Expression and Characterization of a Novelλ-Carrageenase Cgl150A_Wa from Wenyingzhuangia aestuarii

λ-Carrageenan is a highly sulfated polysaccharide alternating of 1,4-O-α-D-galactopyranose-2,6-sulfate(D2S,6S)and 1,3-O-β-D-galactopyranose-2-sulfate(G2S).λ-Carrageenases are desirable tools forλ-carrageenan degradation.Based on the genome mining,a novelλ-carrageenase Cgl150A_Wa was cloned from the bacterium Wenyingzhuangia aestuarii and expressed in Escherichia coli.Cgl150A_Wa was an endo-acting enzyme and exhibited its maximum activity at 30℃and pH 8.0.By employing a glycomics strategy that combined ultra-performance liquid chromatography-mass spectrometry analysis and glycoinformatics,Cgl150A_Wa was proven to degradeλ-carrageenan octaose and hexaose,and the major hydrolysis product of Cgl150A_Wa wasλ-carrageenan tetrose.In addition to the typicalλ-carrageenan motifs,the active center of Cgl150A_Wa might tolerate desulfatedλ-carrageenan motifs.Cgl150A_Wa is a potential biotechnological tool for preparingλ-carrageenan oligosaccharides and structural investigation.

Contribution of phytoliths to total biogenic silica volumes in the tropical rivers of Malaysia and associated implications for the marine biogeochemical cycle

The contribution of phytoliths to total biogenic silica(BSi) volumes in rivers worldwide,and the associated implications for the biogeochemical cycle,require in-depth study.Based on samples from rivers in Peninsular Malaysia,this project investigated the source and characteristics of B Si found in Asian tropical rivers,as well as the process of reverse weathering taking place in these fluvial systems.Results indicated that BSi samples collected in sediments consisted of phytolith,diatom and sponge spicules.Phytoliths,predominantly of the elongate form,comprised 92.8%-98.3% of BSi in the Pahang River.Diatom BSi in this river consisted mainly of pennatae diatoms,but represented a relatively small proportion of the total BSi volume.However,diatom BSi(predominantly of the Centricae form) was more prevalent in the Pontian and Endau Rivers with shares of 68.8% and 79.3% of the total BSi volumes,respectively,than Pahang River.Carbon contents of the BSi particulates ranged from 1.85% to 10.8% with an average of 4.79%.These values are higher than those recorded in other studies to date,and indicate that BSi plays a major role in controlling permanent carbon burial.This study suggests that phytoliths from terrestrial plants are the primary constituents of BSi in the rivers of Peninsular Malaysia,and therefore represent a significant proportion of the coastal silica budget.

Phosphorus speciation,transformation and benthic processes with implications for environmental impacts in the aquaculture area of Rushan Bay

Phosphorus(P)is an essential nutrient for many organisms in the ocean,which plays a central role in the stability of ecosystems and the evolution of the environment.The distribution,occurrence and source-sink process of P in offshore waters are highly influenced by mariculture activities.P transformation in water-sediment system is the key process in P cycling,however,the mechanism is poorly documented in the coastal sea which is influenced by human activities.Based on the comprehensive surveys in the adjacent waters outside Rushan Bay in May,July and August 2014 and February 2015,the form and transformation of P in the suspended particulate matter(SPM)and surface sediment were analyzed.The results showed that contents of total P,authigenic P and organic P of SPM increased with the increase in distance from the shoreline off Rushan Bay,and the detrital-P decreased.The partition coefficient of P between water and SPM was related to the chemical activity of different forms of P,and a higher reactivity of inorganic P associated with SPM was observed.Hypoxia induced by mariculture changes the distribution and morphological composition of P in SPM and sediment in this typical aquaculture area,which can result in a conversion of sink to source of P in the sediment,thereby having a significant impact on the regional nutrient budget and associated with eutrophication.

Exosomes in viral infection:Effects for pathogenesis and treatment strategies

Exosomes are small vesicles that carry molecules from one cell to another.They have many features that make them interesting for research,such as their stability,low immunogenicity,size of the nanoscale,toxicity,and selective delivery.Exosomes can also interact with viruses in diverse ways.Emerging research highlights the significant role of exosomes in viral infections,particularly in the context of diseases like COVID-19,HIV,HBV and HCV.Understanding the intricate interplay between exosomes and the human immune system holds great promise for the development of effective antiviral therapies.An important aspect is gaining clarity on how exosomes influence the immune system and enhance viral infectivity through their inherent characteristics.By leveraging the innate properties of exosomes,viruses exploit the machinery involved in exosome biogenesis to set replication,facilitate the spread of infection,and eliminate immune responses.They can either help or hinder viral infection by modulating the immune system.This review summarizes the recent findings on how exosomes mediate viral infection and how they can be used for diagnosis or therapy.This could lead to new clinical applications of exosomes in disease management.

菲律宾蛤仔过敏原可视化抗体微阵列玻片的检测方法

采用辣根过氧化物酶(HRP)与3,3′,5,5′—四甲基联苯胺(TMB)—H2O2作为信号示踪系统,建立了一种可视化抗体微阵列检测菲律宾蛤仔过敏原的方法。对孵育时间、抗体浓度等免疫条件进行了优化。采用改良双抗体夹心法,将菲律宾蛤仔过敏原的兔多克隆抗体固定于琼脂糖三维芯片上,依次加入待检样品、菲律宾蛤仔鼠多克隆抗体和HRP-羊抗鼠IgG,孵育后加TMB显色,肉眼观察后,用平板扫描仪获取扫描图象,采用GenePixPro6.0软件分析灰度值。试验结果表明,该方法最低可检出10ng/mL的菲律宾蛤仔过敏原,片内平均变异系数(CV)为5.99%,片间为10.3%;香肠及蟹棒中3个不同浓度的加标回收率为73.54%～95.44%;4℃存放4个月内抗体微阵列玻片活性保持稳定。该方法不需要大型精密仪器,结果直观可见,可以发展为对多种过敏原进行同时检测,具有良好的实用和推广价值。

Impact of the water input from the eastern Qiongzhou Strait to the Beibu Gulf on Guangxi coastal circulation

Based on a comparison of synchronized temperature and salinity data collected in the eastern Qiongzhou Strait and at coastal marine stations, this study finds that, in summer, the variation in salinity near the Weizhou Island in Guangxi is similar to that in the eastern and central portions of the Qiongzhou Strait. Additionally, the Beihai Station in Guangxi exhibits a small salinity variation, whereas the Longmen and Bailongwei Stations, both of which are located far from the Qiongzhou Strait, mainly exhibit continental hydrological characteristics in summer. Moreover, a comparison of the multi-year ocean current data from the Qiongzhou Strait and ocean current observations from the Weizhou Island Station and recently installed current-measuring stations shows that the residual current in the Qiongzhou Strait flows westward in winter and summer. The numerical simulation results also indicate that water from the eastern Qiongzhou Strait enters the Beibu Gulf. The characteristics of the temperature and salinity distributions and analyses of the residual currents further confirm that the western Guangdong coastal current is the main source of the westward transport of water in the Qiongzhou Strait. The primary driver of the formation of the western Guangdong coastal current is the westward flow of freshwater from the Zhujiang (Pearl) River. This water enters the Beibu Gulf via the Qiongzhou Strait and enhances the formation of the cyclonic circulation in the northern Beibu Gulf. In summer, the strong influence of the southwesterly wind leads to the formation of a strong northward coastal current along the western coast of the Beibu Gulf. This process promotes the transport of low-salinity diluted water toward the open ocean and the formation of larger- scale cyclonic circulation around Weizhou Island in the eastern Beibu Gulf. The results of this study regarding the effects of the water inflow from the eastern Qiongzhou Strait to the Beibu Gulf on the Guangxi coastal circulation directly challenge conventional conclusions concerning the transport direction of water through the Qiongzhou Strait in winter and summer.

Allelopathic interactions between Prorocentrum micans and Skeletonema costatum or Karenia mikimotoi in laboratory cultures

Algal allelopathy is an ecological/physiological phenomenon that has focused attention on the interactions among algae and the production of algal toxins. We investigated the allelopathic interactions between the dinoflagellate genus Prorocentrum micans and diatom genus Skeletonema costatum and between P. micans and dinoflagellate genus Karenia mikimotoi using bi-algal cultures. Because the effects were species-specific and size-dependent, we evaluated the effect of different initial densities. At low densities of P. micans and high densities of S. costatum inoculated into the same medium, the growth of P. micans was weakly restrained, whereas the growth of S. costatum was significantly suppressed. S. costatum and K. mikimotoi were strongly inhibited by P. micans, in both the bi-algal cultures and enriched filtrates. Direct cell-to-cell contact was not necessary to gain a competitive advantage, thus, our results suggest that P. micans inhibited the growth of S. costatum and K. mikimotoi by the release of allelochemical(s). Last, a mathematical model was used to simulate growth and interactions between P. micans and S. costatum and between P. micans and K. mikimotoi in bi-algal cultures.

Mitochondrial Genome of Episesarma lafondii(Brachyura:Sesarmidae)and Comparison with Other Sesarmid Crabs

Complete mitochondrial genomes(mitogenomes)can provide useful information for phylogenetic relationships,gene rearrangement,and molecular evolution.Here,the complete mitogenome of Episesarma lafondii(Brachyura:Grapsoidea:Sesarmidae)was sequenced through next-generation sequencing technique for the first time.The 15640 bp mitogenome contains the entire set of 37 genes and an AT-rich region.The rearrangements of two tRNA genes(tRNA-His and tRNA-Gln)are compared with that in the pancrustacean ground pattern,and the tandem duplication/random loss model was selected to explain the observed gene rearrangements.The phylogenetic results showed that all sesarmid crabs belong to the same group,wherein the genus Episesarma showed the closest relationship with Clistocoeloma.Furthermore,the monophyly of each family was well supported except for Xanthidae,Gecarcinidae,and Homolidae.The correlation between the phylogeny of Sesarmidae species and the gaps in the QIM region was analyzed.Evidently,the gaps between Q and I(Gap3 and between I and M(Gap4)degenerated with the evolution process.In general,the results will contribute to the in-depth understanding of gene rearrangements in Sesarmidae mitogenomes and provide new insights into the phylogeny of Brachyura.

Phaeocystis globosa Bloom Monitoring: Based on P. globosa Induced Seawater Viscosity Modification Adjacent to a Nuclear Power Plant in Qinzhou Bay, China

The mucus produced by the outbreak of Phaeocystis globosa in the adjacent waters of the Fangchenggang Nuclear Power Plant(FCGNPP) in China has blocked the entrance of the cooling water filtration system of the FCGNPP, and posed a threat to the safe operation of the FCGNPP. At present, there is no related research on whether the changes in seawater viscosity could be used as a new method for monitoring and providing early warning of P. globosa tide. During a complete red tide cycle, the temporal and spatial changes in the hydrological conditions(temperature, salinity, dissolved oxygen), chlorophyll-a(Chl-a), composition and abundance of phytoplankton, number and size of P. globose colonies, concentration of transparent exopolymer particles(TEP) and the seawater viscosity were measured in this study. The results indicate that there was an extremely significant negative correlation between the physical seawater viscosity η_(T,S) and temperature, and the correlation coefficient reached-0.998. The biological seawater viscosity η_(Bio) was positively correlated with the number of P. globosa colonies, and the correlation coefficients is 0.747. Because the increase in phytoplankton abundance, especially during the outbreak of P. globosa and a large amount of mucus produced by the colonies could significantly increase seawater viscosity, we suggest that biological factors were the main reasons for the increase in seawater viscosity. The η_(Bio) was completely consistent with the occurrence process of P. globosa bloom and could be used as a valuable index for P. globosa bloom monitoring.

Spatiotemporal Distribution of Asian Horseshoe Crab Eggs Are Highly Intermingled with Anthropogenic Structures in Northern Beibu Gulf,China

Identification,protection and restoration of spawning habitats are vital for protecting the depleted species.Asian horseshoe crabs are ecologically important macroinvertebrates in coastal and estuarine ecosystems.However,their spawning habitat studies were limited to several reports in tropical regions,possibly due to the lack of modified survey methods,particularly in habitats with a lower density of spawning adults,and/or intermingled with anthropogenic structures.In this study,the year-round egg distribution and spawning habitat baselines of Carcinoscorpius rotundicauda and Tachypleus tridentatus were determined in the northern Beibu Gulf,China.Our findings demonstrated that the peak spawning occurred in June–July and ceased in November–January when the average water temperature dropped below 20℃.Egg aggregations were found<10 cm beneath the sediment surface with regular tidal inundation,regardless of seasonal changes,in the vicinity of natural and artificial structures with elevated,mildly sloping substratum within the high tide zones.The nests were characterized by medium-sized sediment grains(0.5–0.9 mm),high temperatures(31–34℃),low water contents(0.8%–0.9%),and total organic carbon contents(0.5%–0.7%),which might maximize the hatching success.The identified nesting beaches were close to nursery habitats for juveniles,and tidal creeks were present as the possible corridor connecting these two important habitats through the dominant mangrove forests.The findings provide valuable insights in the scope of spawning behavior and nest-site selection of Asian horseshoe crabs under a mixture of natural and artificial structures,which could benefit future management efforts for the exploited spawning populations.

Structural characteristics of central depression belt in deep-water area of the Qiongdongnan Basin and the hydrocarbon discovery of Songnan low bulge

The Qiongdongnan Basin has the first proprietary high-yield gas field in deep-water areas of China and makes the significant breakthroughs in oil and gas exploration.The central depression belt of deep-water area in the Qiongdongnan Basin is constituted by five sags,i.e.Ledong Sag,Lingshui Sag,Songnan Sag,Baodao Sag and Changchang Sag.It is a Cenozoic extensional basin with the basement of pre-Paleogene as a whole.The structural research in central depression belt of deep-water area in the Qiongdongnan Basin has the important meaning in solving the basic geological problems,and improving the exploration of oil and gas of this basin.The seismic interpretation and structural analysis in this article was operated with the 3D seismic of about 1.5×104 km2 and the 2D seismic of about 1×104 km.Eighteen sampling points were selected to calculate the fault activity rates of the No.2 Fault.The deposition rate was calculated by the ratio of residual formation thickness to deposition time scale.The paleo-geomorphic restoration was obtained by residual thickness method and impression method.The faults in the central depression belt of deep-water area of this basin were mainly developed during Paleogene,and chiefly trend in NE–SW,E–W and NW–SE directions.The architectures of these sags change regularly from east to west:the asymmetric grabens are developed in the Ledong Sag,western Lingshui Sag,eastern Baodao Sag,and western Changchang Sag;half-grabens are developed in the Songnan Sag,eastern Lingshui Sag,and eastern Changchang Sag.The tectonic evolution history in deep-water area of this basin can be divided into three stages,i.e.faulted-depression stage,thermal subsidence stage,and neotectonic stage.The Ledong-Lingshui sags,near the Red River Fault,developed large-scale sedimentary and subsidence by the uplift of Qinghai-Tibet Plateau during neotectonic stage.The Baodao-Changchang sags,near the northwest oceanic sub-basin,developed the large-scale magmatic activities and the transition of stress direction by the expansion of the South China Sea.The east sag belt and west sag belt of the deep-water area in the Qiongdongnan Basin,separated by the ancient Songnan bulge,present prominent differences in deposition filling,diaper genesis,and sag connectivity.The west sag belt has the advantages in high maturity,well-developed fluid diapirs and channel sand bodies,thus it has superior conditions for oil and gas migration and accumulation.The east sag belt is qualified by the abundant resources of oil and gas.The Paleogene of Songnan low bulge,located between the west sag belt and the east sag belt,is the exploration potential.The YL 8 area,located in the southwestern high part of the Songnan low bulge,is a favorable target for the future gas exploration.The Well 8-1-1 was drilled in August 2018 and obtained potential business discovery,and the Well YL8-3-1 was drilled in July 2019 and obtained the business discovery.

Simulation of Storm Surge and Wave Due to Typhoon Isewan(5915)

An integrally coupled wave-tide-surge model was developed and then applied to the simulation of the wave-typhoon surge for the typhoon Isewan （typhoon Vera （5915））, which is the strongest typhoon that has struck Japan and caused incalculable damage. An integrally coupled tide-surge-wave model using identical and homogeneous meshes in an unstructured grid system was used to correctly resolve the physics of wave-circulation interaction in both models. All model components were validated independently. The storm surge and wave properties such as the surge height, the significant wave height, wave period and direction were reproduced reasonably under the meteorological forcing, which was reprocessed to be close to the observations. The resulting modeling system can be used extensively for the prediction of the storm surge and waves and the usual barotropic forecast.

Comparative mitochondrial genome analysis of Varunidae and its phylogenetic implications

Complete mitochondrial genomes(mitogenomes)can indicate phylogenetic relationships,as well as useful information for gene rearrangement mechanisms and molecular evolution.Currently,the phylogenetic location of the genus Varuna(Brachyura:Varunidae)has not been well resolved mainly because of limited representatives(only two extant species).Here,we determined a new mitogenome of this genus(Varuna litterata)and added the published mitogenomes to reconstruct the phylogeny of Varunidae.The 16368-bp mitogenome contains the entire set of 37 genes and a putative control region.The characteristics of this newly sequenced mitogenome were described and compared with the other 15 Varunidae mitogenomes.All 16 analyzed mitogenomes have identical gene order and similar molecular features.The sliding window and genetic distance analyses demonstrate highly variable nucleotide diversity,with comparatively low variability of COI and COII,and high variability of ND6.The nonsynonymous/synonymous substitution rates(dN/dS ratio)analysis shows that all 13 PCGs are under purifying selection and ATP8 gene evolves under the least selective pressure.Twelve tRNA genes,two rRNAs,one PCG,and the putative control region are found to be rearranged with respect to the pancrustacean ground pattern gene order.Tandem duplication/random loss model is adopted to explain the large-scale gene rearrangement events occurring in Varunidae mitogenomes.Phylogenetic analyses show that all Varunidae species are placed into one group,and form a sister clade with Macrophthalmidae.Nevertheless,the phylogenetic relationships within Varunidae are not completely consistent based on the two different datasets used in this study.These findings will contribute to a better understanding of gene rearrangement and molecular evolution in Varunidae mitogenomes,as well as provide insights into the phylogenetic studies of Brachyura.

Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

Numerical Simulation of Nutrient and Phytoplankton Dynamics in Guangxi Coastal Bays, China

The increasing riverine pollutants have resulted in nutrient enrichment and deterioration of water quality in the coastal water of Guangxi Province, China. However, the quantitative relationship between nutrient loads and water quality responses, which is crucial for developing eutrophication control strategies, is not well studied. In this study, the riverine fluxes of nutrients were quantified and integrated with nutrient cycling and phytoplankton dynamics by using box models for Guangxi coastal bays. The model concepts and biogeochemical equations were the same; while most model parameters were specific for each bay. The parameters were calibrated with seasonal observations during 2006–2007, and validated with yearly averaged measurements in 2009. The general features of nutrient and phytoplankton dynamics were reproduced, and the models were proved feasible under a wide range of bay conditions. Dissolved inorganic nitrogen was depleted during the spring algal bloom in Zhenzhu Bay and Fangcheng Bay with relatively less nutrient inputs. Phosphorus concentration was high in spring, which decreased then due to continuous phytoplankton consumption. Chlorophyll-a concentration reached its annual maximum in summer, but was the minimum in winter. Eutrophication was characterized by both an increase in nutrient concentrations and phytoplankton biomass in Lianzhou Bay. Either about 80% reduction of nitrogen or 70% reduction of phosphorus was required to control the algal bloom in Lianzhou Bay. Defects of the models were discussed and suggestions to the environmental protection of Guangxi coastal bays were proposed.

Asian Horseshoe Crab Bycatch in Intertidal Zones of the Northern Beibu Gulf:Suggestions for Conservation Management

Bycatch poses a severe challenge to global fishery management.Although studies have focused on marine megafauna bycatch,research on relevant issues regarding invertebrates is limited,particularly for the threatened horseshoe crabs.In this study,the distribution of fishing gears and the bycatch intensity of Asian horseshoe crabs in the intertidal zones of the northern Beibu Gulf that harbors the most abundant juvenile Asian horseshoe crabs populations in China were evaluated.Seven intertidal nursery habitats for Tachypleus tridentatus and Carcinoscorpius rotundicauda were surveyed from May to July in 2021.A transect that crossed the tidal creeks and tidal flats between the high tide embankment/vegetation and low tide line of a given habitat was surveyed during the ebb tides.The type,number,and GPS of fishing gears were recorded when sighted,and the number and prosomal width of each horseshoe crab species were measured.Bycatch intensities differed among habitats,ranging from 0.3 to 18.4 and 1.2 to 22.7 individuals per kilometer of transect for T.tridentatus and C.rotundicauda,respectively.Among the three identified fishing gear types,ground cages and stick net sets caused a stronger bycatch pressure on these two species.Most T.tridentatus bycatch and C.rotundicauda bycatch were large individuals in late juvenile and adult stages.Therefore,the ground cages and stick net sets along/near the tidal creeks,mangrove fringe,and low tide line should be regulated and removed to ensure the functionality of the intertidal zone as the spawning corridor and nursery habitat of Asian horseshoe crabs.

The biogenic silica composition, behavior and budget in the Changjiang Estuary

Three comprehensive surveys were performed in the Changjiang（Yangtze River） Estuary（CJE） to understand the biogenic silica（BSi） composition, behavior and budget. It is indicated that the BSi is composed of phytoliths,phytoplankton and sponges; phytolith BSi has 16 forms and account for 23% to 83% of the bulk BSi in the maximum turbidity zone. The budget shows that the major exogenous BSi source in the water column of the CJE is the riverine input, accounting for 95% of the total BSi input. Dominant processes that maintain BSi levels in the water column are the primary production（55 Gmol/a） and the subsequent BSi sedimentation（46 Gmol/a）; and the BSi pool produced by the primary production represents two point three times the BSi loading of the Changjiang River and 63% of the BSi output, respectively. The net export（26 Gmol/a） of BSi from the CJE to the East China Sea and Yellow Sea roughly equals the riverine BSi loading. The observed total accumulation of BSi is one point seven times larger than the loading of total BSi output, with 53% to 88% of phytolith BSi and their assemblage, indicating that there has already been a ＂filter＂ of terrestrial BSi. The reverse weathering in sediments is an important process for the reactive silica removal in the CJE due to authigenic alterations. It is indicated that the phytolith fluxes in the suspended load represent a significant BSi source in the estuary, and the CJE would act as a net BSi sink.

Infra-Gravity Wave Generation by the Shoaling Wave Groups over Beaches

A physical parameter, fib, which was used to meet the forcing of primary short waves to be off-resonant before wave breaking, has been considered as an applicable parameter in the infra-gravity wave generation. Since a series of modulating wave groups for different wave conditions are perfonned to proceed with the resonant mechanism of infra- gravity waves prior to wave breaking, the amplitude growth of incident bound long wave is assumed to be simply controlled by the normalized bed slope, fib. The results appear a large dependence of the growth rate, a, of incident bound long wave, separated by the three-array method, on the normalized bed slope, fib. High spatial resolution of wave records enables identification of the cross-correlation between squared short-wave envelopes and infra-gravity waves. The cross- shore structure of infra-gravity waves over beaches presents the mechanics of incident bound- and outgoing free long waves with the formation of free standing long waves in the nearshore region. The wave run-up and amplification of infra-gravity waves in the swash zone appear that the additional long waves generated by the breaking process would modify the cross-shore structure of free standing long waves. Finally, this paper would further discuss the contribution of long wave breaking and bottom friction to the energy dissipation of infra-gravity waves based on different slope conditions.

Hydrogen Peroxide Sensor Based on Horseradish Peroxidase Combined with CaCO_3 Microspheres and Gold Nanoparticles

Direct electrochemistry and electrocatalysis of horseradish peroxidase（HRP） were achieved by entrapping the enzyme between CaCO3 microspheres and gold nanoparticles through forming sandwich configuration （CaCO3-HRP-AuNPs）. Polyanion, poly（styrene sulfonate）（PSS）, was hybrid with CaCO3 microspheres to increase the surface negative charges for binding with HRP through electrostatic interaction. After the bioconjugate CaCO3 PSS-HRP was entrapped in chitosan based sol-gel（CS-GPTMS） film, HRP was encapsulated by in situ formation of an outer layer of AuNPs through electrochemical reduction of HAuCl4. The composite film containing AuNPs, CaCO3-PSS-HRP bioconjugates and CS-GPTMS can provide favorable microenvironment for HRP to perform direct electron transfer at glassy carbon electrode（GCE）. HRP retained its bioelectrocatalytic activity and lead to sensitive and fast amperometric response for the determination of H2O2. H2O2 could be detected in a very wide linear range from 5.0×10-6 mol/L to 7.1×10-2 mol/L. The sandwich configuration of CaCO3-biomolecules-AuNPs could serve as a versatile platform for enzyme immobilization and biosensing.