in absorbance at 235 nm. A, AkAly28; B, AkAly33.
Fig. 5. Decrease in the viscosity of sodium alginate solution by the digestion with AkAly28 and AkAly33.
The reaction was carried out at 30 o
C in an Ostwald-type viscometer, in a mixture containing 0.12% sodium
alginate, 0.15 M NaCl, 10 mM sodium phosphate (pH 7.0), 10 U/mL AkAly28 (○) or 7 U/mL AkAly33 (●).
Fig. 6. TLC for the degradation products of poly(M)-rich substrates produced by AkAly28 and AkAly33.
Poly(M)-rich substrate (1.0% (w/v)) in 10 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl
was degraded by AkAly28 (A) and AkAly33 (B) at 30 o
C for 6 h. The aliquots of reaction products (each 2
L) were applied to TLC-60 plate and developed with 1-butanol–acetic acid–water (2:1:1, v:v:v). Total
sugars separated on the plate were visualized by spraying with sulfuric acid in ethanol (A and B, left),
while unsaturated sugars and α-keto acid were detected by TBA staining (A and B, right). M, marker
oligosaccharides; -keto, -keto acid (an open chain form of 2-keto-3-deoxy-gluconaldehyde); ΔM,
unsaturated disaccharide; ΔM2, unsaturated trisaccharide; ΔM3, unsaturated tetrasaccharide; ΔM4,
unsaturated pentasaccharide; ΔM5, unsaturated hexasaccharide.
Fig. 7. Anion-exchange chromatography of the degradation products of Poly(M)-rich substrate. (A)
Poly(M)-rich substrate (1.0% (w/v)) was degraded by 100 U/mL of AkAly28 at 30 o
C and the degradation
products obtained at reaction time 0 h, 1 h and 6 h were subjected to TSK-GEL DEAE-2SW anion-
exchange chromatography. Elution of oligosaccharides was detected with a Shimadzu SPD-20A UV
detector at 235 nm. (B) Poly(M)-rich substrate was degraded by 100 U/mL of AkAly33 as in (A). ΔM,
unsaturated disaccharide; ΔM2, unsaturated trisaccharide.
26. 25
Fig. 8. TLC for the degradation products of unsaturated oligosaccharides produced by AkAly28 and
AkAly33. (A) Unsaturated oligosaccharide substrates, ΔM-ΔM6. (B) The oligosaccharides degraded by
AkAly28 at 30 o
C for 2 h. (C) The oligosaccharides degraded by AkAly33 as in (B). The reaction products
developed on TLC plate were visualized by TBA staining. M, marker; ΔM, unsaturated disaccharide; ΔM2,
unsaturated trisaccharide; ΔM3, unsaturated tetrasaccharide; ΔM4, unsaturated pentasaccharide; ΔM5,
unsaturated hexasaccharide; ΔM6, unsaturated heptasaccharide.
27. 26
Table 1. Summary of the purification of AkAly28 and AkAly33.
Samples
Total protein
(mg)
Specific
activity
(U/mg)
Total activity
(U)
Purification
(fold)
Yield
(%)
Crudea
2163 79.06 171007 1 100
ASb
324.26 390.06 126481 4.93 73.96
DEAEc
8.64 2631.03 22732 33.28 13.29
AkAly33d
3.15 2057.14 6480 26.02 3.79
AkAly28e
1.46 5740.74 8382 72.61 4.90
a
Crude enzyme after the dialysis against 10 mM sodium phosphate (pH 7.0).
b
Fraction precipitated between 60 and 90% saturation of ammonium sulfate.
c
Active fractions obtained by DEAE TOYOPEARL-650M chromatography.
d
AkAly33 purified by Mono-Q column chromatography.
e
AkAly28 purified by Mono-S column chromatography.
28. 27
Table 2. Partial amino-acid sequences of AkAly28 and AkAly33
Peptidesa
Sequencesb
Similar regions of
other enzymes c
AkAly28
N-terminus ASTLWSVGSVPHSTDVSSILGHFAPYYHEWGDDSISTSTK
T-1 KGSFSPLHDKR HdAly, SP2 (51-
61)
T-2 GRFKFK HdAly, SP2 (159-
164)
L-1 MPGLFGGEDGDGAYK HdAly (96-111)
L-2 WNSVSEEVHINTVGK SP2 (170-184)
AkAly33
N-terminus DTVIWSLSSVPLSSDTDVILQNFGPMYHDFGDDSISTSTK
T-3 GMFFSTFFGGSKK HdAly (216-228);
HdAlex (216-228)
L-3 LPGLFGGEK HdAly (96-104);
HdAlex (96-104)
L-4 YDVYFENFGFGIGGK HdAly (80-95);
HdAlex (80-95)
a
T-1 – T-3, tryptic fragments; L-1 – L4, Lysylendopeptidyl fragments. b
Residues conserved among PL-14
enzymes are underlined and a catalytically important lysine in PL-14 enzymes is shown as the bold letter.
c
Residue numbers for similar sequence regions in abalone HdAly and HdAlex, and turban shell SP2 are
shown in the parentheses.